Method for producing fluorescent nanoparticles

Abstract

The present invention relates to providing fluorescent nanoparticles with a target-substance-recognizing substance supported on the surface thereof, wherein the target-substance-recognizing substance has a high level of activity, allowing for highly sensitive measurement of the target substance. These fluorescent nanoparticles have supported on the surface thereof a target-substance-recognizing substance for binding to a target substance. When a 100 μL of liquid containing the fluorescent nanoparticles at a concentration of 100 pmol / L is brought into contact with a substrate on which the target substance has been immobilized over an area of 28.3 mm2 at a density of 0.1 pmol / mm2, and the liquid is stirred for one hour at 4°C, at least 2% of the fluorescent nanoparticles bind to the target substance.

Description

[Technical Field]

[0001] The present invention relates to fluorescent nanoparticles and methods for producing fluorescent nanoparticles. [Background technology]

[0002] In pathological diagnosis, immunohistochemistry (IHC) and in situ hybridization (ISH) are used to detect molecular expression in specimens and to diagnose functional abnormalities such as abnormal gene and protein expression. For example, enzyme-based dye staining methods (DAB staining, etc.) are used for immunostaining.

[0003] However, enzyme-labeled staining such as DAB staining has the problem that the staining density is greatly affected by environmental conditions such as temperature and time, making it difficult to estimate the actual amount of antigen, etc. Therefore, for immunological observation in pathological diagnosis, fluorescent labeling methods using fluorescent labels are also used instead of enzyme-labeled staining.

[0004] This method has the advantage of being more quantitative than DAB staining. The fluorescent labeling method measures the amount of antigen by staining the target antigen using an antibody modified with a fluorescent dye and observing it.

[0005] Conventionally, a staining solution containing fluorescent nanoparticles in which streptavidin as a target substance labeling molecule is directly bound to resin particles containing a fluorescent dye has been known as a fluorescent labeling agent. For example, Patent Document 1 discloses fluorescent nanoparticles bound to streptavidin. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Special Publication No. 2008-543982 Summary of the Invention [Problem to be solved by the invention]

[0007] The fluorescent nanoparticles described in Patent Document 1 have streptavidin bound to their surfaces. The inventors of the present invention have found that using these fluorescent nanoparticles to stain a target substance (a target substance bound to a detection target) can result in low sensitivity in some cases.

[0008] The inventors have thoroughly investigated the reason for this and concluded that the streptavidin (target substance recognition substance) carried on the surface of the fluorescent nanoparticles described in Patent Document 1 may have lost its activity and may not be able to bind to biotin (target substance).

[0009] The present invention has been made in view of the above circumstances, and aims to provide fluorescent nanoparticles having a target substance recognition substance supported on the surface thereof, in which the target substance recognition substance has high activity and can measure the target substance with high sensitivity. Another aim of the present invention is to provide a method for producing such fluorescent nanoparticles. [Means for solving the problem]

[0010] The fluorescent nanoparticles according to one embodiment of the present invention are fluorescent nanoparticles having a target substance recognition substance that binds to a target substance supported on the surface thereof, and the fluorescent nanoparticles are prepared by dissolving 100 μL of a liquid containing the fluorescent nanoparticles at a concentration of 100 pmol / L in a solution containing 0.1 pmol / mm 2 28.3mm at a density of 2 and agitating the resulting mixture at 4° C. for 1 hour, the proportion of the fluorescent nanoparticles that bind to the target substance is 1.5% or more.

[0011] Furthermore, a method for producing fluorescent nanoparticles according to one embodiment of the present invention includes the steps of preparing fluorescent nanoparticles having maleimide groups introduced therein, introducing thiol groups into target substance labeling molecules, and reacting the maleimide groups introduced into the fluorescent nanoparticles with the thiol groups introduced into the target substance labeling molecules, wherein the steps of introducing the thiol groups and reacting the maleimide groups with the thiol groups are carried out at a temperature of 0°C to 8°C. This method is for producing fluorescent nanoparticles having a target substance recognition substance supported on their surface that binds to a target substance. [Effects of the Invention]

[0012] According to the present invention, it is possible to provide fluorescent nanoparticles having a target substance recognition substance supported on the surface thereof, in which the target substance recognition substance has high activity and can measure the target substance with high sensitivity. The present invention also provides a method for producing such fluorescent nanoparticles. [Brief explanation of the drawings]

[0013] [Figure 1] 1A to 1C show an example of a method for producing fluorescent nanoparticles carrying a target substance recognition substance on the surface. [Figure 2] 2A to 2E show the results of immunostaining using fluorescent nanoparticles according to an embodiment of the present invention and fluorescent nanoparticles of a comparative example. [Figure 3] Figure 3A shows the correlation between the performance evaluation of fluorescent nanoparticles using the BCA method and the results of immunostaining, and Figure 3B shows the correlation between the performance evaluation of fluorescent nanoparticles using the biotin plate method and the results of immunostaining. DETAILED DESCRIPTION OF THE INVENTION

[0014] [Fluorescent nanoparticles] The fluorescent nanoparticles according to an embodiment of the present invention include nanoparticles containing a fluorescent dye and a resin, and a target substance recognition substance carried on the surface of the nanoparticles.

[0015] (nanoparticles) The fluorescent nanoparticles according to this embodiment contain nanoparticles as a base material.

[0016] The resin constituting the nanoparticles can be the following thermoplastic or thermosetting resin. Suitable examples of thermoplastic resins include polystyrene, polyacrylonitrile, polyfuran, and similar resins. Suitable examples of thermosetting resins include polyxylene, polylactic acid, glycidyl methacrylate, melamine resin, polyurea, polybenzoguanamine, polyamide, phenolic resin, polysaccharides, and similar resins. Thermosetting resins, particularly melamine resins, are preferred because they can prevent the elution of dyes encapsulated in the nanoparticles even through treatments such as dehydration, clearing, and encapsulation using organic solvents such as xylene.

[0017] The nanoparticles preferably have functional groups on their surfaces for directly or indirectly binding target substance-recognizing substances. As such functional groups, the same functional groups as those used to bind various target substances in the technical field to which the present invention pertains can be used, but for example, epoxy groups or amino groups are preferred.

[0018] The method for preparing nanoparticles having functional groups is not particularly limited. For example, a method can be used in which a (co)monomer having a predetermined functional group on its side chain is (co)polymerized as a monomer for synthesizing the thermoplastic resin or thermosetting resin that constitutes the nanoparticles, or a method can be used in which, after synthesizing the thermoplastic resin or thermosetting resin, the functional group possessed by the resin monomer unit that constitutes it is treated with a reagent to convert it into the predetermined functional group.

[0019] Examples of embodiments for producing nanoparticles using thermoplastic resins include: an embodiment in which glycidyl methacrylate is used as a monomer and copolymerized with styrene to produce nanoparticles of polystyrene-based resins having epoxy groups on the surface; an embodiment in which styrene carboxylic acid or styrene sulfonic acid is copolymerized with styrene to produce nanoparticles of polystyrene-based resins having carboxylic acid or sulfonic acid on the surface; or an embodiment in which aminosulfonic acid is copolymerized with styrene to produce nanoparticles of polystyrene-based resins having amino groups on the surface. The epoxy groups of the glycidyl methacrylate can also be converted to amino groups by a predetermined treatment.

[0020] On the other hand, when producing thermosetting pigment resin particles, an embodiment includes producing nanoparticles of a melamine resin by copolymerizing a melamine resin raw material (for example, MX035 manufactured by Sanwa Chemical Co., Ltd.) as a monomer.

[0021] (fluorescent dye) The fluorescent nanoparticles according to this embodiment contain a fluorescent dye.

[0022] The fluorescent dye may be bound to the nanoparticles by physical or chemical forces, and the fluorescent dye may be incorporated into the nanoparticles, for example, by polymerizing a monomer in a solvent containing the fluorescent dye.

[0023] When forming nanoparticles by polymerizing the monomers that make up the resin and incorporating a fluorescent dye, the fluorescent dye that can be encapsulated in the nanoparticles or fixed to the surface of the particles may be, for example, rhodamine-based dye molecules, BODIPY-based dye molecules, squarylium-based dye molecules, aromatic hydrocarbon-based dye molecules, or combinations thereof.

[0024] Fluorescent dyes such as rhodamine dye molecules are preferred because they have relatively high light resistance, and among these, perylene, pyrene, and perylene diimide, which belong to aromatic hydrocarbon dye molecules, are preferred.

[0025] Examples of rhodamine-based dye molecules include 5-carboxy-rhodamine, 6-carboxy-rhodamine, 5,6-dicarboxy-rhodamine, rhodamine 6G, tetramethylrhodamine, X-rhodamine, Texas Red, Spectrum Red, LD700 PERCHLORATE, derivatives thereof, and the like.

[0026] Examples of BODIPY dye molecules include BODIPY FL, BODIPY TMR, BODIPY 493 / 503, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665 (all manufactured by Invitrogen), and derivatives thereof.

[0027] Examples of squarylium dye molecules include SRfluor 680-Carboxylate, 1,3-Bis[4-(dimethylamino)-2-hydroxyphenyl]-2,4-dihydroxycyclobutenediylium dihydroxide, bis, 1,3-Bis[4-(dimethylamino)phenyl]-2,4-dihydroxycyclobutenediylium dihydroxide, bis, 2-(4-(Diethylamino)-2-hydroxyphenyl)-4-(4-(diethyliminio)-2-hydroxycyclohexa-2,5-dienylidene)-3-oxocyclobut-1-e nolate, 2-(4-(Dibutylamino)-2-hydroxyphenyl)-4-(4-(dibutyliminio)-2-hydroxycyclohexa-2,5-dienylidene)-3-oxocyclobut-1 -enolate, 2-(8-Hydroxy-1,1,7,7-tetramethyl-1,2,3,5,6,7-hexahydropyrido[3,2,1-ij]quinolin-9-yl)-4-(8-hydroxy-1,1,7,7-tetramethyl-2,3,6,7-tetrahydro-1H-pyrido[3,2,1-ij]quinolinium-9(5H)-ylidene)-3-oxocyclobut-1-enolate, and derivatives thereof.

[0028] Examples of aromatic hydrocarbon-based pigment molecules include N,N-Bis-(2,6-diisopropylphenyl)-1,6,7,12-(4-tert-butylphenoxy)-perylene-3,4,9,10-tetracarbonacid diimide, N,N’-Bis(2,6-diisopropylphenyl)-1,6,7,12-tetraphenoxyperylene-3,4:9,10-tetracarboxdiimide, N,N’-Bis(2,6-diisopropylphenyl)perylene-3,4,9,10-bis(dicarbimide), 16,N,N’-Bis(2,6-dimethylphenyl)perylene-3,4,9,10-tetracarboxylic diimide, 4,4’-[(8,16-Dihydro-8,16-dioxodibenzo[a,j]perylene-2,10-diyl)dioxy]dibutyric acid, 2,10-Dihydroxy-dibenzo[a,j]perylene-8,16-dione, 2,10-Bis(3-aminopropoxy)dibenzo[a,j]perylene-8,16-dione, 3,3’-[(8,16-Dihydro-8,16-dioxodibenzo[a,j]perylen-2,10-diyl)dioxy]dipropylamine, 17-BIS(Octyloxy)Anthra[9,1,2-cde-]Benzo[RST]Pentaphene-5-10-Dione, Octadecanoicacid, 5,10-dihydro-5,10-dioxoanthra[9,1,2-cde]benzo[rst]pentaphene-16,17-diylester, Dihydroxydibenzanthrone, Benzenesulfonic acid, 4,4’,4’’,4’’’-[[2,9-bis[2,6-bis(1-methylethyl)phenyl]-1,2,3,8,9,10-hexahydro-1,3,8,10-tetraoxoanthra[2,1,9-def:6,5,10-d’e’f’]diisoquinoline-5,6,12,13-tetrayl]tetrakis(oxy)]tetrakis-,Benzeneethanaminium, 4,4',4'',4'''-[[2,9-bis[2,6-bis(1-methylethyl)phenyl]-1,2,3,8,9,10-hexahydro-1,3,8,10-tetraoxoanthra[2,1,9-def:6,5,10-d'e'f']diisoquinoline-5,6,12,13-tetrayl]tetrakis(oxy)]tetrakis[N,N,N-trimethyl-], and their derivatives.

[0029] (Target substance recognition substance) The fluorescent nanoparticles according to this embodiment have a target substance recognition substance carried on their surface.

[0030] The target substance recognition substance is, for example, a molecule that specifically binds to a target substance bound to a detection target. When the target substance recognition substance binds to the target substance, the detection target is labeled with the fluorescent nanoparticles.

[0031] The target substance recognition substance is not particularly limited, but is, for example, at least one selected from the group consisting of avidin, streptavidin, and neutravidin.

[0032] Examples of combinations of a target substance and a target substance recognition substance that specifically binds to it include combinations of biotin-avidin, biotin-streptavidin, and biotin-neutravidin. Specific binding can be interpreted as a general term in the technical field to which the present invention pertains, depending on the combination, but the binding constant (K A ) or its reciprocal, the dissociation constant (K D ) can also be defined.

[0033] That is, the target substance recognition substance used in the present invention has a binding constant (K A ) is 1×10 5 ~1×1012 The binding constant (K A ) is within this range, the target substance can be treated as a target substance recognition substance that specifically binds to the target substance to be stained.

[0034] (Average particle size) The average particle diameter of the fluorescent nanoparticles is preferably 30 to 300 nm, more preferably 40 to 200 nm, from the viewpoint of enabling suitable observation of bright spots even with a general-purpose fluorescence microscope. If the average particle diameter exceeds 300 nm, the number of bright spots per cell decreases during observation after staining, making it difficult to observe the bright spots. Conversely, if the average particle diameter is less than 30 nm, the number of bright spots per cell increases, making it difficult to observe the bright spots. The average particle diameter can be determined by measuring the major axis of each particle (100 or more) in an image captured with a scanning electron microscope and averaging the measurements.

[0035] (Proportion of fluorescent nanoparticles that bind to target substances) The fluorescent nanoparticles according to this embodiment have a relatively high rate of binding to a target substance because the target substance recognition substance carried on the surface of the nanoparticles has high activity.

[0036] Specifically, 100 μL of liquid containing fluorescent nanoparticles at a concentration of 100 pmol / L was diluted with 0.1 pmol / mm 2 28.3mm at a density of 2 When the fluorescent nanoparticles are brought into contact with a substrate on which a target substance is immobilized over an area of ​​1.5% and stirred for 1 hour at 4° C., the proportion of fluorescent nanoparticles that bind to the target substance on the substrate is 1.5% or more. The proportion of fluorescent nanoparticles that bind to the target substance on the substrate can be evaluated by bringing fluorescent nanoparticles carrying a target substance recognition substance on their surface into contact with the substrate on which the target substance is immobilized and determining the proportion of those bound.

[0037] The target substance recognition substances carried by fluorescent nanoparticles can be either active or inactive. That is, some target substance recognition substances have the ability to bind to the target substance, while others do not. In the above evaluation, a ratio of fluorescent nanoparticles that bind to the target substance of 1.5% or more indicates that there is a large amount of active target substance recognition substances on the surface of the fluorescent nanoparticles.

[0038] By improving the manufacturing process of the fluorescent nanoparticles according to this embodiment, the target substance recognition substance has high activity, and the rate of target substance recognition is improved. This allows for highly sensitive measurements. Furthermore, by keeping the activity of the target substance recognition substance within a certain range, detection sensitivity is improved and detection variability is reduced (detection accuracy is improved).

[0039] In the conventional BCA method, the amount of target substance recognition substance bound to the surface of fluorescent nanoparticles was simply measured as the amount of protein, but the above method makes it possible to measure the activity of the target substance recognition substance supported on the surface of fluorescent nanoparticles.

[0040] The above ratio may be 1.5% or more, but from the viewpoint of more sensitive measurement, it is more preferably 2% or more, even more preferably 2.5% or more, even more preferably 5% or more, and even more preferably 8% or more. The upper limit of the above ratio is preferably 10% or less from the viewpoint of suppressing non-specific binding.

[0041] There are no particular limitations on the method for producing the fluorescent nanoparticles according to this embodiment. For example, the fluorescent nanoparticles according to this embodiment can be produced by the method for producing fluorescent nanoparticles described below.

[0042] [Method of manufacturing fluorescent nanoparticles] The method for producing fluorescent nanoparticles according to this embodiment includes the steps of preparing fluorescent nanoparticles having maleimide groups introduced therein, introducing thiol groups into a target substance recognition substance, and reacting the maleimide groups introduced into the fluorescent nanoparticles with the thiol groups introduced into the target substance labeling molecules. The steps of introducing the thiol groups and reacting the maleimide groups with the thiol groups are carried out at 0°C to 8°C. Each step is described below.

[0043] (Step of preparing fluorescent nanoparticles having maleimide groups introduced therein) In the step of preparing fluorescent nanoparticles having maleimide groups introduced thereinto, fluorescent nanoparticles having maleimide groups introduced thereinto in advance may be obtained, or fluorescent nanoparticles having maleimide groups introduced thereinto may be prepared by introducing maleimide groups thereinto.

[0044] Maleimide groups are preferably introduced into fluorescent nanoparticles by reacting fluorescent nanoparticles to which amino groups have been introduced with a maleimide group-introducing reagent (e.g., NHS-PEG12-maleimide) as shown in Figure 1A. Generally, the introduction of amino groups is considered preferable by first treating the fluorescent nanoparticles with formic acid, as shown in Figure 1A, to add carboxyl groups to their surfaces, and then adding an amino group-introducing reagent (e.g., BAEE) to introduce the amino groups. However, in one embodiment of the present invention, amino groups may be introduced by adding an amino group-introducing reagent to fluorescent nanoparticles that have not been treated with formic acid. Although the reason for this is unclear, omitting the formic acid treatment results in fluorescent nanoparticles with higher target substance-recognizing substance activity and higher sensitivity.

[0045] In addition, by omitting the formic acid treatment, the presence of unreacted carboxyl groups (carboxyl groups without an amino group) prevents the surface of the fluorescent nanoparticles from becoming negatively charged, shifting the surface charge more positively than before, which is expected to improve accessibility to RNAscope probes that have a negative charge derived from nucleic acids.

[0046] Furthermore, as shown in FIG. 1A, after the step of introducing amino groups into the fluorescent nanoparticles and before the step of reacting N-hydroxysuccinimide groups with the amino groups, it is preferable to perform a step of washing the fluorescent nanoparticles with the amino groups introduced with an organic solvent. This washing step is preferably performed so as to remove water using an organic solvent. Specifically, it is preferable to wash the nanoparticles multiple times using washing solutions in which the amount of organic solvent in the washing solution is gradually increased. It is believed that washing in such a way as to remove water can prevent the deactivation of the maleimide group-introducing reagent.

[0047] The amino group-introducing reagent is not particularly limited as long as it can introduce an amino group. Examples of the amino group-introducing reagent include 1,2-Bis(2-aminoethoxy)ethane (BAEE), 1,4-Diaminobutane, 1,10-Diaminodecane, 1,12-Diaminododecane, 1,7-Diaminoheptane, 1,6-Diaminohexane, 1,8-Diaminooctane, 1,5-Diaminopentane, 1,3-Diaminopropane, 1,11-Diaminoundecane, Ethylenediamine, 2,2'-Oxybis(ethylamine), and 1,11-Diamino-3,6,9-trioxaundecane.

[0048] Next, as shown in Figure 1A, the amino group introduced into the fluorescent nanoparticles as described above is reacted with, for example, the N-hydroxysuccinimide group of a compound containing a maleimide group and an N-hydroxysuccinimide group, thereby introducing a maleimide group into the fluorescent nanoparticles.

[0049] The maleimide group-introducing reagent is not particularly limited as long as it can introduce a maleimide group. Examples of the maleimide group-introducing reagent include compounds containing a maleimide group and an N-hydroxysuccinimide group, NHS-PEG-maleimide, TFP-PEG-maleimide, PFP-PEG-maleimide, etc.

[0050] (Step of introducing a thiol group into a target substance recognition substance) The step of introducing a thiol group into the target substance recognition substance is carried out, for example, by reacting a thiol group introduction reagent (e.g., Traut's reagent) with the amino group of the target substance recognition substance, as shown in Figure 1B. From the viewpoint of ensuring high activity of the target substance recognition substance, the step of introducing a thiol group into the target substance recognition substance is preferably carried out at 0 to 8°C, more preferably at 0 to 4°C, and even more preferably at 2 to 4°C.

[0051] The target substance recognition substance is not particularly limited, but is, for example, at least one selected from the group consisting of avidin, streptavidin, and neutravidin.

[0052] The thiol group-introducing reagent is not particularly limited as long as it can introduce a thiol group. Examples of the thiol group-introducing reagent include Traut's reagent (2-iminothiolane), SPDP (N-{6-[3-(2-pyridyldithio)propionamido]hexanoyloxy}sulfosuccinimide), Sulfo-AC5-SPDP (N-{6-[3-(2-pyridyldithio)propionamido]hexanoyloxy}sulfosuccinimide, sodium salt), SATA (N-succinimidyl S-acetylthioacetate), SATP (N-succinimidyl-S-acetylthiopropionate), and SAT(PEG)4 (PEGylated N-succinimidyl S-acetylthioacetate).

[0053] (Step of reacting maleimide group with thiol group) The step of reacting the maleimide group with the thiol group is carried out by reacting, for example, fluorescent nanoparticles to which a maleimide group has been introduced with a target substance recognition substance to which a thiol group has been introduced (FIG. 1C). From the viewpoint of ensuring high activity of the target substance recognition substance, the step of reacting the maleimide group with the thiol group is preferably carried out at 0 to 8°C, more preferably at 0 to 4°C, and even more preferably at 2 to 4°C.

[0054] (effect) According to the fluorescent nanoparticles of the embodiment of the present invention, the activity of the target substance recognition substance carried on the surface thereof is high. Furthermore, the degree of activity can be controlled within a certain range. Furthermore, according to the method for producing fluorescent nanoparticles of the embodiment of the present invention, the activity of the target substance recognition substance carried on the surface thereof can be increased. As a result, the fluorescent nanoparticles of the embodiment of the present invention can improve detection sensitivity and reduce variation in detection results. [Example]

[0055] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0056] [Preparation of fluorescent nanoparticles] Example 1: Low-Temperature Reaction Synthesis of fluorescent nanoparticles (1) Raw material preparation / mixing process 2.5 mg of the fluorescent dye sulforhodamine 101 (Sigma-Aldrich, Texas Red dye) was dissolved in 22.5 mL of pure water. This solution was stirred for 20 minutes while maintaining the temperature at 70°C using a hot stirrer. Next, 1.5 g of the water-soluble melamine resin Nikalac MX-035 (Nippon Carbide Industries Co., Ltd.) was added to the solution, and the mixture was heated and stirred for an additional 5 minutes under the same conditions.

[0057] (2) Polymerization process (formic acid treatment) To the heated solution, 100 μL of formic acid was added, and the mixture was further heated and stirred for 20 minutes while maintaining the solution temperature at 60° C. Thereafter, the solution was allowed to stand and cooled to room temperature.

[0058] The cooled solution was transferred to a centrifuge tube and centrifuged at 12,000 rpm for 20 minutes. The supernatant was removed and the precipitate was collected. The collected precipitate was washed with ethanol and then with water in the same manner.

[0059] (3) Amino group introduction step Amino groups were introduced onto the surface of fluorescent nanoparticles as follows: First, 1 mL of the melamine-based particles (fluorescent nanoparticles) adjusted to 1 nmol / L after the above washing was mixed with 20 μL of 1,2-Bis(2-aminoethoxy)ethane (BAEE), and the mixture was allowed to react at 70°C for 1 hour. In this way, amino groups were introduced onto the fluorescent nanoparticles.

[0060] (4) Cleaning process The reaction mixture was centrifuged at 10,000 G for 20 minutes, the supernatant was removed, and the precipitate was collected. 1 mL of water was then added, and the mixture was centrifuged again at 10,000 G for 20 minutes. The supernatant was removed, and the precipitate was collected. This procedure was repeated two more times, for a total of three water washes. Next, 1 mL of THF was added to the precipitate, and the mixture was centrifuged again at 10,000 G for 20 minutes. The supernatant was removed, and the precipitate was collected.

[0061] The resulting fluorescent nanoparticles were adjusted to a concentration of 3 nmol / L using THF.

[0062] (5) Maleimide group modification of fluorescent nanoparticles NHS-PEG12-maleimide was mixed with this fluorescent nanoparticle solution to a final concentration of 10 mmol / L, and the mixture was allowed to react for 1 hour at room temperature (° C.). In other words, maleimide groups were introduced into the fluorescent nanoparticles in the manner described above.

[0063] This reaction solution was centrifuged at 10,000 G for 20 minutes, the supernatant was removed, and the precipitate was collected. PBS containing 2 mmol / L EDTA was then added to disperse the precipitate. The mixture was then centrifuged again at 10,000 G for 20 minutes, the supernatant was removed, and the precipitate was collected. The fluorescent nanoparticles were washed three times using the series of steps from dispersing the precipitate to centrifugation. Finally, fluorescent nanoparticles were obtained that had the maleimide groups on the particle surface and encapsulated Texas Red dye. Furthermore, the average particle size of the melamine-based particles was measured using a scanning electron microscope using the method described above, and was found to be 150 nm.

[0064] (6) Thiol group modification of streptavidin On the other hand, streptavidin capable of binding to the fluorescent nanoparticles having the maleimide group introduced therein was prepared as follows.

[0065] First, 40 μL of streptavidin (Wako Pure Chemical Industries, Ltd.) adjusted to 1 mg / mL was reacted with 70 μL of Traut's reagent (Thermo Fisher Scientific, Inc.) adjusted to 64 mg / mL for 1 hour at 4° C. In other words, a protected thiol group was introduced into the amino group of streptavidin.

[0066] Thereafter, a free thiol group (-SH) was generated from the protected thiol group by a known hydroxylamine treatment, and the thiol group (-SH) was introduced into streptavidin.

[0067] This streptavidin solution was desalted at 4°C using a gel filtration column (Zeba Spin Desalting Columns: manufactured by Thermo Fisher Scientific) to obtain streptavidin capable of binding to the fluorescent nanoparticles.

[0068] (7) Bonding reaction between maleimide and thiol groups The entire amount of streptavidin was mixed with 1 mL of the fluorescent nanoparticles adjusted to 1 nmol / L using PBS containing 2 mmol / L EDTA, and a reaction to bind the two molecules was carried out at 4° C. for 1 hour. After that, centrifugation and washing were carried out using PBS containing 2 mmol / L EDTA, and only the fluorescent nanoparticles bound to streptavidin were collected, yielding the fluorescent nanoparticles of Example 1.

[0069] (Example 2: Omitting formic acid treatment, water removal and washing, low-temperature reaction) In Example 2, in the above polymerization step (2), the mixture was heated and stirred without adding 100 μL of formic acid.

[0070] In Example 2, the above-mentioned washing step (4) was modified as follows. Specifically, the reaction solution was centrifuged at 10,000 G for 20 minutes, the supernatant was removed, and only the precipitate was collected. Then, 1 mL of water was added, and the mixture was centrifuged again at 10,000 G for 20 minutes, the supernatant was removed, and only the precipitate was collected. Next, 1 mL of a washing solution containing water and THF at a volume ratio of 1:1 was added, and the mixture was centrifuged again at 10,000 G for 20 minutes, the supernatant was removed, and only the precipitate was collected. Next, the mixture was changed to a volume ratio of 1:3, and only the precipitate was collected in the same manner. Next, the mixture was changed to a volume ratio of 0:4, and only the precipitate was collected in the same manner. In this way, washing was performed by gradually increasing the amount of THF in the washing solution to remove water. Otherwise, the fluorescent nanoparticles of Example 2 were obtained in the same manner as in Example 1.

[0071] (Comparative Example 1: Room temperature reaction) In Comparative Example 1, the reaction temperature in the above (6) thiol group modification of streptavidin was room temperature. Also, the reaction temperature in the above (7) binding reaction of maleimide group and thiol group was room temperature. Otherwise, fluorescent nanoparticles of Comparative Example 1 were obtained in the same manner as in Example 1.

[0072] (Comparative Example 2: Formic acid treatment omitted, reaction at room temperature) In Comparative Example 2, in the above (2) polymerization step, 100 μL of formic acid was not added, and the solution temperature was maintained at 60°C while further heating and stirring for 20 minutes. In the above (6) thiol group modification of streptavidin, the reaction temperature was room temperature. In the above (7) binding reaction of maleimide groups and thiol groups, the reaction temperature was room temperature. The fluorescent nanoparticles of Comparative Example 2 were obtained in the same manner as in Example 1, except for the above.

[0073] (Comparative Example 3: Water removal washing, room temperature reaction) In Comparative Example 3, the above-mentioned (4) washing step was modified as follows. Specifically, the reaction solution was centrifuged at 10,000 G for 20 minutes, the supernatant was removed, and only the precipitate was collected. Then, 1 mL of water was added, and the mixture was centrifuged again at 10,000 G for 20 minutes, the supernatant was removed, and only the precipitate was collected. Next, 1 mL of a washing solution containing water and THF at a volume ratio of 1:1 was added, and the mixture was centrifuged again at 10,000 G for 20 minutes, the supernatant was removed, and only the precipitate was collected. Next, the volume ratio of water and THF was changed to 2:2, and only the precipitate was similarly collected. Next, the volume ratio of water and THF was changed to 1:3, and only the precipitate was similarly collected. Next, 1 mL of THF was added, and the precipitate was obtained in the same manner. In this way, washing was performed to remove water using washing solutions with gradually increasing amounts of THF.

[0074] In addition, in the above (6) modification of streptavidin with thiol groups, the reaction temperature was room temperature. In the above (7) binding reaction of maleimide groups and thiol groups, the reaction temperature was room temperature. Otherwise, fluorescent nanoparticles of Comparative Example 3 were obtained in the same manner as in Example 1.

[0075] [Evaluation of fluorescent nanoparticles] Each of the fluorescent nanoparticles obtained as described above was evaluated as follows.

[0076] (Performance evaluation of fluorescent nanoparticles using the BCA method) For each of the fluorescent nanoparticles of Examples 1 and 2 and Comparative Examples 1 to 3, the amount of streptavidin present on the surface of one fluorescent nanoparticle was calculated as the protein amount by the BCA method using Pierce's "Micro BCA Protein Assay kit."

[0077] Specifically, fluorescent nanoparticles before and after streptavidin modification were prepared, and the protein content of each was measured using the BCA method. To measure the protein content, 150 μL of a PBS dispersion of streptavidin-conjugated fluorescent nanoparticles (particle concentration 0.50 nmol / L) and 150 μL of a PBS dispersion of unconjugated fluorescent nanoparticles (particle concentration 0.50 nmol / L) were added to each solution. The bicinchoninic acid (BCA) solution and copper sulfate solution included in the kit were mixed and added, and the absorbance at 562 nm was measured. The absorbance of the unconjugated fluorescent nanoparticles was subtracted as background from the absorbance of the streptavidin-conjugated fluorescent nanoparticles, and the resulting absorbance was compared with a standard curve to determine the net protein content (streptavidin concentration) in the aqueous solution.

[0078] The number of streptavidin molecules per fluorescent nanoparticle was calculated based on the ratio of the protein amount (streptavidin concentration) measured by the BCA method to the amount of fluorescent nanoparticles (fluorescent nanoparticle concentration) used in the measurement. The calculation results are shown in Table 1 below.

[0079] (Performance evaluation of fluorescent nanoparticles using biotin plate method) For each of the fluorescent nanoparticles of Examples 1 to 4 and the Comparative Example, the amount of active streptavidin was measured using a commercially available 96-well plate (G-Biosciences, model number 786-762) on which biotin had been immobilized in advance. This well plate (inner diameter of wells: approximately 6 mm) contained 0.1 pmol / mm 2 28.3mm at a density of 2 is immobilized over an area of

[0080] Specifically, each well of the well plate was washed with PBS (pH 7.5) containing 0.05% Tween 20, and then 200 μL of PBS containing 1% BSA was dispensed into each well. The plate was then left overnight at 4°C. Subsequently, all of the BSA-containing PBS in the wells was aspirated, and 100 μL of a PBS dispersion of streptavidin-modified fluorescent nanoparticles (particle concentration: 0.10 nmol / L) was dispensed. The plate was then placed on a plate shaker (Biosan, model PST-60HL) at 4°C and left for 1 hour while stirring at 500 rpm. The entire PBS dispersion of fluorescent nanoparticles was then aspirated, and 100 μL of PBS was dispensed into each well. The fluorescence intensity at 595 nm in response to 531 nm light irradiation was measured using a plate reader (PerkinElmer, model EnVision 2104). The average fluorescence intensity was calculated from five replicates for each well. The measurement results are shown in the plate reader fluorescence values ​​in Table 1 below.

[0081] The percentage of fluorescent nanoparticles bound to the target substance was calculated based on the plate reader fluorescence values. The calibration curve was prepared by dispensing PBS dispersions of streptavidin-modified fluorescent nanoparticles (particle concentrations: 0 pmol / L, 1.56 pmol / L, 3.12 pmol / L, 6.25 pmol / L, and 12.5 pmol / L) into well plates prepared as described above, stirring the mixture as described above, and then measuring the fluorescence at 595 nm in response to 531 nm light irradiation using a plate reader (PerkinElmer, model number EnVision 2104). The average fluorescence reading for five replicates was used for each well. The percentage of fluorescent nanoparticles bound to the target substance, calculated based on the calibration curve, is shown in Table 1.

[0082] (Immunostaining using fluorescent nanoparticles) Immunostaining was carried out using the fluorescent nanoparticles of Examples 1 to 4 and the Comparative Example.

[0083] Specifically, immunostaining was performed according to the following procedure.

[0084] <IHC staining using staining reagents> An antibody reagent (staining reagent for pathological diagnosis) containing fluorescent nanoparticles and a biotinylated secondary antibody prepared as follows was prepared, and immunostaining was carried out using this staining reagent.

[0085] <Preparation of biotin-modified secondary antibodies> First, 50 μg of anti-rabbit IgG antibody was dissolved in 50 mmol / L Tris-HCl solution (pH 7.5). DTT (dithiothretol) solution was added to the solution to a final concentration of 3 mmol / L. The solution was then reacted at 37°C for 30 minutes. The secondary antibody, reduced with DTT, was purified using a desalting column. 200 μL of the purified antibody was dissolved in 50 mmol / L Tris-HCl solution (pH 7.5) to obtain an antibody solution. Separately, a linker reagent with a 30 Å spacer length, "(+)-Biotin-PEG6-NH-Mal" (PurePEG, product number 2461006-250), was adjusted to 0.4 mmol / L using DMSO. 8.5 μL of this solution was added to the antibody solution, mixed, and reacted at 37°C for 30 minutes.

[0086] This reaction solution was purified using a desalting column, "Zeba Spin Desalting Columns." The absorbance of the desalted reaction solution at a wavelength of 300 nm was measured using a spectrophotometer (Hitachi "F-7000") to calculate the amount of protein contained in the reaction solution. The reaction solution was adjusted to 250 μg / mL with 50 mmol / L Tris solution, and this solution was used as a biotinylated secondary antibody solution.

[0087] <Fluorescent immunostaining method> (1) Deparaffinization process Using the biotinylated secondary antibody, etc., immunostaining and morphological observation staining of human breast cancer-derived cultured cells SK-BR-3 (HER2(3+)) were performed as follows. HER2 IHC Posicon slides (manufactured by Pathology Research Institute, hereafter referred to as control slides) were used as staining slides. These tissue array slides were then deparaffinized.

[0088] (2) Activation treatment process Control slides were deparaffinized and then washed with water. The washed control slides were autoclaved in 10 mmol / L citrate buffer (pH 6.0) at 121°C for 15 minutes to activate the antigens. After the activation process, the control slides were washed with PBS and then blocked for 1 hour using PBS containing 1% BSA.

[0089] (3) Immunostaining process (3-1) Primary antibody reaction The above-mentioned blocked control slide was reacted with a solution of "anti-HER2 rabbit monoclonal antibody (4B5)" manufactured by Roche overnight at 4°C. (3-2) Secondary antibody reaction The control slides that had been subjected to the primary antibody reaction were washed with PBS and then reacted with the biotinylated secondary antibody diluted to 2 μg / mL in PBS containing 1% BSA at room temperature for 30 minutes. (3-3) The control slides that had undergone the secondary antibody reaction were incubated with the fluorescent nanoparticles diluted to 0.02 nmol / L in 1% BSA-containing PBS for 3 hours at room temperature in a neutral pH environment (pH 6.9-7.4). After the incubation, the control slides were washed with PBS.

[0090] (4) Morphological observation staining process After immunostaining, hematoxylin-eosin staining (HE staining) was performed. The immunostained sections were stained with Mayer's hematoxylin solution for 5 minutes for hematoxylin staining. The control slides were then washed with running water at 45°C for 3 minutes. Next, they were stained with 1% eosin solution for 5 minutes for eosin staining.

[0091] (5) Fixation process After the immunostaining and morphological observation staining processes, the tissue sections were washed and dehydrated by immersing them in pure ethanol for 5 minutes four times. They were then immersed in xylene for 5 minutes four times for clearing. Finally, the tissue sections were mounted in mounting medium ("Entelaneu" manufactured by Merck) to prepare control slides for the observation samples.

[0092] (6) Observation and measurement process The control slides that had undergone the fixation process were irradiated with a specified excitation light to emit fluorescence. The control slides in this state were observed and photographed using a fluorescence microscope (Olympus "BX-53") and a microscope digital camera (Olympus "DP73"). The wavelength of the excitation light was set to 575-600 nm by passing it through an optical filter. The wavelength of the fluorescence to be observed was also set to 612-692 nm by passing it through an optical filter. The excitation wavelength conditions for microscope observation and image acquisition were as follows: with excitation at 580 nm, the irradiation energy near the center of the field of view was 900 W / cm 2 The exposure time during image acquisition was arbitrarily set (for example, 4000 μsec) so that the image brightness would not saturate. The number of bright dots (PID score) for SK-BR-3 was taken as the average value for 1000 cells measured using the ImageJ FindMaxima method on images captured at 400x magnification. The observation results are shown in Figure 2. Figures 2A to 2E show the observation results when the fluorescent nanoparticles of Examples 1 and 2 and Comparative Examples 1 to 3 were used, respectively. Table 1 also shows the measurement results of the number of bright dots (PID score) per cell when the fluorescent nanoparticles of Examples 1 and 2 and Comparative Examples 1 to 3 were used.

[0093] [Table 1]

[0094] [About Table 1] In Examples 1 and 2, the step of introducing a thiol group and the step of reacting the thiol group with a maleimide group were carried out at low temperatures, whereas in Comparative Example 1, this reaction was carried out at room temperature. As a result, in Example 1, higher values ​​were obtained in each evaluation (BCA method, biotin plate method, immunostaining, rate of binding to target substance) than in Comparative Example 1. This is thought to be because in Examples 1 and 2, the streptavidin was not inactivated and maintained high activity due to the low temperature reaction.

[0095] In Example 2, amino groups were introduced into the fluorescent nanoparticles without formic acid treatment, and then the nanoparticles were washed in a washing process using a washing solution containing gradually increasing amounts of organic solvent to remove water.Then, the process of introducing thiol groups and the process of reacting the thiol groups with maleimide groups were carried out at low temperatures.As a result, Example 2 obtained higher values ​​in each evaluation than Example 1 and Comparative Examples 1 to 3.

[0096] [About correlation] The correlation between the performance evaluation of fluorescent nanoparticles by the BCA method and the results of immunostaining shown in Table 1 is shown in the graph in Figure 3A. The horizontal axis of the graph in Figure 3A is the PID score (number of bright spots per cell) obtained by immunostaining shown in Table 1, and the vertical axis is the number of streptavidin molecules per fluorescent nanoparticle obtained by the BCA method shown in Table 1.

[0097] The correlation between the performance evaluation of fluorescent nanoparticles using the biotin plate method in Table 1 and the results of immunostaining is shown in the graph in Figure 3B. The horizontal axis of the graph in Figure 3B is the PID score (number of bright spots per cell) obtained by immunostaining shown in Table 1, and the vertical axis is the plate reader fluorescence value obtained by the biotin plate method shown in Table 1.

[0098] Comparing Figure 3A and Figure 3B, Figure 3B shows a higher correlation. In other words, the biotin plate method is able to more accurately evaluate the performance of fluorescent nanoparticles than the BCA method.

[0099] Specifically, for example, as shown in Table 1 above, when the performance of the fluorescent nanoparticles of Comparative Example 3 and the fluorescent nanoparticles of Example 2 was evaluated by the BCA method, the values ​​were 321.9 and 320.8, respectively, which is not much different. However, when immunostaining was actually performed using the fluorescent nanoparticles of Comparative Example 3 and Example 2, the PID scores were 451.7 and 854.8, which is a large difference.

[0100] In contrast, when the performance of the fluorescent nanoparticles of Comparative Example 3 and the fluorescent nanoparticles of Example 2 was evaluated using the biotin plate method, the values ​​were 64089 and 130599, respectively, which is a large difference, and corresponds to PID scores of 451.7 and 854.8.

[0101] In other words, it can be seen that the BCA method cannot correctly evaluate active streptavidin, whereas the biotin plate method can correctly evaluate active streptavidin.

[0102] This application claims priority from Japanese Patent Application No. 2020-049733, filed March 19, 2020. The contents of the specification and drawings of that application are incorporated herein by reference in their entirety. [Industrial Applicability]

[0103] The fluorescent nanoparticles according to this embodiment are highly sensitive and therefore useful for fluorescent imaging and the like.

Claims

1. A method for producing fluorescent nanoparticles having a target substance recognition substance that binds to a target substance carried on the surface thereof, comprising: the fluorescent nanoparticles are characterized in that when 100 μL of a liquid containing the fluorescent nanoparticles at a concentration of 100 pmol / L is brought into contact with a substrate on which the target substance is immobilized at a density of 0.1 pmol / mm 2 over an area of ​​28.3 mm 2 and stirred at 4° C. for 1 hour, the rate of the fluorescent nanoparticles binding to the target substance is 2% or more; preparing fluorescent nanoparticles having maleimide groups introduced therein; introducing a thiol group into the target substance recognition substance; a step of reacting the maleimide group introduced into the fluorescent nanoparticle with the thiol group introduced into the target substance recognition substance; and the step of introducing a thiol group and the step of reacting the maleimide group with the thiol group are carried out at 0°C to 8°C; The step of preparing the fluorescent nanoparticles into which the maleimide group has been introduced comprises the steps of: introducing an amino group into fluorescent nanoparticles that have not been treated with formic acid; and reacting the amino group introduced into the fluorescent nanoparticles with the N-hydroxysuccinimide group of a compound containing a maleimide group and an N-hydroxysuccinimide group. Method for producing fluorescent nanoparticles.

2. The step of preparing the fluorescent nanoparticles having maleimide groups introduced therein includes: The method further comprises a step of washing the fluorescent nanoparticles having the amino groups introduced therein with an organic solvent to remove water, after the step of introducing amino groups into the fluorescent nanoparticles and before the step of reacting the amino groups with the N-hydroxysuccinimide groups. The method for producing fluorescent nanoparticles according to claim 1 .

3. 3. The method for producing fluorescent nanoparticles according to claim 1, wherein the target substance recognition substance is at least one selected from the group consisting of avidin, streptavidin, and neutravidin.

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