JP7798026B2 - Method for producing fluorescent nanoparticles - Google Patents

Description

The present invention relates to fluorescent nanoparticles and a method for producing fluorescent nanoparticles.

Pathological diagnosis involves the use of techniques called immunohistochemistry (IHC) and in situ hybridization (ISH), which involve staining molecules to identify the expression of molecular information in specimens and then observing them to diagnose functional abnormalities such as abnormal gene or protein expression. Immunostaining, for example, involves enzyme-based dye staining (such as DAB staining).

However, enzyme-labeled staining such as DAB staining presents a challenge, as the staining density is significantly affected by environmental conditions such as temperature and time, making it difficult to estimate the actual amount of antigens from the staining density. For this reason, fluorescent labeling methods using fluorescent labels are also used in immunological observations in pathological diagnosis instead of enzyme-labeled staining.

This method is characterized by its superior quantitative capabilities compared to DAB staining. Fluorescent labeling measures the amount of antigen by staining the target antigen using an antibody modified with a fluorescent dye and observing it.

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 for use in fluorescent labeling. For example, Patent Document 1 discloses fluorescent nanoparticles bound to streptavidin.

Special Publication No. 2008-543982

The fluorescent nanoparticles described in Patent Document 1 above have streptavidin bound to their surface. The inventors of the present invention have conducted research and found that using these fluorescent nanoparticles to stain a target substance (a target substance bound to a detection target) can sometimes result in low sensitivity.

The inventors have thoroughly investigated the reasons for this and concluded that in the case of the fluorescent nanoparticles described in Patent Document 1, the streptavidin (target substance recognition substance) supported on their surface may have lost its activity and may not be able to bind to biotin (target substance).

The present invention was made in consideration of the above circumstances, and aims to provide fluorescent nanoparticles having a target substance recognition substance supported on the surface, 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.

Fluorescent nanoparticles according to one embodiment of the present invention are fluorescent nanoparticles carrying on their surface a target substance recognition substance that binds to a target substance, and 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/ ^{mm2 over an area of 28.3 mm2 ,} and the mixture is stirred at 4°C for 1 hour, the percentage of the fluorescent nanoparticles that bind to the target substance is 1.5% or more.

In addition, a method for producing fluorescent nanoparticles according to one embodiment of the present invention includes the steps of preparing fluorescent nanoparticles into which maleimide groups have been introduced, introducing a thiol group into a target substance labeling molecule, and reacting the maleimide group introduced into the fluorescent nanoparticles with the thiol group introduced into the target substance labeling molecule, wherein the steps of introducing the thiol group and reacting the maleimide group with the thiol group are carried out at a temperature between 0°C and 8°C. This is a method for producing fluorescent nanoparticles having a target substance recognition substance supported on their surface that binds to a target substance.

The present invention provides fluorescent nanoparticles carrying a target substance recognition substance on their surface, 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.

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

[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.

(Nanoparticles)
The fluorescent nanoparticles according to this embodiment contain nanoparticles as a base material.

The resins that make up the nanoparticles can be the following thermoplastic or thermosetting resins. Suitable thermoplastic resins include polystyrene, polyacrylonitrile, polyfuran, and similar resins. Suitable thermosetting resins include polyxylene, polylactic acid, glycidyl methacrylate, melamine resin, polyurea, polybenzoguanamine, polyamide, phenolic resin, polysaccharides, and similar resins. Thermosetting resins, especially melamine resins, are preferred because they can prevent the leaching of dyes encapsulated in the nanoparticles, even when subjected to processes such as dehydration, clearing, and encapsulation using organic solvents such as xylene.

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

There are no particular limitations on the method for preparing nanoparticles with functional groups. For example, one method is to (co)polymerize a (co)monomer that already has a specific functional group on its side chain as a monomer for synthesizing the thermoplastic or thermosetting resin that makes up the nanoparticles, or to synthesize the thermoplastic or thermosetting resin and then convert the functional group of the resin monomer unit that makes up the resin into the specific functional group by treating it with a reagent.

Examples of embodiments for producing nanoparticles using a thermoplastic resin include an embodiment in which polystyrene-based resin nanoparticles having epoxy groups on their surfaces are produced by copolymerizing styrene with glycidyl methacrylate as a monomer; an embodiment in which styrene with styrene carboxylic acid or styrene sulfonic acid is copolymerized to produce polystyrene-based resin nanoparticles having carboxylic acid or sulfonic acid on their surfaces; and an embodiment in which styrene with aminosulfonic acid is copolymerized to produce polystyrene-based resin nanoparticles having amino groups on their surfaces. The epoxy groups in the glycidyl methacrylate can also be converted to amino groups by a specific treatment.

On the other hand, when producing thermosetting pigment resin particles, one embodiment involves producing melamine-based resin nanoparticles by copolymerizing a melamine resin raw material (e.g., MX035 manufactured by Sanwa Chemical Co., Ltd.) as a monomer.

(Fluorescent dye)
The fluorescent nanoparticles according to this embodiment contain a fluorescent dye.

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

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 can be, for example, rhodamine-based dye molecules, BODIPY-based dye molecules, squarylium-based dye molecules, aromatic hydrocarbon-based dye molecules, or combinations of these.

Fluorescent dyes such as rhodamine-based dye molecules are preferred because they have relatively high light resistance, and among them, perylene, pyrene, and perylene diimide, which belong to the aromatic hydrocarbon-based dye molecules, are preferred.

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, and derivatives thereof.

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 their derivatives.

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-(dibutyl minio)-2-hydroxycyclohexa-2,5-dienylidene)-3-oxocyclobut-1 -enolate, 2-(8-hydroxy-1,1,7,7-tetramethyl-1,2,3,5,6,7-hexahydropyri-do[3,2,1-ij]quinolin-9-yl)-4-(8-hydroxy-1,1,7,7-tetramethyl-2,3,6,7-tetrahydro-1H-pyri-do[3,2,1-ij]quinolinium-9(5H)-ylidene)-3-oxocyclobut-1-enolate, and derivatives thereof.

Examples of aromatic hydrocarbon dye 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-dii sopropylphenyl) 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, 1 7-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]pentaphe ne-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 derivatives thereof.

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

A 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.

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

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 it can also be defined by an association constant (K _A ) or its reciprocal, the dissociation constant (K _D ).

That is, the target substance recognition substance used in the present invention preferably has a binding constant (K _A ) between itself and the target substance to be stained in the range of 1×10 ⁵ to 1×10 ^12. When 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.

(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 bright spot observation difficult. Conversely, if the average particle diameter is less than 30 nm, the number of bright spots per cell increases, making bright spot observation difficult. 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.

(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.

Specifically, when 100 μL of a liquid containing fluorescent nanoparticles at a concentration of 100 pmol/L is brought into contact with a substrate on which a target substance has been immobilized at a density of 0.1 pmol/ ^mm2 over an area of 28.3 ^mm2 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 has been immobilized and determining the proportion of those bound.

Target substance recognition substances carried by fluorescent nanoparticles can be active or inactive. In other words, some target substance recognition substances have the ability to bind to target substances, while others do not. In the above evaluation, a percentage of fluorescent nanoparticles that bind to target substances of 1.5% or more indicates that there is a large amount of active target substance recognition substance on the surface of the fluorescent nanoparticles.

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

Conventional BCA methods only measure the amount of target substance recognition substance bound to the surface of fluorescent nanoparticles 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.

The above ratio should be 1.5% or more, but from the perspective 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 perspective of suppressing non-specific binding.

The method for producing fluorescent nanoparticles according to this embodiment is not particularly limited. For example, fluorescent nanoparticles according to this embodiment can be produced by the method for producing fluorescent nanoparticles described below.

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

(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.

When introducing maleimide groups into fluorescent nanoparticles, it is preferable to do so 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. Here, it is generally believed that the preferred method for introducing amino groups is to first treat the fluorescent nanoparticles with formic acid, as shown in Figure 1A, to add carboxyl groups to their surfaces, and then add 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 recognition activity and higher sensitivity.

In addition, by omitting the formic acid treatment, the presence of unreacted carboxyl groups (carboxyl groups without an amino group introduced) 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.

Furthermore, as shown in Figure 1A, after the step of introducing amino groups into the fluorescent nanoparticles and before the step of reacting the amino groups with N-hydroxysuccinimide 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 multiple times using a washing solution in which the amount of organic solvent in the washing solution is gradually increased. It is believed that washing in this way to remove water can prevent the deactivation of the maleimide group-introducing reagent.

The above-mentioned amino group introduction reagent is not particularly limited as long as it can introduce an amino group. Examples of amino group introduction reagents include 1,2-Bis(2-aminoethoxy)ethane (BAEE), 1,4-Diaminobutane, 1,10-Diaminodecane, 1,12-Diaminododecane, 1,7-Diaminoheptane, 1,6-Diaminohexane, and 1,8-Diaminohexane. Examples of the diaminobenzoates include diaminominooctane, 1,5-diaminopentane, 1,3-diaminopropane, 1,11-diaminooundecane, ethylenediamine, 2,2'-oxybis(ethylamine), and 1,11-diamino-3,6,9-trioxaundecane.

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.

There are no particular limitations on the maleimide group-introducing reagent, as long as it can introduce a maleimide group. Examples of maleimide group-introducing reagents include compounds containing a maleimide group and an N-hydroxysuccinimide group, NHS-PEG12-maleimide, TFP-PEG-maleimide, PFP-PEG-maleimide, etc.

(Step of introducing a thiol group into a target substance recognition substance)
The step of introducing a thiol group into a 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, this step of introducing a thiol group into the target substance recognition substance is preferably carried out at 0 to 8°C, more preferably 0 to 4°C, and even more preferably 2 to 4°C.

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

There are no particular limitations on the thiol group introduction reagent, as long as it can introduce a thiol group. Examples of thiol group introduction reagents 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), SAT(PEG)4 (PEGylated N-succinimidyl S-acetylthioacetate), and the like.

(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, as shown in Figure 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.

(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.

The invention relating to this embodiment will be described in detail below with reference to examples, but the invention relating to this embodiment is not limited to these examples.

[Preparation of fluorescent nanoparticles]
Example 1: Low-Temperature Reaction
Synthesis of fluorescent nanoparticles
(1) Raw Material Preparation and Mixing Step 2.5 mg of the fluorescent dye sulforhodamine 101 ("Sulforhodamine 101", manufactured by 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 "Nikarac MX-035" (manufactured by 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.

(2) Polymerization step (formic acid treatment)
To the heated solution, 100 μL of formic acid was added, and the solution 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.

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.

(3) Amino group introduction step: Amino groups were introduced onto the surface of the 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.

(4) Washing Step: This 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. This operation 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.

The resulting fluorescent nanoparticles were adjusted to 3 nmol/L using THF.

(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. In other words, maleimide groups were introduced into the fluorescent nanoparticles in the manner described above.

This reaction solution was centrifuged at 10,000 G for 20 minutes, the supernatant was removed, and the sediment was collected. PBS containing 2 mmol/L EDTA was then added to disperse the sediment. The mixture was then centrifuged again at 10,000 G for 20 minutes, the supernatant was removed, and the sediment was collected. The fluorescent nanoparticles were washed three more times using the series of steps from sediment dispersion to centrifugation. Finally, fluorescent nanoparticles with the maleimide groups present on the particle surface and containing Texas Red dye were obtained. 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.

(6) Thiol Group Modification of Streptavidin Meanwhile, streptavidin capable of binding to the maleimide group-introduced fluorescent nanoparticles was prepared as follows.

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. This introduced a protected thiol group to the amino group of streptavidin.

Subsequently, a known hydroxylamine treatment was used to generate a free thiol group (-SH) from the protected thiol group, and the thiol group (-SH) was introduced into streptavidin.

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

(7) Bonding reaction between maleimide group and thiol group 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 of EDTA, and a bond reaction between the two molecules was carried out for 1 hour at 4° C. After that, the mixture was centrifuged and washed using PBS containing 2 mmol/L of EDTA, and only the fluorescent nanoparticles bonded to streptavidin were collected, yielding the fluorescent nanoparticles of Example 1.

(Example 2: Omission of formic acid treatment, water removal washing, low-temperature reaction)
In Example 2, in the above polymerization step (2), heating and stirring were performed without adding 100 μL of formic acid.

In Example 2, 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 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 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 the precipitate was collected. Next, the mixture was changed to a volume ratio of 1:3, and the precipitate was collected in the same manner. Next, the mixture was changed to a volume ratio of 0:4, and 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.

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

(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. Furthermore, in the above (6) thiol group modification of streptavidin, the reaction temperature was room temperature. Furthermore, in the above (7) binding reaction of maleimide groups and thiol groups, the reaction temperature was room temperature. Otherwise, fluorescent nanoparticles of Comparative Example 2 were obtained in the same manner as in Example 1.

(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.

Furthermore, in the above-mentioned (6) modification of streptavidin with thiol groups, the reaction temperature was room temperature. Furthermore, in the above-mentioned (7) binding reaction between maleimide groups and thiol groups, the reaction temperature was room temperature. Otherwise, the fluorescent nanoparticles of Comparative Example 3 were obtained in the same manner as in Example 1.

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

(Performance evaluation of fluorescent nanoparticles by 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."

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, a mixture of the bicinchoninic acid (BCA) solution and copper sulfate solution included in the kit was added to 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), 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.

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 added (fluorescent nanoparticle concentration) in the measurement. The calculation results are shown in Table 1 below.

(Performance evaluation of fluorescent nanoparticles using the 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) had biotin molecules immobilized over an area of 28.3 ^mm at ^a density of 0.1 pmol/mm.

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. The BSA-containing PBS was then 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 fluorescence measurement was repeated five times for each well and the average value was used. The measurement results are shown in Table 1 below as plate reader fluorescence values.

The percentage of fluorescent nanoparticles that bound to the target substance was calculated based on the plate reader fluorescence values and a calibration curve. The calibration curve was created 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 light irradiation at a wavelength of 531 nm using a plate reader (Perkinelmer, model number EnVision 2104). The fluorescence measurement was performed by averaging five replicates for each well. The percentage of fluorescent nanoparticles that bound to the target substance, calculated based on the calibration curve, is shown in Table 1.

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

Specifically, immunostaining was performed using the following procedure.

<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.

<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 (dithiothreitol) solution was then mixed with this 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 then purified using a desalting column. 200 μL of the total amount of purified antibody was dissolved in 50 mmol/L Tris-HCl solution (pH 7.5) to obtain an antibody solution. Meanwhile, a linker reagent with a spacer length of 30 Å, "(+)-Biotin-PEG ₆ -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.

This reaction solution was purified using a Zeba Spin Desalting Column. 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 the biotinylated secondary antibody solution.

<Fluorescent immunostaining method>
(1) Deparaffinization Step Using the biotinylated secondary antibody and other antibodies, 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 slides for staining. These tissue array slides were then deparaffinized.

(2) Activation Step: The 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. The control slides after activation were washed with PBS, and then blocked for 1 hour using PBS containing 1% BSA.

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

(4) Morphological Observation and Staining Step After immunostaining, hematoxylin-eosin staining (HE staining) was performed. The immunostained sections were stained with Mayer's hematoxylin solution for 5 minutes to perform hematoxylin staining. Thereafter, the control slide was washed with running water at 45°C for 3 minutes. Next, the slide was stained with 1% eosin solution for 5 minutes to perform eosin staining.

(5) Fixation process: After the immunostaining process and the morphological observation staining process, the tissue sections were immersed in pure ethanol for 5 minutes four times to wash and dehydrate them. Subsequently, the sections were immersed in xylene for 5 minutes four times to clear the sections. Finally, the tissue sections were mounted using a mounting medium ("Entelaneu" manufactured by Merck) to prepare control slides for the observation samples.

(6) Observation and Measurement Steps: The control slides that had undergone the fixation process were irradiated with a predetermined excitation light to emit fluorescence. The control slides in this state were observed and imaged 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 observed fluorescence 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 such that, with 580 nm excitation, the irradiation energy near the center of the field of view was 900 W/ ^cm² . The exposure time during image acquisition was arbitrarily set (e.g., 4000 μsec) to prevent saturation of image brightness. The number of bright spots (PID score) of SK-BR-3 cells was calculated as the average value of 1000 cells measured using the ImageJ FindMaxima method based 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 spots per cell (PID score) when the fluorescent nanoparticles of Examples 1 and 2 and Comparative Examples 1 to 3 were used.

[Regarding 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, these reactions were 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 due to the low temperature reaction, and therefore retained high activity.

In Example 2, amino groups were introduced to the fluorescent nanoparticles without formic acid treatment, and then the nanoparticles were washed using a cleaning solution containing gradually increasing amounts of organic solvent to remove water. Furthermore, 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 achieved higher values in each evaluation than Example 1 and Comparative Examples 1 to 3.

[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 of 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.

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

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.

Specifically, for example, as shown in Table 1 above, when the performance of the fluorescent nanoparticles of Comparative Example 3 and Example 2 was evaluated using 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 significant difference.

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 64,089 and 130,599, respectively, a large difference, which corresponds to PID scores of 451.7 and 854.8.

In other words, the BCA method is unable to correctly evaluate active streptavidin, whereas the biotin plate method is able to correctly evaluate active streptavidin.

This application claims priority from Japanese Patent Application No. 2020-049733, filed March 19, 2020. The entire contents of the specification and drawings of that application are incorporated herein by reference.

The fluorescent nanoparticles of this embodiment are highly sensitive and therefore useful for fluorescent imaging, etc.

Claims (3)

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/mm2 over an area of 28.3 ^{mm2 ,} and the mixture is 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. 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. 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 .