Immunochromatographic test strip

Abstract

To provide a test strip for immunochromatographic inspection which is suitable for use in antigen inspection against novel coronavirus, has a lower false positive rate than, for example, a conventional kit for immunochromatographic inspection when used for immunochromatographic inspection, and exhibits excellent color development peculiar to metal nanoparticles in positive determination.SOLUTION: A test strip for immunochromatographic test, comprising a labeling substance source-containing pad impregnated with a metal nanoparticle dispersion liquid for immunochromatographic test in which metal nanoparticles are dispersed, the metal nanoparticle dispersion liquid having the following characteristics: The metal nanoparticles have an aliphatic hydroxy acid on the surface. The metallic nanoparticles have an average particle size of 8 to 500nm. The metal nanoparticles are at least one selected from the group consisting of Au, Ag, Pd, Pt, Rh, and Ru. The metal nanoparticles have a particle diameter variation coefficient (CV value) of 0 to 20%.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a test strip for immunochromatographic testing and an immunochromatographic testing kit. [Background technology]

[0002] In recent years, simple diagnostic kits have been developed that can perform a variety of tests in a relatively short time, including positive / negative tests (antigen tests) for pathogen infections such as the novel coronavirus, as well as tests for cancer markers, pregnancy markers, and pesticide residues in food. Simple diagnostic kits utilize specific reactions caused by substances that react specifically with the pathogens being tested. Examples of immunoassays that use antigen-antibody reactions include immunochromatography, radioimmunoassay, enzyme immunoassay, turbidimetric immunoassay, chemiluminescence assay, and surface plasmon resonance assay. These assays can be used to confirm infection in hospitals, clinics, or homes. Immunochromatography, in particular, is easy to operate, inexpensive, and suitable for rapid diagnosis.

[0003] Diagnostic methods using metal nanoparticles are known in in vitro diagnosis, such as immunochromatography and staining of biological materials. Among these metal nanoparticles, gold nanoparticles, for example, exhibit a strong red color due to surface plasmon resonance, enabling simple visual diagnosis. The red color of gold nanoparticles fades little over time and can be observed even when carrying biological materials, making them suitable for use as in vitro diagnostic agents and suitable for use in immunochromatography test kits.

[0004] Known methods for using gold nanoparticles in in vitro diagnostic reagents, such as antigen tests, include immunochromatography, which uses labeled particles in which antibodies are attached to gold nanoparticles. In immunochromatography, when an antigen is present, the labeled particles are allowed to form a complex in which the antigen binds, which is then spread across a mobile phase, and the complex is captured in a test zone carrying the corresponding antibody. Therefore, this method allows the presence or absence of the antigen to be confirmed by the presence or absence of color development in the test zone.

[0005] A known method for immobilizing antibodies on gold nanoparticles to prepare the labeled particles involves dispersing the gold nanoparticles and antibodies in a solvent and then immobilizing them through physical adsorption. However, this method has the problem of aggregation of the gold nanoparticles, and also has the problem of proteins other than antibodies being immobilized on the gold nanoparticles.

[0006] A method using gold nanoparticles modified with a surface treatment agent is known to prevent aggregation of gold nanoparticles and selectively load target antibodies onto gold nanoparticles. Here, the surface treatment agent is a compound that enables the target particles to load antibodies via covalent bonding or other means. For example, Patent Document 1 discloses a surface treatment agent consisting of uncrosslinked dextran or aminodextran having SH groups. Patent Document 2 also discloses gold nanoparticles with alkanethiols, alkanethiol derivatives, dithiol compounds, and trithiol compounds as surface treatment agents. However, the methods described in these documents have the problem that the surface treatment agents used are bulky compounds, making it difficult to fully load antibodies onto the metal nanoparticle surface. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Special Publication No. 2003-536074 [Patent Document 2] Japanese Patent Application Publication No. 6-116602 Summary of the Invention [Problem to be solved by the invention]

[0008] Since the above-mentioned in vitro diagnostic reagents are desired for improved diagnostic accuracy and early detection, they are required to be able to confirm the presence or absence of target antigens, etc., through clear color development and to avoid erroneous diagnostic results due to false positive judgments. Therefore, gold nanoparticles used in in vitro diagnostic reagents are preferably those that have high color development intensity, react with even trace amounts of antigens to produce sufficient color development, and do not result in false positive diagnoses. Furthermore, in vitro diagnosis may require gold nanoparticles of various particle sizes depending on the type of target antigen, etc., so gold nanoparticles that exhibit good color development over a wide range of particle sizes are highly useful as in vitro diagnostic reagents.

[0009] As described above, gold nanoparticles are more suitable as in vitro diagnostic agents when they satisfy the above-mentioned properties. Meanwhile, the gold nanoparticles described in the aforementioned Patent Documents 1 and 2 prevent aggregation of the gold nanoparticles and enable specific loading of the target antibody, but because the surface treatment agent used is a bulky compound, the antibody cannot be sufficiently loaded onto the metal nanoparticle surface. In addition, the particle size distribution of the obtained particles is broad (large CV value), so all of the above properties required to make gold nanoparticles particularly suitable as in vitro diagnostic agents have not been met, and no immunochromatographic test strip equipped with a diagnostic agent satisfying all of the above properties, or a test kit equipped with such a test strip, has been found.

[0010] Therefore, the present invention provides a test strip for immunochromatographic testing that includes metal nanoparticles (or a metal nanoparticle dispersion) for in vitro diagnostics, which are metal nanoparticles suitable as in vitro diagnostic reagents, have high color intensity, and are less likely to produce false positives, and a test kit that includes the test strip. [Means for solving the problem]

[0011] The present inventors have conducted extensive research to solve the above problems and have completed the present invention. The present invention includes the following (1) to (7). (1) A test strip for immunochromatographic testing, comprising a labeling substance source-containing pad impregnated with a metal nanoparticle dispersion for immunochromatographic testing, in which metal nanoparticles are dispersed, and which has the following characteristics [1] to [4]: [1] The metal nanoparticles have an aliphatic hydroxy acid on the surface. [2] The metal nanoparticles have an average particle size of 8 to 500 nm. [3] The metal nanoparticles are at least one selected from the group consisting of Au, Ag, Pd, Pt, Rh, and Ru. [4] The particle size coefficient of variation (CV value) of the metal nanoparticles is 0 to 20%. (2) The test strip for immunochromatographic testing according to (1) above, wherein the test strip for immunochromatographic testing comprises the labeled substance source-containing pad, a sample dropping pad, a membrane filter, an absorbent pad, and a backing sheet, and the membrane filter further comprises a test line and a control line. (3) The test strip for immunochromatographic testing according to (1) or (2) above, wherein the metal nanoparticle dispersion for immunochromatographic testing before being impregnated into the labeled substance source-containing pad has the following characteristics [5] and [6]. [5] When the content of the metal nanoparticles is adjusted to 0.005% by mass, the concentration of free aliphatic hydroxy acid is less than 100 ppm. [6] When the content of the metal nanoparticles is adjusted to 0.005% by mass, the absorbance at a wavelength of 520 nm is 1.0 to 2.2. (4) The test strip for immunochromatographic testing according to any one of (1) to (3) above, wherein, in a particle size distribution obtained by image analysis of the metal nanoparticle dispersion, the particle size at 10% cumulative frequency, from the smallest particle size, is defined as D10, the particle size at 50% cumulative frequency, is defined as D50, and the particle size at 90% cumulative frequency is defined as D90, satisfies the condition represented by the following formula: Formula: 0.9≦[(D10+D90) / 2] / D50≦1.1 (5) The immunochromatographic test strip according to any one of (1) to (4) above, wherein the aliphatic hydroxy acid has 1 to 10 carbon atoms. (6) The test strip for immunochromatographic testing according to any one of (1) to (5) above, wherein the metal nanoparticle dispersion for immunochromatographic testing before being impregnated into the labeled substance source-containing pad has the following characteristic [7]: [7] The metal nanoparticle dispersion for immunochromatographic testing contains nanobubbles having an average bubble diameter of 50 to 500 nm for 10 5 Contains more than 1 / ml. (7) An immunochromatographic test kit comprising the immunochromatographic test strip according to any one of (1) to (6) above. [Effects of the Invention]

[0012] The metal nanoparticle dispersion for immunochromatographic test agents according to the present invention is suitable as an in vitro diagnostic agent, and when subjected to immunochromatographic tests, it has a lower false positive rate than conventional metal nanoparticle dispersions for immunochromatographic test agents and exhibits excellent color development characteristic of metal nanoparticles when a positive determination is made.Furthermore, the metal nanoparticle dispersion for immunochromatographic test agents according to the present invention has a narrower particle size distribution, less aggregation when carrying antibodies, and excellent storage stability compared to conventional metal nanoparticle dispersions for immunochromatographic test agents.

[0013] The immunochromatographic test strip or immunochromatographic test kit equipped with the test strip according to the present invention is suitable for use in, for example, antigen testing for the novel coronavirus. When used in an immunochromatographic test, it has a lower false positive rate than conventional immunochromatographic test kits and exhibits excellent color development characteristic of metal nanoparticles when a positive result is determined.

[0014] Furthermore, the metal nanoparticle dispersion applied to the immunochromatographic test strip of the present invention has a narrower particle size distribution, less aggregation when carrying antibodies, and excellent storage stability compared to the metal nanoparticle dispersion used in conventional immunochromatographic test kits. Therefore, the immunochromatographic test strip of the present invention and a test kit equipped with the test strip are suitable for long-term storage, particularly for large-scale long-term storage in preparation for future pandemic outbreaks. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1(a) is a schematic plan view (main surface) of the test strip of the present invention, and FIG. 1(b) is a cross-sectional view taken along the line AA of FIG. 1(a). [Figure 2] FIG. 2(a) is a schematic plan view (main surface) of the testing kit of the present invention, and FIG. 2(b) is a cross-sectional view taken along line BB of FIG. 2(a). DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention will now be described. The present invention provides a test strip for immunochromatographic testing, which includes a labeling substance source-containing pad impregnated with a metal nanoparticle dispersion for immunochromatographic testing, in which metal nanoparticles are dispersed, and which has the following characteristics [1] to [4]: [1] The metal nanoparticles have an aliphatic hydroxy acid on the surface. [2] The metal nanoparticles have an average particle size of 8 to 500 nm. [3] The metal nanoparticles are at least one selected from the group consisting of Au, Ag, Pd, Pt, Rh, and Ru. [4] The particle size coefficient of variation (CV value) of the metal nanoparticles is 0 to 20%.

[0017] Such a test strip for immunochromatographic testing of the present invention will hereinafter also be referred to as the "test strip of the present invention."

[0018] Furthermore, a metal nanoparticle dispersion for immunochromatographic testing in which metal nanoparticles are dispersed and which has the above-mentioned characteristics [1] to [4] will hereinafter also be referred to as the "dispersion of the present invention." That is, the present invention is a test strip for immunochromatographic testing, which comprises a pad containing a labeling substance source impregnated with the dispersion of the present invention.

[0019] The present invention also relates to an immunochromatographic test kit comprising the test strip of the present invention. Such an immunochromatographic test kit of the present invention will hereinafter also be referred to as the "test kit of the present invention."

[0020] The principle of immunochromatography and the method for detecting the target substance in the test kit of the present invention are not particularly limited, but a representative example is immunochromatography that uses a labeled antibody carrying metal nanoparticles as a labeling substance and a capture antibody that has the property of binding to a complex between the labeled antibody and the target substance.

[0021] In this specification, "immunochromatography" refers to a measurement method for detecting antigenic substances that utilizes the phenomenon in which a test sample gradually flows through a membrane filter (a porous membrane material such as cellulose) while dissolving a reagent due to capillary action.

[0022] The test strip of the present invention will now be described. The test strip of the present invention preferably comprises a labeling substance source-containing pad, a sample drop pad, a membrane filter, an absorbent pad and a backing sheet, and the membrane filter further preferably comprises a test line and a control line.

[0023] The test strip of the present invention will be described with reference to FIG. FIG. 1 shown below shows a preferred embodiment of the test strip of the present invention. The test strip of the present invention is not limited to the embodiment shown in FIG.

[0024] FIG. 1 is a schematic diagram showing an example of the configuration of a test strip of the present invention, in which FIG. 1(a) is a schematic plan view (main surface) and FIG. 1(b) is a cross-sectional view taken along the line AA of FIG. 1(a).

[0025] The test strip 10 of the present invention shown in Figure 1 comprises, from upstream to downstream in the development direction (the direction indicated by arrow F), a pad for dropping a specimen (sample pad) 2, a labeled substance source-containing pad (conjugate pad) 3, a membrane filter 4, and an absorbent pad 5. These components are layered on a backing sheet 6 in the positional relationship shown in Figure 1, and the membrane filter 4 in particular is usually fixed to the backing sheet 6 with an adhesive or an adhesive sheet. The membrane filter 4 has a test line T and a control line C.

[0026] The structure of the test strip of the present invention is not limited as long as it has a pad containing a labeling substance source and can detect antigenic substances using immunochromatography, but typically, it is desirable for the test strip to be for immunochromatographic testing, which has the labeling substance source-containing pad, a sample dropping section, a membrane filter, an absorption pad, and a backing sheet, and further the membrane filter has a test line and a control line. The test kit of the present invention preferably comprises such a test strip of the present invention housed in a housing.

[0027] The dimensions of the test strip of the present invention can be, for example, in the ranges of width 1.5 cm to 3.5 cm, length 5 cm to 30 cm, and thickness (maximum thickness) 2 to 8 mm. The dimensions of the membrane filter can be in the range of 1.5 cm to 3.5 cm in width, 5 cm to 30 cm in length, and 0.5 mm to 2 mm in thickness. The dimensions of the backing sheet can be in the range of 1.5 cm to 3.5 cm in width, 5 cm to 33 cm in length, and 0.5 mm to 2 mm in thickness. The dimensions of the sample dropping pad can be in the range of 1.5 cm to 3.5 cm in width, 5 cm to 3 cm in length, and 0.5 mm to 2 mm in thickness. The dimensions of the labeling substance source-containing pad can be in the range of 1.5 cm to 3.5 cm in width, 5 cm to 4 cm in length, and 0.5 mm to 2 mm in thickness. The absorbent pad has dimensions ranging from 1.5 cm to 3.5 cm in width, 5 cm to 5 cm in length, and 0.5 mm to 2 mm in thickness.

[0028] Furthermore, the immunochromatographic test kit preferably comprises the immunochromatographic test strip housed in a housing (container), and the dimensions of the housing are preferably such that the immunochromatographic test strip can be fitted therein.

[0029] The sample is dropped onto the sample dropping pad. Examples of the sample pad that can be used include a glass fiber pad, a cellulose fiber pad, and a polyester pad.

[0030] The labeled antibody, which is an antibody against the substance to be detected and to which a labeling substance is bound, is immobilized on the labeled substance source-containing pad (conjugate pad). The labeled substance source-containing pad can be prepared, for example, by preparing a suspension containing the labeled antibody, applying the suspension to a suitable absorbent pad (e.g., a glass fiber pad, a cellulose fiber pad, a polyester pad, etc.), and then drying the pad. The dispersion of the present invention is used as the labeling substance.

[0031] The membrane filter is a porous membrane, and can be, for example, a nitrocellulose membrane, a cellulose acetate membrane, a fluororesin membrane, etc. The membrane filter further has a test line (detection portion) and a control line. The membrane filter is the mobile phase, and the metal nanoparticles contained in the dispersion of the present invention impregnated into the labeled substance source-containing pad and the antibody-carrying metal nanoparticles formed by complexing the metal nanoparticles with an antigen are spread on the membrane filter. The test line is used to determine the presence or absence of an antigen. To provide the test line, an antibody (capture antibody) that captures only the antibody-carrying metal nanoparticles that form a complex with the antigen is immobilized on a membrane filter, for example, by coating. The control line is used to confirm whether the metal nanoparticles are properly developed on the membrane filter and develop color. An antibody (also called a control antibody) that specifically recognizes the labeled antibody immobilized on the labeled substance source-containing pad is immobilized on the control line. The immobilization method can be, for example, by coating. The test line is located upstream of the control line (on the sample pad side).

[0032] The absorbent pad serves to absorb excess sample after chromatographic development. Examples of the absorbent pad that can be used include glass fiber pads, cellulose fiber pads, and polyester pads.

[0033] The substance to be detected in the sample dropped onto the sample drop pad reacts with the labeled antibody immobilized on the labeling source-containing pad to form a complex. This complex spreads over the membrane filter and is captured by the capture antibody immobilized on the test line of the membrane filter, thereby causing color development due to the labeled substance of the labeled antibody that accumulates at the test line. Furthermore, excess labeled antibody, etc. is captured by the control antibody immobilized on the control line of the membrane filter, thereby causing color development due to the labeled substance of the labeled antibody that accumulates at the control line. Excess sample that spreads downstream of the control line on the membrane filter is absorbed by the absorbent pad.

[0034] The substance to be detected contained in the sample is a substance that has the property of specifically recognizing and binding to the labeled antibody and the capture antibody, i.e., antigenicity. In other words, the substance to be detected is an antigen. The antigen may or may not have immunogenicity. In the latter case, the substance to be detected is also called a hapten or an incomplete antigen.

[0035] Examples of antigens include, but are not limited to, pathogens such as viruses, viroids, bacteria, and fungi; extracellular endoplasmic reticulum such as exosomes, microvesicles, and apoptotic bodies; proteins, DNA, and RNA derived from the body fluids (blood, serum, saliva, urine, etc.) and hair of test animals; proteins, DNA, and RNA derived from the organs, tissues, and cells of test plants; heavy metals such as mercury, arsenic, aluminum, cadmium, lead, nickel, and tin; and allergens such as pollen from trees and flowers, dust mites, house dust, and food (eggs, wheat, etc.). In particular, the method of the present invention involves measurement using an electron microscope, making it suitable for viruses, viroids, and other organisms that cannot be measured using an optical microscope.

[0036] The type of virus may be a DNA virus or an RNA virus.

[0037] Next, the testing kit of the present invention will be described with reference to FIG. FIG. 2 shown below shows a preferred example of the test kit of the present invention. The testing kit of the present invention is not limited to the embodiment shown in FIG.

[0038] FIG. 2 is a schematic diagram showing an example of the configuration of the testing kit of the present invention, in which FIG. 2(a) is a schematic plan view (main surface) and FIG. 2(b) is a cross-sectional view taken along line BB of FIG. 2(a).

[0039] The testing kit 12 of the present invention shown in Fig. 2 comprises the test strip 10 of the present invention shown in Fig. 1 housed in a housing 11. As shown in Fig. 2(a), the main surface of the housing 11 has a sample dropping opening 21 in the area of ​​the specimen dropping pad 2, and further has display openings 22 above the test line T and control line C of the membrane filter 4.

[0040] In the testing kit of the present invention, the test strip 10 is preferably placed on a solid support (backing sheet 6), such as a plastic adhesive sheet. The solid support is made of a material that does not interfere with the capillary flow of the sample and conjugate. The immunochromatography test strip may also be fixed to the solid support with an adhesive or the like. In this case, the adhesive components are also made of a material that does not interfere with the capillary flow of the sample and conjugate. It is also possible to laminate the insoluble membrane carrier with a polyester film or the like to increase its mechanical strength and prevent evaporation (drying) of water during the assay. The immunochromatography test strip can be stored and mounted in an appropriate container (housing) that takes into consideration the size of the immunochromatography test strip, the method and location of sample addition, the location of the detection portion on the insoluble membrane carrier, and the signal detection method.

[0041] Next, the dispersion of the present invention will be described. The dispersion of the present invention is obtained by dispersing metal nanoparticles in a polar solvent, and hereinafter, the metal nanoparticles dispersed in a polar solvent are also referred to as "metal nanoparticles of the present invention."

[0042] [Types of metal nanoparticles] The metal nanoparticles of the present invention are at least one selected from the group consisting of Au, Ag, Pd, Pt, Rh and Ru. The metal nanoparticles of the present invention are preferably made of Au. Such metal nanoparticles of the present invention are relatively easy to prepare. Furthermore, dispersions of such metal nanoparticles of the present invention in polar solvents are also highly stable. Therefore, in immunochromatographic test reagent applications, they are preferred because they are easy to support antibodies and easily produce the coloring characteristic of metal nanoparticles and, depending on the type, the coloring effect due to plasmon absorption.

[0043] [Average particle size of metal nanoparticles] The metal nanoparticles of the present invention have an average particle size of 8 nm to 500 nm, preferably 8 to 400 nm. Metal nanoparticles of the present invention having an average particle size of 8 nm to 500 nm can be used as in vitro diagnostic agents for immunochromatographic tests, and an average particle size of 8 to 400 nm can exhibit even more sufficient luminescence intensity. An average particle size in the range of 40 to 400 nm is even more preferably recommended. If the average particle size of the metal nanoparticles is less than 8 nm, practical luminescence intensity may not be obtained even if they react with an antigen during immunochromatographic testing due to the small particle size. If the average particle size exceeds 500 nm, the large particle size may cause the dispersion to settle easily, making handling difficult. The particle diameter of the metal nanoparticles of the present invention was measured using an electron microscope. The electron microscope may be a transmission electron microscope or a scanning electron microscope. The measurement method is described in detail below. Note that the measurement methods and calculation methods other than particle diameter measurement are also described below.

[0044] [Shape of metal nanoparticles] The shape of the metal nanoparticles of the present invention is not particularly limited, but spherical or nearly spherical shapes are usually preferred.

[0045] [Coefficient of variation of particle size of metal nanoparticles (CV value)] The particle size coefficient of variation (CV value) of the metal nanoparticles of the present invention is 0 to 20%, and preferably 0 to 15%. Therefore, the metal nanoparticles of the present invention have a highly uniform particle size, and as a result, the dispersion of the present invention exhibits plasmon absorption specific to metal nanoparticles and can exhibit excellent color development. The plasmon absorption refers to the absorption of light in the wavelength range from ultraviolet to near-infrared by metal nanoparticles dispersed in a polar solvent. Furthermore, antibodies are loaded to prepare labeled particles, and loading reduces aggregation, which may result in a lower false positive rate for diagnostic kits.

[0046] [Particle size distribution of metal nanoparticles] In the particle size distribution of the metal nanoparticles of the present invention obtained by image analysis, when the particle size at 10% cumulative frequency is defined as D10, the particle size at 50% cumulative frequency is defined as D50, and the particle size at 90% cumulative frequency is defined as D90, it is desirable that the condition represented by the following formula be satisfied: Formula: 0.9≦[(D10+D90) / 2] / D50≦1.1 When this formula is satisfied, the particle diameter of the metal nanoparticles of the present invention is more uniform, and as a result, they exhibit plasmon absorption specific to metal nanoparticles. Therefore, the metal nanoparticles of the present invention that satisfy the above formula are suitable as labeled particles. Note that, here, labeled particles refer to particles that can specifically bind to a target and generate a signal.

[0047] [Aliphatic hydroxy acids] The metal nanoparticles of the present invention have an aliphatic hydroxy acid on their surfaces, and it is preferable that the aliphatic hydroxy acid forms a layer and covers at least a portion of the surface of the metal nanoparticles of the present invention. Here, the aliphatic hydroxy acid means an aliphatic organic acid having a hydroxyl group and a carboxyl group, and is not particularly limited as long as it is a compound equivalent to an aliphatic hydroxy acid, but polycarboxylic acids are preferred, and dicarboxylic or tricarboxylic acids are particularly preferred. Examples of the dicarboxylic acid include maleic acid, phthalic acid, alginic acid, oxalic acid, tartaric acid, malic acid, succinic acid, gluconic acid, and L-glutamic acid. Examples of the trivalent carboxylic acid include L-ascorbic acid, citric acid, etc. More specifically, examples include disodium citrate, trisodium citrate, etc. An example of a monocarboxylic acid used as the aliphatic hydroxy acid is lactic acid.

[0048] In the dispersion of the present invention, it is preferred that the carboxyl group of the aliphatic hydroxy acid acts on the surface of the metal nanoparticles of the present invention, and the aliphatic hydroxy acid forms a layer on the surface of the metal nanoparticles of the present invention. In this case, the metal nanoparticles of the present invention exhibit high dispersion stability in the dispersion of the present invention. Furthermore, since the steric bulk is low, it is easy to support antibodies when preparing labeled particles.

[0049] The presence of an aliphatic hydroxy acid on the surface of the metal nanoparticles of the present invention contained in the labeling substance source-containing pad and the number of carbon atoms therein can be confirmed as follows. Ten labeled substance-containing pads separated from immunochromatographic test strips were placed in a container, 50 ml of pure water was poured into them, and the pads were left to stand at room temperature for 10 hours. After that, all labeled substance-containing pads in the container were removed, and a mixture of 10 g of the remaining aqueous solution and 10 g of nanosodium hydroxide aqueous solution (concentration 1% by mass) was prepared. The organic components were desorbed from the labeled substance (metal nanoparticles). The mixture was then filtered using an ultrafiltration membrane to separate the metal nanoparticles and filtered water. The filtered water was analyzed using a capillary electrophoresis device (Agilent 7100, manufactured by Otsuka Electronics) to confirm the presence of aliphatic hydroxy acids and their carbon numbers.

[0050] The aliphatic hydroxy acid preferably has 1 to 10 carbon atoms, more preferably 3 to 10 carbon atoms, and even more preferably 4 to 10 carbon atoms. The metal nanoparticles of the present invention having an aliphatic hydroxy acid with such a carbon number are suitable because they are highly compatible with water. If the carbon number is too low, it is difficult for the acid to act on the surface of the metal nanoparticles of the present invention, which may result in a decrease in the dispersion stability of the present invention. On the other hand, if the carbon number is too high, the acid becomes highly hydrophobic, which may result in poor compatibility with water, which may also result in a decrease in the dispersion stability of the present invention.

[0051] The aliphatic hydroxy acid is preferably attached to the surface of the metal nanoparticles of the present invention in the form of a layer by coordinating a carboxyl group. Such a layer of aliphatic hydroxy acid is thought to be formed by the interaction between the carboxyl group and the surface of the metal nanoparticles of the present invention. Even if the dispersion of the present invention is washed with a large amount of water using, for example, an ultrafiltration membrane or a microfilter, or washed by decantation using centrifugation or the like, the aliphatic hydroxy acid is unlikely to separate from the surface of the metal nanoparticles of the present invention. The greater the amount of aliphatic hydroxy acid, the higher the dispersibility in polar solvents and the less likely aggregation occurs when the antibody is supported.

[0052] [Free aliphatic hydroxy acids] As mentioned above, aliphatic hydroxy acids coordinated with metal nanoparticles can exhibit high dispersibility and reduce aggregation during antibody loading. However, free hydroxy acids present in the dispersion of the present invention may reduce the dispersibility of the metal nanoparticles of the present invention. Furthermore, when antibodies are loaded during the preparation of labeled particles, the antibodies are loaded not only on the aliphatic hydroxy acids on the surface of the metal nanoparticles of the present invention but also on the free aliphatic hydroxy acids. The free aliphatic hydroxy acids loaded with the antibodies may change the solubility parameter in the dispersion of the present invention, causing aggregation of the antibody-loaded metal nanoparticles of the present invention. Furthermore, when the dispersion of the present invention is used in an immunochromatographic test reagent, the antibody may react with the free aliphatic hydroxy acids, resulting in a positive result, which may result in a false positive.

[0053] The concentration of free aliphatic hydroxy acid contained in the dispersion of the present invention, which has been adjusted so that the content of the metal nanoparticles of the present invention is 0.005% by mass, is preferably less than 100 ppm, more preferably less than 95 ppm. If the concentration of free aliphatic hydroxy acid is less than 100 ppm, the rate of false positives in an immunochromatographic test reagent prepared using the dispersion of the present invention may be 1.0% or less.

[0054] [Absorbance] In metal nanoparticle dispersions used as immunochromatographic test reagents, the test sensitivity is generally correlated with the absorbance of the metal nanoparticles, and it is known that the higher the absorbance, the higher the sensitivity. In the dispersion of the present invention, when the content of the metal nanoparticles of the present invention is adjusted to 0.005% by mass, the absorbance is preferably 1.0 to 2.2 when measured at a wavelength of 520 nm with a spectrophotometer at an optical path length of 10 mm. When the absorbance is within this range, the labeled particles are highly color-developing, which is preferable. If the absorbance exceeds 2.2, there is a possibility that the particles may be affected by multiple scattering. If the absorbance is less than 1.0, the color development is low, and it tends to be difficult to detect a reaction with a trace amount of antigen.

[0055] The polar solvent in the dispersion of the present invention is not particularly limited, and the polar solvents mentioned in the description of the production method of the present invention below can be used.

[0056] [Concentration of metal nanoparticles] The concentration of metal nanoparticles in the dispersion of the present invention is preferably 0.0001 to 5 mass %, more preferably 0.0005 to 0.1 mass %, and even more preferably 0.001 to 0.1 mass %, calculated as metal.

[0057] [Contains nanobubbles] The dispersion of the present invention contains nanobubbles with an average bubble diameter of 50 to 500 nm. 5 It is preferable that the content is 1 / mL or more. Since these nanobubbles capture impurities, nanobubbles with an average bubble diameter of 50 to 500 nm are used for 10 5 The dispersion of the present invention containing at least 1 / mL of the antibody has excellent color development and dispersibility, and tends to reduce aggregation during antibody immobilization. Examples of impurities that can be captured by nanobubbles include organic impurities derived from raw materials, various bacteria, airborne bacteria, free aliphatic hydroxy acids, and metal impurities. If the average bubble diameter of the nanobubbles that can be contained in the dispersion of the present invention is less than 50 nm, it tends to be difficult to capture impurities, and if the average bubble diameter is more than 500 nm, the nanobubbles tend to have a short lifespan, making the dispersion impractical.

[0058] The lower limit of the number of nanobubbles contained in the dispersion of the present invention is not particularly limited as long as it does not interfere with the use of the dispersion as an immunochromatographic test agent. 5 More preferably, 1.0 x 10 8 The upper limit is not particularly limited, but it is preferably 1.0 × 10 11 cells / mL is preferred, 5.0 × 10 10 1.0 x 10 10 More preferably, it is 1 / mL.

[0059] The type of gas contained in the nanobubbles is not particularly limited, but typically air, oxygen, nitrogen, hydrogen, etc. are used.

[0060] The dispersion of the present invention can be used in an immunochromatographic test reagent that employs immunochromatography. Specifically, in immunochromatography, an immunochromatographic diagnostic kit is used that includes a mobile layer for developing a sample, metal nanoparticles that are provided at one end of the mobile layer and carry antibodies corresponding to an antigen, and a portion for determining the presence or absence of the antigen contained at the other end of the mobile layer. The metal nanoparticles contained in the dispersion of the present invention can be used as these metal nanoparticles.

[0061] [Metal nanoparticle dispersion method] The method for producing the dispersion of the present invention will now be described. The dispersion of the present invention can be produced, for example, by a production method including a seed particle preparation step and a seed particle growth step, which will be described below. The seed particle preparation step is a step of contacting a salt of at least one metal selected from the group consisting of Au, Ag, Pd, Pt, Rh, and Ru with an aliphatic hydroxy acid compound in a polar solvent, and further contacting with a reducing agent as needed, to prepare a seed particle dispersion. The seed particle growth step is a step in which, if necessary, a salt of at least one metal selected from the group consisting of Au, Ag, Pd, Pt, Rh, and Ru, an aliphatic hydroxy acid compound, a reducing agent, and a polar solvent (nanobubble-containing polar solvent) are added to the seed particle dispersion obtained in the seed particle preparation step to grow the seed particles and obtain metal nanoparticles. A manufacturing method including such a seed particle preparation step and a seed particle growth step is also referred to as the manufacturing method of the present invention.

[0062] [Seed particle preparation process] The seed particle preparation step will be described. The seed particle preparation step involves contacting a salt of at least one metal selected from the group consisting of Au, Ag, Pd, Pt, Rh, and Ru with an aliphatic hydroxy acid compound in a polar solvent.

[0063] Examples of the salt of at least one metal selected from the group consisting of Au, Ag, Pd, Pt, Rh and Ru include nitrates, sulfates and chlorides of these metals. Specific examples include chloroauric acid, chloroplatinic acid, dinitrodiammine platinum, palladium chloride, palladium nitrate, silver nitrate, copper nitrate, copper chloride, ruthenium chloride, and ruthenium nitrate. At least one metal selected from the group consisting of Au, Ag, Pd, Pt, Rh, and Ru is suitable for use because it has a low tendency to ionize and is easily reduced to produce fine particles. Furthermore, it tends to be easy to obtain metal nanoparticles with a small coefficient of variation (CV value).

[0064] The concentration of the metal salt in a solution containing a salt of at least one metal selected from the group consisting of Au, Ag, Pd, Pt, Rh, and Ru in a polar solvent is preferably 0.0001 to 5 mass%, more preferably 0.0005 to 0.1 mass%, and even more preferably 0.001 to 0.1 mass%, calculated as the metal. If the concentration of such a metal salt is too low, the yield may decrease, or production efficiency may decrease, resulting in poor economic efficiency. Furthermore, if the concentration of the metal salt is too high, the fine particles tend to aggregate, and uniformly dispersed seed particles may not be obtained. If the concentration is within the above range, seed particles with little aggregation can be prepared.

[0065] The polar solvent for dissolving the salt is not particularly limited, but typically, water, organic solvents such as alcohols and glycols, and mixed solvents thereof can be used. When the polar solvent is a polymer alcohol, depending on the type of metal salt or the mixing ratio of water, the solubility may be insufficient, making it impossible to obtain uniform seed particles or metal nanoparticles with a small particle size variation coefficient. For this reason, when using alcohol, 2-propanol, n-propanol, n-butanol, ethylene glycol, propylene glycol, etc. are suitable.

[0066] Of these polar solvents, it is preferable to use water, especially ultrapure water. Using water as a polar solvent increases the solubility of the aliphatic hydroxy acid compound, allowing the aliphatic hydroxy acid to quickly and sufficiently coordinate to the surface of the seed metal nanoparticles produced when the metal salt is reduced, suppressing the aggregation of the seed particles and enabling the production of more uniform seed particles, ultimately resulting in metal nanoparticles with a small particle size coefficient of variation (CV value).

[0067] The water used in this case preferably has an electrical conductivity of 5 μS / cm or less, more preferably 3 μS / cm or less, and an organic matter content of 25 ppb or less, more preferably 10 ppb or less, in terms of carbon.

[0068] If the electrical conductivity of water used as a polar solvent is high, the aliphatic hydroxy acid compound will be affected by the electrolyte components (especially cations) present in the water, and will form complexes with other cations in the water other than the metal salt, which will not only prevent the compound from functioning as a stabilizer but also reduce coordination with the resulting seed particles, causing the seed particles to aggregate and ultimately resulting in metal nanoparticles with a high CV value. Furthermore, when the antibody is loaded, the antibody and organic impurities may be loaded, causing the antibody-loaded metal nanoparticles to aggregate, which may result in a high false positive rate or poor color development when the final diagnostic kit is completed.

[0069] The polar solvent may be a mixture of water and other polar solvents, and the proportion of water in the mixed solvent is preferably 40% by mass or more, and more preferably 50% by mass or more. It is more preferable that the polar solvent is water alone. If the proportion of water in the polar solvent is less than 40% by mass, the solubility of the polycarboxylic acid compound described below may be insufficient, and the effect of the electrolyte described below may become significant, making it difficult to obtain more uniform seed particles.

[0070] When a solution containing a salt of at least one metal selected from the group consisting of Au, Ag, Pd, Pt, Rh, and Ru is added to a polar solvent, or when the final dispersion of the present invention contains organic matter (meaning organic matter other than the organic solvent used as the polar solvent, such as mold, bacteria, algae, and sugars) as a trace impurity, the organic matter present in the water and the metal salt form a complex, preventing sufficient reduction, similarly causing the seed particles to aggregate, making it difficult to obtain metal nanoparticles of the present invention with a low CV value. Furthermore, during antibody loading, the antibody and organic impurities may interact, causing the antibody-loaded metal nanoparticles to aggregate, which may result in a high false positive rate or poor color development when the final diagnostic kit is produced.

[0071] Examples of electrolyte components that may be present in polar solvents (particularly in water) include salts such as sodium chloride, potassium chloride, magnesium chloride, sodium carbonate, sodium nitrate, and potassium nitrate; cations such as potassium, calcium, and magnesium; and anions such as nitrate, sulfate, carbonate, chloride, bromide, and iodide.

[0072] Water with a low content of electrolyte components and organic matter as trace impurities is commercially available as ultrapure water, and can also be prepared by passing water through a column packed with a known ion exchange resin or adsorbent resin.

[0073] Furthermore, the use of a polar solvent containing nanobubbles may have the effect of capturing organic impurities, improving dispersibility and suppressing aggregation when carrying an antibody. When using ultrapure water containing nanobubbles, the number of nanobubbles in the ultrapure water containing nanobubbles is 1.0 × 10 5 More preferably, 1.0 x 10 8 The upper limit is not particularly limited, but it is preferably 1.0 × 10 11 cells / mL is preferred, 5.0 × 10 10 1.0 x 10 10 Within this range, it is more likely that the metal nanoparticles of the present invention will have a lower CV value.

[0074] The type of gas contained in the nanobubbles is not particularly limited, but typically air, oxygen, nitrogen, hydrogen, etc. are used.

[0075] The seed particle preparation step involves contacting a salt of at least one metal selected from the group consisting of Au, Ag, Pd, Pt, Rh, and Ru with an aliphatic hydroxy acid compound in a polar solvent or a nanobubble-containing polar solvent as described above.

[0076] The aliphatic hydroxy acid compound used herein means an aliphatic organic acid having a hydroxyl group and a carboxyl group, or a salt thereof, and is not particularly limited as long as it is a compound corresponding to the aliphatic hydroxy acid compound, but polycarboxylic acid compounds are particularly preferred, and dicarboxylic or tricarboxylic acid compounds are suitably used. Among these, polycarboxylic acid compounds having 1 to 10 carbon atoms, preferably 3 to 10 carbon atoms, and more preferably 5 to 10 carbon atoms are desirable. Examples of the dicarboxylic acid compound include maleic acid, phthalic acid, alginic acid, oxalic acid, tartaric acid, malic acid, succinic acid, gluconic acid, L-glutamic acid, and alkali metal salts and ammonium salts thereof.

[0077] Examples of the trivalent carboxylic acid compound include L-ascorbic acid, citric acid, and alkali metal salts and ammonium salts thereof. More specifically, examples include disodium citrate and trisodium citrate. These polycarboxylic acid compounds have highly polar functional groups such as carboxyl groups and hydroxyl groups, and therefore easily coordinate with metal ions. They also easily coordinate with the surfaces of metal nanoparticles precipitated by reduction, forming aliphatic hydroxy acids on the particle surfaces, allowing stable seed particles with excellent dispersibility to be obtained. Among these, L-ascorbic acid, citric acid, and sodium citrate are preferably used since they also function as reducing agents. Conventionally used polymeric stabilizers such as polyvinylpyrrolidone and polyvinyl alcohol adsorb to the surface of metal nanoparticles in multiple layers, resulting in significant steric hindrance from the molecules in the upper layer, making it difficult for metal to precipitate on the surface of the metal particles, which can result in uniform particle growth in the process described below.

[0078] The amount of such an aliphatic hydroxy acid compound used will be described below. The ratio ((Ms) / (Mm)) of the number of moles of the aliphatic hydroxy acid compound (Ms) to the number of moles of the metal salt (Mm) is preferably 1.5-10, more preferably 2-8. If the amount of aliphatic hydroxy acid compound used is small and the molar ratio (Ms) / (Mm) is small, the amount of aliphatic hydroxy acid adsorbed onto the surface of the metal nanoparticles will be insufficient, which will lead to aggregation of the particles during reduction, making it difficult to ultimately obtain metal nanoparticles with a low CV value. If the amount of aliphatic hydroxy acid compound used is large and the molar ratio (Ms) / (Mm) is large, the excess aliphatic hydroxy acid may precipitate metal on the surface of the metal nanoparticles, inhibiting particle growth and resulting in the formation and aggregation of new metal nanoparticles. Furthermore, the amount of free aliphatic hydroxy acid may also increase, resulting in a larger amount of free aliphatic hydroxy acid and antibody being loaded when the antibody is loaded, which may increase the false positive rate of the immunochromatography diagnostic kit. Furthermore, the process load in the impurity removal step, which will be described later, may be large, which may cause economic problems.

[0079] The seed particle preparation step involves contacting a salt of at least one metal selected from the group consisting of Au, Ag, Pd, Pt, Rh, and Ru with an aliphatic hydroxy acid compound in a polar solvent, and further contacting the compound with a reducing agent, if necessary.

[0080] When the aliphatic hydroxy acid compound also functions as a reducing agent, it is not necessarily necessary to add a reducing agent. However, when an aliphatic hydroxy acid compound that does not function as a reducing agent is used, a reducing agent may be added as necessary.

[0081] The reducing agent is not particularly limited as long as it can reduce the metal salt, and specific examples include ethanol, methanol, sodium borohydride, α-glucose, ferrous sulfate, and stannous chloride.

[0082] The amount of reducing agent added varies depending on the type of reducing agent. When the reducing agent is other than an alcohol, it is preferable to add the reducing agent so that the ratio (Mr) / (Mm) of the number of moles of the metal salt (Mm) to the number of moles of the reducing agent (Mr) is 1 to 10, preferably 2 to 8. When an alcohol is used as a reducing agent, the reducing agent is preferably added so that the ratio (Mr) / (Mm) of the number of moles of the metal salt (Mm) to the number of moles of the reducing agent (Mr) is 1,000 to 100,000, preferably 10,000 to 50,000. If the molar ratio (Mr) / (Mm) is too small, the reduction may not proceed sufficiently, and uniform particles may not be produced. Conversely, if the molar ratio (Mr) / (Mm) is too large, there may be too much reducing agent, which increases impurities, and the seed particles produced may aggregate.

[0083] The reduction temperature is preferably 0 to 120°C, more preferably 5 to 100°C, when a reducing agent other than citric acid, sodium citrate, or L-ascorbic acid is used. When citric acid, sodium citrate, or L-ascorbic acid is used as a reducing agent instead of adding a separate reducing agent, the reduction temperature is preferably 60 to 120°C, particularly 80 to 100°C. If the reduction temperature is too low, the reduction may take a long time or may be insufficient. If the reduction temperature is too high, the reduction rate is too fast, making it difficult to reproducibly obtain seed particles of the desired particle size, and the particle size tends to be large and the particle size variation coefficient also tends to be large, which may also increase the particle size variation coefficient of the finally obtained metal nanoparticles. Therefore, if the reduction temperature is within the above range, aggregation is reduced and particle size controllability is improved.

[0084] The metal salt, the aliphatic hydroxy acid compound, and the reducing agent added as needed are desirably mixed quickly to form a uniform mixture. Although this varies depending on the type of reducing agent, it is desirable to stir the solution containing the metal salt so that it is in a turbulent state; for example, stirring at a speed of 200 to 1000 rpm, preferably 300 to 800 rpm, is desirable. If the stirring speed is low, the reduction of all the metal salts does not occur simultaneously, and the particle size variation coefficient of the seed particles tends to increase. If the stirring speed is too high, not only is it meaningless, but the solution may scatter and the device may not be efficient.

[0085] The time for adding the solution containing the aliphatic hydroxy acid compound and the reducing agent to the solution containing the metal salt, or vice versa, is preferably 1 second to 10 minutes. If the addition time is longer than 10 minutes, uniform seed particles may not be obtained, resulting in a large CV value. As a method for instantaneous mixing, in addition to stirring, a line mixing method or a micromixing method is also preferred.

[0086] The seed particle preparation step is preferably carried out in an inert gas or reducing gas atmosphere, more preferably in an inert gas atmosphere. Examples of the inert gas include nitrogen and argon. Examples of the reducing gas include hydrogen.

[0087] In the seed particle preparation process, the order of addition is not particularly limited, and the aliphatic hydroxy acid compound and the reducing agent (solution as needed) may be mixed with the solution containing the metal salt, or the solution containing the metal salt and the aliphatic hydroxy acid compound may be mixed with the reducing agent solution.

[0088] By such a seed particle preparation step, a seed particle dispersion can be prepared.

[0089] [Aging process] The seed particle dispersion obtained in the seed particle preparation step is then desirably aged at a temperature range of 50 to 200°C, preferably 70 to 150°C. Such an aging step makes the seed particles uniform, and when they are grown in the subsequent step, it becomes possible to prepare spherical metal nanoparticles with a uniform particle size. Furthermore, aging also reduces unreacted metal salts, making it possible to make the particle size uniform.

[0090] The aging is carried out by heating the dispersion after the preparation of the seed particles to the same temperature as the reaction temperature or to a higher temperature, and then maintaining the mixture for a certain period of time while stirring. If the ripening temperature is low, the stability of the particles may decrease due to the influence of unreacted metal ions or reducing agents. However, even if the ripening temperature is high, particles may fuse together and uniform particles may not be obtained. The aging time is not particularly limited, but is generally 1 to 24 hours. As in the seed particle preparation step, the aging is also preferably carried out in an inert gas or reducing gas atmosphere, usually in an inert gas atmosphere.

[0091] The seed particles contained in the resulting seed particle dispersion preferably have an average particle size of 2 to 20 nm, more preferably 4 to 10 nm. If the size of the seed particles is outside this range, the CV value of the particles obtained by particle growth tends to be large. To adjust the average particle size of the seed particles within the above range, for example, the concentration of the metal salt, the stirring speed, the aging time, etc. may be adjusted. Specifically, although depending on the concentration of the metal salt, a stirring speed of 400 rpm or less tends to produce particles with a large particle size, while a stirring speed of more than 400 rpm tends to produce particles with a small particle size. The average particle size of the seed particles is measured in the same manner as the average particle size of the metal nanoparticles.

[0092] [Seed particle growth process] In the seed particle growth step, a solution containing one or more metal salts, an aliphatic hydroxy acid compound, and a reducing agent is added again, as needed, to the seed particle dispersion obtained in the seed particle preparation step to grow the seed particles and prepare metal nanoparticles.

[0093] The seed particle dispersion may be diluted or concentrated as needed. Dilution can be achieved by adding the polar solvent, while concentration can be achieved by evaporating the polar solvent or by ultrafiltration.

[0094] The concentration of the seed particle dispersion liquid varies depending on the particle size of the seed particles, but is preferably 0.0001 to 5 mass % as metal, and more preferably 0.0005 to 1 mass %. If the concentration of the seed particle dispersion is too low, there will be few seed particles, particle growth will be difficult, new fine particles may be generated, and the particle size coefficient of variation of the obtained metal nanoparticles will tend to be large.If the concentration of the seed particle dispersion is too high, the seed particles may aggregate and grow, resulting in the presence of aggregated particles.

[0095] The metal salt added in the seed particle growth step can be a desired metal salt from among the salts of one or more metals mentioned above. In this case, the metal species may be the same as or different from that of the seed particle.

[0096] The aliphatic hydroxy acid compound, reducing agent, and polar solvent may also be the same as those described above. When the aliphatic hydroxy acid compound or reducing agent remains in the seed particle dispersion, it is preferable that the total number of moles of the aliphatic hydroxy acid compound and reducing agent to be newly added and the number of moles of the metal salt are within the above-mentioned ranges.

[0097] The amount of metal salt added may be such that metal nanoparticles of the desired particle size are obtained, but it is preferable to add the metal salt so that the average particle size of the obtained metal nanoparticles is 8 times or less, preferably 4 times or less, the particle size of the seed particles. If the particle size is 8 times or less, the CV value of the obtained metal nanoparticles can be reduced.

[0098] The rate of addition of the metal salt varies depending on the particle size, concentration, and type of metal salt of the seed particles, but if the metal salt is added in too short a time, the metal salt will not be uniformly adsorbed on the surface of the seed particles, resulting in uneven particle growth of the seed particles and the generation of new fine particles. When preparing uniformly spherical metal nanoparticles, it is desirable to add the metal salt over a certain period of time. Preferably, it is 90 minutes to 48 hours, and more preferably, it is 90 minutes to 24 hours. If the addition time is less than 90 minutes, the addition time is too fast, which may result in the generation of fine particles and a large CV value. If it exceeds 48 hours, the generation of fine particles is suppressed and particles with a small CV value are obtained, but this may not be desirable from the viewpoint of productivity.

[0099] After the particle growth step, the resulting dispersion may be aged under the same aging conditions as those used in the seed particle aging step. This aging step allows the spherical metal nanoparticles to be uniform in size, more spherical, and with less aggregation.

[0100] The metal nanoparticles obtained as described above may be used as seed particles for further growth. That is, a salt of at least one metal selected from the group consisting of Au, Ag, Pd, Pt, Rh, and Ru, an aliphatic hydroxy acid compound, and optionally a reducing agent and a polar solvent may be added to the obtained metal nanoparticles to grow the metal nanoparticles. The metal salt, polar solvent, aliphatic hydroxy acid compound, reducing agent, and reduction conditions are the same as those described above.

[0101] After the metal nanoparticles have grown, a ripening step may be carried out in the same manner. Rather than growing seed particles in a single step, repeated particle growth and ripening processes can produce metal nanoparticles with a more uniform particle size, a higher sphericity coefficient, and less aggregation. Furthermore, by controlling the amount of growth in the particle growth process, the average particle size of the metal nanoparticles can be controlled to any desired size.

[0102] [Impurity removal process] The obtained metal nanoparticles can be used after removing residual salts, reducing agents, and impurities as needed. There are no particular limitations on the method for removing impurities, but specific examples include washing with an ultrafiltration membrane, decantation by centrifugation, ion exchange with an ion exchange resin, and solvent extraction. Washing and decantation of the ultrafiltration membrane are particularly preferred. The molecular weight cutoff of the ultrafiltration membrane is preferably 1,000 to 10,000, more preferably 2,000 to 50,000, depending on the particle size of the metal nanoparticles. Within this range, the nanoparticles do not flow out of the system through the membrane, and only the impurities can be washed away.

[0103] On the other hand, decantation is preferably performed by centrifuging at 1,000 to 10,000 G, although this depends on the particle size. If the pressure is less than 1,000 G, the metal nanoparticles may not settle and may not be able to be washed. If the pressure is more than 100,000 G, the metal nanoparticles will settle, but when dispersing them in a solvent after removing the supernatant, the particles may aggregate strongly and may not disperse. The water used for washing is preferably ultrapure water, more preferably nanobubble-containing ultrapure water. The electrical conductivity of the ultrapure water is preferably 5 μS / cm or less, more preferably 3 μS / cm or less, and the organic matter content, as carbon, is preferably 25 ppb or less, more preferably 10 ppb or less. When using an ultrapure membrane, the amount of ultrapure water used is 0.5 to 100 times, preferably 1 to 50 times, the amount of metal nanoparticle dispersion. This range removes impurities and free aliphatic hydroxy acids, reduces aggregation during antibody immobilization of labeled particles, and provides a metal nanoparticle dispersion that has high color development and a low false positive rate, making it possible to obtain an immunochromatography diagnostic kit.

[0104] When using ultrapure water containing nanobubbles, the number of nanobubbles is 1.0 x 10 5 More preferably, 1.0 x 10 8 The upper limit is not particularly limited, but it is preferably 1.0 × 10 11 cells / mL is preferred, 5.0 × 10 10 1.0 x 10 10 More preferably, it is 1 / mL. The type of gas contained in the nanobubbles is not particularly limited, but typically air, oxygen, nitrogen, hydrogen, etc. are used. [Example]

[0105] The measurement methods used in the examples and comparative examples are described below.

[0106] 1.Measuring the average particle size of metal nanoparticles An electron microscope photograph (magnification 200,000 times, containing 250 or more metal nanoparticles in the same field of view) of a dispersion in which metal nanoparticles are dispersed in a solvent (hereinafter also referred to as "metal nanoparticle dispersion") was taken, and for each of 250 particles randomly selected from the obtained image, the length of the longest part of the primary particle diameter was measured, and this value was taken as the particle diameter of that particle. The arithmetic mean of the 250 particles was then calculated, and the obtained value was taken as the average particle diameter (Ds) of the metal nanoparticles dispersed in the dispersion. In the following examples and comparative examples, a scanning electron microscope (S-2000 model, manufactured by Hitachi, Ltd.) was used as the electron microscope.

[0107] 2. Calculation of the particle size coefficient of variation (CV value) of metal nanoparticles The particle size standard deviation (σ) was calculated from the particle size data of 250 metal nanoparticles obtained in "1. Measurement of the average particle size of metal nanoparticles" above using the following formula. Particle size standard deviation (σ)=√[Σ(Di-Ds) 2 / (n-1)] where Di: particle diameter of individual metal nanoparticles, n=250 Then, using the determined value of the particle diameter standard deviation (σ), the particle diameter coefficient of variation (CV value) was calculated according to the following formula. CV value = particle size standard deviation (σ) / average particle size (Ds) × 100

[0108] 3.Measuring the particle size distribution of metal nanoparticles A cumulative particle size frequency distribution chart was created from the particle size data of 250 metal nanoparticles obtained in "1. Measurement of the average particle size of metal nanoparticles" above, and the particle size at 10% cumulative frequency was defined as D10, the particle size at 50% cumulative frequency as D50, and the particle size at 90% cumulative frequency as D90, in ascending order of particle size.These results were then used to calculate the value of [(D10+D90) / 2] / D50.

[0109] 4. Measurement of free aliphatic hydroxy acid concentration in metal nanoparticle dispersions The metal nanoparticle dispersion was adjusted, with ultrapure water added as needed, to a metal nanoparticle content of 0.005% by mass, and centrifuged for 30 minutes at 1,370,000 rpm (1,000,000 G) at a set temperature of 10°C using a compact ultracentrifuge CS150GXL manufactured by Hitachi Koki Co., Ltd. The concentration of free aliphatic hydroxy acid in the supernatant was then measured using high-performance liquid chromatography.

[0110] 5. Absorbance Measurement of Metal Nanoparticle Dispersion The metal nanoparticle dispersion liquid was adjusted by adding ultrapure water as necessary so that the metal nanoparticle content was 0.005% by mass. The dispersion liquid was placed in a quartz cell with an optical path length of 10 mm, and the absorbance at a wavelength of 520 nm was measured using a UV-visible spectrophotometer (Uvest-V560, manufactured by JASCO Corporation).

[0111] 6. Measurement of carbon content of aliphatic hydroxy acids with metal nanoparticles and carbon number of the same aliphatic hydroxy acids The metal nanoparticle dispersion was adjusted to a metal nanoparticle content of 0.005% by mass, with the addition of ultrapure water as needed. It was then centrifuged for 30 minutes at 1,370,000 rpm (1,000,000 G) using a Hitachi Koki Co., Ltd. CS150GXL compact ultracentrifuge at 10°C and 1,370,000 rpm (1,000,000 G). The precipitate was then collected and placed on a flat filter. To remove impurities, it was washed with 1,000 volumes of ultrapure water. The precipitate was then dried at 120°C, after which the carbon content of the metal nanoparticles was measured using a carbon analyzer (HORIBA EMIA-320V carbon-sulfur analyzer). The carbon number of the aliphatic hydroxy acids was calculated, assuming that all of the carbon was derived from the aliphatic hydroxy acids. Here, the presence of aliphatic hydroxy acids was confirmed by drying the precipitate at 120°C, dissolving the dried precipitate in aqua regia, and analyzing the resulting solution by liquid chromatography, as in the case of measuring the carbon content. In the present invention, the liquid chromatography was performed using an ICS-1100 manufactured by Nippon Dionex Co., Ltd. Furthermore, the carbon content of the measured precipitate was assumed to be entirely attributable to the aliphatic hydroxy acids present on the surface of the metal nanoparticles, and the mass of the aliphatic hydroxy acids was calculated and expressed as a ratio (percentage) to the mass of the metal nanoparticles.

[0112] Regarding the analytical method for unknown metal nanoparticles or metal nanoparticle dispersions, the unknown metal nanoparticles or metal nanoparticle dispersions are first subjected to qualitative analysis to identify the type of metal nanoparticles. While any known means may be used for the qualitative analysis method, for example, an ICP inductively coupled plasma optical emission spectrometry mass spectrometer can be used to identify the metal species of the metal nanoparticles. Next, in the same manner as above, the carbon content of the metal nanoparticles was measured, the number of carbon atoms in the aliphatic hydroxy acid was calculated, the mass of the aliphatic hydroxy acid was calculated, and the ratio (percentage) to the mass of the metal nanoparticles was determined.

[0113] 7. Measurement of metal nanoparticle concentration in metal nanoparticle dispersion The metal nanoparticles were dissolved in aqua regia, and then purified water was added as needed. The contents of Au, Ag, Pd, Pt, Rh, and Ru in the resulting solution were measured using an ICP inductively coupled plasma atomic emission spectrometer (ICP-8500, manufactured by Shimadzu Corporation). Then, assuming that all of these components constituted metal nanoparticles, the concentration (mass %) of metal nanoparticles contained in the metal nanoparticle dispersion was determined.

[0114] 8. Measurement of impurity content The metal nanoparticles were dissolved in aqua regia, and then purified water was added as necessary. The contents of alkali metals (excluding sodium), alkaline earth metals, Fe, Ti, Zn, Mn, Co, Mo, Sn, Al, Zr, Ni, and Cr in the resulting solution were measured using an ICP inductively coupled plasma atomic emission spectrometer (ICP-8500, manufactured by Shimadzu Corporation). Then, assuming that all of these components constitute impurities, the concentration (ppm) of impurities contained in the metal nanoparticle dispersion was calculated.

[0115] 9. Measurement of the average bubble diameter and number of bubbles in a metal nanoparticle dispersion The average bubble diameter of nanobubbles contained in the metal nanoparticle dispersion was measured by measuring the Brownian motion movement speed of bubbles in the metal nanoparticle dispersion using nanotracking analysis. Specifically, approximately 20 mL of the measurement sample (metal nanoparticle dispersion [solid content concentration 0.005% by mass]) was injected into a measuring device (Malvern "NanoSight NS300") while suctioning, and the average bubble diameter and number of bubbles were measured using nanoparticle tracking analysis.

[0116] 10. Average particle size of metal nanoparticles before and after antibody loading The average particle size of the metal nanoparticles before and after antibody loading was the D50 value measured using NANOTRAC WAVE II manufactured by Microtrack Bell Corporation. The antibody was supported on the metal nanoparticles by the method described below.

[0117] [Antibody-carrying] The antibody used was a chorionic gonadotropin antibody, which is used to diagnose pregnancy. The metal nanoparticles obtained in each example and comparative example were concentrated to 0.1% by mass using a rotary evaporator. 1000 μL of the concentrated metal nanoparticles were added with 100 mL of 0.04 mM 1-ethyl-3-(3-dimethylaminopropyl)carboxylimide hydrochloride, and the mixture was allowed to react for 10 minutes. 100 μL of 100 μg / mL chorionic gonadotropin antibody (anti-human monoclonal antibody, Biochemica) was then added and the mixture was allowed to react at room temperature for 2 hours. The carboxylimide hydrochloride and chorionic gonadotropin antibody were buffered in 10 mM borate buffer (pH 8.1). Next, 50 μL of 1% polyethylene glycol 20000 (using 50 mM potassium phosphate buffer (pH 7.5)) and 100 μL of 10% bovine serum albumin (BSA) (using 50 mM potassium phosphate buffer (pH 9.0)) were added and allowed to react for an additional 10 minutes. The mixture was then centrifuged at 3000 G for 15 minutes at 5°C, and the supernatant was removed to obtain the precipitated antibody-supported metal nanoparticles. The resulting antibody-supported metal nanoparticles were ultrasonically dispersed, followed by the addition of 1000 μL of diluent (1.0% BSA, 0.05% polyethylene glycol 20000, 0.1% NaN3, 150 mM NaCl, 20 mM Tris-HCl (pH 8.2)). This centrifugation washing process was repeated five times.

[0118] 11. Measurement of color intensity First, a test diagnostic kit for immunochromatographic diagnosis was prepared. A test line and a control line were created on the membrane, which was the transfer layer. The test line is used to determine the presence or absence of the antigen. A solution containing an anti-hαS antibody shown in Table 1 was applied to the test line as an antibody that captures only the antibody-carrying metal nanoparticles that have formed a complex with the antigen. Specifically, the solutions shown in Table 1 were prepared, and 0.75 μL of this solution was applied to a membrane (nitrocellulose, width 25 mm, length 30 cm) using an antibody applicator (XYZ3060, manufactured by BioDot) at 50 mm / sec, 15 nL / dot, and 0.30 mm pitch to create a test line.

[0119] [Table 1]

[0120] Next, a control line was created to confirm whether the metal nanoparticles were properly developed on the membrane and whether color was generated. A solution containing an anti-hαS antibody shown in Table 2 was applied to the control line as an antibody that captures antibody-carrying metal nanoparticles that do not contain antigen. Specifically, the solutions shown in Table 2 were prepared, and 0.75 μL of this solution was applied to a membrane (nitrocellulose, width 25 mm, length 30 cm) using an antibody applicator (XYZ3060, BioDot) at 50 mm / sec, 15 nL / dot, and 0.30 mm pitch to create a control line.

[0121] [Table 2]

[0122] The membrane with the test and control lines formed was dried in a dryer at 42°C for 1 hour, then dried for another 2 hours at 25°C and 40% humidity. To prevent the antibody-loaded metal nanoparticles from adsorbing to the nitrocellulose membrane, the membrane was first immersed in 0.5% casein (50 mM borate buffer (pH 8.5)) on the test line side, then the entire membrane, and left to stand for 30 minutes at 25°C and 40% humidity. The membrane was then removed from the blocking solution, excess casein solution was removed, and the membrane was then immersed in 0.01% sodium dodecyl sulfate (5 mM phosphate buffer (pH 7.5)) in the same manner as above and left to stand for 30 minutes for static washing. The membrane was then removed and left to stand for 24 hours at 25°C and 40% humidity, after which an absorbent pad was attached and the membrane was cut to a 5 mm width. In this way, a test diagnostic kit for immunochromatographic diagnosis was prepared.

[0123] Then, a mixture of 40 μL of the antigen recombinant hCG (1% BSA, 50 mM phosphate buffer (pH 7.4)) and 4 μL of the metal nanoparticles obtained in each example and comparative example was tested using the test diagnostic kit prepared as described above. The color intensity of the test line on the membrane was then measured using an automatic reaction line reader (Quad Scan: manufactured by Bio Dot). The color intensity in each example and comparative example was evaluated relative to the color intensity in Example 1, which was set at a reference level of 5.

[0124] 12. Immunochromatography test (measurement of false positive rate) A mixture of 40 μL of 1% BSA (50 mM phosphate buffer (pH 7.4)) not containing antigen and 4 μL of the metal nanoparticles obtained in each example and comparative example was tested using the test diagnostic kit prepared as described above. This test was performed 1000 times. The number of kits that developed a color on the test line was then counted, and the false positive incidence rate was calculated from the results as follows: Number of kits that developed a color on the test line / 1000 x 100.

[0125] [Example 1] (Seed particle preparation process) 2.5 g of trisodium citrate dihydrate (Kanto Chemical Co., Ltd.: special grade reagent) was dissolved in 9,800 g of ultrapure water (electrical conductivity: 0.6 μS / cm, organic matter (TOC): 5 ppb). This solution was stirred at 350 rpm under a nitrogen atmosphere using a stirrer. A separate solution of 0.95 g of chloroauric acid tetrahydrate (Wako Pure Chemical Industries, Ltd.: special grade reagent) dissolved in 94.05 g of ultrapure water was added over 10 seconds, and the mixture was aged at 90°C for 1 hour to obtain seed particles (M-1). The concentration of these seed particles was 0.005%.

[0126] (Seed particle growth process) To 500 g of a dispersion of seed particles (M-1) (metal content: 0.0046 mass%), 8,500 g of ultrapure water (electrical conductivity: 0.6 μS / cm, organic matter (TOC): 5 ppb) was added, and a solution of 3.0 g of trisodium citrate (Kanto Chemical Co., Ltd.: special grade reagent) dissolved in 50 g of ultrapure water (electrical conductivity: 0.6 μS / cm, organic matter (TOC): 5 ppb) was added. The mixture was heated to 90°C and stirred at 350 rpm under a nitrogen atmosphere using a stirrer. A solution of 0.95 g of chloroauric acid (Wako Pure Chemical Industries, Ltd.: special grade reagent) dissolved in 95 g of ultrapure water (diluted chloroauric acid) was added over 2 hours. The mixture was then aged at 90°C for 1 hour to precipitate gold on the seed particles, yielding nanoparticles (M-2). 9,500 g of these nanoparticles (M-2) were washed with twice the amount of ultrapure water using an ultrafilter (SIP-1013 manufactured by Asahi Kasei), and the concentration of the metal nanoparticles was adjusted to 0.005 mass %, to obtain metal nanoparticles (M-2F). The physical properties of the metal nanoparticles (M-2F) were measured or tested using the methods 1 to 12. The aliphatic hydroxy acid contained in the metal nanoparticles prepared in Examples 1 to 11, 14, 15, and 16, and Comparative Examples 1 to 5 was citric acid. The results are shown in Table 3.

[0127] [Example 2] The seed particles (M-1) obtained in Example 1 were washed with twice the amount of ultrapure water using an ultrafilter (SIP-1013 manufactured by Asahi Kasei), and the concentration of the metal nanoparticles was adjusted to 0.005 mass % to obtain metal nanoparticles (M-1F). The physical properties of the metal nanoparticles (M-1F) were measured or tested using the methods 1 to 12 above. The results are shown in Table 3.

[0128] [Example 3] The metal nanoparticles (M-1F) obtained in Example 2 were used as seed particles, and were subjected to the same treatment as the seed particle growth step in Example 1, followed by washing with an ultrafilter to obtain metal nanoparticles (M-3F). The physical properties of the metal nanoparticles (M-3F) were measured or tested using the methods 1 to 12 above. The results are shown in Table 3.

[0129] [Example 4] In the seed particle growth step in Example 1, the diluted chloroauric acid solution was added over 2 hours, but in Example 4, the diluted chloroauric acid solution was added over 12 hours. Other than that, the same treatment as in Example 1 was carried out, and metal nanoparticles (M-4F) were obtained after washing with an ultrafilter. The physical properties of the metal nanoparticles (M-4F) were measured or tested using the methods 1 to 12 above. The results are shown in Table 3.

[0130] [Example 5] In the seed particle growth step in Example 1, the diluted chloroauric acid solution was added over 2 hours, but in Example 5, the diluted chloroauric acid solution was added over 48 hours. Other than that, the same treatment as in Example 1 was carried out, and metal nanoparticles (M-5F) were obtained after washing with an ultrafilter. The physical properties of the metal nanoparticles (M-5F) were measured or tested using the methods 1 to 12 above. The results are shown in Table 3.

[0131] [Example 6] While 3.0 g of trisodium citrate was used in the seed particle growth step in Example 1, 1.0 g of trisodium citrate was used in Example 6. Other than that, the same treatment as in Example 1 was carried out, and metal nanoparticles (M-6F) were obtained after washing with an ultrafilter. The physical properties of the metal nanoparticles (M-6F) were measured or tested using the methods 1 to 12 above. The results are shown in Table 3.

[0132] [Example 7] While 3.0 g of trisodium citrate was used in the seed particle growth step in Example 1, 4.0 g of trisodium citrate was used in Example 7. Other than that, the same treatment as in Example 1 was carried out, and metal nanoparticles (M-7F) were obtained after washing with an ultrafilter. The physical properties of the metal nanoparticles (M-7F) were measured or tested using the methods 1 to 12 above. The results are shown in Table 3.

[0133] [Example 8] 10 g of a cation exchange resin (SK-1BH manufactured by Mitsubishi Chemical) was added to 100 g of the dispersion of metal nanoparticles (M-2F) obtained in Example 1, and the mixture was stirred for 1 hour to obtain metal nanoparticles (M-8FI). The physical properties of the metal nanoparticles (M-8FI) were measured or tested using the methods 1 to 12 above. The results are shown in Table 3.

[0134] [Example 9] To 100 g of the dispersion of metal nanoparticles (M-2F) obtained in Example 1, 40 g of amphoteric ion exchange resin (SMNUPB manufactured by Mitsubishi Chemical Corporation) was added and stirred for 6 hours to obtain metal nanoparticles (M-9FI). The physical properties of the metal nanoparticles (M-9FI) were measured or tested using the methods 1 to 12 above. The results are shown in Table 3.

[0135] [Example 10] 8,500 g of ultrapure water (electrical conductivity: 0.6 μS / cm, organic matter (TOC): 5 ppb) was added to 500 g of a dispersion of seed particles (M-1) obtained by the seed particle preparation step of Example 1 (metal content: 0.0046 mass%), and a solution prepared by dissolving 3.0 g of trisodium citrate (manufactured by Kanto Chemical Co., Inc.: special grade reagent) in 50 g of ultrapure water (electrical conductivity: 0.6 μS / cm, organic matter (TOC): 5 ppb) was added, and the temperature was raised to 90° C. Under a nitrogen atmosphere, while stirring at 350 rpm using a stirrer, a solution (diluted metal salt solution) prepared by dissolving 0.69 g of chloroformic acid hexahydrate (Wako Pure Chemical Industries, Ltd.: special grade reagent) and 0.65 g of palladium nitrate dihydrate (Tokuriki Honten Co., Ltd.) in 95 g of ultrapure water was added over 2 hours, and the mixture was then aged at 90°C for 1 hour to precipitate platinum / palladium at a mass ratio of 1 / 1 on the seed particles, yielding Pt / Pd / Au nanoparticles (M-10). 9,500 g of these Pt / Pd / Au nanoparticles (M-10) were washed with twice the amount of ultrapure water using an ultrafilter (SIP-1013 manufactured by Asahi Kasei), and the concentration of the Pt / Pd / Au nanoparticles was adjusted to 0.005%, yielding Pt / Pd / Au nanoparticles (M-10F). The physical properties of the Pt / Pd / Au nanoparticles (M-10F) were measured or tested using the methods 1 to 12 above. The results are shown in Table 3.

[0136] [Example 11] 8,500 g of ultrapure water (electrical conductivity: 0.6 μS / cm, organic matter (TOC): 5 ppb) was added to 500 g of a dispersion of seed particles (M-1) obtained by the seed particle preparation step of Example 1 (metal content: 0.0046 mass%), and 1.5 g of trisodium citrate (manufactured by Kanto Chemical Co., Inc.: special grade reagent) was dissolved in 50 g of ultrapure water (electrical conductivity: 0.6 μS / cm, organic matter (TOC): 5 ppb). The solution was added, heated to 90°C, and stirred at 350 rpm using a stirrer under a nitrogen atmosphere. A solution (diluted metal salt solution) prepared by dissolving 0.44 g of silver nitrate (Wako Pure Chemical Industries, Ltd.: special grade reagent) in 95 g of ultrapure water was added over 2 hours, and the mixture was then aged at 90°C for 1 hour to precipitate silver on the seed particles, yielding Au / Ag / Au nanoparticles (M-11) with a mass ratio of Au / Ag = 1 / 9. 9500 g of these Au / Ag / Au nanoparticles (M-11) were washed with twice the amount of ultrapure water using an ultrafilter (SIP-1013 manufactured by Asahi Kasei), and the concentration of the Au / Ag / Au nanoparticles was adjusted to 0.005%, yielding Au / Ag / Au nanoparticles (M-11F). The physical properties of the Au / Ag / Au nanoparticles (M-11F) were measured or tested using the methods 1 to 12 above. The results are shown in Table 3.

[0137] [Example 12] In the seed particle growth step of Example 1, 3.0 g of trisodium citrate was used, but in Example 12, 1.96 g of tartaric acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was used instead. Otherwise, the same treatment as in Example 1 was carried out, and metal nanoparticles (M-12F) were obtained after washing with an ultrafilter. The physical properties of the metal nanoparticles (M-12F) were measured or tested using the above methods 1 to 12. The aliphatic hydroxy acid contained in the metal nanoparticles prepared in Example 12 was tartaric acid. The results are shown in Table 3.

[0138] [Example 13] In the seed particle growth step of Example 1, 3.0 g of trisodium citrate was used, but in Example 13, 2.30 g of L-ascorbic acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was used instead. Otherwise, the same treatment as in Example 1 was carried out, and metal nanoparticles (M-13F) were obtained after washing with an ultrafilter. The physical properties of the metal nanoparticles (M-13F) were measured or tested using the methods 1 to 12. The aliphatic hydroxy acid contained in the metal nanoparticles prepared in Example 13 was L-ascorbic acid. The results are shown in Table 3.

[0139] [Example 14] (Preparation of nanobubble water) N2 nanobubble water (average bubble diameter: 70 nm, number of bubbles: 240 million / mL) was prepared by contacting ultrapure water with N2 using a swirling flow type bubble generator (HYK-20-SD manufactured by Ligaric Co., Ltd.). Then, instead of the ultrapure water used in the seed particle preparation step and the seed particle growth step of Example 1, N2 nanobubble water was used, and the same treatment as in Example 1 was carried out, and metal nanoparticles (M-14F) were obtained after washing with an ultrafilter. The physical properties of the metal nanoparticles (M-14F) were measured or tested using the methods 1 to 12 above. The results are shown in Table 3.

[0140] [Example 15] In the seed particle growth step of Example 1, gold was precipitated on the seed particles by aging at 90°C for 1 hour, whereas in Example 15, gold was precipitated on the seed particles by aging at 98°C for 1 hour. The remaining steps were the same as in Example 1, and metal nanoparticles (M-15F) were obtained after washing with an ultrafilter. The physical properties of the metal nanoparticles (M-15F) were measured or tested using the methods 1 to 12 above. The results are shown in Table 3.

[0141] [Example 16] In the seed particle growth step of Example 1, gold was precipitated on the seed particles by aging at 90°C for 1 hour, whereas in Example 16, gold was precipitated on the seed particles by aging at 80°C for 1 hour. The remaining steps were the same as in Example 1, and metal nanoparticles (M-16F) were obtained after washing with an ultrafilter. The physical properties of the metal nanoparticles (M-16F) were measured or tested using the methods 1 to 12 above. The results are shown in Table 3.

[0142] [Comparative Example 1] The nanoparticles (M-2) obtained in Example 1 were adjusted with ultrapure water so that the nanoparticle concentration was 0.005% by mass. The physical properties of the nanoparticles (M-2) were measured or tested using the methods 1 to 12 above. The results are shown in Table 3.

[0143] Comparative Example 2 To 500 g of the seed particle (M-1) dispersion obtained in Example 1 (metal content: 0.0046 mass%), 8,500 g of ultrapure water (electrical conductivity: 0.6 μS / cm, organic matter (TOC): 5 ppb) was added, and a solution prepared by dissolving 3.0 g of trisodium citrate (Kanto Chemical Co., Ltd.: special grade reagent) in 50 g of ultrapure water (electrical conductivity: 0.6 μS / cm, organic matter (TOC): 5 ppb) was added. The mixture was heated to 90°C and stirred at 350 rpm under a nitrogen atmosphere using a stirrer. A solution prepared by dissolving 0.95 g of chloroauric acid (Wako Pure Chemical Industries, Ltd.: special grade reagent) in 95 g of ultrapure water (diluted chloroauric acid solution) and ferrous sulfate octahydrate (Kanto Chemical Co., Ltd.) were added. A solution of 2 g of special grade reagent in 18 g of ultrapure water was added to each solution simultaneously over a period of 2 hours, and then the mixture was aged at 90°C for 1 hour to precipitate gold onto the seed particles, yielding nanoparticles (M-R2). 9,500 g of these nanoparticles (M-R2) were washed with twice the amount of ultrapure water using an ultrafilter (SIP-1013 manufactured by Asahi Kasei), and the concentration of the metal nanoparticles was adjusted to 0.005 mass %, yielding metal nanoparticles (M-R2F). The physical properties of the metal nanoparticles (M-R2F) were measured or tested using the methods 1 to 12 above. The results are shown in Table 3.

[0144] Comparative Example 3 10.0 g of trisodium citrate dihydrate (Kanto Chemical Co., Ltd.: special grade reagent) was dissolved in 9800 g of ultrapure water (electrical conductivity: 0.6 μS / cm, organic matter (TOC): 5 ppb). This solution was stirred at 350 rpm under a nitrogen atmosphere using a stirrer. A separate solution of 0.95 g of chloroauric acid tetrahydrate (Wako Pure Chemical Industries, Ltd.: special grade reagent) dissolved in 94.05 g of ultrapure water was added over 10 seconds, and the mixture was aged at 90°C for 1 hour to obtain seed particles (M-R3). The concentration of these seed particles was 0.005% by mass. 9,500 g of these nanoparticles (M-R3) were washed with five times the amount of ultrapure water using an ultrafilter (SIP-1013 manufactured by Asahi Kasei), and the concentration was adjusted to 0.005% by mass to obtain metal nanoparticles (M-R3F). The physical properties of the metal nanoparticles (M-R3F) were measured or tested using the methods 1 to 12 above. The results are shown in Table 3.

[0145] Comparative Example 4 The metal nanoparticles (M-3F) obtained in Example 3 were used as seed particles, and were subjected to the same treatment as the seed particle growth step in Example 1, followed by washing with an ultrafilter to obtain metal nanoparticles (M-R4F-2). The physical properties of the metal nanoparticles (M-R4F-2) were measured or tested using the methods 1 to 12 above. The results are shown in Table 3.

[0146] Comparative Example 5 To 500 g of the seed particle (M-1) dispersion (metal content: 0.0046 mass%) obtained in Example 1, 8,500 g of ultrapure water (electrical conductivity: 0.6 μS / cm, organic matter (TOC): 5 ppb) was added, and a solution prepared by dissolving 3.0 g of trisodium citrate (Kanto Chemical Co., Ltd.: special grade reagent) in 50 g of ultrapure water (electrical conductivity: 0.6 μS / cm, organic matter (TOC): 5 ppb) was added. The mixture was heated to 90°C and stirred at 350 rpm under a nitrogen atmosphere using a stirrer. A solution prepared by dissolving 0.95 g of chloroauric acid (Wako Pure Chemical Industries, Ltd.: special grade reagent) in 95 g of ultrapure water (diluted chloroauric acid solution) was added over 1 minute. The mixture was then aged at 90°C for 1 hour to precipitate gold on the seed particles, yielding nanoparticles (M-R5). 9,500 g of these nanoparticles (M-R5) were washed with twice the amount of ultrapure water using an ultrafilter (SIP-1013 manufactured by Asahi Kasei), and the concentration of the metal nanoparticles was adjusted to 0.005 mass %, yielding metal nanoparticles (M-R5F). The physical properties of the metal nanoparticles (M-R5F) were measured or tested using the methods 1 to 12 above. The results are shown in Table 3.

[0147] [Table 3]

Claims

1. A test strip for immunochromatographic testing, comprising a labeling substance source-containing pad impregnated with a metal nanoparticle dispersion for immunochromatographic testing, in which metal nanoparticles are dispersed, and which has the following characteristics [1] to [4]: [1] The metal nanoparticles have an aliphatic hydroxy acid on the surface. [2] The metal nanoparticles have an average particle size of 8 to 500 nm. [3] The metal nanoparticles are at least one selected from the group consisting of Au, Ag, Pd, Pt, Rh, and Ru. [4] The particle size coefficient of variation (CV value) of the metal nanoparticles is 0 to 20%.

2. 2. The immunochromatographic test strip according to claim 1, wherein the immunochromatographic test strip comprises the labeled substance source-containing pad, a sample drop pad, a membrane filter, an absorbent pad, and a backing sheet, and the membrane filter further comprises a test line and a control line.

3. 3. The test strip for immunochromatographic testing according to claim 1, wherein the metal nanoparticle dispersion for immunochromatographic testing before being impregnated into the labeled substance source-containing pad has the following characteristics [5] and [6]: [5] When the content of the metal nanoparticles is adjusted to 0.005% by mass, the concentration of free aliphatic hydroxy acid is less than 100 ppm. [6] When the content of the metal nanoparticles is adjusted to 0.005% by mass, the absorbance at a wavelength of 520 nm is 1.0 to 2.

2.

4. 3. The test strip for immunochromatographic testing according to claim 1 or 2, wherein, in a particle size distribution obtained by image analysis of the metal nanoparticle dispersion, when the particle size at 10% cumulative frequency is defined as D10, the particle size at 50% cumulative frequency is defined as D50, and the particle size at 90% cumulative frequency is defined as D90, from the smallest particle size, the condition represented by the following formula is satisfied: Formula: 0.9≦[(D10+D90) / 2] / D50≦1.1

5. 3. The immunochromatographic test strip according to claim 1, wherein the aliphatic hydroxy acid has 1 to 10 carbon atoms.

6. 3. The test strip for immunochromatographic testing according to claim 1, wherein the metal nanoparticle dispersion for immunochromatographic testing before being impregnated into the labeled substance source-containing pad has the following characteristic [7]: [7] The metal nanoparticle dispersion for immunochromatographic testing contains nanobubbles having an average bubble diameter of 50 to 500 nm for 10 5 Contains more than 1000 pieces / ml.

7. An immunochromatographic test kit comprising the test strip for immunochromatographic test according to claim 1 or 2.

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