JP7535407B2 - Simple analytical technique that enables visual determination using nanoparticles - Google Patents

Description

The present invention relates to an analytical technology (hereinafter, visual analytical technology) that enables detection of a test target substance by labeling nanoscale particles (hereinafter, nanoparticles) with molecules (hereinafter, affinity molecules) that have affinity for the test target substance (the labeled nanoparticles are hereinafter referred to as nanoparticle probes), and agglomerating the nanoparticles in solution due to binding between the nanoparticle probe and the test target substance, and then detecting the test target substance by the decrease in turbidity caused by the precipitation of the nanoparticles.

A method using gold nanoparticles has been disclosed as an analytical technique that can visually determine the presence of a substance to be tested specifically (Non-Patent Document 1). When gold nanoparticles form aggregates, the wavelength of light that resonates with them shifts to the long wavelength side, and the solution changes from red to purple or blue. Attempts have been made to develop various molecular sensing applications by utilizing the color change that accompanies the aggregation of gold nanoparticles. The most widely used mechanism is a cross-linking reaction in which the substance to be tested acts as a bridge between particles. When gold nanoparticles with many affinity molecules immobilized thereon react at multiple points and bind to each other, an aggregate in which the gold nanoparticles are connected in a three-dimensional mesh pattern is formed. Aggregation and the associated color change occur only when a specific analyte is present, making it possible to sense the substance to be tested.

When the substance to be tested is a gene, a method has been disclosed in which gold nanoparticles labeled with a nucleic acid probe having a sequence complementary to the gene to be tested are used as affinity molecules, and the gold nanoparticles are cross-linked by hybridization between the nucleic acid probe and the target gene, resulting in aggregation of the gold nanoparticles and an accompanying change in color of the reaction solution, and the presence or absence of the target gene can be determined by visually observing this change2 ⁾ .

When the substance to be tested is a protein, a method has been disclosed in which gold nanoparticles labeled with an antibody that uses the target protein as an antigen as an affinity molecule are used (Non-Patent Document 2), and the gold nanoparticles are crosslinked by the binding of the antibody and antigen, resulting in aggregation of the gold nanoparticles and an accompanying change in the color of the reaction solution, and the presence or absence of the protein can be determined from this change (Non-Patent Document 3).

The color change in the above method can be judged well when a spectrophotometer is used, but is not so great that it can be judged reliably by visual inspection. Therefore, the accuracy of the test depends largely on the experience and sense of the person who judges, and there is an issue that sufficient test accuracy cannot be guaranteed.

Other analytical techniques that can specifically determine the presence of a test substance by visual inspection include a method using microparticles (hereinafter referred to as the particle agglutination method). Latex beads are commonly used as microparticles. In this method, similar to the method using gold nanoparticles, affinity molecules are modified on the particle surface, and aggregates are formed by a cross-linking reaction in which the test substance acts as a bridge. This formation causes the latex particles to precipitate, significantly reducing the turbidity of the reaction solution. This reduction in turbidity makes it possible to visually determine the presence or absence of the test substance (Non-Patent Document 4).

Chem Rev. 2012 May 9;112(5):2739-79. Science. 1997 Aug 22;277(5329):1078-81. Clin Biochem. 2004 Jan; 37(1): 27-35. Medical Electronics and Bioengineering 1984, Vol. 22, No. 4, pp. 267-273

In the particle agglutination method, in order to make an accurate visual judgment, it is necessary to provide a clear contrast against the background, so it is desirable for the particles themselves to be colored. However, latex particles obtained by emulsion polymerization are usually white, making visual judgment difficult. In order to solve this problem, a technology has been disclosed for producing colored latex particles by adding a dye to a resin (Patent 3391839). However, if the amount of dye added is increased beyond a certain level, the dye may dissolve from the latex particles and color the reaction solution, and the physical strength of the particles will decrease, so there is a limit to the amount that can be added, making it difficult to produce particles with a darker color tone.

On the other hand, in the particle agglutination method, if the test substance is at the same concentration and there is no difference in the size of the aggregates as a result, it is presumed that precipitation will occur at a lower concentration of the test substance when particles with a higher specific gravity are used than when particles with a lower specific gravity are used, because aggregates with a higher specific gravity will be formed. In addition, it is expected that the more aggregates with a higher specific gravity are formed, the higher the settling speed of the aggregates will be, and the more quickly the turbidity will decrease. Therefore, in the particle agglutination method, when the test substance is not present, it is thought that the test substance can be detected more quickly and with higher sensitivity by using particles with a higher specific gravity, within the range that can ensure a dispersed state until the analysis is completed. However, since the specific gravity of latex particles currently in general use is slightly above 1, it is expected that if particles with a higher specific gravity could be applied to the particle agglutination method, it could contribute to the high sensitivity and speed of the method.

In light of the above, it was inferred that when using the particle agglutination method to visually determine the substance being tested, the two most important requirements for particles are that (1) the particles have a dark color, and (2) if the substance being tested is not present, the particles have a large specific gravity to the extent that they can maintain a dispersed state until the analysis is completed.

In light of the above, the problem to be solved by this invention is to improve the particle agglutination method by applying particles having the above characteristics to the method, thereby enabling the rapid, highly sensitive, and clear determination of the presence or absence of the target substance in a simple manner.

Nanoscale particles (hereinafter, metal composite nanoparticles) having a structure in which multiple metal particles are fixed to resin particles have been disclosed (WO/2016/002742). In the metal composite nanoparticles described in this disclosure, at least a portion of the metal particles are distributed three-dimensionally on the surface layer of the resin particles, and a portion of the three-dimensionally distributed metal particles are partially exposed outside the resin particles, and the remaining portion is encapsulated in the resin particles. Therefore, the resin particles are loaded with a large amount of metal particles that exhibit localized surface plasmon absorption. Therefore, the metal composite nanoparticles are a material that exhibits a darker color than colored latex particles and has excellent visibility, visual judgment, and detection sensitivity. In addition, since the metal particles with a large specific gravity are held three-dimensionally, the specific gravity of the particles is large at 1.2 to 3.0, and the particles may precipitate sharply and in a short time due to the aggregation reaction. Due to these characteristics, it is expected that the metal composite nanoparticles can be suitably applied as particles to be used in particle aggregation methods. However, the above-mentioned metal composite nanoparticles have not yet been verified as particles that can be used in particle aggregation methods, and their effectiveness has not been made clear.

The inventors applied the above-mentioned metal composite nanoparticles to a particle agglutination method to verify whether the presence or absence of the test substance could be visually determined. They discovered that, compared to commonly used latex particles, it was possible to clearly, quickly, and sensitively determine the decrease in turbidity associated with agglutination and precipitation due to the crosslinking reaction between the test substance and affinity molecules. The present invention was made based on this discovery.

The gist of the present invention is as follows.
(1) A method for detecting a test substance, comprising reacting nanoscale particles (hereinafter referred to as "nanoparticles") labeled with molecules (hereinafter referred to as "affinity molecules") having affinity for the test substance in a solution with the test substance, forming nanoparticle aggregates through a cross-linking reaction mediated by the test substance, and determining the presence or absence of the test substance by measuring the decrease in turbidity of the solution due to precipitation of the nanoparticles accompanying the formation of the aggregates.
(2) The method according to (1), wherein the nanoparticles are metal composite nanoparticles.
(3) The method according to (1) or (2), wherein the nanoparticles have a specific gravity of 1.2 to 3.0.
(4) The method according to any one of (1) to (3), wherein the nanoparticles have a color lower in brightness than white.
(5) The method according to (4), wherein the nanoparticles are black in color.
(6) The method according to any one of (1) to (5), wherein the substance to be tested is a nucleic acid having a specific gene sequence, and the affinity molecule is a nucleic acid probe having a sequence complementary to the nucleic acid.
(7) The method according to any one of (1) to (5), wherein the substance to be tested is a protein, and the affinity molecule is an antibody to the protein, or a molecule having affinity for a molecule labeled with the protein.
(8) A kit for detecting a substance to be tested, comprising nanoparticles labeled with affinity molecules, the kit determining the presence or absence of the substance to be tested by reacting the nanoparticles labeled with affinity molecules with the substance to be tested in a solution, forming nanoparticle aggregates through a cross-linking reaction mediated by the substance to be tested, and measuring the decrease in turbidity of the solution due to precipitation of the nanoparticles accompanying the formation of the aggregates.

The present invention makes it possible to quickly, sensitively, and clearly determine the presence or absence of a test target substance using a simple method such as visual inspection.

1 shows a scheme for protein detection using nanoparticles (beads). 1 shows a scheme for gene detection using nanoparticles (beads).

The embodiment of the present invention will be described in detail.

The present invention provides a method for detecting a test substance, which comprises reacting nanoscale particles (hereinafter, "nanoparticles") labeled with molecules (hereinafter, "affinity molecules") having affinity for the test substance in a solution with the test substance, forming nanoparticle aggregates through a crosslinking reaction mediated by the test substance, and determining the presence or absence of the test substance by measuring the decrease in turbidity of the solution due to precipitation of the nanoparticles accompanying the formation of the aggregates.

Examples of test subjects include proteins (including polypeptides, oligopeptides, etc.) such as tumor markers, signal transduction substances, and hormones, nucleic acids (including single-stranded or double-stranded DNA, RNA, polynucleotides, oligonucleotides, PNA (peptide nucleic acid), etc.) or substances having nucleic acids, sugars (including oligosaccharides, polysaccharides, sugar chains, etc.) or substances having sugar chains, lipids, and other molecules. It is preferable that the nucleic acid has a specific gene sequence. Examples of single-stranded DNA include viruses and reverse transcription products (cDNA), and examples of single-stranded RNA include RNA obtained by the NASBA method and ribosomal RNA.

The test target substance may be an analyte contained in a biological sample (e.g., whole blood, serum, plasma, urine, saliva, sputum, nasal or pharyngeal swab, cerebrospinal fluid, amniotic fluid, nipple secretion, tears, sweat, skin exudate, tissue, cell and stool extracts, etc.), other samples (e.g., food extracts, soil, activated sludge or compost extracts, drinking water, sewage, wastewater, river or lake water, seawater, etc.), etc. If necessary, the analyte contained in the sample may be pretreated prior to detection of the test target substance. Pretreatments include chemical treatments using various chemicals such as acids, bases, and surfactants, and physical treatments using heating, stirring, ultrasound, etc. When the analyte is a nucleic acid, nucleic acid amplification may be performed as a pretreatment. The sample may also be appropriately diluted with a solvent (water, saline, buffer solution, etc.) or a water-miscible organic solvent used in a conventional analytical method.

In the present invention, "affinity" refers to the property or tendency of a substance to easily bind to another substance. Examples of molecules that have affinity for the test substance include, when the test substance is a protein, an antibody that uses the test protein as an antigen, and a molecule (e.g., streptavidin, tamavidin) that has affinity for a molecule labeled with the test protein (e.g., biotin). When the test substance is a nucleic acid having a specific gene sequence, examples of such molecules include a nucleic acid probe having a sequence complementary to a specific gene sequence, an aptamer that specifically binds to a specific molecule and is composed of a nucleic acid molecule or peptide, and a molecular imprinting polymer that polymerizes a polymer while incorporating a target template molecule, forming a void of the same shape and size as the template, which is used for molecular recognition.

The nucleic acid probe may be composed of oligodeoxyribonucleotides or oligoribonucleotides. It may also be a chimeric oligonucleotide in which both are present. 2-O-methyl oligoribonucleotides (2'-O-methyl oligoribonucleotides) (oligoribonucleotides in which the nucleoside part at the 5' end is cytidine and the OH group at the 2' position of the cytidine is modified with a methyl group) may also be used. Or, to increase the affinity with RNA, 2'-O-methyl oligoribonucleotides may be present in oligodeoxynucleotides. In addition, Locked Nucleic Acid (LNA), which is an artificial nucleic acid excellent in stability and specificity to a complementary strand, and N-O bond bridged structure type artificial nucleic acid [2',4'-BNANC] may be used. The number of bases of the nucleic acid probe is preferably 5 to 100, more preferably 10 to 30, and particularly preferably 15 to 25. If it exceeds 100, synthesis errors are likely to occur, and the possibility of including a probe having a sequence other than the intended sequence increases, and if it is less than 5, nonspecific hybridization is likely to occur, resulting in large measurement errors. The oligonucleotide of the nucleic acid probe can be manufactured by a normal general oligonucleotide manufacturing method. For example, it can be produced by chemical synthesis, microbial methods using plasmid vectors, phage vectors, etc. (Tetrahedron letters, vol. 22, pp. 1859-1862, 1981; Nucleic acids Research, vol. 14, pp. 6227-6245, 1986). It is preferable to use a currently commercially available nucleic acid synthesizer (e.g., ABI394 (Perkin Elmer, USA)).

Nanoscale particles (nanoparticles) refer to particles including particles having a particle size (diameter) of the nano order (1 nm or more and less than 1000 nm), and the average particle size (diameter) of the nanoparticles is preferably in the range of 20 to 1000 nm, preferably in the range of 50 to 700 nm, and more preferably in the range of 100 to 400 nm. The average particle size can be measured using a disk centrifuge particle size distribution measuring device (CPS Disc Centrifuge DC24000 UHR, manufactured by CPS instruments, Inc.).
The nanoparticles preferably have a darker color than conventional colored latex particles (the light absorption per particle in the visible light region (380 to 750 nm) of nanoparticles is 3 to 12 times that of colored latex particles) and a larger specific gravity than conventional latex particles (the specific gravity of conventional latex particles is about 1.04). Examples of the nanoparticles include nanoscale particles having a structure in which a plurality of metal particles are fixed to a resin particle (hereinafter, metal composite nanoparticles) (for example, WO/2016/002742).

The metal composite nanoparticles include metal particles exposed outside the resin particles and metal particles encapsulated in the resin particles, and at least a portion of these metal particles are preferably distributed three-dimensionally in the surface layer of the resin particles. The amount of metal particles supported is preferably 5 wt% to 70 wt% based on the weight of the metal composite nanoparticles. The amount of metal particles supported is described in the examples described later. 60 wt% to 100 wt% of the metal particles are present in the surface layer of the resin particles, and 5 wt% to 90 wt% of the metal particles present in the surface layer have a portion exposed outside the resin particles. The resin particles are preferably polymer particles having a substituent capable of adsorbing metal ions in their structure. The polymer particles are preferably nitrogen-containing polymer particles. The nitrogen-containing polymer is a resin having nitrogen atoms in the main chain or side chain, and examples thereof include polyamines such as poly-2-vinylpyridine, poly-3-vinylpyridine, and poly-4-vinylpyridine, acrylic resins having nitrogen atoms in the side chains, phenolic resins, and epoxy resins. Examples of metal particles include gold, platinum, palladium, silver, nickel, copper, and alloy particles of these metals, with platinum particles being preferred. The average particle size of the metal particles is preferably within the range of 1 to 80 nm, and is preferably 1 nm or more and less than 70 nm, and more preferably 1 nm or more and less than 50 nm. The average particle size of the metal composite nanoparticles is preferably 1 nm or more and less than 1000 nm, and is preferably 50 nm or more and less than 700 nm, and more preferably 100 nm or more and less than 400 nm. These average particle sizes can be measured using a disk centrifuge particle size distribution measuring device (CPS Disc Centrifuge DC24000 UHR, manufactured by CPS instruments, Inc.). The method for producing the above metal composite nanoparticles is described in WO/2016/002742.

The specific gravity of the nanoparticles is preferably 1.2 to 3.0, more preferably 1.3 to 2.5, and even more preferably 1.5 to 2.1. The specific gravity of metal composite nanoparticles can be calculated by measuring the metal content in the particles and from the specific gravity of the metal alone and the specific gravity of the resin alone. The resin can also be calculated by measuring the specific gravity of a 10 wt % metal-free latex particle dispersion.

The color of the nanoparticles should be a color less bright than white, and is preferably black.
In the present invention, "labeling" is a concept that includes binding and modification, and examples of nanoparticles labeled with affinity molecules include nanoparticles bound to a nucleic acid probe having a sequence complementary to a specific gene sequence when the substance to be tested is a nucleic acid having a specific gene sequence; nanoparticles bound to an antibody that uses the protein to be tested as an antigen when the substance to be tested is a protein; nanoparticles bound to a molecule (e.g., streptavidin, tamavidin) that has affinity for a molecule (e.g., biotin) labeled with the protein to be tested; nanoparticles bound to an aptamer that has affinity for the molecule to be tested; and nanoparticles bound to a molecular imprinting polymer that has affinity for the molecule to be tested.

To label the nanoparticles with the antibody (glycoprotein) of the protein to be tested, for example, a method of labeling the antibody by utilizing the property of proteins adsorbing to the surface of plastics such as polypropylene or polystyrene, a method of modifying the antibody by utilizing passive adsorption of the protein due to electrostatic and hydrophobic interactions between the protein and the surface of the gold or platinum colloid when the nanoparticles are metal composite nanoparticles and the metal nanoparticles are gold or platinum, or a method of introducing a carboxyl group onto the surface of the nanoparticles and covalently binding the antibody through an amide bond between this functional group and a free amino group contained in the antibody.

Molecules (e.g., avidin, streptavidin) that have affinity for the molecule (e.g., avidin) labeled with the test target protein and that are nanoparticles are proteins, just like antibodies, and therefore can be labeled using methods similar to those for labeling antibodies described above.
For labeling nanoparticles with nucleic acid probes, for example, a method of modifying the surface of the nanoparticles with silica, which has an affinity for nucleic acids, and then adsorbing the nucleic acid probe to the silica, a method of introducing succinimidyl groups via carboxyl groups on the nanoparticle surface and reacting these with amino groups introduced into the nucleic acid probe, a method of modifying the surface of the nanoparticles with avidin or streptavidin, and then reacting these with a nucleic acid probe to which biotin, which has a high affinity, has been introduced, and the like are used.

The amount of affinity molecules labeled to nanoparticles depends on the average particle size of the nanoparticles, the amount of molecules to be tested, the sensitivity required for the measurement, etc., but is generally 1 to 1,000 molecules/particle, preferably 5 to 200 molecules/particle, and more preferably 10 to 100 molecules/particle. The amount of labeled affinity molecules can be calculated by measuring the affinity molecule concentrations in the affinity molecule solution before labeling and in the affinity molecule solution recovered after labeling, and calculating the difference between the concentrations.

When the affinity molecule is a protein, a method for measuring the total protein amount can be used, such as ultraviolet spectrophotometry (a spectrophotometric method that utilizes the ultraviolet absorption of proteins), the Bradford method (a colorimetric method that uses Coomassie Brilliant Blue, a triphenylmethane dye), and the Fluorescamine method (a fluorescence measurement method that uses Fluorescamine, which reacts quickly with primary amines in proteins to form derivatives that emit blue-green fluorescence). When the affinity molecule is a nucleic acid probe, such as ultraviolet spectrophotometry (a spectrophotometric method that utilizes the ultraviolet absorption of nucleic acids) and the OliGreen method (a fluorescence measurement method that uses OliGreen, which binds to single-stranded DNA and emits green fluorescence).

The nanoparticles labeled with affinity molecules should be uniformly dispersed in the solution before reacting with the test substance (homogeneous solution system, completely mixed system). The solution in which the nanoparticles labeled with affinity molecules are uniformly dispersed should have an absorbance at 570 nm (measurement cell optical path length: 10 mm) of 0.5 to 10, preferably 1 to 8, and more preferably 2 to 6.
Examples of the solution include pure water, purified water, physiological saline, various buffer solutions, and aqueous solutions containing solvents that have a lower specific gravity than water and are completely miscible with water, such as alcohols, aldehydes, and ketones.
The specific gravity of the solution is preferably 0.8 to 1.2, more preferably 0.9 to 1.1, and even more preferably 0.95 to 1.05.

The concentration of the nanoparticles in the solution may be a concentration at which the decrease in turbidity of the solution due to the crosslinking reaction between the nanoparticles and the test substance can be measured (for example, visually), and may vary depending on the type of nanoparticles, labeling substance (affinity molecule), and test substance. For example, when the test substance is a protein, sugar, or lipid, the concentration of the nanoparticles in the solution may be 10 ⁹ to 10 ¹⁴ particles/mL, preferably 10 ¹⁰ to 10 ¹³ particles/mL, and more preferably 5×10 ¹⁰ to 5×10 ¹² particles/mL. The reaction may be carried out under conditions suitable for a normal antigen-antibody reaction or a reaction between a molecule labeled with a protein and a molecule having affinity thereto, the salt concentration may be 0 to 2.0 M, preferably 0.1 to 1.0 mM, and the pH may be 5 to 9, preferably 6 to 8. The reaction temperature may be 10 to 50° C., preferably 15 to 30° C., and the reaction time may be 1 to 60 minutes, preferably 5 to 20 minutes.

When the test substance is a nucleic acid having a specific gene sequence, the concentration of nanoparticles in the solution is preferably 10 ⁹ to 10 ¹⁴ nanoparticles/mL, more preferably 10 ¹⁰ to 10 ¹³ nanoparticles/mL, and more preferably 5×10 ¹⁰ to 5×10 ¹² nanoparticles/mL. Hybridization between the test substance, that is, the nucleic acid, and the nucleic acid probe can be performed by a conventional known method (Analytical Biochemistry, Vol. 183, pp. 231-244, 1989; Nature Biotechnology, Vol. 14, pp. 303-308, 1996; Applied and Environmental Microbiology, Vol. 63, pp. 1143-1147, 1997). For example, the hybridization conditions are a salt concentration of 0 to 2 molar, preferably 0.1 to 1.0 molar, and a pH of 6 to 8, preferably 6.5 to 7.5. The temperature depends on the dissociation temperature of the nucleic acid probe, but is generally 20 to 80°C, preferably 40 to 70°C, and the reaction time is 1 to 60 minutes, preferably 5 to 20 minutes. (See comments.)

In the present invention, when nanoparticles labeled with affinity molecules are reacted with a test substance in a solution, a crosslinking reaction occurs via the test substance to form aggregates of nanoparticles, which then precipitate, resulting in a decrease in the turbidity of the solution.

When the test substance is a protein, sugar, or lipid, and the affinity molecule is a molecule that has affinity for the molecule (labeled molecule) labeled with the test substance, it is preferable to label one molecule of the test substance with multiple labeled molecules. As a result, a reaction between the molecule labeled with the test substance and the affinity molecule labeled with the nanoparticle occurs at multiple points per molecule of the test substance (crosslinking reaction), forming an aggregate of nanoparticles. With the formation of the aggregate of nanoparticles, the nanoparticles precipitate, and as a result, the turbidity of the solution decreases. A scheme in which a reaction between a molecule labeled with a protein (target protein, for example, Human CD3 epsilon) that is the test substance and a molecule labeled with the nanoparticle occurs at multiple points per molecule of the protein (crosslinking reaction), forming an aggregate of nanoparticles, and the nanoparticles precipitate is shown in FIG. 1. In FIG. 1, the protein is labeled with biotin, and the nanoparticles are labeled with tamavidin. Tamavidin has affinity for biotin.
When the test substance is a nucleic acid having a specific gene sequence, one or more types of nanoparticles labeled with each of two or more types of nucleic acid probes are used, and when the nanoparticles react with the test substance, two or more nanoparticles are crosslinked via one molecule of the test substance, and since there are multiple crosslinks for one nanoparticle, the nanoparticles form an aggregate in which the nanoparticles are three-dimensionally connected in a mesh-like pattern. As the nanoparticles form an aggregate, the nanoparticles precipitate, and the turbidity of the solution decreases. Figure 2 shows a scheme in which a nucleic acid (target gene) having a specific gene sequence, which is the test substance, reacts (hybridizes) with a nucleic acid probe (two types) complementary to the target gene that is labeled on the nanoparticles, the nanoparticles are crosslinked with the target gene, aggregate and precipitate, and the turbidity of the solution decreases. In Figure 2, a succinimidyl group is introduced into the nanoparticles via a carboxyl group on the particle surface, and the succinimidyl group is reacted with an amino group introduced into the nucleic acid probe, thereby labeling the nanoparticles with the nucleic acid probe.

The presence or absence of the substance to be tested can be determined by measuring the decrease in turbidity of the solution due to the crosslinking reaction. If the turbidity of the solution decreases, it is determined that the substance to be tested is present, and if the turbidity of the solution does not decrease, it is determined that the substance to be tested is not present. The turbidity of the solution before the crosslinking reaction is preferably 500 to 4,000, preferably 700 to 4,000, and more preferably 1,000 to 4,000, and the turbidity of the solution after the nanoparticles precipitate and the turbidity decreases due to the crosslinking reaction is preferably 1 to 400, preferably 1 to 200, and more preferably 1 to 50. The difference in turbidity before and after the decrease in the turbidity of the solution is preferably 500 to 4,000, preferably 700 to 4,000, and more preferably 1,000 to 4,000. The turbidity of a solution can be measured by shaking the sample well, placing it in an absorption cell (10 mm), and measuring the intensity of transmitted light at a wavelength of about 600 nm as apparent absorbance. The turbidity of a sample can be calculated by measuring the absorbance of a solution obtained by serially diluting the turbidity standard solution using the above-mentioned measurement method, and using the relationship between the absorbance and turbidity obtained as a calibration curve, from the absorbance of a sample measured separately. In addition, if the absorbance of the sample falls outside the measurement range of the calibration curve, it is possible to measure it accurately by diluting the sample with pure water so that it falls within the measurement range.

The method of the present invention allows the presence or absence of a test target substance to be determined within one hour, preferably within 1 to 60 minutes, and more preferably within 5 to 20 minutes.

The lower limit of the detectable concentration of the test substance by the method of the present invention can be 1 to 100 nM for proteins, sugars, or lipids, and similarly 1 to 100 nM for nucleic acids.

The present invention makes it possible to visually determine the presence or absence of a test target substance quickly, with high sensitivity, and with clarity.

The present invention also provides a kit for detecting a test substance, comprising nanoscale particles labeled with a molecule having affinity for the test substance.

The test substance, the molecule having affinity for the test substance, and the nanoscale particles labeled with the molecule having affinity for the test substance have been described above.

The kit of the present invention may include nanoscale particles labeled with molecules that have affinity for the test target substance, as well as a negative control standard substance, a positive control standard substance, a reaction vessel, a dispersion medium, a gene amplification reagent, etc.

The method and kit of the present invention can be used for genetic testing (detection of pathogens or viruses, genetic polymorphism, mutation analysis, etc.), immunological diagnostic methods (detection of antigens or antibodies, etc.), and the like.

The present invention will now be described in more detail with reference to examples.
[Example 1]
Content: Protein detection by nanoparticle precipitation

<Nanoparticles used>
The nanoparticles used in this example are shown in Table 1 below. Nanoparticles 1 are commercially available latex beads (Micromod, product number: 01-19-202) that have already been labeled with streptavidin. Meanwhile, nanoparticles 2 were prepared in this example.

Table 1. Nanoparticles used in this example

<Absorbance measurement of metal composite nanoparticles>
The absorbance of the metal composite nanoparticles was measured by placing a 0.01 wt % metal composite nanoparticle dispersion (dispersion medium: water) in an optical white plate glass cell (light path length 10 mm) and measuring the absorbance at 570 nm using an instantaneous multi-photometer system (Otsuka Electronics Co., Ltd., MCPD-3700).

<Measurement of solid concentration and metal loading amount>
1 g of the dispersion before the concentration adjustment was placed in a porcelain crucible and heat-treated for 3 hours at 70° C. The weights before and after the heat treatment were measured, and the solid content was calculated by the following formula.
Solid content concentration (wt%) = [weight after drying (g) / weight before drying (g)] x 100

In addition, the sample after the above heat treatment was further heat treated at 500° C. for 5 hours, the weights before and after the heat treatment were measured, and the amount of supported metal was calculated by the following formula.
Metal loading amount (wt%)=
[Weight after heat treatment at 500° C. (g)/Weight before heat treatment at 500° C. (g)]×100

<Measurement of the average particle size of metal composite nanoparticles>
The measurement was performed using a disk centrifuge particle size distribution measuring device (CPS Disc Centrifuge DC24000 UHR, manufactured by CPS instruments, Inc.) The measurement was performed in a state where the metal composite nanoparticles were dispersed in water.

<Synthesis of metal composite nanoparticles>
Aliquat 336 [manufactured by Aldrich] (5.00 g) and triethylene glycol methyl ether methacrylate PEGMA, 10.00 g) were dissolved in 389.5 g of pure water, and then 2-vinylpyridine (2-VP, 48.00 g) and divinylbenzene (DVB, 2.00 g) were added, and the mixture was stirred at 150 rpm and 30 ° C. for 50 minutes under a nitrogen stream, and then at 60 ° C. for 30 minutes. After stirring, 2,2-azobis (2-methylpropionamidine) dihydrochloride (AIBA, 0.50 g) dissolved in 50.00 g of pure water was added dropwise over 2 minutes, and the mixture was stirred at 150 rpm and 60 ° C. for 3.5 hours to obtain resin particles with an average particle size of 200 nm. The mixture was precipitated by centrifugation (9000 rpm, 45 minutes), the supernatant was removed, and the mixture was dispersed again in pure water three times, after which impurities were removed by dialysis.Then, the concentration was adjusted to obtain a 10 wt % resin particle dispersion.

After adding 245 ml of pure water to the above resin particle dispersion (90 ml), 400 mM aqueous chloroplatinic acid solution (90 ml) was added and stirred at 60 rpm and 30°C for 3 hours, and then left at room temperature for 24 hours. After that, the resin particles were precipitated by centrifugation (3100 rpm, 30 minutes), and the supernatant was removed three times to remove excess chloroplatinic acid. The concentration was then adjusted to prepare a 5 wt% platinum ion-adsorbing resin particle dispersion.

Next, the 5 wt% platinum ion-adsorbed resin particle dispersion (55 ml) was added to 3825 ml of pure water, and while stirring at 160 rpm and 3°C, 132 mM dimethylamine borane aqueous solution (110 ml) was dropped over 20 minutes, and then stirred at 160 rpm and 3°C for 1 hour. After that, by stirring at 160 rpm and 25°C for 3 hours, a resin-platinum (metal) composite with an average particle size of 215 nm was obtained. The resin-platinum (metal) composite was precipitated by centrifugation (3100 rpm, 60 minutes), the supernatant was removed, and the composite was dispersed again in pure water three times, and then purified by dialysis to remove impurities. The concentration was then adjusted to obtain a 1 wt% resin-platinum (metal) composite dispersion. The absorbance of the prepared resin-platinum (metal) composite dispersion was measured according to the above method and found to be 0.57. The average particle size of the platinum particles formed was 6 nm, and the amount of platinum carried was 37.1 wt%. In this resin-platinum (metal) composite, the platinum particles included encapsulated platinum particles that were completely encapsulated in the resin particles, partially exposed platinum particles that had a portion embedded in the resin particles and a portion exposed to the outside of the resin particles, and surface-adsorbed platinum particles that were adsorbed on the surfaces of the resin particles, and at least some of the platinum particles were distributed three-dimensionally in the surface layer of the resin particles. In this example, the resin-platinum (metal) composite was used as a metal composite nanoparticle.

<Labeling of tamavidin to metal composite nanoparticles>
0.9 ml of 100 mM borate buffer was added to 0.1 ml (1.0 wt%) of the resin-platinum (metal) composite dispersion, which is the obtained metal composite nanoparticle, and then 10 ml of 1 mg/ml tamavidin solution was mixed and stirred at room temperature for about 3 hours to bind tamavidin (heat-resistant avidin) to the resin-platinum (metal) composite. A bovine serum albumin solution was added to a final concentration of 1%, and the resin-platinum (metal) composite surface was blocked by stirring at room temperature for 2 hours. The resin-platinum (metal) composite was centrifuged at 6000 rpm and 4°C for 5 minutes to recover the resin-platinum (metal) composite, and suspended in a buffer containing 0.1% bovine serum albumin to prepare a tamavidin-labeled resin-platinum (metal) composite dispersion.

<Confirmation of aggregation and precipitation conditions using nanoparticles>
In this example, an experimental system was used that was designed to crosslink nanoparticles through a reaction between streptavidin or tamavidin labeled on nanoparticles and biotin labeled on a protein, resulting in the formation of aggregates of the nanoparticles.

Specifically, 10 μL of 0.1 wt% nanoparticles (latex beads or metal composite nanoparticles) surface-modified with streptavidin or tamavidin, 2 μL of 10×TE Buffer, and an arbitrary amount of biotinylated Human CD3 epsilon protein (manufactured by Acro Biosystems, product number: CDE-H8223) as a test substance were added to a 0.2 ml PCR tube, and then sterilized Milli-Q water was added to make the total amount 20 μL. Note that since the biotinylated Human CD3 epsilon protein used in this example is labeled with multiple biotins per protein molecule, reactions with streptavidin or tamavidin occur at multiple points per protein molecule, forming aggregates in which the nanoparticles are three-dimensionally connected in a mesh-like pattern.
Furthermore, as a negative control experiment, an experiment using non-biotinylated Human CD3 epsilon protein (manufactured by Acro Biosystems, product number: CDE-H5223) was carried out under the same conditions as the experiment using the biotinylated Human CD3 epsilon protein described above, except that the test substance was changed.

The results were evaluated based on the amount of test substance added and the time at which a decrease in turbidity occurred due to nanoparticle precipitation.

The decrease in turbidity was judged by adding 20 μL of a turbidity standard solution with a turbidity of 100 (Kanto Chemical, product number: 40969-23) to a 0.2 ml PCR tube. If the turbidity of the test sample was judged to be lower than that of the target solution by visual inspection, it was judged that the turbidity of the test sample had decreased, and if it was judged to be higher than that of the target solution, it was judged that the turbidity of the test sample had not decreased or that the decrease in turbidity was poor.

<Results>
When latex beads were used as nanoparticles, the only conditions under which a decrease in turbidity due to particle precipitation was visible were the reaction conditions in which the largest amount of the test substance was added (65 ng/tube) and the reaction time was 60 minutes or more.

On the other hand, when metal composite nanoparticles were used as nanoparticles, the decrease in turbidity due to particle precipitation was confirmed under all conditions in which the test substance was added at 12 ng/tube or more and reacted for 10 minutes or more. In addition, in a negative control experiment using non-biotinylated Human CD3 epsilon protein, the decrease in turbidity due to nanoparticle precipitation was not confirmed in any reaction system, indicating that the decrease in turbidity due to nanoparticle precipitation confirmed in this example is due to a crosslinking reaction of nanoparticles caused by a binding reaction between streptavidin or tamavidin and biotin labeled on the protein.

The above results show that when metal composite nanoparticles are applied to a protein detection system using the particle aggregation method, it is possible to improve the detection sensitivity of this method and shorten the analysis time compared to when commonly used latex beads are used.

Table 2. Effect of nanoparticle species on sensitivity and reaction time in protein detection using the particle agglutination method

[Example 2]
Content: Detection of target genes by precipitation of nanoparticles

<Oligo DNA used>
The oligo DNAs used in this example are shown in Table 3. All of the oligo DNAs were manufactured by Tsukuba Oligo Service Co., Ltd. (Ushiku City, Ibaraki Prefecture). Oligo DNAs No. 3 and 4 were terminally modified with amino groups, etc., and purified by HPLC. The rest were purified by gel filtration for the purpose of desalting.

Table 3. List of oligo DNA used

<Introduction of succinimidyl groups into nanoparticles>
Succinimidyl groups were introduced into nanoparticles via carboxyl groups on the particle surface by carrying out the following steps.
0.1 ml of 1 wt % of any nanoparticles was added to a 2 ml microtube, and 0.9 ml of 50 mM 2-morpholinoethanesulfonic acid buffer (hereinafter, MES) pH 6.1 was added, followed by ultrasonic dispersion for about 10 seconds using an ultrasonic cleaner (US-610 (manufactured by SND Co., Ltd.)).

Next, the supernatant was removed by centrifugation, and then 0.5 ml of 50 mM MES buffer (pH 6.1) containing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (hereinafter, EDC) at a concentration of 2 mg/ml and 0.5 ml of 50 mM MES buffer (pH 6.1) containing sodium N-hydroxysulfosuccinimide (hereinafter, solvent-NHS) at a concentration of 2 mg/ml were added, and the mixture was ultrasonically dispersed for about 10 seconds using an ultrasonic cleaner (US-610 (manufactured by SND Co., Ltd.)) and then mixed by inversion for about 1 hour.
Thereafter, the supernatant was removed by centrifugation, and 1.0 ml of 50 mM MES buffer (pH 6.1) was added, followed by ultrasonic dispersion for about 10 seconds using an ultrasonic cleaner (US-610 (manufactured by SND Co., Ltd.)). This process was repeated two more times (a total of three times).

<Modification of Nanoparticles with Nucleic Acid Probes>
The modification of the nanoparticles with the nucleic acid probe was carried out by reacting the succinimidyl group introduced on the particle surface with the amino group modified on the nucleic acid probe, as described in detail below.

1 ml of a nucleic acid probe (No. 3 and 4 oligo DNAs listed in Table 3) with an amino group introduced at the end was added to a 2 ml microtube containing 1 ml of 0.1 wt% succinimidyl group-introduced nanoparticles prepared by the method described above, so that the final concentration was 100 nM, and the reaction was allowed to proceed at room temperature for 2 hours. Only one type of oligo DNA was added and reacted per reaction tube, so a total of four reactions (2 types of nanoparticles x 2 types of oligo DNA) were carried out.

Then, the supernatant was removed by centrifugation, and 1 ml of stop buffer (50 mM MES buffer (pH 6.1) containing ethanolamine at a final concentration of 100 mM) was added, and the mixture was ultrasonically dispersed for about 10 seconds and then mixed by inversion for about 10 minutes.

Next, the supernatant was removed by centrifugation, and 1 ml of blocking buffer (50 mM MES buffer (pH 6.1) containing 1% sodium caseinate) was added, ultrasonically dispersed for about 10 seconds, and then mixed by inversion for about 2 hours.
After that, the supernatant was removed by centrifugation, 1 ml of TE buffer (manufactured by Nippon Gene Co., Ltd.) was added, and ultrasonic dispersion was performed for about 10 seconds. This was repeated again (a total of two times) to obtain a suspension of nanoparticles (nanoparticle probes) labeled with nucleic acid probes.

<Confirmation of aggregation and precipitation conditions using nanoparticles>
In this example, an experimental system was used in which the beads were cross-linked by hybridization between the nucleic acid probes labeled on the nanoparticles and the target gene, resulting in the formation of aggregates of the nanoparticles.

Specifically, 5 μL each of two types of 0.1 wt% nanoparticles (latex beads or metal composite nanoparticles) surface-modified with different nucleic acid probes, 2 μL of 10x TE Buffer, and No. 1 oligo DNA with a sequence complementary to the nucleic acid probe labeled on the nanoparticles as the test substance were added to a 0.2 ml PCR tube to an arbitrary final concentration, and then sterilized Milli-Q water was added to bring the total volume to 20 μL.

The No. 1 oligo DNA used as the target gene in this example has sequences complementary to two different types of nucleic acid probes at one site per molecule, and two types of nanoparticles labeled with one different type of nucleic acid probe are used, so the two nanoparticles are crosslinked via the target gene. In addition, since multiple crosslinks exist for one nanoparticle, the nanoparticles form an aggregate in which they are connected three-dimensionally in a mesh-like pattern.

In addition to the above, a negative control experiment was conducted using No. 2 oligo DNA, which does not have a sequence complementary to the nucleic acid probe, as a non-target gene under the same conditions as the experiment using the target gene above, except that the substance to be tested was changed.

The results were evaluated based on the amount of test substance added and the time at which a decrease in turbidity occurred due to nanoparticle precipitation. The method for determining the decrease in turbidity was the same as in Example 1.

<Results>
When latex beads were used as nanoparticles, the only conditions under which a decrease in turbidity due to particle precipitation was visible were the conditions in which the reaction system had the highest target gene concentration (20,000 nM) and the reaction was continued for 60 minutes or more.

On the other hand, when metal composite nanoparticles were used as the nanoparticles, a decrease in turbidity due to particle precipitation was confirmed under all conditions. When non-target genes were used, no decrease in turbidity due to nanoparticle precipitation was confirmed in any reaction system, indicating that the decrease in turbidity due to nanoparticle precipitation confirmed in this example was due to a cross-linking reaction of nanoparticles caused by hybridization of the target gene and the nucleic acid probe.

The above results show that when metal composite nanoparticles are applied to a gene detection system using the particle agglutination method, it is possible to improve the detection sensitivity of this method and shorten the analysis time compared to when commonly used latex beads are used, just as when proteins are the test substances.

Table 4. Effect of nanoparticle species on sensitivity and reaction time in target gene detection using particle agglutination method

The present invention can be used as an analytical technique that can specifically determine the presence of a substance to be tested.

<Sequence numbers 1 to 4> Shows the base sequences of the oligo DNA used in Example 2.

Claims (5)

A method for detecting a test substance, comprising reacting nanoscale particles (hereinafter referred to as "nanoparticles") having an average particle size of 1 nm or more and less than 1000 nm, which are labeled with a molecule (hereinafter referred to as "affinity molecule") having affinity for the test substance in a solution, forming aggregates of the nanoparticles by a crosslinking reaction mediated by the test substance, and determining the presence or absence of the test substance by measuring a decrease in turbidity of the solution due to precipitation of the nanoparticles accompanying the formation of the aggregates, wherein the nanoparticles have a structure in which a plurality of metal particles are fixed to resin particles, the specific gravity of the nanoparticles is 1.2 to 3.0, and the color of the nanoparticles is a color less bright than white . 2. The method of claim 1 , wherein the nanoparticles are black in color. 3. The method according to claim 1 or 2 , wherein the substance to be tested is a nucleic acid having a specific gene sequence, and the affinity molecule is a nucleic acid probe having a sequence complementary to the nucleic acid. 3. The method according to claim 1, wherein the substance to be tested is a protein, and the affinity molecule is an antibody to the protein, or a molecule having affinity for a molecule labeled with the protein. A kit for use in a method for detecting a test substance, comprising nanoscale particles (hereinafter "nanoparticles") with an average particle diameter of 1 nm or more and less than 1000 nm, which are labeled with a molecule (hereinafter "affinity molecule") having affinity for the test substance, wherein the nanoparticles have a structure in which a plurality of metal particles are fixed to a resin particle, the specific gravity of the nanoparticles is 1.2 to 3.0, and the color of the nanoparticles is less bright than white, the method comprising reacting the nanoparticles labeled with the affinity molecule with the test substance in a solution, forming nanoparticle aggregates by a crosslinking reaction via the test substance, and determining the presence or absence of the test substance by measuring a decrease in turbidity of the solution due to precipitation of the nanoparticles accompanying the formation of the aggregates.