JP6687933B2 - Method for producing water-soluble near-infrared emitting nanoparticles - Google Patents

Description

  The present invention relates to water-soluble near-infrared light emitting nanoparticles using silicon nanoparticles (Si) particles and a fluorescent labeling material using the nanoparticles.

  Actively conducting academic research to elucidate the mechanism of life science and to link it to drug discovery screening by understanding when, where, and with which molecule a specific molecule functions in vivo. It is being appreciated. The most common method of observing biomolecules is a fluorescence imaging method in which a target molecule is fluorescently labeled and the dynamics and position of the molecule and the interaction with other molecules are examined under a fluorescence microscope. In molecular biology experiments, using human or animal tissues in test tubes or incubators to artificially create an environment similar to the body and observe biological reactions is called in-vitro imaging. . On the other hand, observing a biological reaction in a living body or in a cell using a small animal is called in-vivo imaging. In recent years, in-vivo imaging has attracted particular attention. In-vivo fluorescence imaging is a method of photoexciting a fluorescent label in the living body from the outside of the living body and guiding the luminescence from the fluorescent labeling agent to a photodetector installed outside the living body to perform imaging, so that cells in the living body are It is characteristic in that it can be observed non-invasively from the outside without damaging the living body.

  In in-vivo imaging, the excitation light and the emitted light must pass through the living body in order to take out the emitted light to the outside. Ultraviolet light, which is used as excitation light for many fluorescent materials, is not preferable because it is highly absorbed by the living body and hardly penetrates. Further, visible light of 400 to 700 nm is not preferable because hemoglobin and other biological constituents are largely absorbed. On the other hand, at a wavelength of 1000 nm or more, absorption of water becomes strong and the transmittance becomes low, which is not preferable. Therefore, the near infrared wavelength range of 700 to 1000 nm is a preferable wavelength range for in-vivo imaging. The wavelength range is a region called “spectral window of living body” where the transmittance of living body is specifically high. Therefore, in order to perform efficient in-vivo imaging, a fluorescent material that exhibits photoexcitation and emission in the near infrared wavelength region of 700 to 1000 nm is required.

Generally, organic dyes are the most widely used fluorescent labeling materials, but they have the following problems.
・ There are few types of dyes that emit near infrared light.
-Emission quantum yield is not as high as about 10 to 30% (demonstrated in Comparative Example 3).
-Since the Stokes shift between the excitation maximum and the emission maximum is short, there is no way to suppress the background rise due to the excitation light in the fluorescence imaging image. Therefore, it is necessary to increase the contrast of the imaging image by exciting at a wavelength as short as possible rather than at the excitation maximum. In that respect, the emission quantum yield cannot be said to be high at 10 to 30%.
-Photo-deterioration characteristic of organic substances when irradiated with excitation light, low durability due to photodecomposition, and fading due to deterioration are observed. This problem is difficult to solve as long as organic matter is used.

In order to fundamentally solve such a problem (particularly the fourth), it is desirable to replace the light emitting body with an inorganic material. Inorganic nanomaterials containing rare earth ions have been attracting attention as candidate substances. However, these also have the following problems.
・ There are few substances that photoexcite and emit light in the near infrared wavelength range.
-Emission quantum yield is not sufficient, 10% or less, and sensitivity is not sufficient. Here, when the excitation light intensity is increased to increase the detection sensitivity, the excitation light reflected on the unlabeled region in the imaging image lowers the contrast of the imaging image (Non-Patent Document 1).
-Strong excitation light causes phototoxicity problems that are invasive to biomolecules. This problem is the same even when an organic dye is used.

In order to solve the problems that occur in the above two types of phosphors, active research is being conducted on compound semiconductor quantum dot nanoparticles. Materials that can be used in the near-infrared wavelength range are cadmium tellurium, indium arsenide, and lead sulfide from the short wavelength side (700 nm to). These substances have the following characteristics.
・ Cadmium tellurium has excellent light resistance.
-Emission quantum yield of lead sulfide and cadmium tellurium is 40% or more.
・ The emission color can be tuned in the near infrared wavelength range. Furthermore, since the spectral wavelength position depends on the particle size, it is possible to provide a special observation method in which different intermolecular interactions can be observed in parallel in bioimaging.

However, although they have the following problems on the one hand, these problems cannot be changed because they are the basic physical properties of the substance.
-Since the Stokes shift between the excitation maximum and the emission maximum is short, there is no way to suppress the background rise due to the excitation light in the fluorescence imaging image. For this reason, it is necessary to increase the contrast of the imaging image by exciting at a wavelength as short as possible rather than at the excitation maximum. As a result, high emission quantum yield cannot be utilized.
-Many of the atoms that make up these compound semiconductors are restricted by environmental regulations. According to the “Environmental Standards for Water Pollution” of the Ministry of the Environment “Appendix 1 Environmental Standards for the Protection of Human Health” (Non-Patent Document 2), for example, the environmental standards for water pollution of cadmium, lead, and arsenic are: These are extremely small amounts of 0.003 mg / L, 0.01 mg / L, and 0.01 mg / L, respectively. Due to such environmental regulations, these substances cannot be stably used in the future.

  In order to solve the above-mentioned problems with the substances used or proposed so far, it is conceivable to use silicon as the fluorescent labeling material. A sufficiently large silicon crystal is known as a single element semiconductor having an indirect transition type band structure and has a band gap of about 1.14 eV at room temperature. Since it has an indirect transition type band structure, it is necessary to satisfy the law of conservation of momentum in order to cause radiative recombination of photoexcited carriers. In principle, it is impossible to emit fluorescence with high luminous efficiency because its energy is obtained from lattice vibration. However, when the size of the diamond-structured crystal is miniaturized into spherical particles smaller than 8 nm, photoluminescence characteristics having an emission quantum yield of 10,000 times or more as compared with a sufficiently large crystal are exhibited. If the emission quantum yield is generally 1% or more, it can be used as a fluorescent labeling material. Since silicon is known to have extremely lower cytotoxicity than nanoparticles of compound semiconductors, it is expected as a fluorescent labeling material used in the living body (Non-Patent Document 3).

  In order to use silicon nanoparticles as a fluorescent labeling agent in an aqueous system, they are solvated in water, a physiological salt solution, or a phosphate buffered saline solution, are stable for a long period of time, and have an emission quantum yield of 1 when excited by near infrared rays. % Near-infrared emission characteristics are required. However, silicon is oxidized when placed in an environment where oxygen is present. For example, when silicon having a particle diameter of 2 nm is left in the atmosphere, oxidation gradually progresses from the surface of the nanoparticles even at room temperature and becomes silicon oxide in about 2 weeks (demonstrated in Comparative Example 6).

  In order to solve the problem of oxidation and impart water solubility, a method of modifying the surface of nanoparticles with an appropriate organic monolayer has been reported. An unsaturated hydrocarbon molecule having a polar functional group at the end is generally used. Typical polar functional groups are a carboxyl group, an amino group, a sugar ring, a sulfone group, a hydroxyl group, a phosphone group, and an oligonucleotide, and they have high biocompatibility (Patent Documents 1 to 4). In such an unsaturated hydrocarbon molecule, a double bond (or a triple bond) at the terminal opposite to the polar group forms a carbon-silicon covalent bond with the silicon atom on the outermost surface of the nanoparticles through hydrosilylation. It is immobilized on the surface of nanoparticles as molecules. In terms of surface modification, thiol molecules used for surface modification of gold or platinum nanoparticles can also be used. However, in terms of suppressing oxidation, surface modification via a carbon-silicon covalent bond is preferable to sulfur-silicon intermolecular interaction, which is weak chemisorption.

  The emission wavelength of conventional water-soluble silicon nanoparticles is biased in the visible short wavelength region (400 to 500 nm). The visible short wavelength region is a wavelength region in which absorption of biological constituents is large, and thus it cannot be said that it is optimal for imaging not only in-vivo but also in-vitro environment. Regarding water-soluble silicon nanoparticles that can be used in the visible long-wavelength range (500 to 700 nm), the nanoparticles are terminated with a monolayer having a functional group of either a carboxyl group or a nucleotide (Non-Patent Document 4 (Review Paper). )). On the other hand, there are few reports on water-soluble silicon nanoparticles that emit near-infrared light. A few examples are shown below. First, it is known that the emission color can be continuously modulated in the emission wavelength range of 700 to 1100 nm by using silicon as nanoparticles (Non-Patent Document 5 (Review Paper)). In order to synthesize water-soluble silicon nanoparticles that emit light in the near infrared, a method of protecting with micelles constructed with amphipathic molecules has been reported (Non-Patent Document 6).

  However, even if the above-mentioned countermeasures were taken for silicon nanoparticles, the following problems could not be solved.

[Problem 1]
-The quantum yield of light emission in the near infrared wavelength region is 8% or less when excited at the excitation maximum. Since the excitation maximum of near-infrared emission is in the ultraviolet region in terms of electronic structure, the quantum yield of emission is further lowered when photoexcited in the visible long wavelength region which is usually used for imaging. Therefore, a large amount of photon energy was required for excitation, and it was difficult to perform imaging with high contrast.

[Problem 2]
-The full width at half maximum of the emission spectrum is 500 meV or more, which is larger than the full width at half maximum (300 meV or less) of the emission spectrum of the compound semiconductor quantum dot. It is preferable that the multicolor fluorescence imaging utilizing the emission tuning characteristics is performed by using an emission spectrum having a small full width at half maximum and high symmetry, and a full width at half maximum of 500 eV is not suitable.

[Problem 3]
-Since the emission peak of silicon nanoparticles does not depend on the emission wavelength and generally tails toward the long wavelength side, the symmetry is low. The emission maximum in the wavelength range of 700 to 1100 nm depends on the particle size, but the wavelength position at the end point of the emission spectrum is the same regardless of the particle size. The width of the bottom of the spectrum that appears in this way results in a reduction in the contrast property of multicolor fluorescence imaging. For that reason, there has been no example of achieving multicolor fluorescence imaging in the near infrared wavelength region so far. And such a characteristic spectral characteristic is a feature that appears when the indirect transition type semiconductor is made into nanoparticles, and is caused by the zero-phonon transition through the overlap of the electron and hole wave functions in the real space accompanying the miniaturization of crystal particles. It has been thought that.

  This will be described in more detail below. Since the bulk crystal of silicon has an indirect transition type band structure, a wave number selection rule is required for interband recombination of photoexcited carriers (electrons and holes). So in order to satisfy the law of conservation of momentum, the involvement of phonons is needed. However, if the bulk crystal is miniaturized into nanoparticles, the electrons and holes (or excitons or excitons) are confined in the nanocrystals, and the wavefunctions of the electrons and holes in the real space overlap. At the same time, in the wave number space, the spread Δk of the exciton wave number increases due to the uncertainty principle. As a result, the wave number selection rule is broken, and carriers can be recombined between bands without involvement of phonons. However, this transition is fundamentally different from interband recombination in the direct transition band structure. Since the spread of Δk due to atomization is used, the photon energy corresponding to the energy gap difference between the bottom of the conduction band and the top of the valence band reflecting the bulk band structure is included in all fluorescence spectra. As a result, the photon energy corresponding to the end wavelength of the fluorescence spectrum does not depend on the size of the nanocrystal, and coincides with the energy gap at the bottom of the conduction band and the top of the valence band. Therefore, the fluorescence spectrum is tailed toward the long wavelength side.

[Problem 4]
・ For fluorescence imaging of deep cells, it is preferable to excite light in a wavelength range longer than 700 nm, which has high biological permeability, but since silicon nanoparticles hardly absorb light in the wavelength range, excitation efficiency is extremely low. . Therefore, it is necessary to increase the excitation intensity.
-As another problem of using ultraviolet light for excitation, when the object of imaging is a living cell or a living tissue, they are damaged. Also, when the object to be imaged is DNA or protein, there is a possibility that these molecules may be damaged by ultraviolet light, which hinders accurate determination of base sequences and active sites. was there. As a near-infrared excitation (technology for performing near-infrared emission), a phosphor containing a rare earth element as a material that emits infrared light when excited by infrared light (so-called "up-conversion phosphor") is known (non-patent document). Reference 7). When such particles are used as a fluorescent labeling material, near infrared light can be used as excitation light, and therefore, the analyte is not adversely affected like ultraviolet light. However, the particles can obtain arbitrary upconversion emission by selecting the doping amount of rare earth, but in order to obtain different emission colors, it is necessary to select a rare earth material to be doped, and the emission color is There is a problem that it is limited and difficult to select.

  It is also known to use block copolymers for the synthesis of water soluble nanoparticles. For example, since pluronic has excellent characteristics such as low toxicity and good biocompatibility, it is used in medicines, cosmetics and the like (Non-Patent Document 8). There is an attempt to use the nanoparticles whose surface is modified with Pluronic by utilizing them (patent documents 5 and 6).

  It is an object of the present invention to solve the above-mentioned problems of the prior art and to provide water-soluble near-infrared light-emitting silicon nanoparticles with high luminous efficiency and high stability and a fluorescent labeling material using the same.

According to one aspect of the present invention, one or more silicon nanocrystals having a diamond structure, a hydrocarbon film that covers each surface of the silicon nanocrystals, and one that is covered with a hydrocarbon film. Alternatively, a water-soluble silicon nanoparticle provided with a block copolymer composed of a pluronic surfactant that covers a plurality of silicon nanocrystals and emitting light in the near infrared region by irradiation with excitation light is provided.
According to one aspect of the present invention, SiO ₂ enclosing step of making _{HSiO 1.5} was hydrolyzed triethoxysilane, the Si nanocrystals by heat treatment of 1150 ° C. under vacuum to said _{HSiO 1.5} A step of producing a powder, a step of stirring the SiO ₂ powder in an aqueous solution of hydrogen fluoride for 70 to 120 minutes to remove SiO ₂ to produce a hydrogen-terminated Si nanocrystal, the hydrogen-terminated Si Coating the surface of the nanocrystals with an alkyl monomolecular film having 11 to 22 carbon atoms; preparing a nanocrystal toluene dispersion liquid in which the Si nanoparticles coated with the alkyl monomolecular film are dispersed in toluene , subtracting that the triblock copolymer type nonionic surfactants and hydrophilic ethylene oxide and a hydrophobic propylene oxide is solvated in an aqueous acid solution Step of making emissions surfactant component dispersion liquid, a step of mixing the nanocrystalline toluene dispersion before Symbol nonionic surfactant component dispersion liquid, and, the step of removing toluene from the mixture by the mixing A method for producing silicon nanoparticles that emit light in the near infrared region upon irradiation with excitation light.
Here, the concentration of the hydrogen fluoride aqueous solution may be 48%.
The step of coating with the alkyl monomolecular film may be a step of heat-treating the hydrogen-terminated Si nanocrystals in mesitylene containing 1-octadecene at 150 ° C.
Also, the silicon nanoparticles may emit light at a peak wavelength in the range of 700 nm to 1000 nm upon irradiation with excitation light.
Further, the full width at half maximum of the emission spectrum may be 150 meV to 300 meV.
The emission quantum yield of the silicon nanoparticles may be 30% or more.
The Si nanocrystals encapsulated by the Si nanoparticles coated with the alkyl monolayer film may be in the range of 1.9 nm to 6.1 nm.
Also, the Si nanocrystals may be monodisperse particles.
Further, the silicon nanoparticles may emit light in the near infrared region based on two-photon absorption by irradiation with excitation light in the near infrared region.
According to another aspect of the present invention, there is provided a fluorescent labeling material having any of the above silicon nanoparticles.

  According to the present invention, a water-soluble near-infrared light-emitting silicon nanoparticle having a novel structure capable of emitting fluorescence with extremely high efficiency and having a sharp emission spectrum and a fluorescent labeling material using the same are provided. To be done.

The conceptual diagram which shows the structure of water-soluble near-infrared light emitting nanoparticle. The figure which shows the change of a powder X-ray diffraction pattern with etching time. Typical transmission electron microscopy images of octadecane-terminated silicon nanoparticles (left) and nanoparticles after Pluronic shell formation (left). Optical absorption and PL spectra of typical nanoparticles. Typical (a) UV-visible absorption spectrum and (b) emission spectrum of the water-soluble nanoparticles of the present invention in which the emission wavelength can be continuously controlled by particle size (absolute quantum yield of each PL spectrum in percentage are shown in the graph. Fill in). 3 is a photograph showing the stability of a silicon nanocrystal particle terminated with a carboxyl group (Comparative Example 4) with a change in pH. The graph which shows the relationship between the fluorescence maximum wavelength of a near-infrared PL spectrum of water-soluble nanoparticles, and a half value width. The water-soluble nanoparticles used here are the Si nanoparticles of the present invention containing Si nanocrystals which have been subjected to centrifugal separation treatment and chromatography treatment to have high monodispersity and surface homogeneity as an inner shell.

  The silicon nanoparticles of the present invention are water-soluble silicon nanoparticles having a double shell structure (a structure in which the core is double-coated with an inner shell and an outer shell) as shown in FIG. 1, and have a near infrared wavelength of 700 to 1000 nm. It emits strong light in the region. As shown, the core is composed of one or more silicon (Si) nanocrystals, which have a diamond structure. This diamond structure is maintained from the center of the grain to the outermost surface. This results in a high emission quantum yield. In order to prevent the reconstruction of the outermost surface layer that causes amorphization, the anchoring effect of hydrocarbons such as alkyl groups is effective.

  Explaining the anchoring effect, when the particle size of Si nanocrystals is reduced to about 2.5 nm (equivalent to about 850 nm in terms of emission maximum), the number of atoms constituting the surface is overwhelmingly higher than that of the inside. Therefore, in order to stabilize the phase structure of Si nanocrystals, the atoms distort the lattice from the inside of the nanocrystals toward the surface to obtain the most stable structure, and gradually become an amorphous structure over time. Structural relaxation occurs. This relaxation depends on the particle size of the nanocrystal. However, when the organic molecules are present on the surface of the nanocrystals at a high density, the organic molecules become steric hindrance, and the atomic group plays a role of anchor because the atomic group having a large mass is bonded to one surface silicon atom. Structural relaxation due to rearrangement of atoms near the surface is suppressed, and the diamond structure is maintained up to the surface. When an alkyl monomolecular film is used as the hydrocarbon monomolecular film coating the surface of the Si nanoparticles, the hydrocarbon chain length is preferably C1 to C22. Particularly, C11 or more is preferable. Even if the length is longer than C23, the effectiveness is not lost.

  It is merely said that the structural relaxation becomes remarkable when the particle size of the Si nanocrystal is smaller than 2.5 nm, and the structural relaxation occurs even when the particle size is larger than 2.5 nm. Structural relaxation cannot be suppressed by hydrogen atoms, but can be suppressed by hydrocarbons such as alkyl groups. The rate at which structural relaxation occurs depends on the particle size. At the hydrogen termination, the outermost surface of the particles becomes amorphous, so many defects that cause non-radiative recombination are formed. However, the number of defects is reduced by forming an ordered outermost surface by bonding alkyl groups and the like, and the probability of non-radiative recombination is greatly reduced. As a result, efficient light emission can be obtained. Therefore, structural relaxation inhibition should be considered to work for all particles, independent of particle size.

  Moreover, when a hydrocarbon is bonded to the surface of the nanocrystal, Si atoms and point defects on the outermost surface of the nanocrystal are terminated by carbon, which is a non-polar element. The radiative recombination probability of can be increased.

  Each nanocrystal is terminated with a hydrocarbon monolayer as described above. The hydrocarbon film is called an inner shell. The hydrocarbon film is immobilized on the outermost surface of the nanocrystal via a carbon-silicon covalent bond. Note that if the composition of a single molecule contains oxygen, nitrogen, or fluorine, the unshared electron pair existing in each single molecule repels between adjacent molecules during the formation of the single molecule film, so that the single molecule film The molecular density becomes low. As a result, the internal Si nanocrystals are easily oxidized, the above-mentioned anchor effect is not efficiently exhibited, and the stability of the emission color is reduced.

  The inner shell surface is covered with a block copolymer. The block copolymer is called an outer shell. A single inner shell may be contained in one outer shell, or a plurality of inner shells may be contained in one outer shell. The block having lower hydrophilicity among the blocks constituting the block copolymer is chemically adsorbed to the surface of the hydrocarbon film of the inner shell through hydrophobic interaction. On the contrary, the block with higher hydrophilicity is exposed on the outermost surface to give water solubility. The particle diameter of the water-soluble particles depends on the number of crystals included in the inner shell and is approximately 5 to 80 nm. Giving it water solubility makes it stable for a long time in water, physiological salt solutions, and phosphate buffered saline.

  The inclusion of multiple inner shells in one outer shell provides several advantages. For example, as an optical system for monitoring the movement of nanoparticles from outside the body using the fluorescence method, the fluorescence intensity from the particle group is stronger when the particles move in a collective, so fluorescence observation with a simple setup It becomes possible, and the excitation intensity is low. It is also useful for long-term fluorescence monitoring. When the number of particle groups contained in one nanoparticle is very small, monitoring cannot be performed if these particles are discolored. However, if it is composed of a large number of particles, even if some of the particles are discolored, no problem will occur. Also, in terms of monitoring technology, fading of fluorescent particles can be suppressed if the excitation intensity can be lowered.

  Of course, small fluorescent particle groups also have advantages. That is, if the fluorescent particle group is small, the resolution of the imaging becomes high, and the imaging of a minute space becomes possible. If the size of the particles covered with the block copolymer can be controlled, the fluorescent particles can be selectively used according to the purpose of fluorescence monitoring.

  The photoluminescence (PL) of the water-soluble silicon nanoparticles is in the near-infrared wavelength region of 700 to 1000 nm, which is suitable for observing a living tissue level. Since the particles are fluorescent labeling materials having an optimal electronic structure for the two-photon excitation technology, light in the near-infrared wavelength range with high biological permeability can be used for photoexcitation (the wavelength range is referred to as "a spectroscopic window of the living body"). Call). Moreover, the setting of the PL wavelength within the wavelength range of 700 to 1000 nm is achieved by controlling the size of the nanocrystal.

The absolute fluorescence quantum yield of the near infrared emission is a high value of 30% or more. This yield is significantly higher than that of conventional water-soluble Si nanoparticles. It is also higher than the fluorescence quantum yield of commercially available fluorescent dyes. The reason why such a high absolute fluorescence quantum yield can be realized is that the following three items are satisfied for the first time in the structure described above.
-The diamond structure is maintained (photoexcited carrier transition modes are unified).
-The hydrocarbon film suppresses surface oxidation (the formation of the oxide film causes a new transition mode of photoexcited carriers).
The presence of carbon-silicon bonds reduces the number of surface defects that induce non-radiative transitions.

  A quantum yield of more than 30% can also be realized with quantum dots (CdTe, PbSe, PbS, InAs) of compound semiconductor. However, the elements that make up these quantum dots are highly toxic and not suitable for use in living organisms.

  The half width of the PL spectrum of the Si nanoparticles according to the present invention was 150 to 300 meV as shown in FIG. 7. These values are narrower than conventional water-soluble Si nanoparticles. Some of those half-widths are comparable to typical quantum dots PbS that emit near-infrared light. This is because we have achieved precise control of the nanostructure (crystallinity, size, surface layer). Further, the emission spectrum thereof has high symmetry as shown in FIG. 5 and does not have a tail, so that the emission tuning characteristic similar to that of the compound semiconductor quantum dot is achieved. Furthermore, multicolor fluorescence imaging in the near infrared wavelength region is also possible.

  According to the present invention, it was demonstrated for the first time that Si can be used as a fluorescent labeling material for two-photon excitation near infrared bioimaging. In fluorescence imaging of living organisms, the use of two-photon absorption technology enables imaging with high contrast and high detection sensitivity. This is because near-infrared excitation-near-infrared emission can be achieved by utilizing two-photon absorption. Further, because of the two-photon absorption, it becomes possible to excite the silicon nanoparticles at the excitation maximum. That is, near-infrared light can be obtained by emitting near-infrared light from outside the living body. Furthermore, since the emission quantum yield is 30% or more in any wavelength band within the range of 700 to 1100 nm, it is possible to perform imaging with high detection sensitivity with weak excitation light. Since the two-photon absorption technique itself is a well-known matter, its detailed description is omitted.

  Further, the PL wavelength can be finely tuned in the near infrared region. Therefore, in combination with a narrow half-width or an emission spectrum shape that does not draw a tail and that the required excitation wavelength does not change even if the PL wavelength is changed, mutual interaction between different biomolecules under single excitation irradiation It is possible to realize a fluorescent labeling material that allows parallel observation of the action (that is, simultaneous and independent observation). Heretofore, there have been no reports of such marking materials.

  Here, the fact that the emission spectrum is not tailed will be described. In the background art section, this problem is referred to as [problem 3]. However, the inventor of the present application has found that in the near-infrared emission spectrum of diamond-structured silicon nanoparticles, the peak tailing toward the long wavelength side is attributed to the group of particles having different surface polarities and sizes. Therefore, if the origin of the peak that tails to the long wavelength side is based on the electronic structure according to the conventional idea, it is impossible to control it (such as narrowing the half-width of the spectrum), but If it is composed of different particles, it is possible to classify or classify each nanoparticle of the same quality, taking into account the reason why the spectrum half-width is widened. certified.

  Further, the water-soluble labeling material of the present invention has the lowest cytotoxicity as compared with the conventional Si nanoparticles. This uses a pluronic (registered trademark of BASF Corporation (BASF)), which has a high biocompatibility for the outer shell and has amphiphilicity, and self-assembles when the amphipathic molecule forms the outer shell. It was achieved by controlling the change. Specifically, the inner shell is hydrophobic because it is composed of an alkyl monomolecular film having a high molecular density. Therefore, it was possible to self-organize the molecular membrane structure in which the low hydrophilic block of Pluronic strongly interacts with the inner shell, while the hydrophilic block with high bio-affinity is exposed to the outer shell with high density. . Such a structure cannot be formed by simply allowing a pluronic molecule to act on the surface of bare Si nanoparticles, Si nanoparticles terminated with hydrogen atoms, or Si nanoparticles protected by a polar group such as silica.

  Pluronic refers to a triblock copolymer type nonionic surfactant of hydrophilic ethylene oxide (Ethylene Oxide, EO) and hydrophobic propylene oxide (Propylene Oxide, PO), which is currently manufactured by BASF. It is sold by. By changing the EO polymerization degree / PO polymerization degree, the characteristics as a surfactant can be changed. Here, a pluronic surfactant having a molecular weight of 12570 with (EO) 100 (PO) 100 (EO) 100 having an EO polymerization degree of 100 and a PO polymerization degree of 65 was used.

  Furthermore, the nanoparticles of the present invention were well dispersed in pure water, physiological salt solution, and phosphate buffered saline, and were stable for more than half a year. Also in this respect, the nanoparticles of the present invention have desirable characteristics as a fluorescent labeling material used in a living body.

  Hereinafter, the present invention will be described in more detail based on examples. Here, it should be noted that the examples are merely for the purpose of helping understanding of the present invention and do not limit the present invention.

[Experimental Example 1]
In order to obtain fluorescence from Si nanocrystals, it is necessary to control the nanocrystals within a particle size range smaller than 8 nm to obtain a Si crystal single phase having a diamond structure. Therefore, the etching time with the hydrofluoric acid aqueous solution (48%) was controlled between 0 and 5 hours, and the range in which the Si crystal single phase was obtained was estimated. The powder X-ray diffraction method was used for the qualitative determination of the crystal phase.

<Example 1>
HSiO _1.5 obtained by hydrolyzing triethoxysilane was disproportionated at 1150 ° C. under vacuum to obtain SiO ₂ powder including Si nanocrystals. By stirring the powder in aqueous hydrofluoric acid solution (48%) to remove the SiO _2, to obtain a hydrogen-terminated been Si nanocrystals (H-Si). The stirring time was controlled between 70 and 120 minutes. The powder obtained through the centrifugation treatment was dried and then qualitatively analyzed by powder X-ray diffraction.

<Comparative Example 1>
HSiO _1.5 obtained by hydrolyzing triethoxysilane was disproportionated at 1150 ° C. under vacuum to obtain SiO ₂ powder including Si nanocrystals. By stirring the powder in aqueous hydrofluoric acid solution (48%) to remove the SiO _2, to obtain a hydrogen-terminated been Si nanocrystals (H-Si). The stirring time was controlled between 25 and 70 minutes and 120 and 300 minutes, respectively. The powder obtained through the centrifugation treatment was dried and then qualitatively analyzed by powder X-ray diffraction.

<Comparison and examination between Example 1 and Comparative Example 1>
FIG. 2 shows powder X-ray diffraction patterns obtained from Example 1 and Comparative Example 1. SiO ₂ was removed by stirring the powder in an aqueous solution of hydrofluoric acid. The presence of SiO ₂ was judged by the presence or absence of a diffraction peak appearing near 2 theta = 22 °. On the other hand, the presence of Si was judged based on the X-ray diffraction of the (111), (220), and (311) planes that appear near 2 theta = 28 °, 45 °, 56 °. Both were present at the etching time of 25 minutes, and the longer the etching time, the smaller the peak at 2 Theta = 22 ° showing the presence of SiO ₂ . Then, between 70 and 120 minutes, only diffraction peaks derived from Si having a diamond structure were observed. Within this range, the broadening of each diffraction peak with etching time is due to the size down of the Si nanoparticles. On the other hand, when it exceeds 120 minutes, a by-product of Si—F—O system is formed near 2 theta = 17 °. Therefore, when etching is performed using the hydrofluoric acid aqueous solution having the above concentration, it is considered preferable to control the size of the Si nanoparticles within 70 to 120 minutes.

[Experimental Example 2]
The water-soluble fluorescent labeling material proposed by the present invention is composed of Si nanocrystals having a double shell structure. Specifically, it has one or more Si nanocrystals as a core, and each nanocrystal is terminated with a hydrocarbon film in order to stabilize the emission characteristics. It also has a block copolymer that imparts water solubility in the outer shell. In Example 2, a typical synthesis method of a water-soluble fluorescent labeling material (Si nanoparticles solubilized by a double shell structure) and solubility and stability in an aqueous solvent are discussed. In Example 3, the presence of an outer shell is discussed. Does not reduce the emission characteristics.

<Example 2>
HSiO _1.5 obtained by hydrolyzing triethoxysilane was disproportionated at 1150 ° C. under vacuum to obtain SiO ₂ containing Si nanocrystals. By stirring 80 minutes in an aqueous solution of hydrofluoric acid to remove the SiO _2, to obtain a hydrogen-terminated been Si nanocrystals (H-Si). The H—Si nanocrystals were heat-treated at 150 ° C. in mesitylene containing 1-octadecene that had been degassed in advance to obtain Si nanocrystals terminated with an octadecane monomolecular film. The nanocrystalline toluene dispersion was then mixed with the Pluronic dispersion. F127 was used for Pluronic, and the Pluronic dispersion was obtained by solvating in an aqueous acid solution (water: HCl = 10: 1). After mixing for 1 hour, the toluene phase was removed. Unreacted pluronic and hydrogen chloride were removed by dialysis. The product was obtained by drying. The product was dispersed in pure water, a physiological salt solution, and a phosphate buffered saline solution, and then allowed to stand. It was allowed to stand for a predetermined period of time to evaluate dispersibility and stability. The evaluation was carried out visually and based on a fluorescence spectrophotometer.

<Example 3>
HSiO _1.5 obtained by hydrolyzing triethoxysilane was disproportionated at 1150 ° C. under vacuum to obtain SiO ₂ containing Si nanocrystals. By stirring 80 minutes in an aqueous solution of hydrofluoric acid to remove the SiO _2, to obtain a hydrogen-terminated been Si nanocrystals (H-Si). The H—Si nanocrystals were heat-treated at 150 ° C. in mesitylene containing 1-octadecene that had been degassed in advance to obtain Si nanocrystals terminated with an octadecane monomolecular film. The nanocrystalline toluene dispersion was then mixed with the Pluronic dispersion. F127 was used for Pluronic, and the Pluronic dispersion was obtained by solvating in an aqueous acid solution (water: HCl = 10: 1). After mixing for 1 hour, the toluene phase was removed. Unreacted pluronic and hydrogen chloride were removed by dialysis. After drying the product, it was redispersed in non-fluorescent water. A photoluminescence (PL) spectrum and an excitation spectrum were measured using a fluorescence spectrophotometer. Further, the PL quantum yield when excited at the excitation maximum was estimated by the absolute method.

<Example 4>
HSiO _1.5 obtained by hydrolyzing triethoxysilane was disproportionated at 1100 ° C. to 1250 ° C. under vacuum to obtain SiO ₂ containing Si nanocrystals. By stirring 80 minutes in an aqueous solution of hydrofluoric acid to remove the SiO _2, to obtain a hydrogen-terminated been Si nanocrystals (H-Si). The H—Si nanocrystals were heat-treated at 150 ° C. in mesitylene containing 1-octadecene that had been degassed in advance to obtain Si nanocrystals terminated with an octadecane monomolecular film. The nanocrystalline toluene dispersion was then mixed with the Pluronic dispersion. F127 was used for Pluronic, and the Pluronic dispersion was obtained by solvating in an aqueous acid solution (water: HCl = 10: 1). After mixing for 1 hour, the toluene phase was removed. Unreacted pluronic and hydrogen chloride were removed by dialysis. After drying the product, it was redispersed in non-fluorescent water. A photoluminescence (PL) spectrum and an excitation spectrum were measured using a fluorescence spectrophotometer.

<Comparative example 2>
HSiO _1.5 obtained by hydrolyzing triethoxysilane was disproportionated at 1150 ° C. under vacuum to obtain SiO ₂ containing Si nanocrystals. By stirring 80 minutes in an aqueous solution of hydrofluoric acid to remove the SiO _2, to obtain a hydrogen-terminated been Si nanocrystals (H-Si). The H—Si nanocrystals were heat-treated at 150 ° C. in mesitylene containing 1-octadecene that had been degassed in advance to obtain Si nanocrystals terminated with an octadecane monomolecular film. The nanocrystals were dried and then redispersed in non-fluorescent dichloromethane. A photoluminescence (PL) spectrum and an excitation spectrum were measured using a fluorescence spectrophotometer. Further, the PL quantum yield when optically excited at the excitation maximum was estimated by the absolute method.

<Comparative example 3>
The fluorescence quantum yield of the sample produced in the present invention was measured by the absolute method. Using the same instrument, two fluorescent dyes were estimated by absolute method. When Cy7 (product code 15020, Funakoshi Co., Ltd.) was measured, the fluorescence quantum yield was estimated to be 30.3%, and that of HiLyte Fluor 750, Amine (product code 81267, Funakoshi Co., Ltd.) was estimated to be 10.8%. I was stolen. These values were almost in agreement with the fluorescence quantum yields of 30% and 12% published by the respective dye distributors.

<Comparison / examination of Examples 2, 3 and 4 and Comparative Examples 2 and 3>
The sample obtained in Example 2 was easily dispersed in pure water, but was not dispersed in dichloromethane or an alkane-based solvent. On the other hand, the sample obtained in Comparative Example 2 was easily dispersed in dichloromethane or an alkane-based solvent, but was not dispersed in pure water. This difference could be easily discerned visually. From these results, the sample produced in Comparative Example 2 was an octadecane-terminated Si nanocrystal, and exhibited oil solubility due to the presence of the octadecane monomolecular film. The sample prepared in Example 2 was considered to have a nanostructure in which hydrophilic groups were arranged outside by coating Si nanocrystals terminated with octadecane monomolecules with a block copolymer. . FIG. 3 shows typical transmission electron microscope images of these two samples. The distance between nanocrystals is constant regardless of which nanocrystal is viewed. And, there is no region where nanocrystals are bonded to each other. From the above, it is considered that the coating density of the octadecane monomolecular film is high. In addition, in a transmission electron microscope of a sample treated in a pluronic aqueous solution, which is one of block copolymers, particles present in a single nanocrystal, particles in which a plurality of nanocrystals are aggregated, and several tens of nanocrystals are observed. Particles in which individual nanocrystals were aggregated were observed. Due to the presence of the octadecane monolayer, the nanocrystals forming each aggregate maintained a constant distance between the nanocrystals. Therefore, the water-soluble fluorescent labeling material of the present invention is considered to have a nanostructure as shown in FIG. Specifically, the core is composed of one or more Si nanocrystals. The surface of each nanocrystal is terminated by a hydrocarbon chain, and the hydrocarbon film has a high molecular density. Oil-soluble nanoparticles composed of one or more nanocrystals are covered with a block copolymer. In the present invention, Pluronic was used as an example of a block copolymer. The low hydrophilic block of the pluronic molecule chemisorbs to the surface of the nanocrystals modified with the hydrocarbon film through hydrophobic interaction, while the hydrophilic block of the pluronic molecule faces outward to impart water solubility.

  Pluronic-protected water-soluble nanoparticles disperse easily not only in pure water but also in physiological salt solutions and phosphate buffered saline, and there was no change in dispersibility over a long period of more than half a year as long as visually observed. . In addition, since there is no change in the PL spectrum, it can be concluded that the light emitting function is not deteriorated as a result of coating the core that determines the optical characteristics without the inner and outer shells being discarded.

  The PL spectrum and excitation spectrum of the sample obtained in Example 3 are shown in FIG. The PL maximum was observed at 985 nm when photoexcited at the excitation maximum. The excitation maximum was observed at 386 nm. The Stokes shift between excitation and emission was about 600 nm. Such a large Stokes shift is a characteristic feature of silicon nanocrystals having a diamond structure crystal. The full width at half maximum of the PL spectrum was estimated to be 168 nm (220 meV). The fluorescence quantum yield was 31.3%. On the other hand, the fluorescence maximum of the PL spectrum obtained in Comparative Example 1 was 985 nm. The full width at half maximum of the PL spectrum was 167 nm (218 meV). The fluorescence quantum yield was 31.0%. Since 0.3% is included in the experimental error, it was found that the outer shell composed of the block copolymer used in the present invention does not affect the light emission characteristics.

  A typical PL spectrum of water-soluble nanoparticles produced through Example 4 is shown in FIG. The heat treatment time determines the size of 0-valent Si nanocrystals when trivalent Si is disproportionated into 0-valent and 4-valent Si. The higher the heat treatment temperature and the longer the time, the larger the initial crystal diameter. In each sample heat-treated under the same conditions, the longer the stirring time in the hydrofluoric acid aqueous solution, the more the silicon crystals are etched, and the particle size becomes smaller. As a result, the PL spectrum from the nanocrystals shifts to the shorter wavelength side as the stirring time increases. By controlling the particle size in the range of 1.9 to 6.1 nm, the PL spectrum can be continuously controlled in the wavelength range of 700 to 1000 nm. Since the particle size distribution and the half-value width of the PL spectrum in the Si nanocrystals are correlated, the produced Si nanocrystals are classified through a centrifugal separation and column chromatography process to improve the monodispersity and thus the PL spectrum half-width is small. Things are obtained. FIG. 5 shows a typical PL spectrum characteristic. The quantum yield of emission was higher than 30%, and the full width at half maximum of the spectra was in the range of 150 to 300 meV in all PL spectra as shown in FIG. 7. Although the maximum fluorescence wavelength is plotted only up to around 1050 nm in FIG. 7, it was almost the same as around 1050 nm in the range of 1050 to 1100 nm.

[Experimental Example 3]
In order to demonstrate that the fluorescent labeling material of the present invention exhibits higher stability in an aqueous solvent than the conventional single shell structure, the pH dependency was investigated. The stability of the water-soluble particles at each pH was judged by visually observing fluorescence while irradiating 365 nm UV light as excitation light.

<Example 5>
HSiO _1.5 obtained by hydrolyzing triethoxysilane was disproportionated at 1100 ° C. under vacuum to obtain SiO ₂ containing Si nanocrystals. By stirring 100 minutes in hydrofluoric acid aqueous solution to remove the SiO _2, to obtain a hydrogen-terminated been Si nanocrystals (H-Si). The H—Si nanocrystals were heat-treated at 150 ° C. in mesitylene containing 1-octadecene that had been degassed in advance to obtain Si nanocrystals terminated with an octadecane monomolecular film. The nanocrystalline toluene dispersion was then mixed with the Pluronic dispersion. F127 was used for Pluronic, and the Pluronic dispersion was obtained by solvating in an aqueous acid solution (water: HCl = 10: 1). After mixing for 1 hour, the toluene phase was removed. Unreacted pluronic and hydrogen chloride were removed by dialysis. The product was dried, redispersed in ultrapure water, and then enclosed in a quartz cell. Excitation with 365 nm UV light gave red emission.

<Comparative example 4>
HSiO _1.5 obtained by hydrolyzing triethoxysilane was disproportionated at 1100 ° C. under vacuum to obtain SiO ₂ containing Si nanocrystals. By stirring 100 minutes in hydrofluoric acid aqueous solution to remove the SiO _2, to obtain a hydrogen-terminated been Si nanocrystals (H-Si). The H—Si nanocrystals were heat-treated at 150 ° C. in mesitylene containing acrylic acid that had been degassed in advance to obtain Si nanocrystals terminated with a carboxyl group. After redispersion in ultrapure water, it was enclosed in a quartz cell. Excitation with 365 nm UV light gave red emission.

<Comparative Example 5>
HSiO _1.5 obtained by hydrolyzing triethoxysilane was disproportionated at 1100 ° C. under vacuum to obtain SiO ₂ containing Si nanocrystals. By stirring 100 minutes in hydrofluoric acid aqueous solution to remove the SiO _2, to obtain a hydrogen-terminated been Si nanocrystals (H-Si). The H—Si nanocrystals were heat-treated at 150 ° C. in mesitylene containing deaerated allylamine to obtain Si nanocrystals terminated with a carboxyl group. After redispersion in ultrapure water, it was enclosed in a quartz cell. Excitation with 365 nm UV light did not show red photoluminescence.

<Comparison and examination between Example 5 and Comparative Examples 4 and 5>
In the water-soluble nanoparticles obtained from Example 5, even if the pH was changed in the range of 1.5 to 12, the fluorescent red color was uniformly radiated from the entire surface of the solution enclosed in the quartz cell, and did not depend on the change in pH. It was confirmed to be stably dispersed. The carboxyl group-terminated Si nanocrystals obtained in Comparative Example 4 uniformly emitted fluorescence from the entire surface of the solution in the pH range of 1.7 to 9.2 as shown in FIG. Aggregation started, and when pH exceeded 12, particles precipitated and fluorescence from the solution was not seen. The amino group-terminated Si nanocrystals obtained from Comparative Example 5 did not show red fluorescence. This is considered to be because the molecular orbital formed by the interaction of the nitrogen loan pair of the amino group with the outermost surface Si atom was formed between the energy levels formed in the Si nanostructure. Therefore, it is considered that the electron-hole pair generated by photoexcitation occurs not through the HOMO-LUMO transition defined by the Si nanostructure but through the newly formed interface state. As one of the proofs, PL which was considered to be quenched was red-shifted to 850 nm.

[Experimental Example 4]
In order to investigate the effect of suppressing the oxidation of the hydrocarbon monomolecular film forming the inner shell, dry powders of hydrogen-terminated Si nanocrystals and octadecene-terminated Si nanocrystals were exposed to the atmosphere and their reactivity to oxygen was examined. The crystal phase was identified by powder X-ray diffractometry.

<Example 6>
HSiO _1.5 obtained by hydrolyzing triethoxysilane was disproportionated at 1100 ° C. under vacuum to obtain SiO ₂ containing Si nanocrystals. By stirring for 90 minutes in hydrofluoric acid aqueous solution to remove the SiO _2, to obtain a hydrogen-terminated been Si nanocrystals (H-Si). The H—Si nanocrystals were heat-treated at 150 ° C. in mesitylene containing 1-octadecene that had been degassed in advance to obtain Si nanocrystals terminated with an octadecane monomolecular film. When the powder was left for 2 weeks in the air and analyzed by powder X-ray diffraction, there was no change in the diffraction pattern.

<Comparative example 6>
HSiO _1.5 obtained by hydrolyzing triethoxysilane was disproportionated at 1100 ° C. under vacuum to obtain SiO ₂ containing Si nanocrystals. By stirring for 90 minutes in hydrofluoric acid aqueous solution to remove the SiO _2, to obtain a hydrogen-terminated been Si nanocrystals (H-Si). When the powder was allowed to stand in the air for 2 weeks and then analyzed by powder X-ray diffraction, no diffraction showing the crystalline phase of Si was found, and instead, a broad peak showing the formation of silicon oxide was found near 21 °.

<Comparison and examination between Example 6 and Comparative Example 6>
The octadecane monolayer-terminated Si nanocrystals produced in Example 6 did not change in the powder X-ray diffraction pattern even after exposure to the atmosphere for 2 weeks, and therefore had a crystal size that can be estimated from Scherrer's equation. There was no change. This is because, as discussed from the transmission electron microscope image shown in FIG. 3, the terminal carbon of the octadecane molecule is strongly bound to Si on the outermost surface of the nanocrystal by a covalent bond, and further, it occurs between hydrocarbons in the octadecane monolayer. It is considered that the invasion of oxygen was inhibited because the high-density octadecane molecule covered the crystal surface through the hydrophobic interaction. On the other hand, in the hydrogen-terminated Si nanocrystals of Comparative Example 6, oxidation proceeded easily. Hydrogen atoms are smaller than silicon atoms, and active oxygen, which is highly reactive among oxygen species present in the atmosphere, can easily reach the outermost surface of nanocrystals, reacts with silicon, and is oxidized by diffusing into the crystal interior. Probably advanced. Oxidation of hydrogen-terminated Si that is normally placed in the atmosphere also occurs in bulk crystal wafers. Although the thickness of the oxide film is slightly different depending on the season, it is 1.7 to 2.1 nm at room temperature, so that Si nanoparticles exhibiting fluorescence emission characteristics are oxidized into silicon oxide nanoparticles. Is convinced.

[Experimental Example 5]
The cytotoxicity of the fluorescent labeling material of the present invention was investigated. It is shown that high cell viability is achieved when using the nanostructures described in the present invention. We also performed bioimaging by two-photon excitation.

<Example 7>
HSiO _1.5 obtained by hydrolyzing triethoxysilane was disproportionated at 1100 ° C. under vacuum to obtain SiO ₂ containing Si nanocrystals. By stirring 80 minutes in an aqueous solution of hydrofluoric acid to remove the SiO _2, to obtain a hydrogen-terminated been Si nanocrystals (H-Si). The H—Si nanocrystals were heat-treated at 150 ° C. in mesitylene containing 1-octadecene that had been degassed in advance to obtain Si nanocrystals terminated with an octadecane monomolecular film. The nanocrystalline toluene dispersion was then mixed with the Pluronic dispersion. F127 was used for Pluronic, and the Pluronic dispersion was obtained by solvating in an aqueous acid solution (water: HCl = 10: 1). After mixing for 1 hour, the toluene phase was removed. Unreacted pluronic and hydrogen chloride were removed by dialysis. The cytotoxicity test of the water-soluble fluorescent labeling material was performed using Cell Counting Kit-8. A suspension of NIH3T3 cells was distributed in Dulbecco's Modified Eagle Medium (common name: DMEM) medium. The medium was supplemented with 10% fetal bovine serum, 100 units / mL penicillin, and 100 mg / mL streptomycin. This was incubated at 37 ° C. in humidified air containing 5% carbon dioxide for 1 day, then a predetermined amount of a fluorescent labeling agent was added, and incubated at 37 ° C. in humidified air containing 5% carbon dioxide for 1 day. To this, 10 μL of CCK-8 solution was added, and after incubation for 24 hours, cell viability was examined from the absorbance using a microplate reader. When the concentration of the water-soluble fluorescent labeling agent was increased to 25 to 500 μg / mL, the survival rate of NIH3T3 cells was almost 100% up to 300 μg / mL, and the survival rate of 95% or higher was obtained even at a high concentration of 500 μg / mL. Indicated.

<Example 8>
HSiO _1.5 obtained by hydrolyzing triethoxysilane was disproportionated at 1100 ° C. under vacuum to obtain SiO ₂ containing Si nanocrystals. By stirring 80 minutes in an aqueous solution of hydrofluoric acid to remove the SiO _2, to obtain a hydrogen-terminated been Si nanocrystals (H-Si). The H—Si nanocrystals were heat-treated at 150 ° C. in mesitylene containing 1-octadecene that had been degassed in advance to obtain Si nanocrystals terminated with an octadecane monomolecular film. The nanocrystalline toluene dispersion was then mixed with the Pluronic dispersion. F127 was used for Pluronic, and the Pluronic dispersion was obtained by solvating in an aqueous acid solution (water: HCl = 10: 1). After mixing for 1 hour, the toluene phase was removed. Unreacted pluronic and hydrogen chloride were removed by dialysis.

NIH3T3 cells were prepared so as to contain 10 ⁵ cells per mL, and placed on a predetermined glass substrate. 100 μg / mL of water-soluble Si nanoparticles having an emission maximum at 780 nm was allowed to act on this and incubated for 24 hours. After washing with a phosphate buffer several times, it was fixed with paraformaldehyde to prepare a sample for fluorescence imaging.

  Two-photon excitation method was used for fluorescence imaging. When a femtosecond titanium sapphire laser pulse with a wavelength of 790 nm was used as an excitation light source, two-photon excitation emission was separated by a dichroic mirror and a filter, and the sample was excited with photon energy corresponding to 395 nm, and an imaging image of NIH3T3 cells with high fluorescence contrast I caught it.

<Comparative Example 7>
The results of recent cytotoxicity tests of water-soluble Si nanoparticles will be described as comparative examples.
-The MTT test evaluated the pancreatic cancer cytotoxicity of Si nanoparticles protected by phospholipid micelles, and when the nanoparticle concentration exceeded 4 μg / mL, the cell viability began to decrease monotonically and reached 80 at 10 μg / mL. %, Less than 60% at 12 μg / mL (Non-Patent Document 9)
・ The MTS test evaluated the pancreatic cancer cytotoxicity of Si nanoparticles modified with lysine, and the cell viability was 100% at nanoparticle concentrations up to 200 μg / mL, but decreased to 60% at 500 μg / mL. (Non-patent document 10).
When the MTT test evaluated the cytotoxicity of Si nanoparticles terminated with epoxy groups, amino groups, and diol groups on A549 cells and HepG2 human hepatoma-derived cells, the cell viability was almost 100% up to 50 μg / mL. However, when it exceeded 100 μg / mL, the survival rate decreased to 50 to 60%, and when it exceeded 200 μg / mL, it fell to 20% or less (Non-patent Document 11).
When the Hella cytotoxicity of Si nanoparticles terminated with amino groups and carboxyl groups was evaluated by the MTT test, the survival rate decreased to 80% when the concentration of nanoparticles exceeded 60 μg / mL (Non-Patent Document 12).
・ The MTS test evaluated the pancreatic cancer cytotoxicity of a water-soluble fluorescent label in which Si nanoparticles terminated with a carboxy group were encapsulated in high molecular weight polyethylene glycol micelles, and cell survival was observed up to a nanoparticle concentration of 200 μg / mL. The rate was 100%, but it became 60% or less at 500 μg / mL (Non-Patent Document 13).

<Comparison and examination between Examples 7 and 8 and Comparative Example 7>
Cytotoxicity has been studied so far in water-solubilized Si nanoparticles by forming a single shell composed of a monolayer. Conventionally, it has been reported that cytotoxicity for Si nanoparticles is strongly dependent on surface functional groups, thiol groups increase cytotoxicity, and amino groups and ester groups are low cytotoxic groups. . However, it was revealed from Comparative Example 7 that amino groups, ester groups, and the like also have high cytotoxicity in a high-concentration environment of nanoparticles. Even with other nanoparticles known to have high biocompatibility, polyethylene glycol used as a surface protective material with a high molecular weight is more toxic than the fluorescent labeling material of the present invention. Furthermore, it can be said that even if a surface protective layer having a high biocompatibility such as phospholipid is used, the cytotoxicity is high as compared with the water-soluble fluorescent labeling material of the present invention. The biocompatibility of pluronic surfactants is well known, but the low cytotoxicity does not depend only on the molecular chemical properties of pluronic, but the existence of the inner shell causes the thin film structure of the outer shell to It is considered that it was controlled in a self-organizing manner, and as a result, the dense hydrophilic film could be exposed to the outermost surface of the particles.

  In Example 8, fluorescence imaging of cells was performed by two-photon excitation. Since the incident light is 790 nm, it means that the phosphor is excited by the photon energy corresponding to 395 nm. Since 395 nm is a wavelength very close to the excitation maximum from FIG. 3, it means that the phosphor was efficiently excited. Since the fluorescent substance is nanoparticles having a fluorescence maximum at 780 nm, it means that the present imaging was performed in the wavelength range corresponding to the biological spectroscopic window. The important point is that the most widely used wavelength of 790 nm among femtosecond lasers corresponds to the wavelength capable of efficiently photoexciting all the nanoparticles of the present invention. For example, as shown in FIG. 5, the nanoparticles used in Example 8 emit light with an efficiency of more than 40% in absolute fluorescence quantum yield by being optically excited at the excitation maximum, so that the excitation intensity may be weak. It was possible to provide high fluorescence imaging.

  As described above, according to the present invention, it is possible to obtain silicon nanoparticles that emit light efficiently and stably, and can be applied to, for example, a fluorescent labeling material for fluorescent imaging of a living body, and thus, it is possible to use it in industrial Some are highly available.

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Claims (9)

Hydrolyzing triethoxysilane to make HSiO _1.5 ,
Subjecting said HSiO _1.5 to a heat treatment at 1150 ° C. under vacuum to produce SiO ₂ powder containing Si nanocrystals;
Stirring the SiO ₂ powder in a hydrogen fluoride aqueous solution for 70 to 120 minutes to remove SiO ₂ and produce hydrogen-terminated Si nanocrystals;
Coating the surface of the hydrogen-terminated Si nanocrystal with an alkyl monomolecular film having 11 to 22 carbon atoms;
Preparing a nanocrystalline toluene dispersion of the Si nanoparticles coated with the alkyl monolayer in toluene.
The step of preparing a non-ionic surfactant component dispersion liquid obtained by solvating the triblock copolymer type nonionic surfactant in the acid aqueous solution of hydrophilic ethylene oxide and a hydrophobic propylene oxide,
The step of mixing the nanocrystalline toluene dispersion before Symbol nonionic surfactant component dispersion liquid,
And a method for producing silicon nanoparticles that emit light in the near infrared region by irradiation with excitation light, which comprises performing a step of removing toluene from the mixed solution by the above mixing.   The method for producing silicon nanoparticles according to claim 1, wherein the concentration of the hydrogen fluoride aqueous solution is 48%.   The silicon nanoparticle according to claim 1 or 2, wherein the step of coating with the alkyl monolayer is a step of heat-treating the hydrogen-terminated Si nanocrystal at 150 ° C in mesitylene containing 1-octadecene. Production method.   The method for producing silicon nanoparticles according to claim 1, wherein the silicon nanoparticles emit light with a peak wavelength in a range of 700 nm to 1000 nm upon irradiation with excitation light.   The method for producing silicon nanoparticles according to claim 4, wherein the full width at half maximum of the emission spectrum is 150 meV to 300 meV.   The method for producing silicon nanoparticles according to claim 1, wherein the silicon nanoparticles have an emission quantum yield of 30% or more. The Si nanocrystals encapsulated by the Si nanoparticles coated with the alkyl monolayer film are
The size is in the range of 1.9 nm to 6.1 nm,
The method for producing silicon nanoparticles according to claim 1.   The method for producing silicon nanoparticles according to claim 1, wherein the Si nanocrystals are monodisperse particles.   The method for producing silicon nanoparticles according to claim 1, wherein the silicon nanoparticles emit light in the near infrared region based on two-photon absorption by irradiation with excitation light in the near infrared region.