JP6987939B2 - Suspension of gold-coated silver nanoplates and their uses and manufacturing methods - Google Patents

Description

The present invention relates to a suspension of a gold-coated silver nanoplate having a silver nanoplate as a core and a gold layer covering the silver nanoplate, and its use and production method.

Since silver nanoplates absorb light by interacting with light (localized surface plasmon resonance: LSPR), it is known that the suspension exhibits a color depending on the shape of the nanoplate. It is also known that the absorbed light can be changed, that is, the color can be changed by controlling the size and shape of the silver nanoplate. On the other hand, silver nanoplates are dissolved by oxidation and their shape changes. This oxidation-induced change in the shape of the silver nanoplates can cause the intended color change. Therefore, in order to stabilize the silver nanoplate against oxidation, the surface of the silver nanoplate is coated with gold. Patent Document 1 prepares a gold-coated silver nanoplate in the form of a stable suspension by adjusting the concentration and pH of the water-soluble polymer, and labels the detection reagent of the test substance (for example, of the target protein). It is described that it is used as an antibody label used for detection) and applied to detect various test substances with high sensitivity. However, Patent Document 1 does not specifically describe a gold-coated silver nanoplate having a gold layer thicker than 1.0 nm. Further, Non-Patent Documents 1 and 2 and Patent Document 2 describe gold-coated silver nanoplates having a relatively thick gold layer, and it is also described that their stability is excellent, but the particle size It is not described whether a thick gold layer can be stably formed even in a small silver nanoplate.

International Publication No. 2015/182770 International Publication No. 2018/180286

Adv. Funct. Mater (2015), 25, pp. 5435-5443 Adv. Funct. Mater (2011), XX, pp. 1-6, DOI: 10.1002 / adfm. 201110028

Oxidation resistance can be imparted by coating the silver nanoplate with gold, but the protective effect is insufficient with a thin film. On the other hand, when the concentration of gold in the coating process is increased in order to thicken the gold layer, silver has a lower electronegativity than gold, so that it emits electrons and becomes ionized easily, and as a result, silver is released from the silver nanoplate of the core. It elutes and the optical characteristics change before and after coating. In particular, silver nanoplates having a small particle size are susceptible to oxidation because they have a large specific surface area. Furthermore, when the grown gold layer comes into contact with the gold layer of other particles, agglomerates are formed. Therefore, an object of the present invention is to stably provide a gold-coated silver nanoplate having a thick gold layer for a small particle size.

As a result of diligent studies to solve the above problems, the present inventors use a gold ion complexing agent when coating the silver nanoplate with gold, and adjust the balance between the silver concentration and the gold concentration. As a result, it was found that a gold-coated silver nanoplate having a thick gold layer for a small particle size can be stably produced, and the present invention has been completed. That is, the present invention relates to a suspension of gold-coated silver nanoplates shown below, a dried product thereof, and a method for producing the suspension, a method for detecting a test substance using the suspension, and a method for detecting the test substance. It provides a kit.
[1] A suspension of a gold-coated silver nanoplate having a silver nanoplate as a core and a gold layer covering the silver nanoplate.
The average particle size of the silver nanoplate is 55 nm or less, and the gold layer is formed on the main plane and the end face of the silver nanoplate.
The ratio (T / D) of the average thickness (T) of the gold layer on the end face to the average particle size (D) of the silver nanoplate is 0.05 or more.
The suspension has a maximum absorption wavelength in the visible light region, and the ratio of the extinction degree (E _max ) at the _{maximum absorption wavelength to the extinction degree (E 900 ) at the wavelength of 900 nm of the suspension (E max} / E _900). ) Is 5 or more, suspension.
[2] The suspension according to the above [1], wherein the average thickness of the gold layer is 1.5 nm to 10.0 nm.
[3] The suspension according to the above [1] or [2], wherein the silver nanoplate as the core has an aspect ratio of more than 1 and 10 or less.
[4] The suspension according to any one of the above [1] to [3], wherein the maximum absorption wavelength exists in the range of 400 nm to 680 nm.
[5] The suspension according to any one of [1] to [4] above, wherein the degree of quenching at the maximum absorption wavelength is 2 or more.
[6] The suspension according to any one of [1] to [5] above, wherein the gold-coated silver nanoplate carries a specific binding substance to a test substance.
[7] The dried product of the suspension according to any one of the above [1] to [6].
[8] A method for detecting a test substance using the suspension according to the above [6].
The step of contacting the gold-coated silver nanoplate in the suspension with the test substance to form a complex thereof, and
A method comprising the step of detecting the complex.
[9] The gold-coated silver nanoplate in the suspension according to any one of the above [1] to [5] is supported with a specific binding substance to the test substance, and the above [6] is used. The method according to [8] above, further comprising the step of preparing the described suspension.
[10] A kit for detecting a test substance, which comprises the suspension according to any one of the above [1] to [6] or the dried product according to the above [7].
[11] A method for producing a gold-coated silver nanoplate having a silver nanoplate as a core and a gold layer covering the silver nanoplate.
A coating mixture containing a silver nanoplate having an average particle size of 55 nm or less, a gold-containing water-soluble compound, and a gold ion complexing agent is prepared to form the gold layer on the main plane and end face of the silver nanoplate. Including the process
The ratio (T / D) of the average thickness (T) of the gold layer on the end face to the average particle size (D) of the silver nanoplate is 0.05 or more.
In the coating mixture, and the silver concentration (C _Ag) is 0.25mM hereinafter gold concentration (C _Au) is not less than 0.1 mM, and the ratio of C _Au for _{C Ag (C Au / C Ag} ) is A manufacturing method that is 0.50 or more.
[12] The production method according to the above [11], wherein the complexing agent is at least one selected from the group consisting of sulfites, thiosulfates, cyanates, thiosulfates, and thiourea.
[13] A step of preparing a suspension of a gold-coated silver nanoplate having a silver nanoplate as a core and a gold layer covering the silver nanoplate.
A mixed solution of the suspension and the specific binding substance for the test substance is prepared to support the specific binding substance, which comprises a step of contacting the gold-coated silver nanoplate with the specific binding substance. It is a method of manufacturing gold-coated silver nanoplates.
The average particle size of the silver nanoplate is 55 nm or less, and the gold layer is formed on the main plane and the end face of the silver nanoplate.
The ratio (T / D) of the average thickness (T) of the gold layer on the end face to the average particle size (D) of the silver nanoplate is 0.05 or more.
The suspension has a maximum absorption wavelength in the visible light region, and the ratio of the extinction degree (E _max ) at the _{maximum absorption wavelength to the extinction degree (E 900 ) at the wavelength of 900 nm of the suspension (E max} / E _900). ) Is 5 or more,
A production method in which the quenching degree of the suspension at the maximum absorption wavelength is 2 or more, and the concentration of the specific binding substance in the contacting step is 100 μg / mL or less with respect to the volume of the entire mixture.
[14] The extinction degree of the suspension at the maximum absorption wavelength is 100 or less, and / or
The production method according to the above [13], wherein the concentration of the specific binding substance in the contact step is 0.5 μg / mL or more with respect to the total volume of the mixture.

According to the present invention, when the silver nanoplate is coated with gold, a gold ion complexing agent is used, the silver concentration is set to a certain value or less, the gold concentration is set to a certain value or more, and both concentrations are predetermined. By adjusting them so as to satisfy the above relationship, a gold-coated silver nanoplate having a thick gold layer for a small particle size can be stably produced. Therefore, it is possible to produce a gold-coated silver nanoplate having an excellent oxidation resistance and a small particle size. Since the gold-coated silver nanoplate having a small particle size has a maximum absorption wavelength in the visible light region, it is possible to achieve multicoloring of a method for detecting a test substance that requires visual determination such as immunochromatography. Further, since the gold-coated silver nanoplate having a small particle size and a thick gold layer has high dispersibility and can efficiently support a specific binding substance to a test substance, the amount of the specific binding substance used can be increased. It is possible to save money.

The spectroscopic spectrum of the suspension of gold-coated silver nanoplates (Ag @ Au suspensions 1 to 3) is shown. The transmission scanning electron microscope (STEM) observation photograph of the gold-coated silver nanoplate in Ag @ Au suspension 1 is shown. The STEM observation photograph of the gold-coated silver nanoplate in Ag @ Au suspension 2 is shown. The STEM observation photograph of the gold-coated silver nanoplate in Ag @ Au suspension 3 is shown. The spectroscopic spectrum of the suspension of gold-coated silver nanoplates (Ag @ Au suspensions 4 and 5) is shown. The STEM observation photograph of the gold-coated silver nanoplate in Ag @ Au suspension 4 is shown. The STEM observation photograph of the gold-coated silver nanoplate in Ag @ Au suspension 5 is shown. The high-angle scattering annular dark-field scanning transmission microscope (HAADF-STEM) observation photograph of the gold-coated silver nanoplate in Ag @ Au suspension 4 is shown. The spectroscopic spectrum of the suspension of gold-coated silver nanoplates (Ag @ Au suspensions 6 and 7) is shown. The STEM observation photograph of the gold-coated silver nanoplate in Ag @ Au suspension 6 is shown. The STEM observation photograph of the gold-coated silver nanoplate in Ag @ Au suspension 7 is shown. The spectroscopic spectrum of the suspension (Ag @ Au suspension 8) of the gold-coated silver nanoplate is shown. The effect on the spectroscopic spectrum due to the addition of the aqueous sodium chloride solution is shown. The effect on the spectroscopic spectrum due to the addition of the aqueous sodium chloride solution is shown. The spectroscopic spectrum of the suspension (Ag @ Au suspension 9-11) of the gold-coated silver nanoplate is shown. The STEM observation photograph of the gold-coated silver nanoplate in Ag @ Au suspension 9 is shown. The STEM observation photograph of the gold-coated silver nanoplate in the Ag @ Au suspension 10 is shown. The HAADF-STEM observation photograph of the gold-coated silver nanoplate in Ag @ Au suspension 9 is shown. The HAADF-STEM observation photograph of the gold-coated silver nanoplate in the Ag @ Au suspension 10 is shown.

Hereinafter, the present invention will be described in more detail.
The present invention relates to a suspension of a gold-coated silver nanoplate having a silver nanoplate as a core and a gold layer covering the silver nanoplate. As used herein, the term "silver nanoplate" refers to plate-like particles made from silver that have a size on the order of nanometers (nm), and are the upper and lower surfaces of the main plane, as well as the lower surface. , The end face (side surface) between the upper surface and the lower surface is provided. The shape of the upper surface and the lower surface of the silver nanoplate is not particularly limited, and may be, for example, a polygon such as a triangle, a quadrangle, a pentagon, or a hexagon (including a shape with rounded corners) or a circle. .. In the gold-coated silver nanoplate, the gold layer is formed on the main plane and the end face of the silver nanoplate, and a so-called silver nanoplate and a core-shell structure of the gold layer are formed.

The maximum major axis of the main plane of the silver nanoplate is referred to as a particle diameter, which corresponds to the diameter in the case of a circle and the length of the maximum side in the case of a triangle, for example. When the maximum major axis differs between the upper surface and the lower surface of the silver nanoplate, the longer one is defined as the maximum major axis of the silver nanoplate. The average particle size of the silver nanoplates used in the suspension of the present invention is about 55 nm or less, preferably about 50 nm or less, about 40 nm or less, or about 30 nm or less. The lower limit of the average particle size of the silver nanoplate is not particularly limited, but may be, for example, about 1 nm or more, preferably about 10 nm or more. The average thickness of the silver nanoplate is not particularly limited, but may be, for example, about 20 nm or less, preferably about 3 to about 15 nm. The particle size and thickness of the silver nanoplate may be measured by, for example, observation with a scanning electron microscope (SEM), observation with a scanning transmission electron microscope (STEM), or observation with a transmission electron microscope (TEM). The particle size may be measured by a dynamic light scattering type particle size distribution measuring device (DLS). When measuring the particle size of the silver nanoplate using an SEM observation photograph, a STEM observation photograph, or a TEM observation photograph, up and down from a total of 100 points of data obtained by measuring the particle size of 100 arbitrary silver nanoplates. The average particle size may be calculated by preparing 80 points of data excluding 10% and obtaining the average value thereof.

The ratio of the width orthogonal to the maximum major axis to the maximum major axis, that is, the index defined by "maximum major axis / thickness" is called "aspect ratio", and this is used as one of the indexes to express the shape of the nanoplate. Can be done. Since the position of the maximum absorption wavelength changes when the shape of the silver nanoplate changes, the aspect of the silver nanoplate can be said to be an index of the maximum absorption wavelength. In the suspension of the present invention, the aspect ratio of the silver nanoplate is not particularly limited, but may be, for example, larger than about 1, preferably about 1.5 to about 10. When the aspect ratio is in such a range, it is easy to adjust the maximum absorption wavelength due to surface plasmon resonance in the aqueous suspension to the visible light region.

In the gold layer of the gold-coated silver nanoplate, the ratio (T / D) of the average thickness (T) of the gold layer on the end face to the average particle size (D) of the silver nanoplate is about 0.05 or more. , It is preferably formed to be about 0.1 or more, and more preferably about 0.16 or more. In some embodiments, the T / D may be about 0.5 or less, about 0.4 or less, or about 0.3 or less. In some embodiments, the average thickness of the gold layer is from about 1.5 nm to about 10.0 nm or from about 2.0 nm to about 10.0 nm, preferably from about 2.0 nm to about 8.0 nm. When the thickness of the gold layer is within such a range, the oxidation of the silver nanoplate can be more effectively suppressed while maintaining the optical characteristics of the silver nanoplate. The thickness of the gold layer on the end face can be measured using a high-angle scattering annular dark-field scanning transmission electron microscope (HAADF-STEM). Specifically, for 10 arbitrary particles selected from the HAADF-STEM observation photograph, 10% above and below were excluded from the data of a total of 100 points in which the thickness of gold was measured at 10 arbitrary ends of each particle. The average thickness (T) may be calculated by preparing data of 80 points and obtaining the average value thereof.

Further, the thickness of the gold layer on the end face can be calculated from the size of the silver nanoplate before and after the gold coating measured by using a transmission scanning microscope. For example, when the shape of the main plane of the silver nanoplate is circular, the average particle diameter (a) before gold coating and the average particle diameter (b) after gold coating are measured, and the following formula:
T = (ba) / 2
The average thickness (T) can be calculated by. When the shape of the main plane of the silver nanoplate is an equilateral triangle, the average height (c) of the equilateral triangle before gold coating and the average height (d) of the equilateral triangle after gold coating are measured, and the following formula:
T = (dc) / 3
The average thickness (T) can be calculated by.

The color tone of the suspension of the present invention is adjusted by adjusting the size and / or shape of the core silver nanoplate and the thickness of the gold layer, and changing the maximum absorption wavelength of the gold-coated silver nanoplate. It is possible to do. That is, the size and shape of the silver nanoplate are adjusted so that the suspension of the present invention has a maximum absorption wavelength in the visible light region in consideration of the subsequent change in the maximum absorption wavelength due to the gold coating. In some embodiments, the maximal absorption wavelength is present in the range of about 400 nm to about 680 nm.

The gold-coated silver nanoplates in the suspension of the present invention have high particle size uniformity, so that the suspension exhibits a sharp spectroscopic spectrum. That is, the color of the suspension of the present invention is vivid and the saturation is high. The ratio of the extinction degree (E _max) at the maximum absorption wavelength for the extinction degree (E ₉₀₀₎ at a wavelength of 900nm of the suspension (E _max / E ₉₀₀₎ is about 5 or more, preferably about 10 or more. In some embodiments, the suspension of the invention may have a degree of quenching at the maximum absorption wavelength of about 2 or more or about 4 or more, preferably about 2 to about 100, more preferably about 2 to about 50. , Or about 2 to about 20. Although not bound by a specific theory, it is considered that the gold-coated silver nanoplates in the suspension of the present invention have a thick gold layer and therefore can exist stably even at a high concentration.

As the silver nanoplate for preparing the gold-coated silver nanoplate in the suspension of the present invention, those usually used in the art can be adopted without particular limitation, and for example, a commercially available product. May be used, or those manufactured according to a known production method or the method described in Examples described later may be used. On the other hand, when trying to form a relatively thick gold layer that satisfies the above-mentioned conditions on the main plane and the end face of the silver nanoplate, simply increasing the concentration of gold in the coating step makes silver more than gold. However, since the electronegativity is low, electrons are emitted and easily ionized, and as a result, silver is eluted from the silver nanoplate of the core and the optical characteristics change before and after coating. In particular, the silver nanoplates in the suspension of the present invention are susceptible to oxidation because they have a relatively small particle size and a large specific surface area. Furthermore, when the grown gold layer comes into contact with the gold layer of other particles, agglomerates are formed. Therefore, in the step of forming the gold layer on the main plane and the end face of the silver nanoplate, the silver concentration ( _{CA Ag} ) is 0.25 mM or less, and the gold concentration (C _Au ) is 0.1 mM or more. and as the ratio of C _Au for _{C Ag (C Au / C Ag} ) is 0.50 or more, said silver nano plate, gold-containing water-soluble compounds, and coating mixture containing a complexing agent for gold ion preparation Then, let it stand for a predetermined time. By using such a coating mixture, the gold-coated silver nanoplate can be efficiently and stably produced. See below for more details.

In the suspension of the present invention, solid gold-coated silver nanoplates are suspended in a liquid dispersion medium. The dispersion medium can be used without particular limitation as long as it can disperse the gold-coated silver nanoplates, and is, for example, water, an aqueous buffer solution (phosphate buffered saline, Tris hydrochloric acid buffer solution, HEPES buffer solution, etc.). , Alcohols (ethanol, methanol, etc.), ketones (acetone, methyl ethyl ketone, etc.), tetrahydrofuran, etc. may be contained in one or more, and preferably contains water or an aqueous buffer solution suitable for biochemical experiments. The suspension of the present invention may be one in which the gold-coated silver nanoplates are dispersed in the dispersion medium in a stationary state, and in the static state, the gold-coated silver nanoplates are settled, but shake or ultrasonic waves are used. It may be in a distributed state by processing.

The suspension of the present invention may or may not contain a water-soluble polymer. As used herein, the term "water-soluble polymer" refers to a water-soluble substance having a molecular weight of 500 or more, preferably 500 to 1,000,000, and more preferably 500 to 100,000. The term "water-soluble" as used herein means that the polymer dissolves in water in an amount of 0.001% by mass or more under normal temperature and pressure. Examples of the water-soluble polymer include polystyrene sulfonic acid or a sodium salt thereof, polyvinylpyrrolidone (PVP), polyethylene glycol, polyacrylamide, polyvinyl alcohol, polyacrylic acid, polymethacrylic acid, polyallylamine, dextran, and polymethacrylamide. Polyvinylphenol, vinyl polybenzoate, phospholipid polymer, bovine serum albumin (BSA), casein, or bis (p-sulfonatophenyl) phenylphosphine may be used, and these may be used with the gold-coated silver nanoplate. It may be modified with a hydroxyl group, a mercapto group, a disulfide group, an amino group, a carboxyl group or the like which are functional groups to be chemically bonded. Preferably, the water-soluble polymer is polystyrene sulfonic acid or a sodium salt thereof, PVP, or thiol-terminated polyethylene glycol.

The type of water-soluble polymer contained in the suspension of the present invention can be selected according to the use of the suspension. For example, when used in a biological experiment or a cell experiment, polyvinylpyrrolidone, polyethylene glycol, polyvinyl alcohol, or the like having good biocompatibility may be used. When the suspension of the present invention contains a water-soluble polymer, the concentration of the water-soluble polymer dissolved or dispersed in the suspension may be, for example, 100 μM or less, preferably 50 μM or less. , More preferably 10 μM or less. Alternatively, the concentration of the water-soluble polymer may be, for example, 1% by mass or less, preferably 0.5% by mass or less, and more preferably 0.1% by mass or less. When a water-soluble polymer is added to the suspension of the present invention, the dispersion stability of the gold-coated silver nanoplate is improved in the suspension. Without being bound by a specific theory, when the concentration of the water-soluble polymer is 100 μM or less or 1% by mass or less, for example, the present invention is compared with the case where the concentration exceeds 100 μM or 1% by mass. When the suspension of the above was used for the detection of the test substance, the specific binding substance to the test substance was well carried on the gold-coated silver nanoplate, or the specific binding substance to the test substance was carried. Since the gold-coated silver nanoplate forms a stable complex with the test substance, the detection sensitivity of the test substance can be increased as a result.

Methods for measuring the concentration of water-soluble polymers include nuclear magnetic resonance spectroscopy (NMR), size exclusion chromatography (SEC) (eg, gel filtration chromatography (GPC)), and differential thermal / thermal weight measurement (TG-DTA). And so on. In the case of NMR, the suspension of the present invention is dried, and the obtained solid content is dissolved (dispersed) with a heavy solvent for measurement. An internal standard substance (for example, maleic acid) having a known concentration is added to the heavy solvent in advance, and the integrated value of the signal derived from the water-soluble polymer and the integrated value of the signal derived from the internal standard substance in the obtained spectrum are compared. Therefore, the concentration of the water-soluble polymer can be calculated. Further, in the case of GPC, first, a plurality of known water-soluble polymer aqueous solutions are analyzed, and a calibration curve is created from the peak area derived from the water-soluble polymer in the obtained chart. Next, the suspension of the present invention can be measured, and the concentration of the water-soluble polymer can be calculated from the peak area derived from the water-soluble polymer in the obtained chart. Further, in the case of TG-DTA, the water-soluble high molecular weight can be calculated from the weight change when the dry solid content of the suspension of the present invention is measured.

The suspension of the present invention may or may not optionally contain sodium citrate. The inclusion of sodium citrate improves the dispersibility of the gold-coated silver nanoplates in the suspension. When the suspension contains sodium citrate, the concentration of sodium citrate in the suspension may be, for example, 50 mM or less, preferably 25 mM or less, and more preferably 13 mM or less. When the concentration of sodium citrate is 50 mM or less, the gold-coated silver nanoplates in the suspension are less likely to aggregate.

The suspension of the present invention may contain any component as long as it does not adversely affect the gold-coated silver nanoplate. Examples of the optional component include reagents (for example, sodium borohydride and ascorbic acid) and dispersants (for example, polyethylene glycol having a molecular weight of less than 500) used in producing gold-coated silver nanoplates.

In some embodiments, the suspension of the invention can be used to label a specific binding agent to a test substance, i.e., a detection reagent, for the purpose of detecting the test substance. In other words, the gold-coated silver nanoplate can carry a specific binding substance to the test substance. As used herein, the term "supported" means that the gold-coated silver nanoplate is bound to a specific binding substance to a test substance regardless of the mode such as covalent bond, non-covalent bond, or direct or indirect bond. It means that they form a complex. As the carrying method, a normal carrying method can be used without particular limitation, and physical adsorption, chemical adsorption (covalent bond to the surface), chemical bond (covalent bond, coordination bond, ionic bond or metal bond) and the like can be used. Utilizing a method of directly binding the gold-coated silver nanoplate and a specific binding substance to the test substance, or by binding a part of the above-mentioned water-soluble polymer to the surface of the gold-coated silver nanoplate. , A method of directly or indirectly binding a specific binding substance to a test substance to the terminal or main chain or side chain of the water-soluble polymer can be adopted. For example, when the specific binding substance to the test substance is an antibody, the gold-coated silver nanoplate and the antibody solution are mixed, shaken, and centrifuged to carry the antibody-bearing gold-coated silver nanoplate. (Labeled detection reagent) can be obtained as a precipitate. Further, when the gold-coated silver nanoplate and the antibody are supported by electrostatic adsorption, the carrying efficiency of the antibody can be improved by coating the surface of the gold-coated silver nanoplate with polystyrene sulfonic acid having a negative charge. As with the water-soluble polymer, the specific binding substance to the test substance can improve the dispersion stability of the gold-coated silver nanoplate in the suspension of the present invention, and thus the test substance in particular. It is also suitable as a dispersant for gold-coated silver nanoplates carrying a specific binding substance to.

As the specific binding substance to the test substance, the test substance to be detected can be detected by forming a complex with the test substance, and the gold-coated silver nanoplate can be used as a label. Any substance can be used without particular limitation. Specific examples of the combination of the test substance and the specific binding substance to the test substance include an antigen and an antibody that binds to the test substance, an antibody and an antigen that binds to the antibody, a sugar chain or a complex sugar and a lectin that binds to the nucleic acid, and a lectin that binds to the antibody. Sugar chains or complex sugars, hormones or cytokines and their binding receptors, receptors and their binding hormones or cytokines, proteins and their binding nucleic acid aptamers or peptide aptamers, enzymes and their binding substrates, substrates and their binding Enzymes, biotin and avidin or streptavidin, avidin or streptavidin and biotin, IgG and protein A or protein G, protein A or protein G and IgG, T-cell immunoglobulin / mutin domain-containing molecule 4 (Tim4) and phosphatidylserine (PS) ), PS and Tim4, or a first nucleic acid and a second nucleic acid that binds (hybridizes) to the first nucleic acid. The second nucleic acid may be a nucleic acid containing a sequence complementary to the first nucleic acid.

When the test substance is an antigen, the specific binding substance to the antigen may be an antibody. The antibody may be a polyclonal antibody, a monoclonal antibody, a single chain antibody or a fragment thereof that specifically binds to the antigen, and the fragment may be an F (ab) fragment, an F (ab') fragment, and the like. It may be an F (ab') ₂ fragment or an F (v) fragment. The antigen as a test substance may be a lectin such as concanavalin A (ConA), malt aglutinin and lysine, and may be influenza virus, coronavirus, adenovirus, RS virus, rotavirus, human papillomavirus, human immunodeficiency virus and B. It may be a virus such as hepatitis virus, decavirus, dengue virus or a substance contained therein (for example, hepatitis B virus antigen (HBs antigen) or hemagglutinin of influenza virus), aspergillus flavus, chlamydia, syphilis treponema, lytic fungus. , Charcoal bacillus, yellow staphylococcus, diarrhea, Escherichia coli, salmonella, murine typhoid, palatinus, pyogenic and enteritis vibrio, and other pathogenic microorganisms or substances possessed by them (eg, aspergillus flavus afratoxin (B1, B2) , G1, G2 or M1 etc.), verotoxin of intestinal hemorrhagic Escherichia coli or streptidine O of lytic fungi), in the blood of immunoglobulin G (IgG), rheumatic factor and C-reactive protein (CRP). It may be a protein, a glycoprotein such as mutin, insulin, pituitary hormone (eg, growth hormone (GH), corticostimulatory hormone (ACTH), thyroid stimulating hormone (TSH), follicular stimulating hormone). (FSH), luteinizing hormone (LH), prolactin, or melanin cell stimulating hormone (MSH)), thyroid stimulating hormone-releasing hormone (TRH), thyroid hormone (eg, diodesylonine or triiodylonin), villous Gonadotropin, calcium metabolism regulatory hormone (eg, calcitonin or paratormon), pancreatic hormone, gastrointestinal hormone, vasoactive intestinal peptide, follicular hormone (eg, estron), natural or synthetic luteinizing hormone (eg, progesterone), male hormone (eg, progesterone) , Teststerone), and hormones such as corticosteroids (eg, cortisol), and other in vivo substances such as serotonin, urokinase, ferritin, substance P, prostaglandin and cholesterol, prostatic. It may be a tumor marker such as acid phosphatase (PAP), prostate-specific antigen (PSA), alkaline phosphatase, transaminase, trypsin, pepsinogen, α-fet protein (AFP) and cancer fetal antigen (CEA), and blood. It may be a sugar chain antigen such as a liquid antigen, or a marker used for detecting fecal occult blood such as hemoglobin and transferase, and is cross-linked by the action of active factor XIII in the blood coagulation / fibrin system. The stabilized fibrin may be a D-dimer, which is a kind of fibrin degradation product degraded by plasmin, or may be a protein or nucleic acid possessed by extracellular vesicles. When the antigen as the test substance is an in vivo substance such as a hormone or a cytokine, the specific binding substance to the in vivo substance may be not only an antibody but also a receptor, and the antigen as a test substance. When is a sugar chain or a complex sugar having a sugar chain, the specific binding substance to the sugar chain or the complex sugar having a sugar chain may be a lectin as well as an antibody. Further, the antigen as a test substance may be a hapten such as penicillin and cadmium.

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

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

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

When the combination of the test substance and the specific binding substance to the test substance is a protein and a nucleic acid aptamer or a peptide aptamer that binds to the protein, the nucleic acid aptamer is, for example, Shigella sonnet (Bacillus anthracis), Staphylococcus aureus. ), Group D Shigella sonnei, Escherichia colli, Salmonella typhimurium, Streptococcus hemolyticus, Palatyphanthyl bacillus aptamer , Bacteria such as Pseudomonas aeruginosa, Vibrio parahaemoliticus, tumor markers appearing on the cell surface of epithelium such as mutin 1, or DNA aptamers that bind to enzymes such as β-galactosidase and thrombin, or It may be an RNA aptamer that binds to the Tat protein or Rev protein of a human immunodeficiency virus, and the peptide aptamer may be, for example, a peptide aptamer that binds to the neoplastic protein HPV16 E6 of human papillomavirus (HPV).

When the test substance is a vitamin, the specific binding substance for the vitamin may be a vitamin-binding protein such as transcobalamin that binds to vitamin D and transcobalamin that binds to vitamin B12. When the test substance is an antibiotic, the specific binding substance to the antibiotic may be a penicillin-binding protein such as PBP1 and PBP2 that binds to penicillin.

The nucleic acid as a test substance or a substance that binds to the test substance may be, for example, DNA, RNA, oligonucleotide, polynucleotide, or an amplification product thereof.

In another aspect, the invention also relates to a dried product of the suspension described above. The dried product of the present invention can be appropriately prepared from the suspension by a method usually used in the art. The dried product of the present invention is in a suitable form for transporting or storing the gold-coated silver nanoplate. In some embodiments, the dried product is a lyophilized product. When the dried product of the present invention is used, the above-mentioned dispersion medium can be appropriately added and resuspended to reconstitute the suspension.

In another aspect, the invention also relates to a method of detecting a test substance using the suspension described above, wherein the method comprises a specific binding agent to the test substance in the suspension. It includes a step of contacting the supported gold-coated silver nanoplate with the test substance to form a complex thereof, and a step of detecting the complex. The gold-coated silver nanoplate carrying the specific binding substance to the test substance may be prepared immediately before carrying out the method of the present invention. In other words, the method of the present invention carries it on a gold-coated silver nanoplate that does not carry a specific binding agent for the test substance, and carries a gold-coated silver nanoplate that carries the specific binding substance for the test substance. Further may include the step of preparing a suspension of silver nanoplates. In addition, the method of the present invention may further include a step of resuspending a dried product of a suspension of gold-coated silver nanoplates carrying a specific binding substance to the test substance.

The complex can be detected by using a means usually used in the field of detecting a test substance or a means usually used for detecting an agglomerate or a precipitate without particular limitation. For example, the formation of the complex can be measured by dimming degree measurement, absorbance measurement, particle size distribution measurement, particle size measurement, Raman scattered light measurement, color tone change observation, aggregation or precipitation formation observation, immunochromatography method, electrophoresis, and It may be detected by a means selected from the group consisting of flow cytometry.

In another aspect, the invention also relates to a kit for detecting a test substance, including the suspensions or dried products described above. The kit of the present invention may further include a standard product of the test substance, an immunochromatographic test strip (test strip), an instruction manual describing the method of use, and the like.

In another aspect, the invention also relates to a method for producing a gold-coated silver nanoplate having a silver nanoplate as a core and a gold layer covering the silver nanoplate, wherein the method comprises an average particle size. A step of preparing a coating mixture containing a silver nanoplate having a diameter of 55 nm or less, a gold-containing water-soluble compound, and a complexing agent for gold ions to form the gold layer on the main plane and the end face of the silver nanoplate. I'm out. The gold layer can be formed by allowing the coating mixture to stand for a predetermined time. The ratio (T / D) of the average thickness (T) of the gold layer on the end face to the average particle size (D) of the silver nanoplate is about 0.05 or more, preferably about 0.1 or more. Yes, more preferably about 0.16 or more. In some embodiments, the T / D may be, for example, about 0.5 or less, about 0.4 or less, or about 0.3 or less.

In the coating mixture, the silver concentration (C _Ag ) is about 0.25 mM or less, preferably about 0.20 mM or less. In the coating mixture, the gold concentration (C _Au ) is about 0.1 mM or more, preferably about 0.13 mM or more. Then, in the coating mixture, the ratio of C _Au for _{C Ag (C Au / C Ag} ) is from about 0.50 or greater, preferably about 1.00 or more. In some embodiments, C _Au / C _Ag may be, for example, about 3.50 or less or about 3.00 or less. Although not bound by a specific theory, when the silver nanoplate is coated with gold under such conditions, the change in optical properties due to the elution of silver from the silver nanoplate is suppressed, and the change in optical properties is suppressed. Since the generation of agglomerates that may occur during the formation of the gold layer is also suppressed, it is considered that a highly uniform gold-coated silver nanoplate can be efficiently and stably produced.

The "gold-containing water-soluble compound" described in the present specification means a water-soluble compound containing a gold atom as a component. The gold-containing water-soluble compound is not particularly limited as long as it can form a gold layer on the main plane and the end face of the silver nanoplate, and is, for example, gold (III) chloride acid or a sodium salt or potassium salt thereof, or gold cyanide (I). ) Potassium, gold (II) cyanide, or sodium gold (I) sulfite, and the like, preferably gold (III) chloride acid or a sodium salt or potassium salt thereof.

The "gold ion complexing agent" described herein refers to a compound capable of forming a complex with gold ions. The complexing agent is not particularly limited as long as it does not interfere with the formation of a gold layer on the main plane and the end face of the silver nanoplate, and is, for example, sulfate (sodium sulfate, potassium sulfite, ammonium sulfite, etc.), thiosulfate (, etc.). At least selected from the group consisting of sodium thiosulfate, potassium thiosulfate, ammonium thiosulfate, etc.), cyanide (sodium thiosulfate, potassium cyanide, or ammoum cyanide, etc.), thiosulfate, thiourea, chloride salts, etc. It may be one kind, and is preferably a compound having a low ability to form a complex with silver ions such as sulfate or thiosulfate (particularly sodium sulfate or sodium thiosulfate). Without being bound by a particular theory, the complexing agent reduces the chances of contact between the silver nanoplate and the gold ions and gently proceeds with the coating reaction, resulting in a highly homogeneous gold-coated silver nanoplate. It is considered that it can contribute to the stable production of.

The coating mixture may contain any component as long as it does not interfere with the formation of the gold layer. Examples of the optional component include reagents (for example, sodium borohydride and ascorbic acid) and dispersants (for example, polyethylene glycol having a molecular weight of less than 500) used for producing silver nanoplates.

In another aspect, the present invention comprises a step of preparing a suspension of a gold-coated silver nanoplate having a silver nanoplate as a core and a gold layer covering the silver nanoplate, and the suspension and a test. A gold-coated silver nano carrying the specific binding substance, which comprises a step of preparing a mixed solution with the specific binding substance to the substance and bringing the gold-coated silver nanoplate into contact with the specific binding substance. It is also related to the method of manufacturing the plate. In the suspension, the average particle size of the silver nanoplate is about 55 nm or less, preferably about 50 nm or less, about 40 nm or less, or about 30 nm or less. In some embodiments, the average particle size of the silver nanoplates may be about 1 nm or more, preferably about 10 nm or more. The gold layer is formed on the principal plane and the end face of the silver nanoplate. The ratio (T / D) of the average thickness (T) of the gold layer on the end face to the average particle size (D) of the silver nanoplate is about 0.05 or more, preferably about 0.1 or more. Yes, more preferably about 0.16 or more. In some embodiments, the T / D may be, for example, about 0.5 or less, about 0.4 or less, or about 0.3 or less. The suspension has a maximum absorption wavelength in the visible light region, and the ratio of the quenching degree (E _max ) at the _{maximum absorption wavelength to the quenching degree (E 900 ) at a wavelength of 900 nm of the suspension (E max} / E _900). ) Is about 5 or more, preferably about 10 or more.

The extinction degree of the suspension at the maximum absorption wavelength is about 2 or more, preferably about 4 or more. In some embodiments, the quenching degree at the maximum absorption wavelength of the suspension may be about 100 or less, about 50 or less, or about 20 or less. The concentration of the specific binding substance in the contact step is about 100 μg / mL or less, preferably about 50 μg / mL or less, based on the total volume of the mixture. In some embodiments, the concentration of the specific binding agent in the contacting step may be about 0.5 μg / mL or higher, preferably about 1.0 μg / mL or higher.

Since conventional metal nanoparticles tend to aggregate at high concentrations, it is necessary to bring them into contact with each other at a concentration at which the specific binding substance to be carried also acts as a dispersant or a blocking agent. On the other hand, since the gold-coated silver nanoplate in the suspension used in the production method of the present invention can exist stably even at a high concentration, it is possible to reduce the concentration of the specific binding substance to be carried. Conceivable.

Hereinafter, the present invention will be specifically described with reference to Examples, but the scope of the present invention is not limited to these Examples.

1. 1. Preparation Example (1) Preparation of seed particles for silver nanoplates In 200 mL of a 2.5 mM trisodium citrate aqueous solution, 10 mL of a 0.5 g / L polystyrene sulfonic acid (molecular weight 70,000) aqueous solution, and 10 mM hydride. 11.5 mL of an aqueous sodium solution was added with stirring, and then 200 mL of a 0.74 mM aqueous solution of silver nitrate was added at 86 mL / min. After stopping stirring, the obtained solution was allowed to stand in an incubator (30 ° C.) for 60 minutes to prepare an aqueous dispersion (seed suspension) of silver nanoplate seed particles.

(2) Preparation of silver nanoplate aqueous dispersion 45 mL of 10 mM ascorbic acid aqueous solution and 72 mL of the seed suspension were added to 2000 mL of distilled water with stirring. Then, 120 mL of a 7.4 mM silver nitrate aqueous solution was added at 26 mL / min. To the obtained solution, 20 mL of a 250 mM trisodium citrate aqueous solution was added, and stirring was stopped. The obtained solution was allowed to stand in an incubator (23 ° C.) under an air atmosphere for 20 hours to prepare a silver nanoplate aqueous dispersion 1 (AgPL suspension 1). The silver concentration of this AgPL suspension 1 was calculated to be 0.41 mM. The average particle size of the prepared silver nanoplate was 24.8 nm. The average particle size was obtained by observing the AgPL suspension 1 with a scanning transmission electron microscope (STEM) and measuring the maximum major axis of 100 arbitrary silver nanoplates. It was calculated by calculating the average value of the data of 80 points.

In addition, AgPL suspension 2 and AgPL suspension 3 were prepared by the same method except that the amount of the seed suspension used was changed to 300 mL or 24 mL, respectively. The average particle size of the prepared silver nanoplates was 13.2 nm (AgPL suspension 2) and 48.4 nm (AgPL suspension 3), respectively.

(3) Gold coating treatment of silver nanoplates 14 mL of a 24 mM gold chloride aqueous solution, 8 mL of a 200 mM sodium hydroxide aqueous solution, and 103 mL of a 10 mM sodium sulfite aqueous solution were mixed with 150 mL of distilled water to prepare a growth solution 1. .. Then, in 312 mL of distilled water, 312 mL of AgPL suspension 1, 69 mL of a 5 mass% polyvinylpyrrolidone aqueous solution, 14 mL of a 500 mM ascorbic acid aqueous solution, 14 mL of a 500 mM sodium hydroxide aqueous solution, and 3 mL of a 100 mM sodium sulfite aqueous solution were added. 275 mL of the growth solution 1 was mixed and allowed to stand at room temperature for 24 hours to prepare an aqueous dispersion 1 (Ag @ Au suspension 1) of a gold-coated silver nanoplate. Then, Ag @ Au suspension 2 and Ag @ Au suspension 3 were prepared by the same method except that the growth solution 1 was diluted 1.33 times or 2-fold and used. FIG. 1 shows a spectral spectrum obtained by appropriately diluting Ag @ Au suspensions 1 to 3 and measuring with a spectrophotometer V-770 (manufactured by JASCO Corporation). Then, STEM observation photographs of gold-coated silver nanoplates in Ag @ Au suspensions 1 to 3 are shown in FIGS. 2 to 4, respectively.

In addition, Ag @ Au suspension was used except that AgPL suspension 2 or AgPL suspension 3 was used instead of AgPL suspension 1 and growth solution 1 diluted 2.5 times or 2 times was used, respectively. Ag @ Au suspension 4 and Ag @ Au suspension 5 were prepared in the same manner as in Liquid 1, respectively. FIG. 5 shows a spectroscopic spectrum obtained by appropriately diluting Ag @ Au suspension 4 or 5 and measuring with a spectrophotometer V-770 (manufactured by JASCO Corporation). The STEM observation photographs of the gold-coated silver nanoplates in the Ag @ Au suspensions 4 and 5 are shown in FIGS. 6 and 7, respectively, and the HAADF-STEM observations of the gold-coated silver nanoplates in the Ag @ Au suspension 4 are shown. The photograph is shown in FIG.

As a comparative example, the Ag @ Au suspension was used in the same manner as the Ag @ Au suspension 1 except that the AgPL suspension 1 was doubled (624 mL) and distilled water was not used during the gold coating reaction. 6 was prepared. Further, instead of the growth solution 1, a growth solution 2 (prepared by mixing 25 mL of distilled water with 28 mL of a 24 mM gold chloride aqueous solution, 16 mL of a 200 mM sodium hydroxide aqueous solution, and 206 mL of a 10 mM sodium sulfite aqueous solution) was used. The Ag @ Au suspension 7 was prepared in the same manner as the Ag @ Au suspension 6 except that it was used. FIG. 9 shows a spectroscopic spectrum obtained by appropriately diluting Ag @ Au suspension 6 or 7 and measuring with a spectrophotometer V-770 (manufactured by JASCO Corporation). Then, STEM observation photographs of the gold-coated silver nanoplates in the Ag @ Au suspensions 6 and 7 are shown in FIGS. 10 and 11, respectively.

Then, according to the method for preparing suspension A1 described in Example 1 of Patent Document 1 (International Publication No. 2015/182770), an aqueous dispersion of a gold-coated silver nanoplate was prepared, and the pH was adjusted with an aqueous sodium hydrogen carbonate solution. Adjusted to 7.3 to prepare Ag @ Au suspension 8. FIG. 12 shows a spectroscopic spectrum obtained by appropriately diluting the Ag @ Au suspension 8 and measuring it with a spectrophotometer V-770 (manufactured by JASCO Corporation).

(4) Characteristics of gold-coated silver nanoplates Ag @ Au suspensions 1 to 8 were observed with a transmission scanning microscope, and the average thickness of the gold layer on the end face of the gold-coated silver nanoplates in each suspension was calculated. Specifically, for the convenience of calculation, a gold-coated silver nanoplate having a circular shape is selected, the average particle size is measured, and the difference from the average particle size measured before the gold coating is divided by two. , The average thickness of the gold layer on the end face was calculated. Table 1 shows the silver concentration and gold concentration of the mixed solution during the gold coating operation, the average particle size of the core silver nanoplate and the average thickness of the gold layer at the end face, and the maximum absorption wavelength and the maximum of the gold-coated silver nanoplate. The absorption wavelength or the degree of extinction at 900 nm are collectively shown.

The Ag @ Au suspensions 1 to 5 all exhibited bright colors and showed sharp spectral spectra (FIGS. 1 and 5). In addition, uniform particles were formed in all suspensions (FIGS. 2-4 and 6-8). On the other hand, the colors exhibited by Ag @ Au suspensions 6 and 7 were turbid, and in their spectral spectra, light absorption was observed even on the wavelength side longer than the maximum absorption wavelength (FIG. 9). In the Ag @ Au suspensions 6 and 7, many aggregated and connected particles were also observed (FIGS. 10 and 11). Light absorption was also observed on the longer wavelength side than the maximum absorption wavelength, and the saturation was reduced as a whole, probably because of the influence of such agglomerates. Therefore, in order to uniformly produce gold-coated silver nanoparticles having a thick gold layer and to produce a highly saturated suspension, it is not enough to add a gold complexing agent to increase the gold concentration. , It is necessary to lower the silver concentration and adjust so that the silver concentration and the gold concentration satisfy the predetermined relationship.

2. 2. Oxidation resistance test examples To Ag @ Au suspensions 1 to 8, an aqueous sodium chloride solution was added so that the final concentration was 5%, and the change in color tone before and after the addition was visually observed. In addition, the spectral spectra of Ag @ Au suspensions 3 and 8 before and after the addition of the aqueous sodium chloride solution were measured and compared. The results are shown in Table 2 and FIGS. 13 and 14.

Since the gold-coated silver nanoplates of Ag @ Au suspensions 1 to 7 had a thick gold layer, the color tone did not change before and after the addition of sodium chloride, and the spectral spectrum did not change (Table 2 and FIG. 13). .. On the other hand, since the gold-coated silver nanoplate of Ag @ Au suspension 8 has a thin gold layer, the silver nanoplate of the core is easily oxidized by the addition of sodium chloride, and its shape changes, resulting in a spectroscopic spectrum and a color tone. It has changed significantly (Table 2 and FIG. 14).

In order to confirm that the oxidation resistance improving action of the gold-coated silver nanoplates of the examples is not related to the dispersion stabilizing action of the water-soluble polymer (polyvinylpyrrolidone), after washing the gold-coated silver nanoplates, the gold-coated silver nanoplates are washed. An oxidation resistance test with sodium chloride was carried out. Specifically, gold-coated silver nanoplates in Ag @ Au suspensions 1 to 8 are precipitated by centrifugation, the supernatant is removed, and a 0.5 mM trisodium citrate aqueous solution is added to precipitate gold. The coated silver nanoplates were redistributed. The same operation was repeated twice to prepare Ag @ Au suspensions 1'to 8'. The concentration of each Ag @ Au suspension was adjusted so that the degree of quenching at the maximum absorption wavelength was 2.0. Sodium chloride aqueous solution was added to each of these suspensions so that the final concentration was 5%, and the change in color tone before and after the addition was visually observed. The results are shown in Table 3.

Cleaning to remove most of the water-soluble polymers did not change the results of the oxidation resistance test. That is, in Ag @ Au suspensions 1 to 7, the color tone of the suspension did not change before and after the addition of sodium chloride due to the thick gold layer, and in Ag @ Au suspension 8 with a thin gold layer, the color tone of the suspension did not change. The addition of sodium chloride easily oxidized the silver nanoplates in the core, changing their shape and significantly changing the color tone (Table 3).

3. 3. Immunochromatographic test example The gold-coated silver nanoplate in Ag @ Au suspension 3 was precipitated by centrifugation, the supernatant was removed, and then a 0.5 mM trisodium citrate aqueous solution was added to precipitate the gold-coated silver nanoplate. Was dispersed. The same operation was repeated twice, and the concentration of the Ag @ Au suspension was adjusted so that the quenching degree at the maximum absorption wavelength was 2.0. The resulting mixture was obtained by mixing 1 mL of this suspension with 0.1 mL of anti-human chorionic gonadotropin (hCG) antibody solution diluted to a concentration of 50 μg / mL with 5 mM borate buffer (pH 7.5). Was allowed to stand at room temperature for 60 minutes. 0.05 mL of 80 μM polyethylene glycol solution (in 5 mM borate buffer) was added thereto, and the obtained suspension was allowed to stand at room temperature for 30 minutes. Then, 0.100 mL of 0.5 mM bovine serum albumin solution (in 5 mM borate buffer) was added, and the obtained suspension was allowed to stand at room temperature for 60 minutes. This was centrifuged to precipitate a complex of the antibody and a gold-coated silver nanoplate, and the supernatant was removed. Then, 0.40 mL of 20 mM Tris-hydrochloric acid buffer (containing 8 μM polyethylene glycol, 0.25 mM BSA, and 150 mM NaCl) was added to the complex and redispersed, and the extinction of the maximum absorption wavelength was 1. The developing solution 3 was prepared by adjusting the concentration so as to be 0.0. Further, in the same manner, developing liquids 4 and 5 were prepared from Ag @ Au suspensions 4 and 5, respectively.

An anti-hCG antibody was linearly fixed as a capture antibody in the center of an immunochromatographic test paper (manufactured by Fordix Co., Ltd.) equipped with a strip-shaped nitrocellulose membrane to which a water absorption pad was attached to one end. Immerse the end of the immunochromatographic test strip without the water absorption pad in 50 μL of test solution containing or not containing 200 mIU / mL hCG (10 mM phosphate buffered saline containing 0.25 mM BSA). Was developed on an immunochromatographic test paper. Next, 60 μL of any of the developing liquids 3 to 5 was developed. Finally, the coloration of the portion where the capture antibody was immobilized was evaluated visually or by luminance analysis. In the brightness analysis, the immunochromatographic test paper after unfolding the developing liquids 3 to 5 is scanned by a scanner (device name: CanoScan LiDE500F, manufacturer: Canon Co., Ltd.), and the detection line (the part where the captured antibody is fixed) and the detection line are scanned. Image-J (Open source and public image processing software developed by Wayne Rasband at the National Institutes of Health; http: // It was quantified using imagej.nih.gov/ij/). More specifically, the minimum luminance of each portion was measured 5 times each, and the median of the obtained numerical values was adopted as the minimum luminance of the detection line or the control region. Then, the minimum luminance of the detection line was subtracted from the minimum luminance of the control region to obtain the luminance difference. The results are shown in Table 4.

（目視判定の基準）
＋＋：鮮明な着色あり
＋ ：着色あり
− ：着色なし

(Criteria for visual judgment)
++: With vivid coloring ＋: With coloring −: Without coloring

The detection antibody could be carried on a gold-coated silver nanoplate having a thick gold layer and used for an immunochromatography method. By adopting silver nanoplates with different particle sizes for the core, the test substance could be labeled with various color tones.

4. Other Preparation Examples and Test Examples According to the method described in item 1 (2) above, AgPL suspension 4 and AgPL suspension are carried out in the same manner except that the amount of seed suspension used is changed to 100 mL or 42 mL. Liquids 5 were prepared respectively. The average particle size of the prepared silver nanoplates was 20.2 nm (AgPL suspension 4) and 32.8 nm (AgPL suspension 5), respectively.

According to the method described in item 1 (3) above, Ag @ Au suspension except that AgPL suspension 4 was used instead of AgPL suspension 1 and growth solution 1 diluted 1.7 times was used. Ag @ Au suspension 9 was prepared in the same manner as in turbid liquid 1. Further, Ag @ was used in the same manner as Ag @ Au suspension 1 except that AgPL suspension 5 was used instead of AgPL suspension 1 and the growth solution 1 diluted 1.4 times was used. Au suspension 10 was prepared. Then, Ag @ was used in the same manner as Ag @ Au suspension 1 except that AgPL suspension 2 was used instead of AgPL suspension 1 and the growth solution 1 diluted 2.9-fold was used. Au suspension 11 was prepared. FIG. 15 shows a spectral spectrum obtained by appropriately diluting Ag @ Au suspensions 9 to 11 and measuring with a spectrophotometer V-770 (manufactured by JASCO Corporation). The STEM observation photographs of the gold-coated silver nanoplates in the Ag @ Au suspensions 9 and 10 are shown in FIGS. 16 and 17, respectively, and the HAADF-STEM observation photographs of the gold-coated silver nanoplates are shown in FIGS. 18 and 19, respectively. show.

According to the description of the above item 1 (4), the average thickness of the gold layer on the end face of the gold-coated silver nanoplates in the Ag @ Au suspensions 9 to 11 was calculated. Table 5 shows the silver concentration and gold concentration of the mixed solution during the gold coating operation, the average particle size of the core silver nanoplate and the average thickness of the gold layer at the end face, and the maximum absorption wavelength and the maximum of the gold-coated silver nanoplate. The absorption wavelength or the degree of extinction at 900 nm are collectively shown.

The Ag @ Au suspensions 9 to 11 all exhibited vivid colors and showed a sharp spectroscopic spectrum (FIG. 15). In addition, uniform particles were formed in any of the suspensions 9 to 11 (FIGS. 16 to 19).

An aqueous sodium chloride solution was added to each of the Ag @ Au suspensions 9 to 11 so that the final concentration of sodium chloride was 5%, and the change in color tone before and after the addition was visually observed. Since the gold-coated silver nanoplates of Ag @ Au suspensions 9 to 11 had a thick gold layer, the color tone did not change before and after the addition of sodium chloride (Table 6).

In addition, the gold-coated silver nanoplates were washed in the same manner as in item 2 above to prepare Ag @ Au suspensions 9'to 11'. The concentration of each Ag @ Au suspension was adjusted so that the degree of quenching at the maximum absorption wavelength was 2.0. Sodium chloride aqueous solution was added to each of these suspensions so that the final concentration was 5%, and the change in color tone before and after the addition was visually observed. The results are shown in Table 7.

Even if most of the water-soluble polymer (polyvinylpyrrolidone) was removed by a washing operation, the color tone of the suspension did not change before and after the addition of sodium chloride due to the thick gold layer, and the result of the oxidation resistance test. Did not change (Table 7).

5. Example of method for supporting a specific binding substance to a test substance A gold-coated silver nanoplate in an Ag @ Au suspension 10 is precipitated by centrifugation, the supernatant is removed, and then a 0.5 mM trisodium citrate aqueous solution is added. And the precipitated gold-coated silver nanoplates were dispersed. The same operation is repeated twice, and an aqueous solution of trisodium citrate is added so that the quenching degree at the maximum absorption wavelength becomes 2, 4, or 8, and the Ag @ Au suspension 10 (2), 10 ( 4) and 10 (8) were prepared. The suspension having a high concentration was appropriately diluted, and then the degree of quenching was measured, and the degree of quenching of the undiluted solution was calculated by multiplying by the dilution ratio.

1 mL Ag @ Au suspension 10 of 50 μg / mL anti-hCG antibody solution (5 mM borate buffer, pH 7.5) so that the final concentration of antibody is as shown in Table 6 below. (2) was added to 10 (4) or 10 (8), and the mixed solution was allowed to stand at room temperature for 60 minutes. 50 μL of 80 μM polyethylene glycol solution (in 5 mM borate buffer) was added thereto, and the obtained suspension was allowed to stand at room temperature for 30 minutes. Then, 100 μL of 0.5 mM bovine serum albumin solution (in 5 mM borate buffer) was added, and the obtained suspension was allowed to stand at room temperature for 60 minutes. At this time, there was no change in the color tone of each suspension, and the dispersed state was also good. On the other hand, when a suspension of gold nanocolloids (spherical gold nanoparticles; particle diameter 40 nm), which is often used as a marker for immunochromatography tests, is used and the same operation is performed, the degree of quenching at the maximum absorption wavelength is 2. When the final concentration of the antibody is 0 and the final concentration of the antibody is 1.25 μg / mL or the quenching degree at the maximum absorption wavelength is 4.0 and the final concentration of the antibody is 2.50 μg / mL, the spherical gold nanoparticles are aggregated and immunochromatographic. It was not ready for testing. The antibody is usually supported on the spherical gold nanoparticles under the conditions that the quenching degree at the maximum absorption wavelength is 1.0 and the final concentration of the antibody is 5 μg / mL.

Each suspension of gold-coated silver nanoplates was centrifuged to precipitate gold-coated silver nanoplates carrying anti-hCG antibodies and the supernatant was removed. To the precipitated gold-coated silver nanoplate, 0.40 mL of 20 mM Tris-hydrochloric acid buffer (containing 8 μM polyethylene glycol, 0.25 mM BSA, and 150 mM NaCl) was added and redispersed to quench the maximum absorption wavelength. Adjust the concentration so that the degree is 1.0, and develop solutions 10 (2) -1, 10 (2) -2, 10 (4) -1, 10 (8) -1, and 10 for the immunochromatographic test. (8) -2 was prepared. Table 8 summarizes the supporting conditions of the anti-hCG antibody and the corresponding developing solution.

As described in item 3 above, except that the developing liquids 10 (2) -1, 10 (2) -2, 10 (4) -1, 10 (8) -1, and 10 (8) -2 were used. An immunochromatographic test was performed to detect hCG in the same manner. The results are shown in Table 9.

In general, agglomeration of metal nanoparticles can be a problem in the step of carrying an antibody on metal nanoparticles (see the results of spherical gold nanoparticles in Table 8). In order to prevent the aggregation of the metal nanoparticles, the suspension needs to have a protein concentration of a certain level or higher. Therefore, when the concentration of the metal nanoparticles is increased, it is required to increase the concentration of the antibody accordingly. However, surprisingly, when a gold-coated silver nanoplate having a small particle size and a thick gold layer is used, the antibody when the gold-coated silver nanoplate is thickened despite the increase in specific surface area. Aggregation did not occur even when the concentration was lowered. In addition, even when the gold-coated silver nanoplates were subjected to the supporting step under different antibody concentration conditions, there was no significant difference in the detection sensitivity when used in the immunochromatographic test. Therefore, it is considered that the gold-coated silver nanoplate having a small particle size and a thick gold layer has excellent dispersibility and high efficiency of carrying an antibody.

From the above, when the silver nanoplate is coated with gold, a gold ion complexing agent is used, the silver concentration is set to a certain value or less, the gold concentration is set to a certain value or more, and the two concentrations have a predetermined relationship. It was found that by adjusting them to meet the requirements, a gold-coated silver nanoplate having a thick gold layer for a small particle size can be uniformly produced. Therefore, it is possible to produce a gold-coated silver nanoplate having an excellent oxidation resistance and a small particle size. Since the gold-coated silver nanoplate having a small particle size has a maximum absorption wavelength in the visible light region, it is possible to achieve multicoloring of a method for detecting a test substance that requires visual determination such as immunochromatography. Further, since the gold-coated silver nanoplate having a small particle size and a thick gold layer has high dispersibility and can efficiently support a specific binding substance to a test substance, the amount of the specific binding substance used can be increased. It is possible to save money.

Claims (14)

A suspension of a gold-coated silver nanoplate having a silver nanoplate as a core and a gold layer covering the silver nanoplate.
The average particle size of the silver nanoplate is 55 nm or less, and the gold layer is formed on the main plane and the end face of the silver nanoplate.
The ratio (T / D) of the average thickness (T) of the gold layer on the end face to the average particle size (D) of the silver nanoplate is 0.05 or more.
The suspension has a maximum absorption wavelength in the visible light region, and the ratio of the extinction degree (E _max ) at the _{maximum absorption wavelength to the extinction degree (E 900 ) at the wavelength of 900 nm of the suspension (E max} / E _900). ) Is 5 or more, suspension. The suspension according to claim 1, wherein the gold layer has an average thickness of 1.5 nm to 10.0 nm. The suspension according to claim 1 or 2, wherein the silver nanoplate as the core has an aspect ratio of more than 1 and 10 or less. The suspension according to any one of claims 1 to 3, wherein the maximum absorption wavelength exists in the range of 400 nm to 680 nm. The suspension according to any one of claims 1 to 4, wherein the quenching degree at the maximum absorption wavelength is 2 or more. The suspension according to any one of claims 1 to 5, wherein the gold-coated silver nanoplate carries a specific binding substance to a test substance. The dried product of the suspension according to any one of claims 1 to 6. A method for detecting a test substance using the suspension according to claim 6.
The step of contacting the gold-coated silver nanoplate in the suspension with the test substance to form a complex thereof, and
A method comprising the step of detecting the complex. The suspension according to claim 6 is prepared by carrying a specific binding substance to the test substance on the gold-coated silver nanoplate in the suspension according to any one of claims 1 to 5. 8. The method of claim 8, further comprising the steps of A kit for detecting a test substance, which comprises the suspension according to any one of claims 1 to 6 or the dried product according to claim 7. A method for producing a gold-coated silver nanoplate having a silver nanoplate as a core and a gold layer covering the silver nanoplate.
A coating mixture containing a silver nanoplate having an average particle size of 55 nm or less, a gold-containing water-soluble compound, and a gold ion complexing agent is prepared to form the gold layer on the main plane and end face of the silver nanoplate. Including the process
The ratio (T / D) of the average thickness (T) of the gold layer on the end face to the average particle size (D) of the silver nanoplate is 0.05 or more.
In the coating mixture, and the silver concentration (C _Ag) is 0.25mM hereinafter gold concentration (C _Au) is not less than 0.1 mM, and the ratio of C _Au for _{C Ag (C Au / C Ag} ) is A manufacturing method that is 0.50 or more. The production method according to claim 11, wherein the complexing agent is at least one selected from the group consisting of sulfites, thiosulfates, cyanides, thiosulfates, and thioureas. A step of preparing a suspension of a gold-coated silver nanoplate having a silver nanoplate as a core and a gold layer covering the silver nanoplate.
A mixed solution of the suspension and the specific binding substance for the test substance is prepared to support the specific binding substance, which comprises a step of contacting the gold-coated silver nanoplate with the specific binding substance. It is a method of manufacturing gold-coated silver nanoplates.
The average particle size of the silver nanoplate is 55 nm or less, and the gold layer is formed on the main plane and the end face of the silver nanoplate.
The ratio (T / D) of the average thickness (T) of the gold layer on the end face to the average particle size (D) of the silver nanoplate is 0.05 or more.
The suspension has a maximum absorption wavelength in the visible light region, and the ratio of the extinction degree (E _max ) at the _{maximum absorption wavelength to the extinction degree (E 900 ) at the wavelength of 900 nm of the suspension (E max} / E _900). ) Is 5 or more,
A production method in which the quenching degree of the suspension at the maximum absorption wavelength is 2 or more, and the concentration of the specific binding substance in the contacting step is 100 μg / mL or less with respect to the volume of the entire mixture. The extinction of the suspension at the maximum absorption wavelength is 100 or less and / or
13. The production method according to claim 13, wherein the concentration of the specific binding substance in the contact step is 0.5 μg / mL or more with respect to the total volume of the mixture.

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