WO2010004774A1 - Silicon nanoparticle suspension, and labeling agent for biological substance - Google Patents

Abstract

Disclosed is a silicon nanoparticle suspension which is applicable in vivo because any fluorinated compound is removed therefrom, and which has excellent brightness. Also disclosed is a labeling agent for a biological substance, which is safe for living bodies and can be produced by using the silicon nanoparticle suspension. The silicon nanoparticle suspension is produced through the steps of: producing a silicon oxide film containing a silicon atom on a base plate; subjecting the silicon oxide film to a heat treatment (annealing); subjecting the resulting silicon oxide film to the treatment with hydrofluoric acid; and removing a fluorine ion from hydrofluoric acid and separating the base plate, wherein the total content of fluorinated compounds in the silicon nanoparticle suspension is 100 ppm or less in terms of a fluorine ion content.

Description

Silicon nanoparticle suspension and biological material labeling agent

The present invention relates to a silicon nanoparticle suspension and a biological material labeling agent that is safe for a living body using the silicon nanoparticle suspension.

The silicon nanoparticle phosphor is manufactured by performing high-frequency sputtering, and then performing heat treatment and HF treatment (see, for example, Patent Documents 1 and 2). In these patents, in order to remove fluoride ions remaining on the surface of the silicon oxide film containing silicon nanoparticles by HF treatment, a method of immersing in a solution is employed.

Also, as quantum dots, compound semiconductors such as III-IV compound semiconductors and II-IV compound semiconductors are widely known. However, these compounds have a great impact on the environment, and it is necessary to give sufficient consideration to handling and disposal during use. For this reason, in consideration of the environment and living organisms, silicon quantum dots are used that are superior in safety and have less environmental impact than these compound semiconductors.

However, conventionally, in the silicon nanoparticle suspension manufacturing process, attention is not paid to impurities remaining due to acid treatment, etc., and it does not mean safety when used as a suspension, only particles. (See, for example, Patent Document 3).

By simply immersing the silicon nanoparticle-containing silicon oxide film after the HF treatment in the solution, the fluoride adsorbed physically on the surface can be sufficiently removed, but the fluorine that has penetrated into the film. The chemicals are not removed. Therefore, after that, the silicon nanoparticle is taken out as a suspension, and a dangerous level of fluoride is left to be used for biolabeling. In other words, it is extremely dangerous for a living body to use a silicon nanoparticle suspension synthesized by a high-frequency sputtering method as a fluorescent substance for biomarking.

In addition, when a fluorescent substance is used for labeling a biological substance, there is a problem that it is not clearly understood how much fluoride is contained and how harmful it is for a living body.
JP 2006-70089 A Japanese Patent Application Laid-Open No. 2004-296781 JP 2005-172429 A

Accordingly, an object of the present invention is to provide a silicon nanoparticle suspension that can be applied in vivo by removing the fluoride and has excellent emission luminance, and further uses the silicon nanoparticle suspension. Thus, a biological material labeling agent that is safe for the living body is provided.

The above object of the present invention is achieved by the following configuration.

1. A process of producing a silicon atom-containing silicon oxide film on a substrate, subjecting the silicon atom-containing silicon oxide film to heat treatment (annealing), hydrofluoric acid treatment, removing fluorine ions from the hydrofluoric acid, and separating the substrate The silicon nanoparticle suspension manufactured in (1), wherein the total content of fluoride in the silicon nanoparticle suspension based on fluorine ions is 100 ppm or less.

2. 2. The silicon nanoparticle suspension according to 1 above, wherein the total content of fluoride in the silicon nanoparticle suspension based on the fluorine ions is 50 ppm or less.

3. 3. The silicon nanoparticle suspension according to 2 above, wherein the total content of fluoride in the silicon nanoparticle suspension based on the fluorine ions is 15 ppm or less.

4. 4. The silicon nanoparticle suspension according to any one of 1 to 3, wherein the step of removing fluorine ions from the hydrofluoric acid is immersed in high-temperature and high-pressure water.

5. 5. The silicon nanoparticle suspension according to 4 above, wherein the temperature in the high-temperature and high-pressure water is 100 to 300 ° C. and the pressure is 0.1 to 10 MPa.

6. 6. The silicon nanoparticle suspension according to 5, wherein the temperature is 200 to 250 ° C. and the pressure is 0.1 to 10 MPa.

7. 7. The silicon nanoparticle suspension according to 6 above, wherein the temperature is 200 to 250 ° C. and the pressure is 1 to 5 MPa.

8. 8. A biological material labeling agent, wherein the silicon nanoparticle suspension according to any one of 1 to 7 and a molecular labeling material are bonded via an organic molecule.

According to the present invention, it is possible to provide a silicon nanoparticle suspension that can be applied in vivo by removing the fluoride and has excellent emission luminance, and further to the living body using the silicon nanoparticle suspension. It was possible to provide a safe biological material labeling agent.

Hereinafter, the present invention will be described in detail.

The silicon nanoparticle suspension of the present invention is characterized in that the total content of fluoride in the silicon nanoparticle suspension based on fluorine ions is 100 ppm or less.

The silicon nanoparticle suspension of the present invention is generally obtained through the following steps.

(A) A silicon atom-containing silicon oxide film is formed on a semiconductor substrate (for example, a silicon substrate) using a high-frequency sputtering method,
(B) heat-treating (annealing) the silicon atom-containing silicon oxide film to form silicon nanoparticles in the silicon oxide film;
(C) The silicon oxide film is treated with hydrofluoric acid to remove the silicon oxide to expose the silicon nanoparticles,
(D) The exposed silicon nanoparticles are immersed in pure water and subjected to a stirring process to remove adhering hydrofluoric acid and separate the semiconductor substrate to obtain a suspension of silicon nanoparticles.

In the present invention, high-temperature and high-pressure treatment of pure water is preferably performed in place of the immersion and stirring treatment in pure water in (d) above. Specifically, for example, it is preferable to use an autoclave. The temperature is preferably 100 to 300 ° C, more preferably 200 to 250 ° C. The pressure is preferably from 0.1 to 10 MPa, more preferably from 1 to 5 MPa.

Note that this treatment is not limited to the inside of the autoclave as long as it can be immersed in high-temperature and high-pressure water. High-temperature, high-pressure water includes so-called subcritical and supercritical water, regardless of gas or liquid state.

Regarding the temperature and time of the heat treatment (annealing) in the above (b), it is preferably 1000 to 1150 ° C. for 45 to 90 minutes, more preferably 1000 to 1100 ° C. for 50 to 80 minutes.

In the hydrofluoric acid treatment in the above (c), a hydrofluoric acid aqueous solution or hydrofluoric acid vapor is used, but hydrofluoric acid vapor is preferred, and the temperature and time of the hydrofluoric acid vapor to be exposed are preferably 3 to 30 at 30 to 60 ° C. Minutes, more preferably at 35 to 50 ° C. for 5 to 15 minutes.

The formation of the silicon atom-containing silicon oxide film is performed by, for example, a high-speed sputtering method (for example, Japanese Patent Application Laid-Open No. 2004-296781).

In addition, forming a silicon atom-containing silicon oxide film on a semiconductor substrate is generated by (1) evaporating the opposing raw material semiconductor by the first high-temperature plasma generated between the electrodes and generating an electrodeless discharge in a reduced-pressure atmosphere. It is also possible to pass through the second high temperature plasma (for example, Japanese Patent Laid-Open No. 6-279015) and (2) laser ablation method (for example, Japanese Patent Laid-Open No. 2004-356163).

In a high-speed sputtering apparatus, argon gas is introduced into a vacuum chamber, the argon gas is ionized, and the ionized argon ions are converted into a silicon chip and quartz glass (a silicon chip is formed on a quartz glass as a predetermined material). The atoms and molecules emitted from the target material are deposited on the semiconductor substrate to form a silicon atom-containing silicon oxide (SiO _x ) film.

Examples of the target material in the high-speed sputtering method include silicon chip and quartz glass, and silicon nanoparticles having various particle sizes can be obtained by controlling the area ratio of the silicon chip and quartz glass. The area ratio between the silicon chip and the quartz glass is in the range of 1 to 50%.

The particle size can also be controlled by changing the high-frequency power and gas pressure, which are sputtering conditions. The high frequency power is 10 to 500 W, and the gas pressure is 1.33 × 10 ⁻² to 1.33 × 10 Pa.

The average particle size of the silicon nanoparticles according to the present invention is preferably 1 nm or more and 10 nm or less. More preferably, they are 3 nm or more and 8 nm or less, Especially preferably, they are 3.5 nm or more and 6 nm or less.

In the silicon nanoparticle suspension produced by the method for producing a silicon nanoparticle suspension of the present invention, the content of fluoride in the silicon nanoparticle suspension based on fluorine ions is 100 ppm or less, preferably 50 ppm or less, More preferably, it is 15 ppm or less. When the content of the fluoride is 100 ppm or less, the biological safety of the biological material labeling agent prepared from the silicon nanoparticle suspension is improved, and the emission luminance of the silicon nanoparticle suspension itself is also improved.

Fluoride content of the silicon nanoparticle suspension is measured using a fluorine ion meter (manufactured by Toko Chemical Co., Ti-5101).

The silicon nanoparticle suspension of the present invention can be applied to a biological material labeling agent. Further, by adding the silicon nanoparticle labeling agent (biological material labeling agent) of the present invention to a living cell or living body having a target (tracking) substance, the target substance binds or adsorbs to the conjugate or adsorbent. By irradiating excitation light of a predetermined wavelength and detecting fluorescence of a predetermined wavelength generated from the fluorescent semiconductor fine particles according to the excitation light, fluorescence dynamic imaging of the target (tracking) substance can be performed. That is, the biomaterial labeling agent of the present invention can be used for bioimaging methods (technical means for visualizing biomolecules constituting the biomaterial and dynamic phenomena thereof).

Since the surface of the above-mentioned silicon nanoparticles is generally hydrophobic, for example, when used as a biological material labeling agent, the water dispersibility is poor as it is, and there is a problem that particles aggregate, It is preferable to hydrophilize the surface of the silicon nanoparticles.

As a method of hydrophilization treatment, for example, there is a method of chemically and / or physically binding a surface modifier to the particle surface after removing the lipophilic group on the surface with pyridine or the like. As the surface modifier, those having a carboxyl group and an amino group as hydrophilic groups are preferably used, and specific examples include mercaptopropionic acid, mercaptoundecanoic acid, aminopropanethiol and the like. Specifically, for example, 10 ⁻⁵ g of Si / SiO ₂ type nanoparticles are dispersed in 10 ml of pure water in which 0.2 g of mercaptoundecanoic acid is dissolved, and stirred at 40 ° C. for 10 minutes to treat the surface of the shell. By doing so, the surface of the shell of the inorganic nanoparticles is modified with a carboxyl group.

The biosubstance labeling agent is obtained in a structure in which at least the above-described hydrophilized silicon nanoparticles and the molecular labeling substance that binds to the biomolecule at the end are bound. An organic molecule having a functional group that becomes a linking group and binds to the molecular labeling substance may be interposed between the hydrophilic silicon nanoparticles and the molecular labeling substance.

The biological material labeling agent can label the biological material by specifically binding and / or reacting with the target biological material. Examples of the molecular labeling substance include nucleotide chains, antibodies, proteins, and cyclodextrins.

In the biological substance labeling agent, silicon nanoparticles subjected to hydrophilization treatment and a molecular labeling substance are bound by organic molecules. The organic molecule is not particularly limited as long as it is an organic molecule capable of binding silicon nanoparticles and a molecular labeling substance. For example, albumin, myoglobin, casein, and the like are part of the protein, and are a kind of protein. It is also preferable to use avidin together with biotin.

Especially, it is used at the end that binds to the molecular labeling substance. Further, in order to bind to the molecular labeling substance, a binding functional group, for example, an amino group or a carboxyl group may have a mercapto group or a maleimide group at the terminal. Furthermore, you may have a link group which connects the terminal by the side of a silicon nanoparticle and the terminal by the side of a molecule | numerator in an organic molecule. The link group is not particularly limited as long as it is organic, but preferably has hydrophilicity, may have a hydrophilic functional group, and an ethylene glycol chain is also preferably used. The form of bonding is not particularly limited, and examples thereof include covalent bonding, ionic bonding, hydrogen bonding, coordination bonding, physical adsorption, and chemical adsorption. A bond having a strong bonding force such as a covalent bond is preferable from the viewpoint of bond stability.

As a specific example, silicon nanoparticles are hydrophilized with mercaptoundecanoic acid and biotin is used as an organic molecule. In this case, the carboxyl groups of the hydrophilized silicon nanoparticles are preferably covalently bonded to biotin, Is further selectively bound to a molecular labeling substance (such as an antibody) to which avidin is bound to obtain a biological substance labeling agent.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

Example 1
(Film formation by sputtering)
Ar gas is introduced into the vacuum chamber, and Ar gas ions ionized by the high-frequency controller are collided with a target material made of Si chip and quartz glass. These emitted atoms and molecules are deposited on a semiconductor substrate to form a silicon oxide film in which silicon atoms are mixed in the silicon oxide film.

(Annealing treatment)
The obtained silicon atom-containing silicon oxide film containing silicon atoms is rapidly heated to 1000 ° C. in an Ar atmosphere and subjected to heat treatment to agglomerate the silicon atoms in the film to nano size to oxidize silicon nanoparticles. A silicon film is formed. Here, the heat treatment time is 60 minutes, and the temperature is 1000 ° C.

(Hydrofluoric acid treatment)
The obtained silicon oxide film containing silicon nanoparticles was exposed to a hydrofluoric acid vapor at 40 ° C. for 10 minutes.

(Cleaning process)
The cleaning treatment using high-temperature and high-pressure water was performed in an autoclave. The temperature was raised to 300 ° C., compressed air was introduced from the outside, and the pressure was 1 MPa. The processing time is 10 minutes.

Examples 2-7
In Example 1, cleaning treatment was performed in the same manner except that the temperature and pressure described in Table 1 were used.

Example 8 (comparison)
Residual fluorine ions were removed by immersing the silicon oxide film containing silicon nanoparticles after hydrofluoric acid treatment in pure water at 25 ° C. for 10 minutes.

(Heat oxidation treatment)
The silicon nanoparticle-containing silicon oxide film after cleaning is naturally oxidized. Natural oxidation is the preservation of a silicon oxide film containing silicon nanoparticles in air.

(Separation of silicon nanoparticles and dispersion in liquid)
A silicon nanoparticle suspension can be obtained by introducing a silicon oxide film containing naturally oxidized silicon nanoparticles into a solution and performing ultrasonic treatment for 10 minutes. The brightness | luminance of the silicon nanoparticle suspension shown in Table 1 is the brightness | luminance measured after this process. A 146 nm vacuum ultraviolet lamp (manufactured by Ushio) was used as a light source, a sample was set in a vacuum chamber, irradiated from a fixed distance at a vacuum degree of 1.33 × 10 Pa, and excitation luminescence was measured with a luminance meter. The luminance value is shown as a relative value when the sample of Example 8 is 100.

(Production of silicon nanoparticle labeling agent)
Silicon nanoparticle suspension prepared in Example 1-8, to 10ml of pure water were dissolved mercaptoundecanoic acid 0.2 g, redispersed 1 × ^{10 -5} g min (equivalent amount), 40 ° C., By stirring for 10 minutes, nanoparticles having a hydrophilic surface were obtained.

Thereafter, 25 mg of avidin was added to each aqueous solution of silicon nanoparticles whose surface was hydrophilized, and stirred at 40 ° C. for 10 minutes to prepare avidin-conjugated nanoparticles.

The obtained avidin-conjugated nanoparticle solution was mixed with a biotinylated oligonucleotide with a known base sequence and stirred to prepare an oligonucleotide labeled with the nanoparticle.

When the above labeled oligonucleotides were dropped and washed on a DNA chip on which oligonucleotides having various base sequences were immobilized, the oligonucleotides on the DNA chip having base sequences complementary to the labeled oligonucleotides were washed. Only the spot was confirmed to emit light of different colors depending on the particle size of the silicon nanoparticles by ultraviolet irradiation.

From this, it was confirmed that oligonucleotide labeling with silicon nanoparticles according to the present invention was possible.

(Biological safety test)
The biosafety of the silicon nanoparticle labeling agent is evaluated using trypan blue staining. A 0.3% trypan blue solution is diluted with PBS (−) and placed in a test board together with the silicon nanoparticle labeling agent and HERA cells. By counting the number of cells stained blue, the ratio of viable cells after trypan blue staining is calculated. The higher the percentage of living cells, the higher the safety.

As shown in Table 1, by obtaining the silicon nanoparticle suspension having the structure of the present invention, it is excellent in luminance and high detectability is obtained as a biological material labeling agent. Moreover, since the proportion of living cells is high in the biological safety test, an excellent biological substance labeling agent can be provided.

Claims (8)

A process of producing a silicon atom-containing silicon oxide film on a substrate, subjecting the silicon atom-containing silicon oxide film to heat treatment (annealing), hydrofluoric acid treatment, removing fluorine ions from the hydrofluoric acid, and separating the substrate The silicon nanoparticle suspension produced in (1), wherein the total content of fluoride in the silicon nanoparticle suspension based on fluorine ions is 100 ppm or less. 2. The silicon nanoparticle suspension according to claim 1, wherein the total content of fluoride in the silicon nanoparticle suspension based on the fluorine ions is 50 ppm or less. 3. The silicon nanoparticle suspension according to claim 2, wherein the total content of fluoride in the silicon nanoparticle suspension based on the fluorine ions is 15 ppm or less. The silicon nanoparticle suspension according to any one of claims 1 to 3, wherein the step of removing fluorine ions from the hydrofluoric acid is immersed in high-temperature and high-pressure water. The silicon nanoparticle suspension according to claim 4, wherein the temperature in the high-temperature and high-pressure water is 100 to 300 ° C and the pressure is 0.1 to 10 MPa. 6. The silicon nanoparticle suspension according to claim 5, wherein the temperature is 200 to 250 ° C. and the pressure is 0.1 to 10 MPa. The silicon nanoparticle suspension according to claim 6, wherein the temperature is 200 to 250 ° C and the pressure is 1 to 5 MPa. A biological material labeling agent, wherein the silicon nanoparticle suspension according to any one of claims 1 to 7 and a molecular labeling material are bonded via an organic molecule.