WO2021186813A1

WO2021186813A1 - Fluorescent nanoparticles and method for manufacturing fluorescent nanoparticles

Info

Publication number: WO2021186813A1
Application number: PCT/JP2020/046420
Authority: WO
Inventors: 義一栗原; 祐輝三宅; 中山　慎; 治▲高▼ 李
Original assignee: コニカミノルタ株式会社
Priority date: 2020-03-19
Filing date: 2020-12-11
Publication date: 2021-09-23
Also published as: JPWO2021186813A1

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

Fluorescent nanoparticles and methods for producing fluorescent nanoparticles

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

In pathological diagnosis, immunohistochemistry (IHC) and in situ hybridization (ISH) are used to perform staining targeting molecules to confirm the expression of molecular information in specimens, resulting in functional abnormalities such as abnormal gene and protein expression. Observations have been made to diagnose. For immunostaining, for example, a dye staining method using an enzyme (DAB staining or the like) is used.

However, staining with an enzyme label such as DAB staining has a problem that it is difficult to estimate the actual amount of antigen or the like from the staining concentration because the staining concentration is greatly affected by environmental conditions such as temperature and time. Therefore, in immunological observation in pathological diagnosis, a fluorescent labeling method using a fluorescent label is also performed instead of staining with an enzyme label.

This method is characterized by being superior in quantitativeness compared to DAB staining. The fluorescent labeling method measures the amount of antigen by observing the target antigen by staining it with an antibody modified with a fluorescent dye.

Conventionally, a dyeing 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 used for fluorescent labeling. For example, Patent Document 1 discloses fluorescent nanoparticles to which streptavidin is bound.

Special Table 2008-543982

Streptavidin is bound to the surface of the fluorescent nanoparticles described in Patent Document 1 above. As a result of studies by the present inventors, it has been found that the sensitivity may be low when the target substance (target substance bound to the detection target) is stained with the fluorescent nanoparticles.

The present inventors have diligently investigated the reason, and in the fluorescent nanoparticles described in Patent Document 1, streptavidin (target substance recognizing substance) supported on the surface thereof has lost its activity, and biotin (target substance) has been used. I thought it was because there were cases where it could not be combined.

The present invention has been made in view of the above circumstances, and the target substance recognizing substance has high activity in the fluorescent nanoparticles on which the target substance recognizing substance is supported on the surface, and the target substance can be measured with high sensitivity. It is an object of the present invention to provide fluorescent nanoparticles that can be produced. Another object of the present invention is to provide a method for producing the fluorescent nanoparticles.

The fluorescent nanoparticles according to an embodiment of the present invention are fluorescent nanoparticles in which a target substance recognizing substance that binds to a target substance is supported on the surface, and is a 100 μL liquid containing the fluorescent nanoparticles at a concentration of 100 pmol / L. and the fluorescent nano 0.1 pmol / mm the target substance over an area of 28.3 mm ² at a density of ² into contact with a substrate immobilized, binding to the target substance when allowed to stir 1 hour at 4 ° C. The proportion of particles is 1.5% or more.

Further, the method for producing fluorescent nanoparticles according to an embodiment of the present invention includes a step of preparing fluorescent nanoparticles into which a maleimide group has been introduced, a step of introducing a thiol group into a target substance-labeled molecule, and the fluorescent nanoparticles. The step of reacting the maleimide group introduced in the above with the thiol group introduced into the target substance-labeled molecule, the step of introducing the thiol group, and the step of reacting the maleimide group with the thiol group. Is a method for producing fluorescent nanoparticles in which a target substance recognizing substance that binds to a target substance is supported on the surface, which is carried out at 0 ° C. to 8 ° C.

According to the present invention, among fluorescent nanoparticles on which a target substance recognizing substance is supported on a surface, the target substance recognizing substance has high activity, and it is possible to provide fluorescent nanoparticles capable of measuring the target substance with high sensitivity. Can be done. The present invention can also provide a method for producing the fluorescent nanoparticles.

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

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

(Nanoparticles)
The fluorescent nanoparticles according to the present embodiment include nanoparticles as a mother body.

The following thermoplastic resin or thermosetting resin can be used as the resin constituting the nanoparticles. As the thermoplastic resin, for example, polystyrene, polyacrylonitrile, polyfuran, or a similar resin can be preferably used. As the thermosetting resin, for example, polyxylene, polylactic acid, glycidyl methacrylate, melamine resin, polyurea, polybenzoguanamine, polyamide, phenol resin, polysaccharide or similar resin can be preferably used. Thermosetting resins, particularly melamine resins, are preferable in that elution of the dye contained in the nanoparticles can be suppressed even by treatments such as dehydration, permeation, and encapsulation using an organic solvent such as xylene.

It is preferable that the nanoparticles are provided with a functional group for at least directly or indirectly binding the target substance recognizing substance to the surface. As such a functional group, the same functional group as in the case of binding various target substances to each other in the technical field to which the present invention belongs can be used, but for example, an epoxy group or an amino group is preferable.

The method for preparing the nanoparticles having a functional group is not particularly limited, but for example, a predetermined functional group is previously side-chained as a monomer for synthesizing a thermoplastic resin or a thermosetting resin constituting the nanoparticles. After (co) polymerizing the (co) monomer contained in the resin or synthesizing a thermoplastic resin or a thermosetting resin, the functional groups of the resin monomer units constituting the resin are treated with a reagent to obtain the predetermined functional groups. A method of conversion can be used.

In the example of the embodiment in which nanoparticles are produced using a thermoplastic resin, polystyrene resin nanoparticles having an epoxy group on the surface are produced by copolymerizing glycidyl methacrylate with styrene as a monomer. Or an embodiment in which styrene carboxylic acid or styrene sulfonic acid is copolymerized with styrene to produce polystyrene-based resin nanoparticles having carboxylic acid or styrene acid on the surface, or amino sulfonic acid is copolymerized with styrene. An embodiment of producing polystyrene-based resin nanoparticles having an amino group on the surface thereof is included. The epoxy group contained in the glycidyl methacrylate can also be converted into an amino group by a predetermined treatment.

On the other hand, in the case of producing thermosetting dye resin particles, there is an embodiment in which nanoparticles of a melamine resin are produced by copolymerizing using a melamine resin raw material (for example, MX035 manufactured by Sanwa Chemical Co., Ltd.) as a monomer. Be done.

(Fluorescent dye)
The fluorescent nanoparticles according to the present embodiment include a fluorescent dye.

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

Examples of the fluorescent dye to be encapsulated in the nanoparticles or fixed to the surface of the nanoparticles when forming nanoparticles while incorporating the fluorescent dye by polymerizing the monomers constituting the resin include the following rhodamine-based dye molecules and BODIPY-based. Dye molecules, squarylium-based dye molecules, aromatic hydrocarbon-based dye molecules, or combinations thereof can be used.

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

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 are included.

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. BODIPY 650/665 (manufactured by Invitrogen) and derivatives thereof are included.

Examples of squalylium-based dye molecules include SRfluor 680-Carboxylate, 1,3-Bis [4- (dimethylamino) -2-hydroxyphenyl] -2,4-dihydroxycyclobutenediylium dihydroxide, bis1,3-. ) Phenyl] -2,4-dihydroxycyclobutenediylium dihydroxide, bis, 2- (4- (Diethylamino) -2-hydroxyphenyl) -4- (4- (diethylminio) -2-hydroxylicylic -1-enlate, 2- (4- (Dibutylamino) -2-hydroxyphenyl) -4- (4- (dibutylimino) -2-hydroxycyclohexa-2,5-dienyllide) -3-oxyclobut-1-enlate, 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-tetramhydro-1H-pyrido [3,2,1-ij] quinolinium-9 (5H) -ylidene) -3-oxocyclobut-1-enlate, Derivatives thereof and the like are included.

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-teracarbonacid dimide, N, N'-Bis (2,6-diisotropyperylene) -1,6,7,12-teraphenoxyperylene-3,4: 9,10-terracarboxdimide, 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-terracarboxyldi-midide, 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 , 2,10-Bis (3-aminopropoxy) dibenzo [a, j] perylene-8,16-dione, 3,3'-[(8,16-Dihydro-8,16-dioxodibenzo [a, j] perylene- 2,10-diyl) perylene] dipropylamine, 17-BIS (Octyloxy) Anthra [9,1,2-cde-] Benzo [RST] Perylene-5-10-Dione, Octadecanoicacid, 5,10-dihydro -Dioxoanthra [9,1,2-cde] benzo [rst] pentaphene-16,17-diylester, Dihydroxydibenzanthrone, Benzenesulphonic acid, 4,4', 4'', 4'''-[[2,9-bis [2,9-bis [2,9-bis [2,9-bis 2,6-bis (1-methylylene) perylene] -1,2,3,8,9,10-hexahydro-1,3,8,10-terraoxoanthra [2,1,9-def: 6,5,10 -D'e'f'] diisoquinoline-5,6,12,13-tetrayl] tetracis (oxy)] tet rakes-, Benzeneethanaminium, 4,4', 4'', 4'''-[[2,9-bis [2,6-bis (1-methylyl) phenyl] -1,2,3,8,9, 10-hexahydro-1,3,8,10-tetraokistra [2,1,9-def: 6,5,10-d'e'f'] diisoquinoline-5,6,12,13-tetrayl] tetracis (oxy) )] Tetrakis [N, N, N-trimethyl-], derivatives thereof and the like are included.

(Target substance recognition substance)
A target substance recognizing substance is supported on the surface of the fluorescent nanoparticles according to the present embodiment.

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

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

Examples of the combination of the target substance and the target substance recognizing substance that specifically binds to the target substance include a combination of biotin-avidin, biotin-streptavidin, and biotin-neutravidin. To specifically bind, in response to such a combination, it is possible to interpret as a generic term in the art of the present invention, association constant (K _A) or dissociation constant which is the inverse ( It can also be defined by _KD).

That is, the target substance recognition substance to be used in the present invention, the binding constant between a target substance to be dyed object (K _A) is preferably in the range of ^{1 × 10 5 ~ 1 × 10} 12. If binding constants (K _A) is within the range, the target substance can be handled as a target substance recognition substance that specifically binds to a target substance to be dyed object.

(Average particle size)
The average particle size of the fluorescent nanoparticles is preferably 30 to 300 nm, more preferably 40 nm to 200 nm, from the viewpoint that bright spots can be preferably observed even with a general-purpose fluorescence microscope. When the average particle size exceeds 300 nm, the number of bright spots per cell decreases during observation after staining, making it difficult to observe bright spots. Conversely, when the average particle size is less than 30 nm, per cell. This is because the number of bright spots increases and it becomes difficult to observe the bright spots. The average particle size can be taken as the average value by measuring the major axis of each particle (100 or more) shown in the image taken by the scanning electron microscope.

(Ratio of fluorescent nanoparticles bound to the target substance)
The fluorescent nanoparticles according to the present embodiment have a relatively high rate of binding to the target substance because the target substance recognizing substance supported on the surface thereof has high activity.

Specifically, 100 pmol / concentration of L to 100μL of liquid containing fluorescent nanoparticles, 4 is brought into contact with the substrate target substance is immobilized over an area of 28.3 mm ² at a density of 0.1 pmol / mm ² ° C. The proportion of fluorescent nanoparticles bound to the target substance on the substrate when stirred for 1 hour is 1.5% or more. The ratio of the fluorescent nanoparticles bound to the target substance on the substrate is determined by bringing the fluorescent nanoparticles on the surface of the target substance recognition substance into contact with the substrate on which the target substance is immobilized. It can be evaluated by.

The target substance recognition substance supported on the fluorescent nanoparticles may be active or inactive. That is, the target substance recognition substance may or may not have the ability to bind to the target substance. In the above evaluation, the ratio of the fluorescent nanoparticles bound to the target substance is 1.5% or more, which indicates that there are many active target substance recognizing substances on the surface of the fluorescent nanoparticles.

By improving the production process of the fluorescent nanoparticles according to the present embodiment, the target substance recognizing substance has high activity and the rate of recognizing the target substance is improved. This makes it possible to perform highly sensitive measurements. Also. By setting the degree of activity of the target substance recognition substance within a certain range, the detection sensitivity is improved and the variation in detection is reduced (the detection accuracy is improved).

In the conventional BCA method, only the amount of the target substance recognizing substance bound to the surface of the fluorescent nanoparticles is measured as the amount of protein, but in the above method, the target substance recognizing substance supported on the surface of the fluorescent nanoparticles is recognized. The activity of a substance can be measured.

The above ratio may be 1.5% or more, but from the viewpoint of measuring with higher sensitivity, it is more preferably 2% or more, further preferably 2.5% or more, and 5% or more. Is more preferable, and 8% or more is further preferable. The upper limit of the above ratio is preferably 10% or less from the viewpoint of suppressing non-specific binding.

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

[Manufacturing method of fluorescent nanoparticles]
The method for producing fluorescent nanoparticles according to the present embodiment includes a step of preparing fluorescent nanoparticles into which a maleimide group has been introduced, a step of introducing a thiol group into a target substance recognizing substance, and a step of introducing the fluorescent nanoparticles into the fluorescent nanoparticles. It comprises a step of reacting a maleimide group with a thiol group introduced into the target substance-labeled molecule. The step of introducing the thiol group and the step of reacting the maleimide group with the thiol group are carried out at 0 ° C. to 8 ° C. Hereinafter, each step will be described.

(Step of preparing fluorescent nanoparticles with maleimide groups introduced)
In the step of preparing the maleimide group-introduced fluorescent nanoparticles, the maleimide group-introduced fluorescent nanoparticles may be obtained in advance, or the maleimide group is introduced into the fluorescent nanoparticles to introduce the maleimide group. Nanoparticles may be made.

When a maleimide group is introduced into the fluorescent nanoparticles, it can be carried out by reacting the maleimide group-introducing reagent (for example, NHS-PEG12-maleimide) with the fluorescent nanoparticles into which the amino group has been introduced as shown in FIG. 1A. preferable. Here, in general, in order to introduce an amino group, fluorescent nanoparticles are first treated with formic acid as shown in FIG. 1A, a carboxyl group is added to the surface thereof, and then an amino group introducing reagent (for example, BAEE) is used. It is considered preferable to introduce an amino group by adding it. However, in one embodiment of the present invention, an amino group-introducing reagent may be added to the fluorescent nanoparticles that have not been treated with formic acid to introduce an amino group. By omitting the formic acid treatment, although the reason is not clear, it is possible to obtain fluorescent nanoparticles having higher activity of the target substance recognizing substance and high sensitivity.

In addition, by omitting the formic acid treatment, the presence of unreacted carboxy groups (carboxy groups into which no amino group has been introduced) suppresses the surface of the fluorescent nanoparticles from being negatively charged, and the surface charge is suppressed. Is expected to be tilted to the positive side as compared with the conventional case, and the accessibility to the probe of RNAscape having a negative charge derived from nucleic acid is expected to be improved.

Further, as shown in FIG. 1A, after the step of introducing an amino group into the fluorescent nanoparticles and before the step of reacting the amino group with an N-hydroxysuccinimide group, the fluorescent nano having an amino group introduced. It is preferable to carry out a step of washing the particles with an organic solvent. This washing step is preferably performed so as to remove water using an organic solvent. Specifically, it is preferable to perform cleaning a plurality of times using a cleaning solution in which the amount of the organic solvent in the cleaning solution is gradually increased. It is considered that the deactivation of the maleimide group-introducing reagent can be suppressed by washing by removing water in this way.

The above amino group introduction reagent is not particularly limited as long as an amino group can be introduced. Examples of amino group-introducing reagents include 1,2-Bis (2-aminoethoxy) ethane (BAEE), 1,4-Diaminobutane, 1,10-Diaminodekane, 1,12-Diaminododekane, 1,7-Diaminoheptane, 1, 6-Diaminohexane, 1,8-Diaminooctane, 1,5-Diaminopenentane, 1,3-Diaminopropane, 1,11-Diaminodykane, Ethylenediamine, 2,2'-Oxybis (ethylamine), 1,2'-Oxybis (ethylamine), 1, 9-trioxanedecane and the like are included.

Next, as shown in FIG. 1A, the amino group introduced into the fluorescent nanoparticles as described above includes, for example, the N-hydroxysuccinimide group of a compound containing a maleimide group and an N-hydroxysuccinimide group. Introduces a maleimide group into the fluorescent nanoparticles by reacting.

The maleimide group-introducing reagent is not particularly limited as long as the maleimide group can be introduced. 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 and the like.

(Step of introducing a thiol group into a target substance recognition substance)
The step of introducing a thiol group into the target substance recognizing substance is performed, for example, by reacting the amino group of the target substance recognizing substance with a thiol group introducing reagent (for example, a trout reagent), as shown in FIG. 1B. The step of introducing a thiol group into the target substance recognizing substance is preferably carried out at 0 to 8 ° C., and is preferably carried out at 0 to 4 ° C. from the viewpoint of ensuring that the target substance recognizing substance has high activity. More preferably, it is carried out at 2 to 4 ° C.

The thiol group-introducing reagent is not particularly limited as long as a thiol group can be introduced. Examples of thiol group-introducing reagents include trout reagent (2-Iminothiolane), SPDP (N- {6- [3- (2-Pyridyldithio) PEGylamide] 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 -Acetylthioactate) and the like are included.

(Step of reacting maleimide group and thiol group)
The step of reacting the maleimide group with the thiol group is carried out, for example, by reacting the fluorescent nanoparticles into which the maleimide group has been introduced with the target substance recognizing substance into which the thiol group has been introduced, as shown in FIG. 1C. (Fig. 1C). The step of reacting the maleimide group with the thiol group is preferably carried out at 0 to 8 ° C., and more preferably at 0 to 4 ° C. from the viewpoint of ensuring that the target substance recognizing substance has high activity. Preferably, it is more preferably carried out at 2-4 ° C.

(effect)
According to the fluorescent nanoparticles according to the embodiment of the present invention, the activity of the target substance recognition substance supported on the surface thereof is high. Moreover, the degree of activity can be kept in a certain range. Further, according to the method for producing fluorescent nanoparticles according to the embodiment of the present invention, the activity of the target substance recognizing substance supported on the surface thereof can be increased. Thereby, according to the fluorescent nanoparticles according to the embodiment of the present invention, it is possible to measure the improvement of the detection sensitivity and the reduction of the variation in the detection result.

Hereinafter, the invention according to the present embodiment will be described in detail with reference to the examples, but the invention according to the present embodiment is not limited to these examples.

[Preparation of fluorescent nanoparticles]
(Example 1: Low temperature reaction)
<Synthesis of fluorescent nanoparticles>
(1) Raw Material Preparation / Mixing Step 2.5 mg of the fluorescent dye sulforhodamine 101 (“Sulforhodamine 101”, Sigma-Aldrich, TexasRed dye) was dissolved in 22.5 mL of pure water. The solution was stirred with a hot stirrer for 20 minutes while maintaining at 70 ° C. Next, 1.5 g of the water-soluble melamine resin "Nicarac MX-035" (manufactured by Nippon Carbide Industries, Ltd.) was added to the solution, and the mixture was further heated and stirred for 5 minutes under the same conditions.

(2) Polymerization step (formic acid treatment)
100 μL of formic acid was added to the heated solution, and the mixture was further heated and stirred for 20 minutes while maintaining the solution temperature at 60 ° C. Then, the solution was left to cool to room temperature.

The cooled solution was transferred to a centrifuge tube and centrifuged at 12000 rpm for 20 minutes with a centrifuge. The supernatant of the centrifuged solution was removed and only the sediment was recovered. The recovered sediment was washed with ethanol and then washed with water in the same manner.

(3) Amino group introduction step As shown below, an amino group was introduced on the surface of the fluorescent nanoparticles. First, 1 mL of melamine-based particles (fluorescent nanoparticles) adjusted to 1 nmol / L after the washing was mixed with 20 μL of 1,2-Bis (2-aminoethoxy) ethane (BAEE) and reacted at a temperature of 70 ° C. for 1 hour. .. In this way, the amino group was introduced into the fluorescent nanoparticles.

(4) Washing step This reaction solution was centrifuged at 10000 G for 20 minutes to remove the supernatant and collect only the sediment. Then, 1 mL of water was added, and centrifugation was performed again at 10000 G for 20 minutes to remove the supernatant and collect only the sediment. This operation was performed two more times, and a total of three water washes were performed. Next, 1 mL of THF was added to the sediment, and centrifugation was performed again at 10000 G for 20 minutes to remove the supernatant and recover the sediment.

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

(5) Modification of Maleimide Group of Fluorescent Nanoparticles NHS-PEG12-maleimide was mixed with a solution of these fluorescent nanoparticles so as to have a final concentration of 10 mmol / L, and reacted at room temperature for 1 hour. That is, the maleimide group was introduced into the fluorescent nanoparticles as described above.

This reaction solution was centrifuged at 10000 G for 20 minutes to remove the supernatant and collect only the sediment. Then, PBS containing 2 mmol / L of EDTA was added to disperse the precipitate. Then, again, centrifugation was performed at 10000 G for 20 minutes to remove the supernatant, and only the sediment was recovered. The fluorescent nanoparticles were washed three more times by a series of operations from the dispersion treatment of the sediment to the centrifugation. Then, fluorescent nanoparticles in which the maleimide group was present on the particle surface and contained a Texas Red dye were obtained. Further, when the average particle size of the melamine-based particles was measured by the above-mentioned method using a scanning electron microscope, the average particle size was 150 nm.

(6) Modification of Streptavidin to Thiol Group On the other hand, streptavidin capable of binding to fluorescent nanoparticles into which the maleimide group was introduced was prepared as follows.

First, 40 μL of streptavidin (manufactured by Wako Pure Chemical Industries, Ltd.) adjusted to 1 mg / mL was reacted with 70 μL of a trout reagent (manufactured by Thermo Fisher) adjusted to 64 mg / mL at 4 ° C. for 1 hour. That is, a thiol group protected against the amino group of streptavidin was introduced.

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

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

(7) Bonding reaction of maleimide group and thiol group The total amount of this streptavidin and 1 mL of the fluorescent nanoparticles adjusted to 1 nmol / L above were mixed with PBS containing 2 mmol / L of EDTA, and the conditions were 1 hour at 4 ° C. Then, the reaction of binding both molecules was carried out. Then, the mixture was centrifuged and washed with PBS containing 2 mmol / L of EDTA, and only the fluorescent nanoparticles to which streptavidin was bound were recovered to obtain the fluorescent nanoparticles of Example 1.

(Example 2: Formic acid treatment omitted, water removal washing, low temperature reaction)
In Example 2, in the above-mentioned (2) polymerization step, 100 μL of formic acid was not added and the mixture was heated and stirred.

Further, in Example 2, the above (4) cleaning step was changed as follows. That is, the reaction solution was centrifuged at 10000 G for 20 minutes, the supernatant was removed to collect only the sediment, then 1 mL of water was added, and the reaction solution was centrifuged again at 10000 G for 20 minutes to remove the supernatant. Only the sediment was collected. Next, 1 mL of a washing solution having a volume ratio of water and THF of 1: 1 was added, and centrifugation was performed again at 10000 G for 20 minutes to remove the supernatant and collect only the sediment. Next, the volume ratio of water and THF was set to 1: 3, and only the sediment was recovered in the same manner. Next, the sediment was recovered in the same manner with water and THF set to a volume ratio of 0: 4. In this way, washing was performed so as to remove water using a washing liquid in which the amount of THF was gradually increased. Except for this, 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 was set to room temperature in the above-mentioned (6) Streptavidin thiol group modification. Further, in the above-mentioned (7) bonding reaction of a maleimide group and a thiol group, the reaction temperature was set to room temperature. Fluorescent nanoparticles of Comparative Example 1 were obtained in the same manner as in Example 1 except for the above.

(Comparative Example 2: Formic acid treatment omitted, room temperature reaction)
In Comparative Example 2, in the polymerization step (2) described above, 100 μL of formic acid was not added, and the solution was further heated and stirred for 20 minutes while maintaining the solution temperature at 60 ° C. Further, in the above-mentioned (6) thiol group modification of streptavidin, the reaction temperature was set to room temperature. Further, in the above-mentioned (7) bonding reaction of a maleimide group and a thiol group, the reaction temperature was set to room temperature. Fluorescent nanoparticles of Comparative Example 2 were obtained in the same manner as in Example 1 except for the above.

(Comparative Example 3: Water removal washing, room temperature reaction)
In Comparative Example 3, the above (4) cleaning step was changed as follows. That is, the reaction solution was centrifuged at 10000 G for 20 minutes, the supernatant was removed to collect only the sediment, then 1 mL of water was added, and the reaction solution was centrifuged again at 10000 G for 20 minutes to remove the supernatant. Only the sediment was collected. Next, 1 mL of a washing solution having a volume ratio of water and THF of 1: 1 was added, and centrifugation was performed again at 10000 G for 20 minutes to remove the supernatant and collect only the sediment. Next, the volume ratio of water and THF was set to 2: 2 and only the sediment was recovered in the same manner. Next, the volume ratio of water and THF was set to 1: 3, and only the sediment was recovered in the same manner. Next, 1 mL of THF was added, and a precipitate was obtained in the same manner. In this way, washing was performed so as to remove water using a washing liquid in which the amount of THF was gradually increased.

Further, in the above-mentioned (6) Streptavidin thiol group modification, the reaction temperature was set to room temperature. Further, in the above-mentioned (7) bonding reaction of a maleimide group and a thiol group, the reaction temperature was set to room temperature. Fluorescent nanoparticles of Comparative Example 3 were obtained in the same manner as in Example 1 except for the above.

[Evaluation of fluorescent nanoparticles]
Each fluorescent nanoparticle 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 nanoparticles was determined by the BCA method using "Micro BCA Protein Assay kit" of Pierce. Calculated as the amount of protein.

Specifically, fluorescent nanoparticles before streptavidin modification and fluorescent nanoparticles after streptavidin modification were prepared, and the amount of protein was measured by the BCA method for each. In the measurement, 150 μL of PBS dispersion of fluorescent nanoparticles bound with streptavidin (particle concentration 0.50 nmol / L) and 150 μL of PBS dispersion of fluorescent nanoparticles not bound with streptavidin (particle concentration 0.50 nmol / L). The bicinconic acid (BCA) solution and the copper sulfate solution included in the kit were mixed and added to each of the above, and the absorbance at 562 nm was measured. The absorbance of the fluorescent nanoparticles before streptavidin modification is subtracted from the absorbance of the fluorescent nanoparticles before streptavidin modification as the background, and the absorbance is compared with the standard curve to determine the net amount of protein in the aqueous solution (concentration of streptavidin). It was determined.

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

(Performance evaluation of fluorescent nanoparticles by biotin plate method)
Amount of streptavidin having activity for each of the fluorescent nanoparticles of Examples 1 to 4 and Comparative Example using a commercially available 96-well plate (manufactured by G-Biosciences, model number 786-762) in which biotin was immobilized in advance. Was measured. In this well plate (inner diameter of the well is about 6 mm), biotin molecules are immobilized at a density of ^{0.1 pmol / mm 2} over an area ^{of 28.3 mm 2.}

Specifically, after washing each well of the well plate with PBS containing 0.05% Tween 20 (pH 7.5), 200 μL of PBS containing 1% BSA was dispensed into each well to prepare the plate. It was allowed to stand overnight in an environment of 4 ° C. Subsequently, all the BSA-containing PBS in the well was aspirated, 100 μL of a PBS dispersion of streptavidin-modified fluorescent nanoparticles (particle concentration 0.10 nmol / L) was dispensed, and a plate shaker installed in an environment of 4 ° C. A plate was placed on (Biosan, model number PST-60HL) and left for 1 hour with stirring at 500 rpm. Further, the entire amount of the PBS dispersion of fluorescent nanoparticles was aspirated, 100 μL of PBS was dispensed into the wells, and the fluorescence value at 595 nm with respect to light irradiation at a wavelength of 531 nm was measured with a plate reader (manufactured by PerkinElmer, model number EnVision2104). .. In the fluorescence measurement, the average value for 5 times was repeatedly used for each well. The measurement results are shown in the plate reader fluorescence values in Table 1 below.

Further, from the above plate reader fluorescence value, the proportion of fluorescent nanoparticles bound to the target substance was determined based on the calibration curve. The calibration curve was prepared on a well plate prepared in the same manner as above, and a PBS dispersion of fluorescent nanoparticles modified with streptavidin (particle concentration 0 pmol / L, 1.56 pmol / L, 3.12 pmol / L, 6.25 pmol / L). , 12.5 pmol / L) was dispensed, stirred in the same manner as above, and then the fluorescence value at 595 nm with respect to light irradiation at a wavelength of 531 nm was measured with a plate reader (manufactured by PerkinElmer, model number EnVision2104). .. In the fluorescence measurement, the average value for 5 times was repeatedly used for each well. Table 1 shows the ratio of fluorescent nanoparticles bound to the target substance determined based on the calibration curve.

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

Specifically, immunostaining was performed according to the following procedure.

≪IHC staining using staining reagents≫
An antibody reagent (staining reagent for pathological diagnosis) having fluorescent nanoparticles and a biotinylated secondary antibody prepared as follows was prepared, and immunostaining was performed using this staining reagent.

<Preparation of biotin-modified secondary antibody>
First, 50 μg of anti-rabbit IgG antibody was dissolved in a 50 mmol / L Tris-HCl solution (pH 7.5). A DTT (dithiothretrol) solution was mixed with the solution so as to have a final concentration of 3 mmol / L. Then, the solution was reacted at 37 ° C. for 30 minutes. Then, a DTT-reduced secondary antibody was 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. On the other hand, a linker reagent "(+)-Biotin-PEG _6- NH-Mal" (manufactured by PurePEG, product number 246106-250) having a spacer length of 30 angstroms was used in 0.4 mmol / DMSO. It was adjusted to be L. 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 by subjecting it to a desalting column "Zeba Spin Desalting Colors". The absorption of the desalted reaction solution at a wavelength of 300 nm was measured with a spectroscopic altimeter (“F-7000” manufactured by Hitachi) to calculate the amount of protein contained in the reaction solution. The reaction solution was adjusted to 250 μg / mL with a 50 mmol / L Tris solution, and the solution was used as a solution of the biotinylated secondary antibody.

《Fluorescent immunostaining method》
(1) Deparaffinizing Step Using the above biotinylated secondary antibody and the like, immunostaining and morphological observation staining of human breast cancer-derived cultured cells SK-BR-3 (HER2 (3+)) were performed as follows. As a slide for staining, a HER2 IHC positive control slide (manufactured by Pasoroji Research Institute, hereinafter referred to as a control slide) was used. The tissue array slides were deparaffinized.

(2) Activation treatment step The control slide was deparaffinized and then washed by replacing it with water. The washed control slide was autoclaved in 10 mmol / L citrate buffer (pH 6.0) at 121 ° C. for 15 minutes to activate the antigen. The control slides after the activation treatment were washed with PBS, and the washed control slides were blocked with PBS containing 1% BSA for 1 hour.

(3) Immunostaining treatment step (3-1) Primary antibody reaction A solution of "anti-HER2 rabbit monoclonal antibody (4B5)" manufactured by Roche was reacted overnight at 4 ° C. with the above-mentioned blocking-treated control slide. rice field.
(3-2) Secondary antibody reaction The control slide subjected to the primary antibody reaction was washed with PBS and then reacted with the biotinylated secondary antibody diluted to 2 μg / mL with PBS containing 1% BSA for 30 minutes at room temperature. rice field.
(3-3)
The above-mentioned fluorescent nanoparticles diluted to 0.02 nmol / L with PBS containing 1% BSA were added to the control slide subjected to the secondary antibody reaction at room temperature in a neutral pH environment (pH 6.9 to 7.4). The reaction was carried out for 3 hours under the conditions of. The control slides after the reaction were washed with PBS.

(4) Morphological observation staining step After immunostaining, hematoxylin / eosin staining (HE staining) was performed. The immunostained sections were stained with Meyer hematoxylin solution for 5 minutes to perform hematoxylin staining. Then, the control slide was washed with running water at 45 ° C. for 3 minutes. Next, eosin staining was performed by staining with a 1% eosin solution for 5 minutes.

(5) Fixation Treatment Step The tissue sections that had completed the immunostaining step and the morphological observation staining step were washed and dehydrated by immersing them in pure ethanol for 5 minutes four times. Subsequently, the operation of immersing in xylene for 5 minutes was performed four times to perform transparency. Finally, a tissue section was encapsulated using an encapsulant (“Enteran New” manufactured by Merck & Co., Inc.) to prepare a control slide slide of a sample for observation.

(6) Observation / Measurement Step The control slide after the immobilization treatment step was irradiated with a predetermined excitation light to emit fluorescence. The control slide in that state was observed and imaged with a fluorescence microscope (“BX-53” manufactured by Olympus Corporation) and a digital camera for a microscope (“DP73” manufactured by Olympus Corporation). The wavelength of the excitation light was set to 575 to 600 nm by passing it through an optical filter. The wavelength of fluorescence to be observed was also set to 612 to 692 nm by passing through an optical filter. The conditions of the excitation wavelength at the time of microscopic observation and image acquisition were such that ^{the irradiation energy near the center of the visual field was 900 W / cm 2 when excited at 580 nm.} The exposure time at the time of image acquisition was arbitrarily set (for example, set to 4000 μsec) so that the brightness of the image was not saturated, and the image was taken. The number of bright spots (PID score) of SK-BR-3 was taken as the average value of 1000 cells measured by the ImageJ FindMaxima method based on the image taken at 400 times. The observation results are shown in FIG. 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 shows the measurement results of the number of bright spots (PID score) per cell when the fluorescent nanoparticles of Examples 1 and 2 and Comparative Examples 1 to 3 were used.

[About Table 1]
In Examples 1 and 2, the step of introducing the thiol group and the step of reacting the thiol group with the maleimide group were carried out at a low temperature, 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 the target substance) than in Comparative Example 1. It is considered that this is because streptavidin was not inactivated and had high activity in Examples 1 and 2 because the reaction was carried out at a low temperature.

In Example 2, an amino group was introduced into the fluorescent nanoparticles without treatment with formic acid, and the washing was carried out by using a washing liquid in which the amount of the organic solvent was gradually increased in the washing step so as to remove water, and further thiol. The step of introducing the group and the step of reacting the thiol group with the maleimide group were carried out at a low temperature. As a result, in Example 2, a higher value was obtained in each evaluation than in any of Example 1 and Comparative Examples 1 to 3.

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

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

Comparing FIG. 3A and FIG. 3B, FIG. 3B has a higher correlation. That is, it can be seen that the biotin plate method can correctly evaluate the performance of the 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 the fluorescent nanoparticles of Example 2 were evaluated by the BCA method, the values were 321.9 and 320, respectively. It is 8, and there is not much difference. However, when immunostaining is actually performed using the fluorescent nanoparticles of Comparative Examples 3 and 2, the PID scores are 451.7 and 854.8, which are significantly different.

On the other hand, when the performance of the fluorescent nanoparticles of Comparative Example 3 and the fluorescent nanoparticles of Example 2 were evaluated by the biotin plate method, the values were 64089 and 130599, respectively, and there was a large difference, which was the PID score. It corresponds to 451.7 and 854.8.

That is, it can be seen that the BCA method cannot correctly evaluate the active streptavidin, whereas the biotin plate method can correctly evaluate the active streptavidin.

This application claims priority based on Japanese Patent Application No. 2020-049733 filed on March 19, 2020. All the contents described in the application specification and drawings are incorporated in the specification of the present application.

The fluorescent nanoparticles according to this embodiment are useful for fluorescence imaging and the like because of their high sensitivity.

Claims

Fluorescent nanoparticles on which a target substance recognition substance that binds to a target substance is supported on the surface.
The 100μL of liquid containing the fluorescent nanoparticles at a concentration of 100 pmol / L, is brought into contact with the substrate on which the target substance is immobilized over an area of 28.3 mm 2 at a density of 0.1pmol / mm 2, 1 at 4 ° C. The proportion of the fluorescent nanoparticles bound to the target substance when agitated for a time is 1.5% or more.
Fluorescent nanoparticles.
The fluorescent nanoparticles according to claim 1, wherein the target substance recognizing substance is at least one selected from the group consisting of avidin, streptavidin and neutravidin.
The fluorescent nanoparticles according to claim 1 or 2, wherein the target substance is biotin.
A method for producing fluorescent nanoparticles in which a target substance recognition substance that binds to a target substance is supported on the surface.
The process of preparing fluorescent nanoparticles with maleimide groups introduced,
The step of introducing a thiol group into the target substance recognition substance and
A step of reacting the maleimide group introduced into the fluorescent nanoparticles with the thiol group introduced into the target substance recognizing substance, and
Have,
The step of introducing the thiol group and the step of reacting the maleimide group with the thiol group are carried out at 0 ° C. to 8 ° C.
Method for producing fluorescent nanoparticles.
The step of preparing the fluorescent nanoparticles into which the maleimide group has been introduced is
The process of introducing an amino group into fluorescent nanoparticles that have not been treated with formic acid,
It comprises a step of 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.
The method for producing fluorescent nanoparticles according to claim 4.
The step of preparing the fluorescent nanoparticles into which the maleimide group has been introduced is
After the step of introducing an amino group into the fluorescent nanoparticles and before the step of reacting the amino group with the N-hydroxysuccinimide group, the fluorescent nanoparticles introduced with the amino group are subjected to an organic solvent. Further having a step of washing and removing water,
The method for producing fluorescent nanoparticles according to claim 5.
The method for producing fluorescent nanoparticles according to any one of claims 4 to 6, wherein the target substance recognizing substance is at least one selected from the group consisting of avidin, streptavidin and neutravidin.