JP2017095744A - Composition for detecting test substance containing gold nanorod and usage thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a tool for a biosensor having a wide variation of color tones and appropriate for applying to a multicolor analysis, and also to provide a tool for a biosensor with high color tone stability and improved operability.SOLUTION: A composition for detecting a test substance contains a gold nanorod having an aspect ratio (long axis/short axis) of larger than 1.00.SELECTED DRAWING: Figure 15

Description

  The present invention relates to a composition for detecting a test substance, which includes gold nanorods or gold nanorods carrying a specific binding substance for the test substance, and a test substance detection method using them.

Light absorption derived from localized surface plasmon resonance (LSPR) based on noble metal nanoparticles has been applied to advanced biosensing technology, and the absorption wavelength depends on the shape of the nanoparticle and the characteristics of the metal. It depends on. Various metal nanoparticles have been studied in this technical field, and gold nanorods are also attracting attention in the development of highly sensitive biosensors (Patent Document 1, Non-Patent Document 1). Gold nanorods have a maximum absorption wavelength in the visible to near-infrared region, and the position of the maximum absorption wavelength can be manipulated by controlling the shape. Various methods for producing gold nanorods have been studied (Patent Documents 2 to 6, Non-Patent Documents 2 and 3).
On the other hand, spherical gold nanoparticles (or colloidal gold particles) and silver nanoplates are being developed for application to biosensors. Recently, spherical (gold flat sugar-like) gold nanoparticles having protrusions have been developed and applied to immunochromatography (Patent Document 7). Also, silver nanoplates have been studied for application to detection methods such as immunochromatography (Patent Document 8).

Japanese Patent No. 4512876 International Publication No. 2006/066572 Japanese Patent No. 4529160 Japanese Patent No. 4573153 Japanese Patent No. 4636454 Japanese Patent No. 4665499 International Application No. PCT / JP2015 / 065658 Japanese Patent No. 5358648

Cao J, et al., Sensors and Actuators B (2014), Vol. 195, pp. 332-351 Mehtala JG, et al., Langmuir (2014), Vol. 30, No. 46, pp. 13727-13730 Tapan SK, et al., Journal of the American Chemical Society (2004), Vol. 126, pp. 8648-8649

  However, the maximum absorption wavelength of gold nanoparticles is limited to around 530 nm and is not suitable for application to multicolor analysis. Although the above-described gold-peeled gold nanoparticles can adjust the maximum absorption wavelength, further tools for biosensors are required. The gold-coated silver nanoplates can be adjusted for maximum absorption wavelength, but tend to be oxidized in the atmosphere. Color stability of the immunochromatography detection line and a conjugate pad containing a detection reagent are prepared. The resistance to drying was insufficient. Furthermore, in order to centrifuge the gold-coated silver nanoplate, a high rotational speed of about 25000 rpm is required, and the operability is poor. Therefore, an object of the present invention is to provide a biosensor tool that is rich in variations in color tone and suitable for application to multi-color analysis. Another object of the present invention is to provide a biosensor tool having high color tone stability and improved operability.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that gold nanorods are excellent as a biosensor tool, and have completed the present invention.
That is, the present invention provides a composition for detecting a test substance shown below, a method for detecting the test substance, and a kit for detecting the test substance.
[1] A composition for detecting a test substance, comprising gold nanorods,
An aspect ratio (major axis / minor axis) of the gold nanorod is greater than 1.00.
[2] The composition according to [1], wherein the gold nanorod has an aspect ratio (major axis / minor axis) of 3.00 or less.
[3] The composition according to [1] or [2], wherein the gold nanorod has an aspect ratio (major axis / minor axis) of 2.00 or less.
[4] The composition according to any one of [1] to [3], wherein a length of a major axis or a minor axis of the gold nanorod is 5 to 500 nm.
[5] The composition according to any one of [1] to [4], wherein a length of a major axis or a minor axis of the gold nanorod is 10 to 200 nm.
[6] The composition according to any one of [1] to [5], wherein the gold nanorod has a surface area of 118 to 1,170,000 nm ² .
[7] The composition according to any one of [1] to [6], wherein a surface area of the gold nanorod is in a range of 472 to 188,000 nm ² .
[8] The composition according to any one of [1] to [7], wherein a volume of the gold nanorod is in a range of 98.2 to 98,100,000 nm ³ .
[9] The composition according to any one of [1] to [8], wherein a volume of the gold nanorod is in a range of 786 to 6,280,000 nm ³ .
[10] The composition according to any one of [1] to [9], further including a water-soluble polymer.
[11] The composition according to [10], wherein the water-soluble polymer is selected from the group consisting of polystyrene sulfonic acid, polyvinyl pyrrolidone, and thiol-terminated polyethylene glycol.
[12] The composition according to [10] or [11], wherein the water-soluble polymer is polystyrene sulfonic acid.
[13] The composition according to any one of [1] to [12], wherein the gold nanorod carries a specific binding substance for the test substance.
[14] The combination of the test substance and the specific binding substance includes an antigen and an antibody that binds to the antigen, an antibody and an antigen that binds to the antigen, a sugar chain or a complex carbohydrate and a lectin that binds to the lectin, and a sugar chain that binds to the lectin or Complex carbohydrates, hormones or cytokines and receptors that bind to them, receptors and hormones or cytokines that bind to them, proteins and nucleic acid or peptide aptamers that bind to them, enzymes and substrates that bind to them, substrates and enzymes that bind to them, biotin And avidin or streptavidin, avidin or streptavidin and biotin, IgG and protein A or protein G, protein A or protein G and IgG, and a first nucleic acid and a second nucleic acid that binds to it. The composition according to [13] above.
[15] A method for detecting the test substance using the composition according to [13] or [14],
Mixing the composition with the test substance to form a complex of the test substance and a gold nanorod carrying the specific binding substance; and
A method comprising the step of detecting the complex.
[16] Formation of the complex is performed by measuring extinction, absorbance, turbidity, particle size distribution, particle size, Raman scattering, color change, observation of aggregation or precipitation, immunochromatography, The method according to [15], wherein the detection is performed by means selected from the group consisting of electrophoresis and flow cytometry.
[17] The [13] or [14], wherein the gold nanorod in the composition according to any one of the above [1] to [12] is loaded with a specific binding substance for the test substance. The method according to [15] or [16] above, further comprising the step of preparing the composition.
[18] A kit for use in the method according to any one of [15] to [17], comprising the composition according to any one of [1] to [14].

  According to the present invention, the use of gold nanorods having an aspect ratio (major axis / minor axis) of greater than 1.00 can widen variations in color tone. Therefore, it is possible to provide a biosensor tool suitable for application to multicolor analysis. The gold nanorods are stable against oxidation since the material is gold, and since the shape is a rod, there are few protrusions on the surface and there are many flat portions, so that the carrying ability of a specific binding substance such as an antibody is excellent. Furthermore, it has a feature that it is easy to handle because the number of rotations required for centrifugation is small. Therefore, it is possible to provide a biosensor tool having high color stability and improved operability.

The optical characteristic of the aqueous suspension of gold nanorod seed particles is shown. The optical characteristic of the aqueous suspension of gold nanorod A (purple) is shown. A transmission scanning electron microscope (STEM) observation photograph (A) and a scanning electron microscope (SEM) observation photograph (B) of the gold nanorod A (purple) are shown. The optical characteristic of the aqueous suspension of gold nanorods B (cyan) is shown. The STEM observation photograph (A) and SEM observation photograph (B) of gold nanorod B (cyan) are shown. The optical characteristic of the aqueous suspension of gold nanorod C (blue) is shown. The STEM observation photograph (A) and SEM observation photograph (B) of gold nanorod C (blue) are shown. The optical property of the aqueous suspension of the seed particle of a silver nanoplate is shown. The SEM observation photograph of the seed particle of silver nanoplate is shown. The optical characteristic of the aqueous suspension a of a silver nanoplate is shown. The optical characteristic of the water suspension of gold-coated silver nanoplate D is shown. The SEM observation photograph of the gold-coated silver nanoplate D is shown. The optical characteristic of the aqueous suspension of spherical gold nanoparticles is shown. The procedure of an immunochromatographic test is shown. The result of the brightness | luminance analysis of an immunochromatography test is shown. The result of the dry tolerance test after an immunochromatography test is shown.

Hereinafter, the present invention will be described in more detail.
The composition for detecting a test substance of the present invention is characterized by containing gold nanorods having an aspect ratio (major axis / minor axis) of more than 1.00.

“Gold nanorods” described in this specification refers to shape anisotropic gold nanoparticles, such as triangular columns, quadrangular columns, pentagonal columns, hexagonal columns (including shapes with rounded corners) and cylinders. It has a rod-like structure such as The bottom surface may be a flat surface, a convex surface or a concave surface, and the side in the height direction may be a straight line or not a straight line. Further, an index defined by a ratio of the maximum major axis of a particle to a width orthogonal to the maximum major axis (maximum major axis / width orthogonal to the maximum major axis) is referred to as an aspect ratio, which can be used to represent the shape of the particle. . The maximum major axis of the gold nanorod of the present invention corresponds to the height of the gold nanorod, and may be referred to as “major axis” in the present specification. The width orthogonal to the maximum major axis of the gold nanorod corresponds to the diameter of the bottom surface of the gold nanorod, and may be referred to as a “short axis” in the present specification. The gold nanorod has an aspect ratio (major axis / minor axis) greater than 1.00. Since it can be said that the particles whose width orthogonal to the height is not less than the height have a plate-like structure, they are not included in the definition of the gold nanorod of the present invention.
The gold nanorods of the present invention can exhibit a color tone that cannot be achieved with spherical gold nanoparticles by having an aspect ratio (major axis / minor axis) greater than 1.00. The gold nanorod may be a cylinder type in which the major axis and the minor axis are close to each other. Since the long nano gold rod has a maximum absorption wavelength in the visible light region, it can be advantageously used in a detection method for visually determining the presence or absence of a test substance. For example, when the aspect ratio (major axis / minor axis) of the gold nanorod is greater than 1.00 and less than or equal to 3.00, the maximum absorption wavelength can be adjusted in the range of about 530 nm to about 800 nm in the aqueous dispersion. If the aspect ratio (major axis / minor axis) of the gold nanorod is greater than 1.00 and less than or equal to 2.00, the maximum absorption wavelength can be adjusted in the range of about 530 nm to about 670 nm in the aqueous dispersion. it can. The above-mentioned aqueous dispersion of gold nanorods having the maximum absorption wavelength exhibits red, magenta, purple, amber, blue, cyan, and light cyan colors, and the color density is controlled by the concentration of the gold nanorods in the aqueous dispersion. Is possible.
The gold nanorods of the present invention may be prepared in a larger size than conventional gold nanorods. When the size is increased, the absorbance per particle is increased, and gold nanorods having excellent coloring power can be prepared. The length of the major axis or minor axis of the gold nanorod is, for example, 5 to 500 nm, preferably 10 to 200 nm, and more preferably 20 to 100 nm. Further, the surface area and volume of the gold nanorod may be calculated by assuming that the shape of the gold nanorod is a cylinder, assuming that the short axis is the diameter of the bottom circle and the long axis is the height. In this case, the surface area of the gold nanorod may be, for example, 118 to 1,170,000 nm ² , preferably 472 to 188,000 nm ² , more preferably 1,890 to 47,100 nm ² . The volume of the gold nanorods in this case may be, for example, 98.2 to 98,100,000 nm ³ , preferably 786 to 6,280,000 nm ³ , more preferably 6,290 to 785, 000 nm ³ . In addition, gold nanorods having a size in such a range have better dispersion stability than gold nanorods having a size exceeding that range.
The gold nanorod of the present invention can be produced by a known production method adopted in the field of gold nanorods. For example, gold nanorods may be manufactured by the methods described in the above-mentioned Patent Documents 1 to 6 or Non-Patent Documents 1 to 3.

  The composition for detecting a test substance of the present invention contains the gold nanorod. The amount of gold nanorods in the composition may be, for example, 50% by mass or less, preferably 1 to 0.000015% by mass, particularly preferably 0.1 to 0.1% by mass with respect to the total mass of the composition. 0.00015% by mass.

  The composition for detecting a test substance containing the gold nanorod of the present invention can be used for labeling a specific binding substance, that is, a detection reagent, to the test substance. In other words, the gold nanorod of the present invention can carry a specific binding substance for a test substance. The “support” described in the present specification is a complex in which a gold nanorod and a specific binding substance for a test substance are bound to each other regardless of a form such as a covalent bond, a non-covalent bond, or a direct or indirect bond. It means that is formed. As the loading method, a normal loading method can be used without any particular limitation, and physical adsorption, chemical adsorption (covalent bond to the surface), chemical bond (covalent bond, coordinate bond, ionic bond or metal bond), etc. Using this method, the gold nanorods can be directly bonded to a specific binding substance for the test substance, or a part of the water-soluble polymer described below can be bonded to the surface of the gold nanorods, and the end of the water-soluble polymer can be bonded. Alternatively, a method of binding a specific binding substance for the test substance directly or indirectly to the main chain or side chain can be employed. For example, when the specific binding substance for the test substance is an antibody, the gold nanorod of the present invention and the antibody solution are mixed, shaken, and centrifuged to obtain a gold nanorod carrying an antibody (labeled). Detection reagent) can be obtained as a precipitate. In addition, when the gold nanorod of the present invention and an antibody are supported by electrostatic adsorption, the efficiency of supporting the antibody can be improved by coating the surface of the gold nanorod with polystyrene sulfonic acid having a negative charge.

  As a specific binding substance for the test substance of the present invention, a test substance to be detected can be detected by complex formation with the test substance, and gold nanorods can be used as a label. If there is, it can be used without any particular limitation. Specific examples of a combination of a test substance and a specific binding substance for the test substance include an antigen and an antibody that binds to the antigen, a sugar chain or a complex carbohydrate and a lectin that binds to the antigen, a lectin and a sugar chain or complex carbohydrate that binds to the lectin, Hormone or cytokine and receptor that binds to it, receptor and hormone or cytokine that binds to it, protein and nucleic acid or peptide aptamer that binds to it, enzyme and substrate that binds to it, substrate and enzyme that binds to it, biotin and avidin or strepto Examples include avidin, avidin or streptavidin and biotin, IgG and protein A or protein G, protein A or protein G and IgG, or a first nucleic acid and a second nucleic acid that binds (hybridizes) thereto. The second nucleic acid may be a nucleic acid including a sequence complementary to the first nucleic acid.

When the test substance is an antigen, the specific binding substance for the antigen may be an antibody. The antibody may be a polyclonal antibody, a monoclonal antibody, a single chain antibody or a fragment thereof that specifically binds to the antigen, and the fragment includes an F (ab) fragment, an F (ab ′) fragment, It may be an F (ab ′) ₂ fragment or an F (v) fragment. Antigens as test substances may be lectins such as concanavalin A (ConA), malt agglutinin and ricin, and include influenza virus, adenovirus, RS virus, rotavirus, human papilloma virus, human immunodeficiency virus and hepatitis B virus. Such as hepatitis B virus antigen (HBs antigen) or influenza virus hemagglutinin, Aspergillus flavus, chlamydia, syphilis treponema, streptococcus, anthrax, Staphylococcus aureus, Pathogenic microorganisms such as Shigella, Escherichia coli, Salmonella, Salmonella typhi, Paratyphi, Pseudomonas aeruginosa and Vibrio parahaemolyticus, or substances possessed by them (for example, aflatoxins (B1, B2, G1, G, Aspergillus flavus) Or M1), enterotorrhagic Escherichia coli verotoxin or lytic streptridine O), and blood proteins such as immunoglobulin G (IgG), rheumatoid factor and C-reactive protein (CRP), Or a glycoprotein such as mucin, insulin, pituitary hormone (eg, growth hormone (GH), adrenocorticotropic hormone (ACTH), thyroid stimulating hormone (TSH), follicle stimulating hormone (FSH), Luteinizing hormone (LH), prolactin, or melanocyte stimulating hormone (MSH)), thyroid stimulating hormone releasing hormone (TRH), thyroid hormone (eg diiodothyronine or triiodothyronine), chorionic gonadotropin, calcium metabolism Regulatory hormones (eg calcitonin or parathol Pancreatic hormone, gastrointestinal hormone, vasoactive intestinal peptide, follicular hormone (eg, estrone), natural or synthetic luteinizing hormone (eg, progesterone), male hormone (eg, testosterone), and corticosteroid (eg, It may be a hormone such as cortisol, and may be other in vivo substances such as serotonin, urokinase, ferritin, substance P, prostaglandin and cholesterol, prostatic acid phosphatase (PAP), prostate specific antigen (PSA) ), Alkaline phosphatase, transaminase, trypsin, pepsinogen, α-fetoprotein (AFP) and carcinoembryonic antigen (CEA), or a sugar chain antigen such as a blood group antigen. It may be a marker used for detection of fecal occult blood, such as insulin and transferrin, and is a fibrin degradation product in which stabilized fibrin cross-linked by active factor XIII action is degraded by plasmin in the blood coagulation / fibrinolysis system. It may be a kind of D-dimer. When the antigen as the test substance is an in vivo substance such as a hormone or cytokine, the specific binding substance for the in vivo substance may be not only an antibody but also a receptor. Is a sugar chain or a complex carbohydrate having a sugar chain, the specific binding substance for the sugar chain or the complex carbohydrate having a sugar chain may be not only an antibody but also a lectin. In addition, the antigen as the test substance may be a hapten such as penicillin and cadmium.

  When the test substance is an antibody, the specific binding substance for the antibody may be an antigen. The antigen may be the whole antigen or a fragment thereof that specifically binds to the antibody, or a fusion substance in which they are bound to another carrier. The antibody as a test substance may be an autoantibody such as an anti-cyclic citrullinated peptide (CCP) antibody or an antiphospholipid antibody, and is an antibody against a foreign antigen such as an anti-chlamydia antibody, an anti-HIV antibody or an anti-HCV antibody. May be.

  When the test substance is a sugar chain, the specific binding substance for the sugar chain may be a lectin. The lectin may be a galectin, a C-type lectin, a legume lectin or a fragment thereof that specifically binds to the sugar chain. The sugar chain as the test substance may be a monosaccharide or a polysaccharide, and may be a complex carbohydrate in which they are bound to a protein or lipid. For example, when the test substance is a sugar chain containing mannose, a leguminous lectin concanavalin A (ConA) can be used as a specific binding substance for the sugar chain.

  When the test substance is a lectin, the specific binding substance for the lectin may be a sugar chain. The sugar chain may be a monosaccharide, polysaccharide, complex carbohydrate or a fusion substance in which they are bound to another carrier that specifically binds to the lectin. The lectin as the test substance may be a galectin, a C-type lectin or a legume lectin. For example, when the test substance is leguminous lectin concanavalin A (ConA), a sugar chain containing mannose can be used as a specific binding substance for the lectin.

  When the combination of the test substance and the specific binding substance for the test substance is a protein and a nucleic acid aptamer or peptide aptamer that binds to the protein, the nucleic acid aptamer is, for example, Bacillus anthracis, Staphylococcus aureus ), D group Shigella sonnei, Escherichia coli, Salmonella typhimurium, Streptococcus hemoliticus, Salcto trophium (S) Pseudomonas aeruginosa (Pseu DNA aptamer that binds to bacteria such as Omonas aeruginosa), Vibrio parahaemolyticus, tumor markers appearing on the surface of epithelial cells such as mucin 1, or enzymes such as β-galactosidase and thrombin, or human immunodeficiency virus It may be an RNA aptamer that binds to Tat protein or Rev protein, and the peptide aptamer may be, for example, a peptide aptamer that binds to human papillomavirus (HPV) oncoprotein HPV16 E6.

When the test substance is a vitamin, the specific binding substance for the vitamin may be a vitamin-binding protein such as transcalciferin that binds vitamin D and transcobalamin that binds vitamin B12. When the test substance is an antibiotic, the specific binding substance for the antibiotic may be a penicillin-binding protein such as PBP1 and PBP2 that binds to penicillin.
The nucleic acid as the test substance or the substance that binds to the test substance may be, for example, DNA, RNA, oligonucleotide, polynucleotide, or an amplification product thereof.

The gold nanorod carrying a specific binding substance for the test substance of the present invention can bind to the corresponding test substance and detect it. The method for detecting a test substance of the present invention comprises the step of mixing the composition of the present invention with a test substance to form a complex of the test substance and a gold nanorod carrying a specific binding substance for the test substance, And a step of detecting the complex. The gold nanorod carrying a specific binding substance for the test substance of the present invention may be prepared immediately before carrying out the method for detecting the test substance of the present invention. In other words, the method for detecting a test substance of the present invention may further include a step of causing a gold nanorod to carry a specific binding substance for the test substance.
The complex can be detected by using a means usually used in the field of detection of a test substance or a means usually used for detecting an aggregate or a precipitate without particular limitation. For example, the formation of the complex may be performed by quenching measurement, absorbance measurement, turbidity measurement, particle size distribution measurement, particle size measurement, Raman scattered light measurement, color change observation, aggregation or precipitation formation observation, immunochromatography method, Detection may be by means selected from the group consisting of electrophoresis and flow cytometry.

Absorbance refers to the intensity of light absorption of a parallel light beam that passes through the object (absorbance in a narrow sense). In actual measurement, light loss due to reflection or scattering on the surface of the object is also considered. (Absorbance in a broad sense). Absorbance in a broad sense, which means the intensity of light loss due to all factors such as light absorption, reflection and scattering, is specifically referred to herein as extinction. When a complex of a test substance and a gold nanorod carrying a specific binding substance for the test substance is formed, the quenching degree or absorbance in the wavelength region around the maximum absorption wavelength of the gold nanorod is reduced.
Extinction measurement or absorbance measurement may be performed in a wavelength region in the range of 200 to 2500 nm, or may be performed in the maximum absorption wavelength of gold nanorods in the range of 430 to 2000 nm. The extinction degree or absorbance may be measured by an ultraviolet-visible spectrophotometer (for example, UV-visible near-infrared spectrophotometer MPC3100UV-3100PC, Shimadzu Corporation) that is usually used for these measurements.

When a complex of a test substance and a gold nanorod carrying a specific binding substance for the test substance is formed, the gold nanorods are cross-linked via the test substance, and the particle diameter becomes larger than that of a single gold nanorod. As the particle size of the particles dispersed in the composition of the present invention increases, the absorption, reflection or scattering of light incident on the composition increases, and the composition exhibits turbidity. Absorption, reflection, or scattering of light in a composition exhibiting turbidity can be measured as extinction or absorbance, and the extinction or absorbance that increases depending on the degree of turbidity is also referred to as turbidity.
In the composition of the present invention, when a plurality of particles are present adjacent to each other by forming a complex of a test substance and a gold nanorod carrying a specific binding substance for the test substance, a dipole-dipole mutual Due to the action, a wavelength region in which the extinction degree or absorbance increases depending on the formation of the complex exists on the longer wavelength side than the maximum absorption wavelength of the gold nanorod (Nano Lett., Vol. 4, No. 9). , (2004), p.1627-1631). The complex may be detected by measuring the extinction degree or absorbance in this wavelength region as turbidity. For example, when the maximum absorption wavelength of the gold nanorod is 560 nm, turbidity measurement may be performed in the wavelength region of 660 to 840 nm, and when the maximum absorption wavelength of the gold nanorod is 620 nm, the 720 to 900 nm Turbidity measurement may be performed in the wavelength region. The turbidity may be measured by an ultraviolet-visible spectrophotometer (for example, UV-visible near-infrared spectrophotometer MPC3100UV-3100PC, Shimadzu Corporation) or a turbidimeter that is usually used for the measurement.

In the immunochromatography method, gold nanorods gather at the detection site (line shape). At this time, when the gold nanorod absorbs light in the visible range, the formation of the complex can be recognized as a color. The result of this test can be quantified by visual judgment and luminance difference analysis. In the visual determination, the presence or absence of the detection line after the immunochromatographic test is visually confirmed, and the lowest test substance concentration at which the detection line can be confirmed can be set as the detection sensitivity. In the luminance analysis, the lowest test substance concentration when the absolute value of the value obtained by subtracting the luminance difference 2 from the following luminance difference 1 is 2 (detection limit luminance difference) or more can be used as the detection sensitivity.
1. 1. Difference in luminance between a detection line and a portion other than the detection line (control region) in an immunochromatographic test using a developing solution not containing a test substance. Luminance difference between the detection line on the immunochromatographic carrier and the part other than the detection line (control area) during the immunochromatographic test using a developing solution containing the test substance. Also, in the immunochromatographic test using gold nanorods that absorb near-infrared light The detection can be confirmed by irradiating the detection part with near infrared light after the test and measuring the absorbance at that time.

  A kit for use in the method for detecting a test substance of the present invention comprises the composition of the present invention. The kit of the present invention may further include a standard test substance, an immunochromatographic test paper, or an instruction manual describing the method of use.

The composition of the present invention may further contain a water-soluble polymer. The “water-soluble polymer” described in the present specification refers to a water-soluble substance having a molecular weight of 500 to 1,000,000, preferably 500 to 100,000. “Water-soluble” described in the present specification means that the polymer is dissolved in water by 0.001% by mass or more under normal temperature and normal pressure. Examples of the water-soluble polymer include polystyrene sulfonic acid, polyvinyl pyrrolidone (PVP), polyethylene glycol, polyacrylamide, polyvinyl alcohol, polyacrylic acid, polymethacrylic acid, polyallylamine, dextran, polymethacrylamide, polyvinylphenol, poly Vinyl benzoate, bovine serum albumin (BSA), casein, or bis (p-sulfonatophenyl) phenylphosphine may be used, and these include a hydroxyl group that is a functional group chemically bonded to the gold nanorod of the present invention, It may be modified with a mercapto group, disulfide group, amino group, carboxyl group or the like. Preferably, the water-soluble polymer is polystyrene sulfonic acid, PVP, or thiol-terminated polyethylene glycol. You may use the commercially available dispersing agent used for the paint, the ink, etc. containing these water-soluble polymers as a water-soluble polymer mix | blended with the composition of this invention.
The kind of water-soluble polymer contained in the composition of the present invention can be selected according to the use of the composition. For example, when used for biological experiments or cell experiments, polyvinyl pyrrolidone, polyethylene glycol, polyvinyl alcohol, or the like having good biocompatibility may be used.
When the composition of the present invention contains a water-soluble polymer, the concentration of the water-soluble polymer dissolved or dispersed in the composition (number of moles of water-soluble polymer per liter of the composition) is: For example, it is 50 μM or less, preferably 25 μM or less, particularly preferably 10 μM or less.
Although this invention is not restrained by a specific theory, when a water-soluble polymer is mix | blended with the composition of this invention, the dispersion stability of a gold nanorod will improve in the said composition. In addition, when the concentration of the water-soluble polymer is 50 μM or less, for example, the specific binding substance for the test substance is favorably supported on the gold nanorod compared to the case where the concentration exceeds 50 μM, or Since the gold nanorod carrying the specific binding substance for the test substance forms a stable complex with the test substance, the detection sensitivity of the test substance can be increased as a result.

Examples of methods for measuring the concentration of water-soluble polymers include nuclear magnetic resonance spectroscopy (NMR), size exclusion chromatography (SEC), gel filtration chromatography (GPC), and suggested thermo / thermogravimetry (TG-DTA). It is done.
In the case of NMR, the composition of the present invention is dried, and the obtained solid content is dissolved (dispersed) in a heavy solvent and measured. Add an internal standard substance (eg, maleic acid) of known concentration to the heavy solvent in advance, and compare the integrated value of the signal derived from the water-soluble polymer and the integrated value of the signal derived from the internal standard substance in the obtained spectrum. Thus, the concentration of the water-soluble polymer can be calculated.
In the case of GPC, a plurality of water-soluble polymer aqueous solutions having a known concentration are first analyzed, and a calibration curve is created from the peak area derived from the water-soluble polymer in the obtained chart. Next, the composition of the present invention is measured, and the concentration of the water-soluble polymer can be calculated from the peak area derived from the water-soluble polymer in the obtained chart.
Furthermore, in the case of TG-DTA, the water-soluble high molecular weight can be calculated from the weight change when the dry solid content of the composition of the present invention is measured.

  The composition of the present invention may contain any component as long as it does not adversely affect the gold nanorods. Examples of optional components include reagents used in producing gold nanorods (for example, sodium borohydride and ascorbic acid), dispersants (for example, trisodium citrate, hexadecyltrimethylammonium bromide), and the like. .

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, the scope of the present invention is not limited to these Examples.

Example 1
(1) Production of gold nanorod seed particle aqueous suspension 30 g of 0.1 mM hexadecyltrimethylammonium bromide (CTAB) aqueous solution, 1 g of 0.01 M chloroauric acid aqueous solution, 0.01 M sodium borohydride aqueous solution 4 g was added while stirring. The obtained solution was allowed to stand in an incubator (30 ° C.) for 120 minutes to prepare an aqueous suspension of seed particles of gold nanoparticles. The optical characteristics of the prepared water suspension (stock solution) are shown in FIG. The optical properties were measured using an ultraviolet-visible near-infrared spectrophotometer MPC3100UV-3100PC manufactured by Shimadzu Corporation under the conditions of optical path length: 1 cm and measurement wavelength: 190-1300 nm.

(2) Production of gold nanorod A (color tone: purple)
To 238 g of 0.05 M CTAB aqueous solution, 10 g of 0.01 M chloroauric acid aqueous solution, 1.5 g of 0.01 M silver nitrate aqueous solution and 1.6 g of 0.1 M L-ascorbic acid aqueous solution were added with stirring. Furthermore, 0.011 g of a seed particle aqueous suspension of gold nanorods prepared in (1) was added. The obtained solution was allowed to stand in an incubator (30 ° C.) for 100 hours to prepare an aqueous suspension of gold nanorods A. The aqueous suspension of gold nanorods A was purple. FIG. 2 shows the optical characteristics of an aqueous suspension obtained by diluting the prepared suspension with ultrapure water to 4 volumes. The wavelength exhibiting the maximum absorption was 596 nm (extinction degree 0.66). The optical properties were measured using an ultraviolet-visible near-infrared spectrophotometer MPC3100UV-3100PC manufactured by Shimadzu Corporation under the conditions of optical path length: 1 cm and measurement wavelength: 190-1300 nm. When the gold nanorod A in the gold nanorod A aqueous suspension was observed by SEM, the average minor axis length of the gold nanorod was 61 nm, the average major axis length was 75 nm, and the aspect ratio was 1.23. Further, when the gold nanorod A is regarded as a cylinder (the short axis is the diameter of the bottom circle), the surface area is about 20,218 nm ² and the volume is about 219,185 nm ³ . FIG. 3 shows the results of observation of the gold nanorod A with a transmission scanning electron microscope (STEM) or a scanning electron microscope (SEM) using an ultrahigh resolution analytical scanning electron microscope SU-70 (manufactured by Hitachi, Ltd.).

(3) Preparation of PSS-coated gold nanorod A The aqueous suspension of gold nanorod A prepared in (2) is centrifuged (2,000 × g, 30 ° C., 10 minutes) to precipitate gold nanorod A. , 2.9 mL of the supernatant was removed. Thereafter, the gold nanorod A at the bottom of the centrifuge tube was redispersed with 2.9 mL of a 0.14 wt% poly (sodium-p-styrenesulphonate) (PSS) aqueous solution to prepare a PSS-coated gold nanorod intermediate a1 aqueous suspension. . The PSS-coated gold nanorod intermediate a1 aqueous suspension was centrifuged (2,000 × g, 30 ° C., 10 minutes) to precipitate the PSS-coated gold nanorod intermediate a1, and 2.9 mL of the supernatant was removed. Thereafter, the PSS-coated gold nanorod intermediate a1 at the bottom of the centrifuge tube was redispersed by adding 2.9 mL of a 0.14 wt% PSS aqueous solution to prepare a PSS-coated gold nanorod intermediate a2 aqueous suspension. The PSS-coated gold nanorod intermediate a2 aqueous suspension was centrifuged (2,000 × g, 30 ° C., 10 minutes) to precipitate the PSS-coated gold nanorod intermediate a2, and 2.9 mL of the supernatant was removed. Thereafter, the PSS-coated gold nanorod intermediate a2 was redispersed by adding 2.9 mL of a 0.14 wt% PSS aqueous solution to prepare a PSS-coated gold nanorod A aqueous suspension. The pH of the PSS-coated gold nanorod A suspension (hereinafter, suspension A) was 6.3 at room temperature (20 ° C.). For the pH measurement, a twin pH meter B-212 (glass electrode method) manufactured by Horiba, Ltd. was used.
The color tone of the 4-fold diluted solution of Suspension A was purple.
The gold content of the suspension A was 0.0079% by mass relative to the total mass of the suspension.

Example 2
(1) Production of gold nanorod B (color tone: cyan)
To 238 g of 0.05 M CTAB aqueous solution, 10 g of 0.01 M chloroauric acid aqueous solution, 1.5 g of 0.01 M silver nitrate aqueous solution and 1.6 g of 0.1 M L-ascorbic acid aqueous solution were added with stirring. Furthermore, 0.105 g of a seed particle aqueous suspension of gold nanorods prepared in (1) of Example 1 was added. The resulting solution was allowed to stand in an incubator (30 ° C.) for 100 hours to prepare an aqueous suspension of gold nanorods B. The aqueous suspension of gold nanorod B exhibited cyan. FIG. 4 shows the optical characteristics of an aqueous suspension obtained by diluting the prepared suspension with ultrapure water to 4 volumes. The wavelength exhibiting the maximum absorption was 620 nm (extinction degree 0.57). The optical properties were measured using an ultraviolet-visible near-infrared spectrophotometer MPC3100UV-3100PC manufactured by Shimadzu Corporation under the conditions of optical path length: 1 cm and measurement wavelength: 190-1300 nm. When the gold nanorod B in the gold nanorod B aqueous suspension was observed by SEM, the average minor axis length of the gold nanorod B was 34 nm, the average major axis length was 55 nm, and the aspect ratio was 1.62. Further, when the gold nanorod B is considered to be cylindrical (the short axis is the diameter of the bottom circle), the surface area is about 7,691 nm ² and the volume is about 49,936 nm ³ . FIG. 5 shows the results of observation of the gold nanorod B with a transmission scanning electron microscope (STEM) or a scanning electron microscope (SEM) using an ultrahigh resolution analytical scanning electron microscope SU-70 (manufactured by Hitachi, Ltd.).

(2) Production of PSS-coated gold nanorods B Centrifugation (2,000 × g, 30 ° C., 10 minutes) of 3 mL of the aqueous suspension of gold nanorods B produced in (1) to precipitate the gold nanorods B , 2.9 mL of the supernatant was removed. Thereafter, the gold nanorod B at the bottom of the centrifuge tube was redispersed with 2.9 mL of a 0.14 wt% poly (sodium-p-styrenesulphonate) (PSS) aqueous solution to prepare a PSS-coated gold nanorod intermediate b1 aqueous suspension. . The PSS-coated gold nanorod intermediate b1 aqueous suspension was centrifuged (2,000 × g, 30 ° C., 10 minutes) to precipitate the PSS-coated gold nanorod intermediate b1, and 2.9 mL of the supernatant was removed. Thereafter, PSS-coated gold nanorod intermediate b1 at the bottom of the centrifuge tube was redispersed by adding 2.9 mL of a 0.14 wt% PSS aqueous solution to prepare an aqueous suspension of PSS-coated gold nanorod intermediate b2. The PSS-coated gold nanorod intermediate b2 aqueous suspension was centrifuged (2,000 × g, 30 ° C., 10 minutes) to precipitate the PSS-coated gold nanorod intermediate b2, and 2.9 mL of the supernatant was removed. Thereafter, 2.9 mL of a 0.14 wt% PSS aqueous solution was added and redispersed in the PSS-coated gold nanorod intermediate b2 to prepare a PSS-coated gold nanorod B aqueous suspension. The pH 7.0 of the PSS-coated gold nanorod B suspension (hereinafter, suspension B) was room temperature (20 ° C.). For the pH measurement, a twin pH meter B-212 (glass electrode method) manufactured by Horiba, Ltd. was used.
The color tone of the 4-fold diluted solution of Suspension B was cyan.
The gold content of the suspension B was 0.0079% by mass relative to the total mass of the suspension.

Example 3
(1) Production of gold nanorod C (color tone: blue)
To 238 g of 0.05 M CTAB aqueous solution, 10 g of 0.01 M chloroauric acid aqueous solution, 1.5 g of 0.01 M silver nitrate aqueous solution and 1.6 g of 0.1 M L-ascorbic acid aqueous solution were added with stirring. Further, 0.053 g of a seed nanoparticle aqueous suspension of gold nanorods prepared in (1) of Example 1 was added. The obtained solution was allowed to stand for 100 hours in an incubator (30 ° C.) to prepare an aqueous suspension of gold nanorods C. The aqueous suspension of gold nanorods C was blue. FIG. 6 shows the optical characteristics of a water suspension obtained by diluting the prepared suspension with ultrapure water to 4 volumes. The wavelength exhibiting the maximum absorption was 610 nm (extinction degree 0.59). The optical properties were measured using an ultraviolet-visible near-infrared spectrophotometer MPC3100UV-3100PC manufactured by Shimadzu Corporation under the conditions of optical path length: 1 cm and measurement wavelength: 190-1300 nm. When the gold nanorod C in the gold nanorod C aqueous suspension was observed by SEM, the average minor axis length of the gold nanorod was 39 nm, the average major axis length was 58 nm, and the aspect ratio was 1.49. Further, when the gold nanorod C is regarded as a cylinder (the short axis is the diameter of the bottom circle), the surface area is about 9,495 nm ² and the volume is about 69,286 nm ³ . FIG. 7 shows the results of observation of the gold nanorod C by transmission scanning electron microscope (STEM) or scanning electron microscope (SEM) with an ultrahigh resolution analytical scanning electron microscope SU-70 (manufactured by Hitachi, Ltd.).

(2) Production of PSS-coated gold nanorods C 3 mL of the aqueous suspension of gold nanorods C produced in (1) was centrifuged (2,000 × g, 30 ° C., 10 minutes) to precipitate the gold nanorods C. , 2.9 mL of the supernatant was removed. Thereafter, the gold nanorod C at the bottom of the centrifuge tube was redispersed with 2.9 mL of a 0.14 wt% poly (sodium-p-styrenesulphonate) (PSS) aqueous solution to prepare a PSS-coated gold nanorod intermediate c1 aqueous suspension. . The PSS-coated gold nanorod intermediate c1 aqueous suspension was centrifuged (2,000 × g, 30 ° C., 10 minutes) to precipitate the PSS-coated gold nanorod intermediate c1, and 2.9 mL of the supernatant was removed. Thereafter, PSS-coated gold nanorod intermediate c1 at the bottom of the centrifuge tube was redispersed by adding 2.9 mL of a 0.14 wt% PSS aqueous solution to prepare a PSS-coated gold nanorod intermediate c2 aqueous suspension. The PSS-coated gold nanorod intermediate c2 aqueous suspension was centrifuged (2,000 × g, 30 ° C., 10 minutes) to precipitate the PSS-coated gold nanorod intermediate c2, and 2.9 mL of the supernatant was removed. Thereafter, 2.9 mL of a 0.14 wt% PSS aqueous solution was added and redispersed in the PSS-coated gold nanorod intermediate c2 to prepare a PSS-coated gold nanorod C aqueous suspension. The pH 7.0 of the PSS-coated gold nanorod C suspension (hereinafter referred to as suspension C) was room temperature (20 ° C.). For the pH measurement, a twin pH meter B-212 (glass electrode method) manufactured by Horiba, Ltd. was used.
The color tone of the 4-fold diluted solution of Suspension C was blue.
The gold content of the suspension C was 0.0079% by mass relative to the total mass of the suspension.

Comparative Example 1
(1) Preparation of seed particles of silver nanoplate 20 mL of 2.5 mM trisodium citrate aqueous solution, 1 mL of 0.5 g / L 70,000 molecular weight polystyrene sulfonic acid aqueous solution and 1.2 mL of 10 mM sodium borohydride aqueous solution Then, 50 mL of 0.5 mM aqueous silver nitrate solution was added with stirring at 20 mL / min. The obtained solution was allowed to stand for 60 minutes in an incubator (30 ° C.) to prepare an aqueous suspension of silver nanoplate seed particles. The optical characteristics of the prepared water suspension (stock solution) are shown in FIG. The optical properties were measured using an ultraviolet-visible near-infrared spectrophotometer MPC3100UV-3100PC manufactured by Shimadzu Corporation under the conditions of optical path length: 1 cm and measurement wavelength: 190-1300 nm. The wavelength exhibiting the maximum absorption was 396 nm (extinction degree 3.3), which is light absorption derived from LSPR of spherical silver nanoparticles. In addition, the extinction degree of this invention is the value of the light absorbency when measuring a suspension with a spectrophotometer. Moreover, the result of having performed the scanning electron microscope (SEM) observation with ultra high resolution analytical scanning electron microscope SU-70 (made by Hitachi, Ltd.) is shown in FIG. The particle diameter was mainly plate-like particles having a size of 3 nm or more and less than 10 nm.

(2) Preparation of silver nanoplate To 200 mL of ultrapure water, 4.5 mL of 10 mM ascorbic acid aqueous solution was added, and 4 mL of silver nanoplate seed particle aqueous suspension prepared in (1) was further added. To the resulting solution, 120 mL of 0.5 mM aqueous silver nitrate solution was added with stirring at 30 mL / min. Stirring was stopped 4 minutes after the addition of the aqueous silver nitrate solution, 20 mL of a 25 mM trisodium citrate aqueous solution was added, and the resulting solution was allowed to stand for 100 hours in an incubator (30 ° C.) under atmospheric conditions. A nanoplate aqueous suspension a was prepared. FIG. 10 shows the optical characteristics of an aqueous suspension obtained by diluting the prepared suspension with ultrapure water to 4 volumes. The wavelength exhibiting the maximum absorption was 526 nm (extinction degree 1.2). The optical properties were measured using an ultraviolet-visible near-infrared spectrophotometer MPC3100UV-3100PC manufactured by Shimadzu Corporation under the conditions of optical path length: 1 cm and measurement wavelength: 190-1300 nm. When the silver nanoplate in the water suspension a was observed by SEM, the average particle diameter of the silver nanoplate was 31 nm, the average thickness was 8 nm, and the aspect ratio was 3.8. For the analysis of the SEM observation photograph, an ultra-high resolution analytical scanning electron microscope SU-70 manufactured by Hitachi, Ltd. was used.

(3) Preparation of gold-coated silver nanoplate D (color tone: magenta)
9.1 mL of a 0.125 mM polyvinylpyrrolidone (PVP) (molecular weight: 40,000) aqueous solution was added to 120 mL of the silver nanoplate aqueous suspension prepared in (2), and a 0.5 M ascorbic acid aqueous solution 1. After adding 6 mL, 9.6 mL of 0.14 mM chloroauric acid aqueous solution was added at 0.5 mL / min with stirring. The obtained solution was allowed to stand in an incubator (30 ° C.) for 24 hours to prepare an aqueous suspension of gold-coated silver nanoplate D (hereinafter, suspension d) in which the surface of the silver nanoplate was coated with gold. did.
The main dispersion medium of the suspension d was water.
The gold-coated silver nanoplate D contained in the suspension d is a mixture of polygonal and circular plates including a triangular shape with an average maximum length (particle diameter) of the main surface of 31 nm, and an average thickness. Was 8 nm. Further, the average gold thickness in the gold-coated silver nanoplate D contained in the suspension d was 0.30 nm.
The pH of the suspension d was 4.0 at room temperature (20 ° C.). For the pH measurement, a twin pH meter B-212 (glass electrode method) manufactured by Horiba, Ltd. was used.
The silver content of the suspension d was 0.0016% by mass relative to the total mass of the suspension.

(4) pH adjustment of suspension d To 1.95 mL of suspension d obtained in (3), 200 mL of PBS buffer 0.05 mL and 190 mM sodium carbonate aqueous solution 0.025 mL were added with stirring. A pH-adjusted gold-coated nanoplate D suspension (hereinafter, suspension D) was prepared. FIG. 11 shows the optical characteristics of an aqueous suspension obtained by diluting the prepared suspension with ultrapure water to a volume of 3 times. The wavelength exhibiting the maximum absorption was 531 nm (extinction degree 1.2). The optical properties were measured using an ultraviolet-visible near-infrared spectrophotometer MPC3100UV-3100PC manufactured by Shimadzu Corporation under the conditions of optical path length: 1 cm and measurement wavelength: 190-1300 nm.
The main dispersion medium of Suspension D was water.
The gold-coated silver nanoplate contained in the suspension D is a mixture of a polygonal plate including a triangular shape and a circular plate, the average of the maximum length of the main surface is 31 nm, and the average thickness Was 8 nm. Further, the surface area was 2,289 nm ² and the volume was 6,038 nm ³ on the assumption that all of the gold-coated silver nanoplates were circular plates.
In addition, the average gold thickness in the gold-coated silver nanoplate contained in the suspension D was 0.30 nm. FIG. 12 shows the results of scanning electron microscope (SEM) observation using an ultra-high resolution analytical scanning electron microscope SU-70 (manufactured by Hitachi, Ltd.).
The color tone of the 3-fold diluted solution of Suspension D was magenta.
The pH of the suspension D was 7.3 at room temperature (20 ° C.).
The silver content of the suspension D was 0.0015% by mass with respect to the total mass of the suspension.

Comparative Example 2
(1) Preparation of PVP-stabilized spherical gold nanoparticle suspension (suspension e) 0.125 mM polyvinylpyrrolidone (PVP) in 12 mL of commercially available spherical gold nanoparticles (particle diameter: 40 nm, concentration: 0.0068 wt%) ) (Molecular weight: 40,000) aqueous solution 0.91 mL was added with stirring. The obtained solution was allowed to stand in an incubator (30 ° C.) for 24 hours to prepare a PVP-stabilized spherical gold nanoparticle suspension (hereinafter referred to as suspension e).

(2) Adjusting pH of suspension e To 1.95 mL of suspension e obtained in (1), 0.05 mL of 200 mM PBS buffer and 0.025 mL of 190 mM sodium carbonate aqueous solution were added with stirring. A pH-adjusted PVP-stabilized spherical gold nanoparticle suspension E (hereinafter, suspension E) was prepared. FIG. 13 shows the optical characteristics of an aqueous suspension obtained by diluting the prepared suspension to 3.8 volumes with ultrapure water. The wavelength exhibiting the maximum absorption was 522 nm (extinction degree 0.38). The optical properties were measured using an ultraviolet-visible near-infrared spectrophotometer MPC3100UV-3100PC manufactured by Shimadzu Corporation under the conditions of optical path length: 1 cm and measurement wavelength: 190-1300 nm.
The main dispersion medium of Suspension E was water.
The spherical gold nanoparticles contained in the suspension E had an average particle size of 40 nm.
The color tone of the suspension E was red.
The pH of Suspension E was 7.0 at room temperature (20 ° C.).
The gold content of the suspension E was 0.0061% by mass with respect to the total mass of the suspension.

Test example 1
The test results of the suspensions of Examples and Comparative Examples were evaluated using an immunochromatographic test of the following procedure.
(1) Preparation of developing solution used for immunochromatographic test (support of specific binding substance on PSS-coated gold nanorods (labeling of detection reagent))
Solution of antibody (product name: Goat anti HBsAg, manufacturer: Arista Biologicals, Inc.) (detection antibody in immunochromatography) to hepatitis B virus antigen (HBs antigen) at a concentration of 50 μg / mL in 1 mM PBS (−) buffer 0.2 mL and 1.8 mL of PSS-coated gold nanorod suspension (Suspension A, B, or C) were mixed, and the resulting mixture was shaken at room temperature for 60 minutes. Next, 0.044 mL of a solution of SUNBRIGHT ME-020SH (molecular weight: 2,000, manufactured by NOF CORPORATION), which is a polyethylene glycol modified with a thiol group at a concentration of 0.8 mM, in 1 mM PBS (−) buffer was added. And the resulting suspension was shaken at room temperature for 30 minutes. Next, 0.100 mL of a solution of 0.5 mM bovine serum albumin (product name: Bovine Serum Albumin, abbreviation: BSA, molecular weight: 66 kDa, manufacturer: Sigma-Aldrich) in 1 mM PBS (−) buffer was added, The resulting suspension was shaken for 30 minutes at room temperature. Subsequently, centrifugation (2,000 × g, 30 ° C., 10 minutes) was performed to precipitate the antibody-PSS-coated gold nanorod complex, and 1.90 mL of the supernatant was removed. Thereafter, the antibody-PSS-coated gold nanorod complex was redispersed by adding 0.40 mL of 1 mM PBS (−) buffer containing 4.9 μM BSA, and UV-visible near-infrared manufactured by Shimadzu Corporation. A spectrophotometer MPC3100UV-3100PC was used to adjust the extinction degree to 2.0 to prepare a developing solution.

Table 1 below shows the relationship between the PSS-coated gold nanorod suspension used, the produced developing solution, and the color tone thereof.

(2) Preparation of developing solution used for immunochromatographic test (support of specific binding substance on gold-coated silver nanoplate (labeling of detection reagent))
Solution of antibody (product name: Goat anti HBsAg, manufacturer: Arista Biologicals, Inc.) (detection antibody in immunochromatography) to hepatitis B virus antigen (HBs antigen) at a concentration of 50 μg / mL in 1 mM PBS (−) buffer 0.2 mL and 1.8 mL of gold-coated silver nanoplate suspension D were mixed, and the resulting mixture was shaken at room temperature for 60 minutes. Next, 0.044 mL of a solution of SUNBRIGHT ME-020SH (molecular weight: 2,000, manufactured by NOF CORPORATION), which is a polyethylene glycol modified with a thiol group at a concentration of 0.8 mM, in 1 mM PBS (−) buffer was added. And the resulting suspension was shaken at room temperature for 30 minutes. Next, 0.100 mL of a solution of 0.5 mM bovine serum albumin (product name: Bovine Serum Albumin, abbreviation: BSA, molecular weight: 66 kDa, manufacturer: Sigma-Aldrich) in 1 mM PBS (−) buffer was added, The resulting suspension was shaken for 30 minutes at room temperature. Subsequently, centrifugation (26,000 × g, 30 ° C., 10 minutes) was performed to precipitate the antibody-gold-coated silver nanoplate complex, and 1.90 mL of the supernatant was removed. Thereafter, the antibody-gold-coated silver nanoplate complex was redispersed by adding 1.90 mL of 1 mM PBS (−) buffer containing 4.9 μM BSA, and UV-visible near-red, manufactured by Shimadzu Corporation. Using an outer spectrophotometer MPC3100UV-3100PC, the extinction degree was adjusted to 2.0, and a developing solution D (color tone: magenta) was produced.

(3) Preparation of developing solution used for immunochromatographic test (support of specific binding substance on spherical gold nanoparticles (labeling of detection reagent))
Solution of antibody (product name: Goat anti HBsAg, manufacturer: Arista Biologicals, Inc.) (detection antibody in immunochromatography) to hepatitis B virus antigen (HBs antigen) at a concentration of 50 μg / mL in 1 mM PBS (−) buffer 0.2 mL and 1.8 mL of spherical gold nanoparticle PVP stabilization suspension E were mixed, and the resulting mixture was shaken at room temperature for 60 minutes. Next, 0.044 mL of a solution of SUNBRIGHT ME-020SH (molecular weight: 2,000, manufactured by NOF CORPORATION), which is a polyethylene glycol modified with a thiol group at a concentration of 0.8 mM, in 1 mM PBS (−) buffer was added. And the resulting suspension was shaken at room temperature for 30 minutes. Next, 0.100 mL of a solution of 0.5 mM bovine serum albumin (product name: Bovine Serum Albumin, abbreviation: BSA, molecular weight: 66 kDa, manufacturer: Sigma-Aldrich) in 1 mM PBS (−) buffer was added, The resulting suspension was shaken for 30 minutes at room temperature. Subsequently, centrifugation (4200 × g, 30 ° C., 10 minutes) was performed to precipitate the antibody-spherical gold nanoparticle complex, and 1.90 mL of the supernatant was removed. Thereafter, the antibody-spherical gold nanoparticle complex was redispersed by adding 1.90 mL of 1 mM PBS (−) buffer containing 4.9 μM BSA, and UV-visible near-infrared manufactured by Shimadzu Corporation. A spectrophotometer MPC3100UV-3100PC was used to adjust the extinction degree to 2.0, and a developing solution E (color tone: red) was produced.

(4) Immunochromatographic test According to the procedure shown in FIG. 14, an immunochromatographic test using hepatitis B virus antigen (HBs antigen) as a test substance was immobilized on the anti-HBs antibody (capture antibody) in a straight line (detection line in FIG. 14). The immunochromatographic test paper was used.
The immunochromatographic test paper used was purchased from a contract manufacturing company for immunochromatographic test paper (Biodevice Technology Co., Ltd.). When the capture antibody was fixed linearly, a capture antibody adjusted to a concentration of 1 g / mL with 5 mM PBS (−) buffer was used.
The first developing solution (the developing solution used in FIG. 14A) is a solution of HBs antigen (product name: HBsAg Protein (Subtype adr), manufacturer: Fitzgerald Industries International Inc.) in 1 mM PBS (−) buffer. there were. As the first developing solution, solutions having HBs antigen concentrations of 6.0 μM, 0.6 μM, 0.06 μM, 0.006 μM, and 0M (blank) were prepared.
The second developing solution was 1 mM PBS (−) buffer.
The third developing solution (the developing solution used in FIG. 14C) was the developing solution A described above.
The specific test procedure was as follows.
On the immunochromatographic test paper, 15 μL of the first developing solution was developed (FIG. 14A). By developing the first developing solution, the HBs antigen is captured by the capture antibody immobilized on the detection line of the test paper (FIG. 14B).
Next, 30 μL of the second developing solution was developed, and the excess antigen on the immunochromatographic test paper was washed.
Finally, 60 μL of the third developing solution was developed (FIG. 14 (c)). By the development of the third developing solution, the detection antibody (antibody-PSS-coated gold nanorod complex) is bound to the HBs antigen captured on the detection line of the test paper (FIG. 14 (d)).
The same procedure was repeated using each first developing solution having a different HBs concentration.

(5) Evaluation by visual determination of immunochromatographic test Coloring of PSS-coated gold nanorods in the detection line after the development of the third developing solution (color of suspension, ie purple when using developing solution A, developing solution B, and The presence or absence of HBs antigen was determined by visually confirming (blue when C was used). The results are shown in Table 2 below.
+: Coloring in the detection line could be confirmed.
−: Coloring in the detection line could not be confirmed.

(6) Evaluation by luminance analysis of immunochromatographic test Scan the immunochromatographic test paper after developing the third developing solution (device name: Cano Scan LiDE500F, manufacturer: Canon Inc.), and minimize the detection line (capture antibody immobilization part) Image analysis software (Image-J: open source and public image processing software developed by Wayne Rasband at the National Institutes of Health (http: //) imagej.nih.gov/ij/)). As the minimum luminance, five different luminances in the detection line and the control region were measured once, and the median value obtained was adopted. The brightness difference (the minimum brightness of the detection line−the minimum brightness of the control area) was calculated, and the results are shown in the following Table 3 and FIG. Note that A to E in FIG. 15 mean the developing solutions A to E, respectively.
The numerical value in the column of each developing solution indicates a luminance difference.

(7) Drying tolerance test after immunochromatographic test The HBs antigen concentration in the first developing solution is 6.0 µM, and the immunochromatographic test paper after developing the third developing solution is dried in a room at a temperature of 20 ° C and a humidity of 50%. It was. At the start of drying (0 hours), 24 hours, 48 hours, 168 hours, 720 hours, 720 hours, and 2160 hours after the start of drying, the immunochromatographic test paper was scanned (apparatus name: Cano Scan LiDE500F, manufacturer: Canon Inc.). Then, the obtained image was taken in with Adobe Photoshop of bitmap image editing software, and CIELAB D50 of the detection line and the target area (detection line or part other than the detection line) was measured. The color difference (ΔE) between the detection line and the target area was calculated from the following equation 1.
(Expression 1) Color difference calculation formula ΔE = √ (ΔL ² + Δa ² + Δb ² )
ΔL = L ₁ −L ₂
Δa = a ₁ −a ₂
Δb = b ₁ −b ₂
(In the Lab color space which is a kind of complementary color space, the L value indicates lightness, the a value and the b value indicate the strength of the color tone. When the b value is positive, it indicates yellowishness, and when it is negative, it indicates blue-purple, where L ₁ is the L value of the detection line, L ₂ is the L value of the control area, and a ₁ is the detection line. A value of the control region, a ₂ is the a value of the control region, b ₁ is the b value of the detection line, and b ₂ is the b value of the control region.

The color difference calculation results are shown in the following Table 4 and FIG. Note that A to E in FIG. 16 mean developing solutions A to E, respectively.

The physical properties of metal nanoparticles in the suspensions prepared in Examples 1 to 3 and Comparative Examples 1 and 2 and the evaluation results are summarized in Table 5 below.

  Even when the third developing solution containing gold nanorods not supporting an antibody was used, no luminance difference was generated (data not shown), whereas the third developing solution containing gold nanorods supporting an antibody was not used. When used, clear lines that were visually identifiable were observed and significant brightness differences were also measured. This is because the gold nanorod carrying the anti-HBs antibody formed a complex with the HBs antigen captured by the capture antibody in the detection line. It has been found that even when gold nanorods are applied to immunochromatography, a test substance can be detected with the same sensitivity as gold-coated silver nanoplates or spherical gold nanoparticles. Although only one color detection reagent can be prepared with spherical gold nanoparticles, detection reagents of a plurality of colors can be prepared by using gold nanorods having an aspect ratio (major axis / minor axis) of more than 1.00. It was possible to expand the color variation. Further, the gold nanorods were stable against drying (oxidation), and the centrifugal force necessary for centrifugation was also weak. Therefore, according to the present invention, it is possible to provide a biosensor tool that is suitable for application to multicolor analysis, has high color stability, and has improved operability.

Claims (18)

A composition for detecting a test substance, comprising gold nanorods,
An aspect ratio (major axis / minor axis) of the gold nanorod is greater than 1.00.   The composition according to claim 1, wherein the gold nanorod has an aspect ratio (major axis / minor axis) of 3.00 or less.   The composition according to claim 1 or 2, wherein the gold nanorod has an aspect ratio (major axis / minor axis) of 2.00 or less.   The composition according to any one of claims 1 to 3, wherein a length of a major axis or a minor axis of the gold nanorod is 5 to 500 nm.   The composition according to any one of claims 1 to 4, wherein a major axis or a minor axis of the gold nanorod is 10 to 200 nm. The composition according to any one of claims 1 to 5, wherein a surface area of the gold nanorod is in a range of 118 to 1,170,000 nm ² . The surface area of the gold nanorods, in the range of 472～188,000Nm ^2, composition according to any one of claims 1 to 6. The composition according to any one of claims 1 to 7, wherein a volume of the gold nanorods is in a range of 98.2 to 98,100,000 nm ³ . The composition according to any one of claims 1 to 8, wherein the gold nanorods have a volume in the range of 786 to 6,280,000 nm ³ .   The composition according to any one of claims 1 to 9, further comprising a water-soluble polymer.   11. The composition of claim 10, wherein the water soluble polymer is selected from the group consisting of polystyrene sulfonic acid, polyvinyl pyrrolidone, and thiol terminated polyethylene glycol.   The composition according to claim 10 or 11, wherein the water-soluble polymer is polystyrene sulfonic acid.   The composition according to any one of claims 1 to 12, wherein the gold nanorods carry a specific binding substance for the test substance.   The combination of the test substance and the specific binding substance is an antigen and an antibody that binds to it, an antibody and an antigen that binds to it, a sugar chain or a complex carbohydrate and a lectin that binds to it, a lectin and a sugar chain or a complex carbohydrate that binds to it Hormones or cytokines and receptors that bind to them, receptors and hormones or cytokines that bind to them, proteins and nucleic acid or peptide aptamers that bind to them, enzymes and substrates that bind to them, substrates and enzymes that bind to them, biotin and avidin or Claims selected from the group consisting of streptavidin, avidin or streptavidin and biotin, IgG and protein A or protein G, protein A or protein G and IgG, and a first nucleic acid and a second nucleic acid bound thereto. 14. The composition according to 13. A method for detecting the test substance using the composition according to claim 13 or 14, comprising:
Mixing the composition with the test substance to form a complex of the test substance and a gold nanorod carrying the specific binding substance; and
A method comprising the step of detecting the complex.   The formation of the complex includes quenching measurement, absorbance measurement, turbidity measurement, particle size distribution measurement, particle size measurement, Raman scattered light measurement, color change observation, aggregation or precipitation formation, immunochromatography, electrophoresis, and 16. The method of claim 15, wherein the detection is by means selected from the group consisting of flow cytometry.   The step of preparing the composition according to claim 13 or 14 by further supporting a specific binding substance for the test substance on the gold nanorod in the composition according to any one of claims 1 to 12. 17. A method according to claim 15 or 16, comprising.   A kit for use in the method according to any one of claims 15 to 17, comprising the composition according to any one of claims 1 to 14.