JP2009280841A - Film containing semiconductor nanoparticles, semiconductor nanoparticles and biological labeling agent using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a film containing semiconductor nanoparticles, capable of achieving a luminescence of substantially uniform, high luminescence intensity regardless of particle size, and to provide the semiconductor nanoparticles and a biological labeling agent using the semiconductor nanoparticles. <P>SOLUTION: The film containing the semiconductor nanoparticles is formed by a sputtering method, has a film thickness of 0.5-10 μm and contains crystallized semiconductor nanoparticles of 5.0×10<SP>6</SP>to 1.0×10<SP>8</SP>pieces per μm<SP>3</SP>. The semiconductor nanoparticles are taken out from the film. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a semiconductor nanoparticle-containing film, a semiconductor nanoparticle characterized by being taken out into a dispersion medium, and a biological material labeling agent using the semiconductor nanoparticle.

  Recent advances in nanotechnology suggest the possibility of using nanoparticles for detection, diagnosis, sensing and other applications. In addition, nanoparticle complexes that interact with biological systems have recently gained widespread interest in the fields of biology and medicine. These complexes are considered promising as new intravascular probes for both sensing (eg imaging) and therapeutic purposes (eg drug delivery).

  In general, a material that exhibits a quantum confinement effect in a nanometer-sized semiconductor material is called a “quantum dot”. Such a quantum dot is a small lump within about 10 and several nanometers in which several hundred to several thousand semiconductor atoms are gathered, but when absorbing energy from an excitation source and reaching an energy excited state, the energy of the quantum dot Releases energy corresponding to the band gap. Therefore, it is considered that by adjusting the size or material composition of the quantum dots, it is possible to adjust the energy band gap and use energy in various levels of wavelength bands.

  However, quantum dots have a crystal structure and the property that the band gap changes depending on the particle size, and the emission wavelength changes with the change of the band gap. This leads to spectral variations. In order to avoid this, there is a fundamental problem such as complicated operations such as classifying particles of a single spectrum.

By the way, silicon (Si) is the material most often used as an electronic material, and has been highly expected as an element for producing nanometer-level devices. Various methods for producing Si nanoparticles have been studied and proposed. Has been. For example, Patent Documents 1 and 2 disclose a method of extracting Si nanoparticles by producing a Si atom group in a SiO ₂ film by sputtering and performing a heat treatment and hydrofluoric acid process.

However, in this method, there is a problem that it is difficult to obtain a certain light emission intensity by changing the number density of Si atoms in the SiO ₂ film and the particle diameter of the Si nanoparticles.

On the other hand, research and development in this semiconductor nanoparticle-related technology field, especially research and development aimed at application in the fields of biology and medicine, has just begun, and there are many problems to be solved. (For example, refer to Patent Documents 3 and 4).
JP 2006-70089 A JP 2007-63378 A JP 2004-99349 A JP-A-2005-314408

  The present invention has been made in view of the above problems, and a solution to the problem is to provide a semiconductor nanoparticle-containing film and semiconductor nanoparticles in which the emission intensity is almost uniform and high emission can be obtained regardless of the particle size. That is. Moreover, it is providing the biological material labeling agent using the said semiconductor nanoparticle.

  The above-mentioned problem according to the present invention is solved by the following means.

1. It is a semiconductor nanoparticle containing film | membrane formed by sputtering method, Comprising: Film thickness is 0.5-10 micrometers, The crystallized semiconductor nanoparticle is 5.0 * 10 < ⁶ > -1.0 * in the said film | membrane. A semiconductor nanoparticle-containing film containing 10 ⁸ particles / μm ³ .

  2. A semiconductor nanoparticle extracted from the semiconductor nanoparticle-containing film according to 1 above into a dispersion medium.

  3. 3. The semiconductor nanoparticle according to 2, wherein the semiconductor nanoparticle is extracted from the semiconductor nanoparticle-containing film according to 1 and contains at least one of silicon (Si) or germanium (Ge).

  4). 4. The semiconductor nanoparticle according to 2 or 3, wherein the semiconductor nanoparticle is extracted from the semiconductor nanoparticle-containing film according to 1 above, and the surface thereof is hydrophilized.

  5). 2. A biological material labeling agent, comprising a semiconductor nanoparticle extracted from the semiconductor nanoparticle-containing film according to the above item 1 and a molecular labeling substance bonded via an organic molecule.

  6). 6. The biological substance labeling agent according to 5 above, wherein the molecular labeling substance is a nucleotide chain.

  7. 7. The biological material labeling agent according to 5 or 6, wherein the organic molecule that binds the semiconductor nanoparticles and the molecular labeling material is biotin and avidin.

  By the above means of the present invention, it is possible to provide a semiconductor nanoparticle-containing film and semiconductor nanoparticles in which the emission intensity is substantially uniform and high emission can be obtained regardless of the particle diameter. In addition, a biological material labeling agent using the semiconductor nanoparticles can be provided.

The semiconductor nanoparticle-containing film of the present invention is a semiconductor nanoparticle-containing film formed by a sputtering method, and has a film thickness of 0.5 to 10 μm. 0.0 × 10 ^{6 to} 1.0 × 10 ⁸ pieces / μm ^{3 are} contained. This feature is a technical feature common to the inventions according to claims 1 to 7.

  In the present invention, the semiconductor particles according to the present invention are preferably semiconductor nanoparticles extracted from the semiconductor nanoparticle-containing film into a dispersion medium. Moreover, it is preferable that the said semiconductor nanoparticle is an aspect containing at least one of silicon (Si) or germanium (Ge).

  The semiconductor nanoparticles according to the present invention can be applied to a biological material labeling agent. For this purpose, the surface of the semiconductor nanoparticles is preferably hydrophilized.

  When the semiconductor nanoparticles according to the present invention are applied to a biological material labeling agent, the biological material labeling agent in a mode in which the semiconductor nanoparticles and the molecular labeling material are bonded via an organic molecule is preferable. In that case, it is preferable that the molecular labeling substance is a nucleotide chain. Moreover, it is preferable that the organic molecule which couple | bonds the said semiconductor nanoparticle and a molecular labeling substance is biotin and avidin.

  Hereinafter, the present invention, its components, and the best mode and mode for carrying out the present invention will be described in detail.

(Semiconductor nanoparticles)
The semiconductor nanoparticles according to the present invention can be formed using various semiconductor materials.

  Examples of the semiconductor material that can be used in the present invention include the following. For example, a semiconductor compound of Group IV, II-VI, and III-V of the periodic table of elements can be used.

  Among the II-VI group semiconductors, MgS, MgSe, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, ZnS, ZnSe, ZnTe, CdS, CdSe, HgS, HgSe and HgTe can be mentioned.

  Among group III-V semiconductors, GaAs, GaN, GaPGaSb, InGaAs, InP, InN, InSb, InAs, AlAs, AlP, AlSb, and AlS can be cited.

  Among the Group IV semiconductors, Ge, Pb and Si can be mentioned, but Ge and Si are more preferable, and Si is most suitable because it has almost no toxicity.

(Semiconductor nanoparticle-containing film and semiconductor nanoparticle production method)
The semiconductor nanoparticle-containing film of the present invention and the semiconductor nanoparticles of the semiconductor nanoparticles are preferably produced by a production method having the following steps.
Step (1): Step of producing a film containing semiconductor nanoparticles in a gas and performing a heat treatment Step (2): Step of taking out the semiconductor nanoparticles produced in (1) above into a liquid Step (3): Above The step of applying surface modification to the semiconductor nanoparticles prepared in (1) or the semiconductor nanoparticles taken out in the dispersion medium in (2) The above step (1) is performed by, for example, silicon oxide (Si / SiO ₂ sputtering). A step of forming a Si atom group in the SiO ₂ ) film and a step of producing Si nanoparticles by heat treatment of the atom group.

  Specifically, an inert gas (for example, Ar gas) is introduced into the vacuum chamber, and the inert gas (Ar gas) ions ionized by the high-frequency controller are collided with the target material made of Si chip and quartz glass. These released atoms and molecules are deposited on a semiconductor substrate to form a film in which Si atoms are mixed in the silicon oxide film. Next, the obtained silicon oxide film containing Si atoms is rapidly heated to a predetermined temperature in an inert gas (Ar gas) atmosphere and subjected to heat treatment to aggregate the Si atoms in the film to a desired particle size. Let

The step (2) includes a step of applying Si nanoparticles in the SiO ₂ film to hydrofluoric acid vapor and a step of extracting particles by applying ultrasonic waves.

  Specifically, surface treatment is performed by exposing the obtained silicon oxide film containing Si nanoparticles to hydrofluoric acid vapor at about 40 ° C. Next, natural oxidation or overheating oxidation treatment is performed. Thereafter, the silicon nanoparticle-containing silicon oxide film is put into a dispersion medium (for example, ethanol) and subjected to ultrasonic treatment for a predetermined time.

  In the step (2), various known solvents can be used as the dispersion medium, and alcohols such as ethyl alcohol, sec-butyl alcohol, and t-butyl alcohol, pure water, and the like can be used.

  The step (3) includes a step of applying a nanoparticle oxide film and surface modification.

  In the step (3), a double bond-containing compound having a functional group having bioaffinity at the terminal can be used for the surface modification. In the present invention, various well-known capping agents can be used. For example, allylamine, diallylamine, vinylpyridine, vinylpyrrolidone, vinylpyrrolidine, vinylcarbazolevinylimidazole, N-vinylacetamide and the like are preferable.

In the present invention, the semiconductor nanoparticle-containing film is a semiconductor nanoparticle-containing film formed by sputtering, and has a film thickness of 0.5 to 10 μm. Crystallized semiconductor nanoparticles are contained in the film. , 5.0 × 10 ^{6 to} 1.0 × 10 ⁸ pieces / μm ³ .

If the number of semiconductor nanoparticles in the film is 5.0 × 10 ⁶ or less, the number of semiconductor constituent atoms (for example, Si atoms) is too small, so the number of particles per unit volume varies depending on the location in the film. End up. On the other hand, when the density is 1.0 × 10 ⁸ or more, the density of semiconductor constituent atoms (for example, Si atoms) in the film is too high, and it is difficult to obtain crystal particles and it is difficult to obtain uniform emission intensity.

In the present invention, the method of controlling the number of semiconductor nanoparticles in the film is controlled in the sputtering process so that the distribution of semiconductor nanoparticle constituent atoms (for example, Si atoms) present in the film is uniform. . This is performed by controlling the composition ratio (Si / SiO ₂ ratio) of the target in the sputtering process, the sputtering time, the atmospheric gas (eg, Ar gas) pressure, the heat treatment temperature, and the heat treatment time. In the case of Si nanoparticles, the Si / SiO ₂ ratio is 3 to 30%, and the Ar gas pressure is adjusted within the range of 0.2 to 1.5 Pa, thereby adjusting the Si particle density in the film. Can be controlled. Moreover, a film thickness can be controlled by performing sputtering time for 60 to 300 minutes.

  In addition, the film prepared above is heat-treated so that the temperature is uniformly applied three-dimensionally, and the heat treatment temperature at that time requires a high temperature of 1000 ° C. or higher for crystallization, Perform between 1200 ° C. The heat treatment time is between 30 minutes and 120 minutes. By controlling the heat treatment time, the number of crystallized nanoparticles can be controlled within the above range.

  In the present invention, the film thickness of the semiconductor nanoparticle-containing film can be measured with a commercially available film thickness measuring device, for example, a stylus type surface shape measuring device Dektak 6M (manufactured by ULVAC). The lower limit of the film thickness is preferably 0.1 μm, more preferably 0.5 μm, from the viewpoint of mechanical strength and light absorption ability. On the other hand, the upper limit value of the film thickness is preferably 10 μm in the film formation and the hydrofluoric acid treatment solution treatment after the film formation.

The method for measuring (counting) the number of semiconductor nanoparticles is to take out a cross-section of the produced film, observe it with a high-resolution transmission electron microscope (HR-TEM), and measure it from a TEM observation image. The number of crystallized semiconductor nanoparticles with visible lattices is counted. The observation surface is obtained by cutting out the film surface / film cross section, observing 50 nm × 50 nm, taking out the number, and converting the number in 1 μm ³ .

  The semiconductor nanoparticles in the film are extracted by applying the prepared semiconductor nanoparticle-containing film to a hydrogen fluoride solution, dissolving the film, and then extracting the particles into water or ethanol by ultrasonic treatment.

  The emission intensity of the dispersion of semiconductor nanoparticles extracted from the semiconductor nanoparticle-containing film was measured with a Hitachi spectrofluorometer F-7000 (manufactured by Hitachi High-Technologies).

  In the present invention, the particle size of the semiconductor nanoparticles can be controlled by repeating the oxidation and hydrofluoric acid treatment on the surface of the semiconductor nanoparticles. At that time, more precise control is possible by changing the heating temperature and the heating time.

  The average particle diameter of the semiconductor nanoparticles is preferably 1 to 10 nm from the viewpoint of production of a semiconductor nanoparticle label with enhanced luminescent color and biomolecule detectability. Furthermore, the average particle size is preferably in the range of 1 to 8 nm. The emission color of the semiconductor nanoparticle phosphor is determined by the particle diameter, and the smaller the particle diameter, the shorter the wavelength. Therefore, from the viewpoint of preventing mixing of various emission colors, the standard deviation of the particle size distribution of the semiconductor nanoparticle phosphor is preferably 20% or less with respect to the average particle size.

  In the present invention, the average particle size of the semiconductor nanoparticles must originally be determined in three dimensions, but it is difficult because it is too fine, and in reality it must be evaluated with a two-dimensional image. Therefore, a transmission electron microscope (TEM) is required. It is preferable to obtain the average by taking a large number of images by changing the shooting scene of the electron micrograph using. Therefore, in the present invention, the average particle diameter is a diameter obtained by taking an electron micrograph using a TEM, measuring a cross-sectional area of a sufficient number of particles, and setting the measured value as an area of a corresponding circle. Obtained as the diameter, the arithmetic average was taken as the average particle diameter. The number of particles photographed with a TEM is preferably 20 or more, and more preferably 100 particles are photographed.

  In the present application, “the light emission intensity is uniform” means that the difference is ± 20% with respect to a certain reference light emission. In the case of more than that, the difference in emission intensity is too large and varies for each particle size.

  The fact that the emission intensity of the semiconductor nanoparticles is uniform regardless of the emission wavelength can be suitably used for applications such as LEDs and displays.

  In addition, the solution in which the semiconductor nanoparticles according to the present invention are dispersed can be used for applications such as bioanalysis and medical fields such as a label because of uniform emission intensity at multiple wavelengths.

  The light source of the excitation light is not limited as long as it satisfies the desired wavelength and intensity conditions. Various lasers such as a Ne laser and various LEDs can be used.

  The wavelength of the excitation light depends on the type and particle size of the semiconductor nanoparticles, but usually 200 to 1000 nm is used.

(Application example)
The semiconductor nanoparticles of the present invention can be applied to single molecule analysis in various technical fields by taking out from the film. For example, in the single molecule observation method, multiple types of molecules may be identified simultaneously by labeling multiple types of molecules with semiconductor nanoparticles having different emission spectra and irradiating the molecules with excitation light. it can. The applicable types of molecules include structural isomers having the same chemical composition but different chemical structures.

  In the following, typical application examples will be described.

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

[Hydrophilic treatment of semiconductor nanoparticle aggregates]
Since the surface of the semiconductor nanoparticles described above is generally hydrophobic, for example, when used as a biological material labeling agent, the water dispersibility is poor as it is, and there are problems such as aggregation of particles. The surface of the semiconductor nanoparticles is preferably subjected to a hydrophilic treatment.

As a hydrophilic treatment method, 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 / amino group as a hydrophilic group are preferably used, and specific examples include mercaptopropionic acid, mercaptoundecanoic acid, aminopropanethiol and the like. Specifically, for example, 10 ⁻⁵ g of Ge / GeO 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 can be modified with a carboxyl group.

[Biological substance labeling agent]
The biological material labeling agent according to the present invention can be obtained by bonding the above-described hydrophilic treated semiconductor nanoparticles, the molecular labeling substance and the organic molecule.

<Molecular labeling substance>
The biological material labeling agent according to the present invention enables the labeling of the biological material by specifically binding and / or reacting with the target biological material.

  Examples of the molecular labeling substance include nucleotide chains, antibodies, antigens and cyclodextrins.

<Organic molecule>
In the biological material labeling agent according to the present invention, hydrophilic semiconductor nanoparticles and a molecular labeling substance are bound by an organic molecule. The organic molecule is not particularly limited as long as it is an organic molecule capable of binding a semiconductor nanoparticle and a molecular labeling substance. For example, among proteins, albumin, myoglobin, casein, etc., and avidin, which is a kind of protein, are used together with biotin. It is also preferably used. The form of the bond is not particularly limited, and examples thereof include a covalent bond, an ionic bond, a hydrogen bond, a coordination bond, 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.

  Specifically, when the semiconductor nanoparticles are hydrophilized with mercaptoundecanoic acid, avidin and biotin can be used as organic molecules. In this case, the carboxyl group of the hydrophilized nanoparticle is preferably covalently bonded to avidin, avidin is further selectively bonded to biotin, and biotin is further bonded to a molecular labeling substance to form a biological material labeling agent.

<Example 1>
(Film formation by sputtering)
Ar gas (0.5 Pa) was introduced into the vacuum chamber, and Ar gas ions ionized by the high frequency controller were collided with a target material made of Si chip and quartz glass. These released atoms and molecules were deposited on a semiconductor substrate to form a film in which Si atoms were mixed in the silicon oxide film. At this time, the amount of Si / SiO ₂ was adjusted as shown in Table 1.

(Annealing treatment)
The obtained silicon oxide film containing Si atoms was rapidly heated to a temperature shown in Table 1 in an Ar atmosphere and subjected to heat treatment to aggregate the Si atoms in the film to a nano size. The heat treatment time is shown in Table 1.

(Hydrofluoric acid treatment)
The obtained silicon nanoparticle-containing silicon oxide film was subjected to surface treatment by exposing it to 40 ° C. hydrofluoric acid vapor.

(Heat oxidation treatment)
The silicon oxide film containing Si nanoparticles after hydrofluoric acid treatment was subjected to natural oxidation and overheating oxidation treatment. The temperature and time of the heat oxidation treatment are shown in Table 1.

(Separation of Si nanoparticles and dispersion in liquid)
The silicon oxide film containing silicon nanoparticles subjected to natural oxidation or superheat oxidation was put into ethanol and subjected to ultrasonic treatment for 10 minutes.

(Measurement of emission spectrum)
The obtained Si nanoparticles dispersed in ethanol were irradiated with excitation light having a wavelength of 350 nm, and the generated fluorescence spectrum was measured. The emission spectrum was performed using a Hitachi Fluorometer F-7000. For near-infrared light emission with an emission wavelength of 700 nm or more, measurement was performed using a spectrometer (SWNIR) with Hamamatsu UV-VIS as a light source.

  The measurement results are shown in Table 1. Note that the emission intensity was measured according to Sample No. The relative light intensity of 2 was used as a reference.

  As is clear from the results shown in Table 1, it can be seen that the emission intensity of the sample according to the present invention is almost uniform and high regardless of the particle diameter. Similar results were obtained when germanium (Ge) was used instead of silicon (Si).

<Example 2>
Sample No. obtained in Example 1 above. 1 semiconductor nanoparticle: 25 mg of avidin was added to an ethanol dispersion of 1.0 × 10 ⁻⁵ mol / l and stirred at 40 ° C. for 10 minutes to prepare an avidin conjugate nanoparticle.

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

  When the above labeled (labeled) oligonucleotide is dropped and washed on a DNA chip on which oligonucleotides having various base sequences are immobilized, the oligonucleotide has a complementary base sequence to the labeled (labeled) oligonucleotide. Only the spot of was emitted with excitation light of 810 nm.

  This confirmed the labeling (labeling) of the oligonucleotide with the nanoparticles. That is, it can be seen from this result that a biological material labeling agent using the semiconductor nanoparticles according to the present invention can be provided.

Claims (7)

It is a semiconductor nanoparticle containing film | membrane formed by sputtering method, Comprising: Film thickness is 0.5-10 micrometers, The crystallized semiconductor nanoparticle is 5.0 * 10 < ⁶ > -1.0 * in the said film | membrane. A semiconductor nanoparticle-containing film containing 10 ⁸ particles / μm ³ . A semiconductor nanoparticle extracted from the semiconductor nanoparticle-containing film according to claim 1 in a dispersion medium. 3. The semiconductor nanoparticle according to claim 1, wherein the semiconductor nanoparticle is extracted from the semiconductor nanoparticle-containing film according to claim 1 and contains at least one of silicon (Si) and germanium (Ge). . 4. The semiconductor nanoparticle according to claim 2, wherein the semiconductor nanoparticle is extracted from the semiconductor nanoparticle-containing film according to claim 1 and the surface thereof is subjected to a hydrophilic treatment. A biological substance labeling agent, wherein the semiconductor nanoparticles extracted from the semiconductor nanoparticle-containing film according to claim 1 and a molecular labeling substance are bonded via an organic molecule. The biological substance labeling agent according to claim 5, wherein the molecular labeling substance is a nucleotide chain. The biological substance labeling agent according to claim 5 or 6, wherein the organic molecule that binds the semiconductor nanoparticles and the molecular labeling substance is biotin and avidin.