JP2010105896A - Method for producing silica nanoparticle containing functional organic molecule having carboxy group, silica nanoparticle obtained thereby and labeling reagent using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a silica nanoparticle, in which the efficiency is improved when a functional organic molecule having a carboxy group, such as a fluorescent pigment molecule and a light absorbing pigment molecule, is fetched in the silica nanoparticle, and to provide the silica nanoparticle to be produced by the producing method, a method for fetching the functional organic molecule and a labeling reagent which is excellent in reproducibility of measured results and allows an ultramicro-target substance to be analyzed quantitatively with high sensitivity. <P>SOLUTION: The method for producing the silica nanoparticle containing the functional organic molecule having at least one carboxy group includes: a step (a) of amide-bonding the functional organic molecule having at least one carboxy group and at least one active ester group to at least one amino group of organoalkoxysilane; and a step (b) of hydrolyzing both of an amide-bonded compound obtained at the step (a) and tetraalkoxysilane and subjecting the obtained organosilanol and silanol to a polycondensation reaction to prepare the silica nanoparticle containing the functional organic molecule. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for producing nanometer-sized silica nanoparticles. More specifically, the present invention relates to a method for producing nanometer-sized silica nanoparticles used as a labeling reagent for detecting or quantifying a specific substance such as protein, silica nanoparticles obtained by the method, and a labeling reagent using the same.

  When water is added to a tetraalkoxysilane typified by tetraethoxysilane (hereinafter sometimes referred to as TEOS) and hydrolyzed, siloxane bonds (Si-O bonds) are formed by condensation polymerization. (For example, refer nonpatent literature 1). Then, if a dye molecule having fluorescence or color development properties is bound to the inside or the surface of the silica particle, a labeling reagent capable of optically detecting the presence can be obtained.

As a method of incorporating organic dye molecules into the inside of silica particles, a dye molecule having an N-hydroxysuccinimide (NHS) ester bond that easily reacts with an amino group is converted to 3-amino which is a kind of silane coupling agent having an amino group. By combining with the amino group of the propyltriethoxysilane (APS) molecule, hydrolyzing the ethoxy group of the compound consisting of the dye molecule and the APS molecule thus obtained and polymerizing with silica, the result is that silica and There is a method in which a dye molecule is bonded by an amide bond (see, for example, Patent Document 1).
As an apparatus for detecting a fluorescent label, an Ar ⁺ ion laser having a wavelength of 488 nm is generally used as a light source, and fluorescein dyes (carboxyfluorescein, fluorescein isothiocyanate) are effectively excited at this wavelength. (FITC) etc.).

In the method of incorporating the organic dye molecule described above, the carboxyl group of carboxyfluorescein is combined with the amino group of APS and incorporated into the silica particles. However, in this case, the incorporation efficiency is lower than other dyes such as rhodamine dyes. was there. If the uptake efficiency is low, a large amount of dye must be added to take in the same amount of dye, and in this case, the dye that has not been taken up in silica is wasted. The NHS ester of the dye described above is very expensive, and waste of the dye directly leads to an increase in the cost of the fluorescent reagent as a product.
In addition, when fluorescent dye molecules are incorporated into silica particles, the fluorescence intensity decreases due to concentration quenching when the distance between adjacent fluorescent dye molecules becomes narrow. Therefore, in order to achieve a stronger fluorescence intensity, it is as uniform as possible inside the silica particles. It is desirable to incorporate dye molecules. However, in the case of a dye molecule having a low uptake efficiency, the dye molecule tends to be unevenly distributed on the particle surface because it is slowly taken into the silica after the silica particles are formed first. This also causes a problem that a strong fluorescence intensity cannot be obtained when a dye molecule having a low uptake efficiency is used.
EP1036763B1 publication Journal of Colloid and Interface Science, 26, 62-69 (1968)

  In view of the above problems, the object of the present invention is to provide a method for producing silica nanoparticles that improves the efficiency of incorporation of functional organic molecules such as fluorescent dye molecules and light-absorbing dye molecules into the silica nanoparticles. An object of the present invention is to provide a method for producing silica nanoparticles in which the fluorescence intensity of the fluorescein dye contained therein is enhanced by alkalizing. Another object of the present invention is silica nanoparticles that are produced by them and contain the functional organic molecules at a high concentration and can be used as a highly sensitive labeling reagent, and have an average particle size of nanometer size. And providing silica nanoparticles having a narrow particle size distribution and a uniform particle size. Furthermore, an object of the present invention is to provide a labeling reagent that is excellent in reproducibility of measurement results and enables highly sensitive quantitative analysis of a very small amount of target substance.

The above problems have been achieved by the following means.
(1) A method for producing silica nanoparticles containing functional organic molecules, comprising the following steps (a) and (b):
(A) amide-bonding a functional organic molecule having at least one carboxyl group with an organoalkoxysilane having at least one amino group represented by the following general formula 1 and at least one positively chargeable site; And (b) a silica nanoparticle containing the functional H organic molecule obtained by hydrolyzing the amide bond compound and tetraalkoxysilane obtained in the step (a), respectively, and subjecting the resulting organosilanol and silanol to a polycondensation reaction. Preparing the particles,
General formula 1
H ₂ NR ¹ Si (OR ² ) ₃
(In the formula, R ¹ is a substituent having at least one positively-chargeable moiety, and is a divalent substituent that is single-bonded to the amino group (H ₂ N) and silicon atom (Si) in the formula, respectively) OR ² represents an alkoxyl group having 1 to 6 carbon atoms.)
(2) The method for producing silica nanoparticles containing functional organic molecules according to (1), further comprising the following step (c) after the step (b):
(C) Further, tetraalkoxysilane is further added to the reaction solution to cause hydrolysis, the resulting silanol, the organosilanol remaining in the reaction, and the silica nanoparticles obtained in the step (b) A step of forming a silica shell layer on the surface of the silica nanoparticles by a polycondensation reaction,

(3) The functional organic molecule is an organic dye molecule, and R ^{1 of the} organoalkoxysilane has 5 carbon atoms having an amino group and a bivalent branched hydrocarbon group having 5 carbon atoms or a secondary amine. The method for producing silica nanoparticles according to (1) or (2), which is a divalent linear hydrocarbon group of
(4) The functional organic molecule is 5 (6) -carboxyfluorescein, and the hydrolysis and polycondensation reaction in the steps (b) and (c) are performed in a water-ethanol mixed solvent in the presence of ammonia. The method for producing silica nanoparticles according to any one of (1) to (3),
(5) At the time of preparing the silica nanoparticles in the step (b) or forming the shell in the step (c), an organoalkoxysilane having at least one amino group is hydrolyzed together with the tetraalkoxysilane. The method for producing silica nanoparticles according to (4), wherein the silanol and organosilanol are subjected to a condensation polymerization reaction,

(6) Silica nanoparticles obtained by the production method according to any one of (1) to (5), and (7) a labeling reagent prepared using the silica nanoparticles according to (6). It is to provide.
In the present specification and claims, “silica nanoparticles” refer to colloidal silica particles having an average particle size of 1000 nm or less. When the silica nanoparticles are used as a labeling reagent, the particle diameters suitable for use depending on the detection substance and the detection method are various, but are often in the range of 20 to 500 nm, and the silica nanoparticles are in this range. It is preferable.

The method for producing silica nanoparticles of the present invention can improve the amount (efficiency) of incorporation of the functional organic molecules having at least one carboxyl group into the silica nanoparticles.
The method for producing silica nanoparticles of the present invention exhibits strong fluorescence characteristics in an alkaline environment such as 5 (6) -carboxyfluorescein contained in the silica nanoparticles by maintaining the inside of the prepared silica nanoparticles alkaline. It is possible to enhance the fluorescence intensity of the organic fluorescent dye.

The silica nanoparticles of the present invention contain functional organic molecules such as fluorescent dye molecules and light absorbing dye molecules at a high concentration, and can be used as a highly sensitive labeling reagent.
The labeling reagent of the present invention can be used for highly sensitive analysis.

First, the manufacturing method of the silica nanoparticle containing the functional organic molecule of this invention is demonstrated.
The method for producing silica nanoparticles containing functional organic molecules of the present invention (hereinafter simply referred to as “the production method of the present invention”) comprises the following steps (a) and (b).
(A) a functional organic molecule having at least one carboxyl group and at least one amino group represented by the general formula 1 and at least one amino group of the organoalkoxysilane having a positively chargeable site (B) hydrolyzing the amide bond compound obtained in step (a) and tetraalkoxysilane, respectively, and subjecting the resulting hydrolyzate to organosilanol and silanol to condensation polymerization. The process of making it react and preparing the silica nanoparticle containing the said functional organic molecule.

  Specific examples of the functional organic molecule include an organic dye molecule, a light-absorbing dye molecule, a magnetic molecule, a radiolabeled molecule, a pH-sensitive dye molecule having at least one carboxyl group that can be used for binding to an amino group. Although it is mentioned, it is preferable that it is an organic pigment | dye molecule | numerator which has at least 1 carboxyl group which can be used for a coupling | bonding with an amino group. Examples of the organic dye molecules include organic fluorescent dyes. Here, 5 (6) -carboxyfluorescein and 5 (6) -carboxyTAMRA, which are the organic fluorescent dyes, will be described.

Note that NHS esters of both dyes are commercially available from Invitrogen. When such an NHS ester is used, the step (a) can be realized only by mixing with an organoalkoxysilane having an amino group in a solvent such as DMF.
When amide-bonded with 3-aminopropyltriethoxysilane (hereinafter referred to as APS), which is a silane coupling agent having one amino group, is incorporated into silica nanoparticles, the incorporation efficiency of 5 (6) -carboxyfluorescein is 5 ( 6) It is 1/20 or less of carboxy TAMRA. The cause is that 5 (6) -carboxyfluorescein amide-bonded with APS does not have a positive charging site, whereas 5 (6) -carboxyTAMRA amide-bonded with APS has a positive charging site. it is conceivable that. The TAMRA molecule has two nitrogen atoms bonded to a six-membered ring, and these pull out surrounding hydrogen atoms as amines to induce a bias in the π-electrons of the six-membered ring, resulting in an excess of positive charges as a whole. Actually, when a hydrogen atom of a carboxyl group connected to another six-membered ring is extracted by the nitrogen atom, a negatively charged site due to an ionized carboxyl group and a positively charged site due to a bias of π electrons are formed in pairs. Conceivable. On the other hand, a fluorescein molecule does not have a nitrogen atom, and a positively charged site is not formed in this way.
There are only negatively charged sites where the silanols are ionized on the surface of ordinary silica nanoparticles, and an attractive force acts on the cation, but there is a pair of charged sites such as the TAMRA molecule. The attractive force derived from the electric dipole also works. If the APS-derived site forms a siloxane bond with the surface of the silica nanoparticle while the 5 (6) -carboxy TAMRA molecule amide-bonded to the APS is adsorbed to the surface of the silica nanoparticle by this attractive force, the TAMRA molecule remains as it is in the silica nanoparticle. Is taken in. This difference in the presence or absence of attractive force is considered to produce the difference in the above-mentioned uptake efficiency.
However, in the present invention, by using the organoalkoxysilane represented by the above general formula 1 having at least one positively chargeable group B, the amide-linked 5 (6) -carboxyfluorescein can be used. It is possible to increase the efficiency of incorporation of functional organic molecules having no positively charged sites into silica nanoparticles.

In the divalent substituent R ¹ of the organoalkoxysilane represented by the general formula 1, at least one site that can be positively charged (hereinafter sometimes simply referred to as B group) is an amino group (first group). Monoamine), secondary amines, tertiary amines and the like. As the substituent R ¹ , a linear or branched chain having 3 to 9 carbon atoms, a linear or branched divalent hydrocarbon group having an amino group, a secondary amine or a tertiary amine. And a divalent hydrocarbon group.
Specific examples of the organoalkoxysilane represented by the general formula 1 include the following compound 1, compound 2 (N- (2-aminoethyl) -3-aminopropyltriethoxysilane), compound 3, and the like.

Compound 2 is commercially available from Shin-Etsu Chemical Co., Fluorochem Co., etc. Compound 3 can be subjected to a condensation reaction between amino acid lysine and APS using a condensing agent (for example, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC)).
The amide bond reaction (step (a)) between an active ester group such as NHS ester of the functional organic molecule and the amino group of the organoalkoxysilane is DMSO (dimethyl sulfoxide) or DMF (N, N-dimethylformamide). It can carry out by melt | dissolving in solvents, such as, and reacting, stirring at room temperature (for example, 25 degreeC) conditions. Although there is no restriction | limiting in particular as reaction time, It is preferable to make it react for 0.5 to 2 hours.
In that case, the quantitative relationship between the functional organic molecule used in the reaction and the organoalkoxysilane is preferably equivalent in terms of mole. Since functional organic molecules having NHS ester groups are generally expensive, it is desirable to reduce waste as much as possible. However, when the proportion of the organoalkoxysilane is high, organoalkoxysilane having an unreacted amino group is incorporated into silica particles. Silica particles may have a tendency to agglomerate in pure water.

Examples of the tetraalkoxysilane used in step (b) of the production method and the like of the present invention include tetraethoxysilane (TEOS), tetramethoxysilane and the like, and TEOS is preferable.
In the step (b) of the production method and the like of the present invention, if the amount of the tetraalkoxysilane contained in the reaction solvent is too large, particles are likely to coalesce, and conversely if too low, the particle size distribution is widened. There is a tendency. Specifically, it is preferably 0.1 to 2.0% by volume, and more preferably 0.2 to 1.0% by volume.
In the step (b) of the production method and the like of the present invention, the amount of the amide bond compound obtained in the step (a) to be contained in the reaction solvent is as a mixed molar ratio with the tetraalkoxysilane: It is preferable to make it react in the range of 100-1: 5, and it is more preferable to make it react in the range of 1: 50-1: 10.
The content of the functional organic molecule in the obtained silica nanoparticles can be controlled by the amount of the amide bond compound to be dissolved or contained.

The reaction solvent is preferably a mixed solvent of water and a hydrophilic solvent, and examples of the hydrophilic solvent include ethanol, methanol and the like, and ethanol is preferable. The volume ratio of the water and the hydrophilic solvent is preferably a mixed solvent of 1:20 to 1: 3, more preferably a mixed solvent of 1: 6 to 1: 4.
The concentration of aqueous ammonia contained in the reaction solvent used in step (b) of the production method and the like of the present invention is preferably 1 to 28% by mass from the viewpoint of controlling the average particle size of the target silica nanoparticles. 2 to 14% by mass is more preferable.

The higher the ammonia content (ammonia concentration), the higher the nucleation frequency of the silica particles and the growth rate of the silica particles, but the dependence on the ammonia concentration is higher for the growth rate of the silica particles. Therefore, the average particle diameter of the target silica nanoparticles can be adjusted by adjusting the ammonia concentration based on the difference (difference) in the ammonia concentration dependency.
Although there is no restriction | limiting in particular as temperature conditions of formation of the said silica nanoparticle in the said process (b), It is preferable to carry out on 0-60 degreeC temperature conditions. Although there is no restriction | limiting in particular as reaction time, It is preferable to make it react for 1 to 24 hours.

  About the said process (b) in this invention, the case where TEOS is used as tetraalkoxysilane is illustrated in the following scheme.

The tetraalkoxysilane after leaving by any hydrolysis by the action of ammonia four alkoxyl groups was contained in the reaction solvent, to form a siloxane bond engaged dehydration condensation, eventually formula SiO ₂ It changes to the silica represented by.
The amide bond compound obtained in the step (a) also has three alkoxyl groups, which are similarly eliminated by hydrolysis, so that it is possible to form a siloxane bond with silica. .
In general, silica refers to a three-dimensional structure composed of a silicon atom and an oxygen atom based on a siloxane bond (Si—O bond). In the present invention, the silica is composed of a silicon atom and an oxygen atom containing the organosiloxane component as described above. It is assumed that the following three-dimensional structure is included.

However, it can be said that the amide bond compound obtained in the step (a) is generally less likely to be taken into silica as compared with tetraalkoxysilane, because the bonded functional organic molecule portion has steric hindrance. However, this is not the case when an attractive interaction acts with silica. If it is adsorbed on the silica surface by attractive interaction, the Si (OR ² ) ₃ part reacts with silica to form a siloxane bond in the meantime, and if the silica particle grows as it is, the entire functional organic molecule part becomes the silica particle. It will be taken in inside, and the taking-in efficiency will become high according to the strength of attractive interaction.
Silanol groups (Si—OH) that do not lead to the formation of siloxane bonds exist on the surface of the silica particles, and these are easily negatively charged. For this reason, the positively chargeable group B is attracted to silica by the attractive force between the B group and the silanol group when approaching to a short distance. Since the adsorption force in such a case is not so strong, it may be detached again by thermal vibration. However, if the Si (OR ² ) ₃ part reacts to form a siloxane bond while adsorbing, it will no longer be released, so even if the positively charged group B exists, Uptake efficiency can be increased.
In addition, since an attractive force derived from an electric dipole also acts on a molecule having a pair of positive and negative charged sites, the efficiency of incorporation into silica nanoparticles can be increased in the same manner as a molecule having only a positively chargeable group.

In general, the larger the amount of the functional organic molecule incorporated, the better. However, the organic fluorescent dye has an optimum amount of incorporation because there is a decrease in fluorescence intensity due to concentration quenching. The amount is about 3 × 10 ¹⁷ molecules per ml, which corresponds to about 13000 molecules when converted to silica particles having a particle size of 100 nm. In the silica nanoparticles of the present invention, the density in the silica particles can be achieved with a smaller amount of the organic fluorescent dye used than in the conventional method, and as a result, the use of the expensive organic fluorescent dye can be kept low.

In the manufacturing method of this invention, it is preferable that the following process (c) is further included after the said process (b).
(C) Further, tetraalkoxysilane was additionally contained in the reaction solution for hydrolysis, and the resulting silanol, the organosilanol remaining in the reaction solution, and the step (b) were obtained. A step of forming a silica shell layer on the surface of the silica nanoparticles by subjecting the silica nanoparticles to a condensation polymerization reaction.

In the step (b), even when the amide bond compound remains in the reaction solution, when the tetraalkoxysilane concentration in the reaction solution decreases, the precipitation and dissolution of silica in the silica nanoparticles reach an equilibrium, The incorporation of the amide bond compound into the silica nanoparticles can be terminated.
Therefore, the tetraalkoxysilane is newly contained in the reaction solution by the step (c) and the particle formation reaction is restarted, thereby inducing the incorporation of the amide bond compound and the above per silica nanoparticle prepared. The content of functional organic molecules can be increased.

The thickness of the shell formed at this time is preferably thinner from the viewpoint of increasing the content of the functional organic molecule per colloidal weight. However, in order to stably incorporate the functional organic molecules into the particles, a thickness about that size is required. That is, when a functional organic molecule having a size of about 1 nm is incorporated, the thickness of the shell is preferably about 1 to 2 nm.
The amount of the tetraalkoxysilane used as a silane coupling agent necessary to form a 1 nm thick shell varies depending on the particle size of the colloid. For example, when the average particle size of the colloid is 30 nm, the amount of the tetraalkoxysilane used to form a 1 nm thick shell is 10% of the amount of the tetraalkoxysilane used in the step (b). On the other hand, when the average particle diameter of the colloid is 300 nm, the amount of tetraalkoxysilane in the amount of 1% may be used to form a shell having the same thickness.

However, when an organic fluorescent dye is used as the functional organic molecule, the silica shell layer obtained in the step (c) has a thickness that can prevent self-quenching caused by FRET (Fluorescence resonance energy transfer). It is preferable to have.
At the end of the particle formation reaction (silica colloid formation reaction), the concentration of silanol, which is a raw material for silica nanoparticles, is reduced, while the concentration of the functional organic molecules that are not high in efficiency of absorption is not so reduced. Therefore, the concentration of the functional organic molecule is higher in the vicinity of the surface of the silica nanoparticles formed at the end of the silica colloid formation reaction than in the central part. This also applies to the shell layer formed in the step (c), where the functional organic molecule concentration is low inside the shell layer and the concentration is high outside.
If the shell layer is too thin, the distance between the portion where the organic fluorescent dye component near the surface of the silica nanoparticles is unevenly distributed and the portion where the organic fluorescent dye component is unevenly distributed in the adjacent shell layer is too close, and FRET is As a result, self-quenching occurs between the organic fluorescent dye components, resulting in a decrease in fluorescence intensity (see, for example, Physical Review B 73, 245302 (2006)). In order to effectively suppress FRET, it is preferable that the organic fluorescent dyes are separated by an interval of 10 nm or more. Therefore, the thickness of the shell layer is preferably 10 nm or more.
The amount of the tetraalkoxysilane further contained in the reaction solution to form a layer of 10 nm or more varies depending on the particle size of the silica nanoparticles formed at that time, and is not particularly limited. For example, the average particle size is 30 nm. In order to form a 10 nm layer on the silica nanoparticles, 100 to 120 parts by mass is preferable with respect to 100 parts by mass of the tetraalkoxysilane used in the step (b), and 10 nm on silica particles having an average particle diameter of 100 nm. In order to form a layer, 30-36 mass parts is preferable with respect to 100 mass parts of said tetraalkoxysilane used in the said process (b).

Although there is no restriction | limiting in particular as the formation temperature conditions of the said shell layer in the said process (c), It is preferable to carry out on the temperature conditions of 0-60 degreeC. Although there is no restriction | limiting in particular as reaction time, It is preferable to make it react for 1 to 24 hours.
The content of the functional organic molecule per silica nanoparticle obtained may be further increased by repeating the step (c) two or more times in the production method of the present invention. However, it is troublesome to repeat the step (c) many times, and the particle size distribution of the silica nanoparticles synthesized thereby increases, so that the number of repetitions of the step (c) can be increased as much as possible. That's not to say. Actually, in order to form a silica shell layer containing 1 to 6 layers of the functional organic molecules on the surface of the silica nanoparticles, the step (b) is preferably performed 1 to 6 times.

In the production method and the like of the present invention, when a fluorescein dye such as 5 (6) -carboxyfluorescein is used as the functional organic molecule, the silica nanoparticles are prepared in the step (b) or the step (c). Silanol and organosilanol obtained by hydrolyzing organoalkoxysilane having at least one amino group together with the tetraalkoxysilane and the amide bond fluorescein dye obtained in the step (a) at the time of shell formation are subjected to a condensation polymerization reaction. It is preferable to form silica nanoparticles or shells for the following reason.
Fluorescein dyes such as 5 (6) -carboxyfluorescein are known to increase fluorescence intensity in an alkaline environment. In the present invention, by taking advantage of this point and maintaining alkalinity inside the prepared silica nanoparticles, it is possible to achieve higher fluorescence intensity than that achieved by including a fluorescein dye by a conventional method.

Silica particles obtained from a tetraalkoxysilane or the like by a wet method are generally porous, and many of the dye molecules incorporated in the particles are also affected by the solvent. Therefore, most of the dyes inside the colloidal silica particles dispersed in a neutral solvent such as pure water are in a neutral environment, and the fluorescein dyes cannot exhibit strong fluorescence characteristics. On the other hand, when the organoalkoxysilane having the amino group (such as the aforementioned APS or compounds 1 to 3) is added when preparing the silica particles, the inside of the prepared silica nanoparticles can be alkalized. Amino groups tend to draw protons (H ⁺ ) out of the solvent in order to alleviate the uneven distribution of electrons, and the remaining hydroxide ions (OH ⁻ ) remain in the vicinity to make the internal environment of the silica nanoparticles alkaline. Present. The organoalkoxysilane component having this amino group is strongly bonded to the silica component constituting the silica particles by a siloxane bond, and therefore does not flow out by washing the particles.
Examples of the organoalkoxysilane having an amino group include 3-aminopropyltriethoxysilane (APS), the aforementioned compounds 1-3, 3- [2- (2-aminoethylamino) ethylamino] propyl-triethoxysilane, N-2 (aminoethyl) 3-aminopropylmethyldimethoxysilane, 3-aminopropyltrimethoxysilane and the like can be mentioned, and APS is preferable.

The more the amount of the organoalkoxysilane having the amino group in the step (b) or (c) is used, that is, the more the amino group in the silica nanoparticle is, the stronger the inside of the particle becomes more alkaline and the fluorescence intensity of the fluorescein dye. Becomes higher. However, when the amount of the organoalkoxysilane having an amino group is large, many amino groups, that is, positively charged sites are supplied also to the surface of the silica nanoparticles. Silanol groups, which are the remnants of the synthesis reaction, are present as negatively charged sites on the surface of silica particles prepared from tetraalkoxysilane and the like, and if a close number of amino groups are present on the particle surface (belong to different particles). ) Attracting force works between positive and negative charged parts. In that case, the silica particles are aggregated to form an aggregate, and when the degree becomes severe, the colloid is gelled. Even if it does not go so far, the presence of amino groups on the particle surface that can be positively charged sites lowers the negative zeta potential of silica particles and approaches the zero point, which is a factor that reduces the dispersibility in water turn into.
In order to solve this problem, a silica shell layer not containing an amino group may be provided outside the particle surface where the amino group exists.

  The silica shell layer containing no amino group is prepared by subjecting the reaction solution of the step (b) or (c) to a solvent substitution treatment using a new reaction solvent containing no organoalkoxysilane having the amino group. After redispersing the prepared silica nanoparticles, the tetraalkoxysilane is further hydrolyzed, and the resulting silanol and the silica nanoparticles obtained in the step (b) or (c) are subjected to a condensation polymerization reaction. A silica shell layer containing no amino group can be formed on the surface of the silica nanoparticles.

In the present invention, the solvent substitution means that the reaction solution of the step (b) or (c) containing the organoalkoxysilane having an amino group is a new ammonia water containing no organoalkoxysilane having the amino group. Substitution with a solvent means that, in detail, washing operations such as ethanol washing and water washing, vacuum distillation and the like are not performed.
As a specific solvent replacement treatment operation, after the completion of the step (b) or (c), the reaction solution obtained was precipitated by weak centrifugation at 200 × g to 2000 × g for 5 to 20 minutes. Thereafter, the supernatant is immediately removed, and the resulting precipitate is redispersed in the new reaction solvent, or the limit of YM-10, YM-100 (both trade names, manufactured by Millipore), etc. Examples of the method include ultrafiltration using a filtration membrane and then redispersion in the new reaction solvent.
The thickness of the shell is the counter ion in the dispersion corresponding to the positively charged sites of the silica particles (if silica particles with amino groups are dispersed in pure water, the amino group counter ions are hydroxide It is sufficient that the dispersion range of (which becomes ions) is confined inside the sliding surface of the particles, that is, about 10 nm.

  The amount of the organoalkoxysilane having an amino group to be reacted with the tetraalkoxysilane is determined as an amount by which the colloid can be stably maintained when a silica shell layer not containing the amino group is formed. . The amount varies depending on the type of organoalkoxysilane to be used, but when a silica colloid is prepared by combining the APS with the TEOS, the mixed molar ratio of APS to TEOS is preferably 10% or less, preferably 5%. The following is more preferable. Of course, from the viewpoint of increasing the fluorescence intensity of the fluorescein dye, it is preferable that the amount of the organoalkoxysilane having an amino group used is large.

Next, “silica nanoparticle surface modification” will be described.
Silica is generally known to be chemically inert and easy to modify. The silica nanoparticles of the present invention can also easily bind a desired substance to the surface.
In the silica nanoparticles of the present invention, it is preferable to bind or adsorb a substance that recognizes a desired target biomolecule to the surface.
When the silica nanoparticles contain a fluorescent dye molecule or a light absorbing dye molecule as the functional organic molecule, a target biomolecule in a specimen (eg, any cell extract, lysate, medium / culture solution, solution, buffer) (Including physiologically active substances) can be labeled with fluorescent or light absorbing dyes.
Examples of the substance that recognizes the target biomolecule that modifies the surface of the silica nanoparticles include antibodies, antigens, peptides, DNA, RNA, sugar chains, ligands, receptors, chemical substances, and the like.
Here, molecular recognition refers to (1) hybridization between DNA molecules or DNA-RNA molecules, (2) antigen-antibody reaction, (3) reaction between enzyme (receptor) and substrate (ligand), etc. A specific interaction between molecules.

Here, a ligand refers to a substance that specifically binds to a protein, such as a substrate that binds to an enzyme, a coenzyme, a regulatory factor, a hormone, or a neurotransmitter, as well as a low molecular weight molecule or ion. Also includes high molecular weight materials.
The chemical substances are not limited to natural organic compounds, but include artificially synthesized physiologically active compounds and environmental hormones.
That is, a substance that recognizes a target biomolecule whose surface has been modified with silica nanoparticles itself becomes a receptor site, and for example, hybridization using antigen-antibody reaction, biotin-avidin reaction, and complementarity of base sequences. Specific binding to a target biomolecule can be performed using specific molecular recognition such as.
The surface modification such as adsorption by the biomolecule on the surface of the silica nanoparticle of the present invention is performed by mixing the colloid of the silica nanoparticle and the biomolecule solution in the presence or absence of a condensing agent or a crosslinking agent. Is preferably performed.
For example, the biomolecule can be adsorbed on the surface of the silica nanoparticle by mixing the colloid of the silica nanoparticle and the biomolecule solution in the absence of a condensing agent or the like.

Specific examples in the case of using the condensing agent or the crosslinking agent include N- (6-maleimidocaproyloxy) succinimide (EMCS), glutaraldehyde, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC). Etc.
Regarding the reaction conditions such as the number of equivalents of the condensing agent or crosslinking agent used for surface modification, the colloidal dispersion medium, the type and volume of the solvent of the biomolecule solution, and the reaction temperature, there are no particular restrictions as long as the surface modification proceeds. Absent.
After the surface modification, separation of the silica nanoparticles from the biomolecules that are not bound or adsorbed to the silica nanoparticles can be performed by centrifugation or ultrafiltration.
After the surface modification of the silica nanoparticles with the biomolecule, blocking treatment may be performed with an arbitrary blocking agent such as PEG or BSA from the viewpoint of further preventing the above-mentioned non-specific adsorption.
Confirmation of whether the surface modification of the biomolecule has been made can be performed by performing a general protein quantification method (for example, UV method, Lowry method) on the biomolecule contained in the solution obtained by removing particles from the mixed solution by centrifugation or ultrafiltration. , Bradford method), and the amount of the reduced biomolecule can be quantified.

Next, the labeling reagent of the present invention will be described.
The labeling reagent of the present invention uses the silica nanoparticles of the present invention. It is preferable to give a fluorescent or light-absorbing dye label using the silica nanoparticles. Further, the surface of the silica nanoparticle is modified with a substance that recognizes the target biomolecule, such as an antibody or hormone, by modifying the surface of the silica nanoparticle, and the target biomolecule is to be evaluated by an apparatus for detecting optical properties or by visual inspection. It can be used as a labeling reagent that makes it possible to evaluate whether or not it exists.
Specific examples of the labeling reagent of the present invention include a biomolecule detection reagent, a biomolecule quantification reagent, a biomolecule separation reagent, a biomolecule recovery reagent, or an immunostaining reagent.
The target biomolecule can be used as an analytical reagent for detecting, quantifying, separating or recovering. Moreover, when the molecular recognition with the said target biomolecule is an antigen-antibody reaction, it can be set as the reagent for immuno-staining which uses the said silica nanoparticle.

Hereinafter, the present invention will be described in more detail based on examples. The present invention is not limited to these examples.
Example 1 (Preparation of silica nanoparticles of the present invention)
1. Step (a)
Powdered 5 (6) -carboxyfluorescein-NHS ester was dissolved in DMF to a concentration of 10 mM (0.2 ml). The dye solution was divided into two parts, one with APS equivalent to the dye as a comparative example, and the other with N- (2-aminoethyl) -3-aminopropyl equivalent to the dye as an example of the present invention. Triethoxysilane (hereinafter referred to as “AEAPS”) (manufactured by Fluorochem) was added, and the mixture was shaken and stirred at room temperature (25 ° C.) for 1 hour to obtain an amide bond compound. APS, fluorescein-AEAPS.)
The obtained dye solution was diluted 5000 times with a 10 mN aqueous sodium hydroxide solution and light at an optical path length of 1.0 cm (hereinafter the same applies) using an absorptiometer V650 (trade name, manufactured by JASCO Corporation). Photoluminescence (PL) spectrum measurement was performed using an absorption spectrum measurement and a fluorescence spectrophotometer FP-6500 (trade name, manufactured by JASCO Corporation). FIG. 1 is a diagram showing optical absorption spectra and PL spectra of fluorescein-APS and fluorescein-AEAPS in a sodium hydroxide aqueous solution at a concentration of 1 μM. The light absorption is expressed as absorbance (dimensionless), and the unit of PL is expressed as arbitrary unit (au). The same applies hereinafter.
As is clear from FIG. 1, no difference more than the preparation error was observed in the spectral results of both samples.

2. Step (b)
14% by mass of ammonia water was diluted 5-fold with ethanol to prepare a solution for preparing a silica colloid. Into this, TEOS (0.05 ml) for a volume ratio of 0.5% and a dye solution of carboxyfluorescein-APS or fluorescein-AEAPS were added and stirred for 2 hours to perform silica nanoparticle formation reaction.
Here, the fluorescein-APS dye solution and the fluorescein-AEAPS dye solution prepared in the step (a) were used, and each dye was used as an individual sample. The input amount of each dye solution (10 mM) was twice the input amount of TEOS in terms of volume.
After completion of the reaction, the dispersion was placed in a conical tube and centrifuged at 5000 × g for 10 minutes to extract the prepared colloidal silica particles. Thereafter, colloidal washing was performed twice with ethanol and four times with pure water by the same centrifugal separation to obtain silica nanoparticles (10 g / l × 1.3 ml).
The concentration (g / l) of the obtained silica nanoparticles is obtained by measuring a mass obtained by separating a part of the silica nanoparticle dispersion liquid, collecting only the silica nanoparticles contained therein, and drying the silica nanoparticles. (The same applies hereinafter).
The silica nanoparticles obtained after washing had already greatly different color development intensity at that stage, and it was found that fluorescein-AEAPS had much higher uptake efficiency.
Fluorescein-APS-containing silica nanoparticles as comparative examples after washing and fluorescein-AEAPS-containing silica nanoparticles as examples of the present invention were dispersed in pure water, and light absorption spectrum measurement and PL spectrum measurement were performed. FIG. 2 is a diagram showing light absorption spectra and PL spectra (both spectra per silica nanoparticle concentration of 1 g / l) of a fluorescein-APS-containing silica nanoparticle colloidal dispersion and a fluorescein-AEAPS-containing silica nanoparticle colloidal dispersion.
As is clear from FIG. 2, fluorescein-AEAPS as an example of the present invention is incorporated into silica nanoparticles 10 times more in terms of absorbance intensity and 7 times in terms of fluorescence intensity than fluorescein-APS as a comparative example. It was confirmed.
The particle size distribution of the prepared silica nanoparticles was the same at 200 ± 20 nm.

Example 2 (Preparation of silica nanoparticles having a shell layer of the present invention)
1. Step (b)
4% by mass of aqueous ammonia was diluted 5-fold with ethanol, and the entire solution was heated to 50 ° C. Among them, TEOS (0.05 ml) for a volume ratio of 0.5% and 1. The dye solution of fluorescein-AEAPS prepared in the step (a) was added and stirred for 3 hours to carry out a silica nanoparticle formation reaction.
The input amount of the dye solution was twice the input amount of TEOS in terms of volume (the concentration of the dye solution is the same as in Example 1).
2. Step (c): Formation of First Shell Layer Containing APS Thereafter, 25 vol% TEOS and 2 vol% APS were additionally added to the initial TEOS input amount, and stirring was further continued. At this time, heating of the reaction solution was stopped and slow cooling was started, and then the reaction solution temperature was lowered to room temperature (25 ° C.) by 1 hour. Thereafter, stirring was continued for about 20 hours. After completion of the reaction, the dispersion was placed in a conical tube and centrifuged at 10,000 × g for 15 minutes to precipitate the prepared silica nanoparticles at the bottom of the tube. The supernatant containing the remaining pigment and APS was removed to obtain silica nanoparticles having a first shell layer containing APS.
3. Step (c): Formation of second shell layer not containing APS Next, a shell-forming reaction solution comprising 4% by mass ammonia water and ethanol (ammonia water: ethanol = 1: 4) prepared separately was added. The precipitated colloid was redispersed by ultrasonic irradiation. This colloidal dispersion was returned to the reaction vessel again, and 25% by volume of TEOS was added again to the initial TEOS input amount. Then, stirring was continued at room temperature for 2 hours to form a second shell layer having a high TEOS purity. Thereafter, the colloid was extracted and washed with ethanol twice and with pure water four times by the same centrifugation to obtain silica nanoparticles having a second shell layer not containing APS (10 g / l × 1.2 ml). .
The particle size distribution of the prepared silica nanoparticles was the same at 50 ± 10 nm.

4). Optical properties The optical properties of the obtained silica nanoparticles were evaluated.
3 and 4 show colloidal dispersions in which silica nanoparticles (particle size distribution 50 ± 10 nm) having the first shell layer containing APS and the second shell layer not containing APS obtained above are dispersed in pure water. It is a figure which shows the light absorption spectrum and PL spectrum (all are the spectrums per silica nanoparticle density | concentration of 1 g / l) of a liquid (henceforth an alkalized colloid dispersion liquid).
In the figure, as a comparison, a colloidal dispersion (hereinafter referred to as a non-alkalized colloidal dispersion) in which silica nanoparticles (particle size distribution 50 ± 10 nm) containing no shell and having two layers are dispersed in pure water, Light absorption spectrum and PL spectrum (both spectra per 1 g / l of silica nanoparticles) of a colloidal dispersion (hereinafter referred to as alkali dispersion of non-alkalized colloid) dispersed in 10 mN sodium hydroxide aqueous solution (NaOH) Show.
From these results shown in FIGS. 3 and 4, the light absorption spectrum and fluorescence intensity of the alkalized colloidal dispersion changed from the characteristics of the non-alkalized colloid dispersion to the characteristics of the alkali dispersion of the non-alkalized colloid. You can see that you are approaching. From this, it is clear that inclusion of APS in the first shell in the alkalinized colloid realizes alkalinization inside the colloidal particle, and inclusion of APS in the silica particle results in fluorescence of the fluorescein dye. It is confirmed that it has the effect of increasing strength.

5). Evaluation of dispersibility Subsequently, the zeta potential of the colloidal dispersion was measured, and the dispersibility was further evaluated. The zeta potential was measured using Zeta Sizer Nano (trade name; manufactured by Malvern) as a measuring device. Evaluation of dispersibility was performed by observing colloidal particle images with an SEM (scanning electron microscope; manufactured by Hitachi, Ltd.).
FIG. 5 is a diagram showing the zeta potential of silica nanoparticles in pure water. In the figure, a is a peak indicating the zeta potential of silica nanoparticles having a first shell layer containing APS and no second shell layer containing no APS (hereinafter referred to as APS surface uncoated particles), b Is a peak indicating the zeta potential of silica nanoparticles having a first shell layer containing APS and a second shell layer not containing APS (hereinafter referred to as APS surface-coated particles).
From this result shown in FIG. 5, it can be seen that the absolute value of the zeta potential of the APS surface uncoated particles is lower than that of the APS surface coated particles. This is because the negative zeta potential inherent to silica particles has shifted in the positive direction due to the effect of amino groups derived from APS present on the particle surface in the APS surface uncoated particles, but also in the APS surface coated particles. This indicates that the amino group derived from APS is covered with the second shell layer and located inside the particle, and the influence on the zeta potential of the particle is lost.

FIG. 6 is a view showing an SEM image of APS surface-coated particles. The scale bar in FIG. 6 indicates 300 nm (magnification of 100,000 times). In the figure, silica particles that appear white are adjacent to each other, but because the arrangement is flat, this is the result of the particles gathering together when the dispersion medium evaporates and the droplet interface recedes. It can be said that there is. Therefore, it can be seen that the silica particles were dispersed alone in the dispersion.
FIG. 7 is a view showing an SEM image of APS surface uncoated particles. In addition, the scale bar in FIG. 7 shows 300 nm (magnification 100,000 times). In the figure, it can be seen that the silica particles that appear white aggregate to form a three-dimensional network structure. The particles collected by receding the droplet interface cannot have such a three-dimensional structure. Therefore, this is an agglomerated structure already formed when the particles were present in the dispersion medium. It can be said.
From the above SEM observation results, the dispersibility of the particles is lowered when the amino group derived from APS is present on the particle surface, and the dispersibility can be recovered by coating the surface with a silica shell layer containing no amino group. I understand.

Example 3
(Adsorption of antibody to colloid of silica nanoparticles)
1 mL of 50 mM KH ₂ PO ₄ (pH 6.5) and 9 mL of colloid of silica nanoparticles of Example 2 (10 mg / mL) were added to the centrifuge tube, and the mixture was gently stirred. 1 mL (60 μg / mL) of an anti-hCG antibody (Anti-hCG clone codes / 5008, manufactured by Medix Biochemical) was added to the centrifuge tube with stirring, and the mixture was allowed to stand at room temperature for 1 hour. 0.55 mL of 1% by mass of PEG (polyethylene glycol, molecular weight 20000, manufactured by Wako Pure Chemical Industries, Ltd.) was added thereto and stirred gently, and 1.1 mL of 10% BSA was further added and stirred gently.
The mixed solution was centrifuged at 12,000 × g for 15 minutes, and the supernatant was removed leaving about 1 mL, and the precipitate was dispersed in the remaining supernatant. To this dispersion, 20 mL of a storage buffer (20 mM Tris-HCl (pH 8.2), 0.05% PEG 20000, 1% BSA, 0.1% NaN ₃ ) was added, centrifuged again, and the supernatant was about 1 mL. The residue was removed and the precipitate was dispersed in the remaining supernatant (colloid A).

(Molecular recognition test)
Subsequently, 100 μl of silica nanoparticle colloid (colloid A) surface-modified with the anti-hCG antibody was placed in one of the wells of a 96-well microplate. Next, a solution (50 mM KH ₂ PO ₄ , pH 7.0) containing 1 mg / mL of an anti-IgG antibody (Anti Mouse IgG, manufactured by Dako) was prepared. FIG. 8 is a plan view of the strip 1 used in the molecular recognition test. Membrane 4 (Hi-Flow Plus 120 membrane, manufactured by MILLIPORE) coated with the above solution at a coating amount (about 1 mm width) of 0.75 μL / cm in a line shape at a position 3 about 15 mm from one end 2 is 5 mm wide. To be a strip 1 (length 25 mm).
The end of the strip 1 was immersed in a colloid of surface-modified silica nanoparticles with an anti-hCG antibody placed in one of the wells of the 96-well microplate and left for 1 hour.
As is clear from FIG. 8, it was confirmed that the portion 3 where the anti-IgG antibody was applied in a line was colored red, and silica nanoparticles having the surface modified with the anti-hCG antibody were formed. Moreover, it turns out that the silica nanoparticle of this invention is suitable as an analytical reagent.

FIG. 1 is a diagram showing a light absorption spectrum (per concentration of 1 μM) and a photoluminescence (PL) spectrum of fluorescein-APS and fluorescein-AEAPS in an aqueous sodium hydroxide solution. FIG. 2 is a diagram showing light absorption spectra and PL spectra (both spectra per silica nanoparticle concentration of 1 g / l) of a fluorescein-APS-containing silica particle colloid dispersion and a fluorescein-AEAPS-containing silica particle colloid dispersion. FIG. 3 is a diagram showing light absorption spectra of a non-alkalized colloid dispersion and an alkalized colloid dispersion. FIG. 4 is a diagram showing PL spectra of a non-alkalized colloid dispersion and an alkalized colloid dispersion. FIG. 5 is a diagram showing the zeta potential of silica nanoparticles in pure water. FIG. 6 is a view showing an SEM image of APS surface-coated particles. FIG. 7 is a view showing an SEM image of APS surface uncoated particles. FIG. 8 is a plan view of the strip used in the molecular recognition test of Example 3. FIG.

Claims (7)

The manufacturing method of the silica nanoparticle containing a functional organic molecule characterized by including the following process (a) and (b).
(A) The amino group and amide of the organoalkoxysilane having at least one amino group represented by the following general formula 1 and at least one positively chargeable site: And (b) hydrolyzing the amide bond compound and tetraalkoxysilane obtained in step (a), respectively, and subjecting the resulting organosilanol and silanol to condensation polymerization reaction, The process of preparing the silica nanoparticle to contain.
General formula 1
H ₂ NR ¹ Si (OR ² ) ₃
(In the formula, R ¹ is a divalent substituent having at least one positively chargeable moiety, and is a single bond with each of the amino group (H ₂ N) and the silicon atom (Si) in the formula 2 Represents a valent substituent, and OR ² represents an alkoxyl group having 1 to 6 carbon atoms.) The method for producing silica nanoparticles containing functional organic molecules according to claim 1, further comprising the following step (c) after the step (b).
(C) Further, tetraalkoxysilane is further added to the reaction solution to cause hydrolysis, and the resulting silanol, the organosilanol remaining in the reaction solution, and the silica nanoparticle obtained in the step (b) are obtained. A step of forming a silica shell layer on the surface of the silica nanoparticles by subjecting the particles to a condensation polymerization reaction. The functional organic molecule is an organic dye molecule, and R ^{1 of the} organoalkoxysilane is a divalent branched hydrocarbon group having 5 carbon atoms having an amino group or a divalent hydrocarbon having 5 carbon atoms having a secondary amine. The method for producing silica nanoparticles according to claim 1, wherein the method is a linear hydrocarbon group.   The functional organic molecule is 5 (6) -carboxyfluorescein, and the hydrolysis and polycondensation reaction in the steps (b) and (c) is performed in the presence of ammonia in a water-ethanol mixed solvent. The manufacturing method of the silica nanoparticle of any one of Claims 1-3.   The silanol obtained by hydrolyzing an organoalkoxysilane having at least one amino group together with the tetraalkoxysilane during the preparation of the silica nanoparticles in the step (b) or the shell formation in the step (c) The method for producing silica nanoparticles according to claim 4, wherein organosilanol is subjected to a condensation polymerization reaction.   The silica nanoparticle obtained by the manufacturing method of any one of Claims 1-5.   A labeling reagent prepared using the silica nanoparticles according to claim 6.