JP4854547B2 - Silver fine particle and nucleic acid complex and method for producing the same - Google Patents

Description

  The present invention relates to a complex of silver fine particles and a nucleic acid and a method for producing the same. The present invention also relates to a fluorescent stain containing the complex used in the bio field.

As a method of identifying a specific tissue by fluorescent staining of a biological sample, there is a technique using a low molecular fluorescent dye itself, a polymer fine particle containing or bound with a fluorescent dye, or a light emitting semiconductor fine particle (quantum dot) (for example, Non-patent document 1: NANOTECHNOLOGY 16 (2): R9-R25 FEB 2005).
However, when an organic dye is used, there is a problem of fading of the fluorescent dye due to excitation light, and in the case of fluorescent polymer fine particles having a particle size of 100 nm or less, there is a problem that the fluorescence intensity is insufficient. Semiconductor fine particles that emit light even with a particle size of several nanometers are attracting a great deal of attention because they are small in size and have a high quantum yield. However, since cadmium is used, the problem is that they are highly toxic to cells. (For example, Non-Patent Document 2: Langmuir, 23, 1974-1980, (2007)).

On the other hand, when a molecule is adsorbed on the surface of an aggregate of metal nanoparticles such as gold, silver, or copper, the Raman scattering cross section of the adsorbed molecule is enhanced by about 10 ⁶ to 10 ¹⁴ times as compared with a normal Raman scattering cross section, which is strong. It has been clarified by laser spectroscopic measurement that it shows Raman scattering (for example, Non-Patent Document 3: J. Raman Spectrosc. 36, 3421-3427 (2005)).
However, a single metal nanoparticle obtained by a conventional production method hardly shows strong Raman scattering or light emission, which is not preferable as a staining agent when a biological sample is observed with an optical microscope. In addition, in the case of a conventional method for producing fine metal particles, it is difficult to remove a reducing agent, a dispersing agent, and the like, and it cannot be used as a stain for a biological sample from the viewpoints of luminescence and safety (for example, non-patent) Sentence 4: J. Phys. Chem. 86, 3391-3395 (1982); Non-Patent Document 5: Mater. Lett. 60, 834-838 (2006)).

NANOTECHNOLOGY 16 (2): R9-R25 FEB 2005 Langmuir, 23, 1974-1980, (2007) J. Raman Spectrosc. 36, 3421-3427 (2005) J. Phys. Chem. 86, 3391-3395 (1982) Mater. Lett. 60, 834-838 (2006)

  Under the circumstances as described above, there is a demand for metal fine particles having properties such as fluorescence exceeding a necessary level even with a relatively small particle size, little influence of fading and low toxicity, and a method for producing the same.

  The present inventors have found that a complex formed by combining a specific silver fine particle and a nucleic acid satisfies the above-mentioned properties, and a method by which such a complex can be easily produced. completed. That is, the present invention provides the following composite of silver fine particles and nucleic acid, a method for producing the same, a fluorescent staining agent containing the complex, and the like.

(1) A complex of silver fine particles having a diameter of 5 nm to 500 nm and a nucleic acid.
(2) The composite according to (1), wherein the diameter is 5 nm to 50 nm.
(3) The composite according to (1) or (2), wherein the silver fine particles have a plurality of crystal structures.
(4) The composite according to (3), wherein the silver fine particles have a mosaic structure.
(5) The complex according to any one of (1) to (4), wherein the nucleic acid is DNA or RNA consisting of 5 to 50 bases containing adenine and / or guanine.
(6) The complex according to (5), wherein the nucleic acid is single-stranded DNA or RNA containing adenine or guanine.
(7) The complex according to (6), wherein the nucleic acid is selected from 5 to 50-mer of adenine.
(8) The complex according to (6) above, wherein the nucleic acid is selected from guanine 5 to 50-mers.
(9) The complex according to any one of (5) to (8), wherein the DNA or RNA has a functional group at its terminal.
(10) The complex according to (9), wherein the functional group is biotin or thiol.
(11) The complex according to any one of (1) to (10), wherein the silver particles and the nucleic acid are complexed in a state where the nucleic acid is present inside and / or outside of the silver fine particles.
(12) The complex according to any one of (1) to (11), wherein the surface is covered with the nucleic acid.
(13) The complex according to any one of (1) to (12), which shows Raman scattering.
(14) The complex according to (13), which exhibits fluorescence due to an interaction between the nucleic acid and the silver fine particles.
(15) A fluorescent staining agent comprising the complex according to any one of (1) to (14) above.
(16) A method for producing a complex of silver fine particles having a diameter of 5 nm to 500 nm and a nucleic acid, using a solution containing silver ions and a nucleic acid.
(17) The method according to (16) above, wherein silver ions are reduced in a solution containing the silver ions and the nucleic acid.
(18) The method according to (17), wherein the reduction is performed by adding a reducing agent.
(19) The method according to (17) above, wherein the reduction is performed by ultraviolet irradiation.
(20) A silver particle and nucleic acid complex obtained by the method according to any one of (16) to (19) above.

The complex according to a preferred embodiment of the present invention has advantages such as exhibiting fluorescence more than a necessary level even with a relatively small particle size, little influence of fading, and low toxicity. Therefore, such a complex is suitable as a fluorescent stain used in the bio field.
Further, according to the method for producing a complex of the present invention, a complex of silver fine particles and nucleic acid having the above properties can be easily produced.

1. SUMMARY OF THE INVENTION The present invention relates to a silver fine particle / nucleic acid complex, a method for producing the same, a fluorescent stain containing the complex, and the like.
In order to control the emission wavelength of ordinary fluorescent dyes and luminescent particles, it is necessary to control the molecular structure, particle size, and the like. However, since the silver fine particle / nucleic acid complex of the present invention utilizes light emission (Raman scattering) that causes a wavelength shift with respect to excitation light, there is no need to perform special structure control. In addition, since nucleic acid (for example, DNA) is used as a dispersant, the biocompatibility is high and cell death is hardly induced even when introduced into cells.
In the production method of the present invention, usually, nucleic acid (DNA, RNA, etc.), which is a biopolymer, and silver ions are dissolved in an aqueous solution, and silver ions are reduced by reducing the silver ions with a reducing agent or light irradiation. Produces a complex that is complexed with DNA. Nucleic acid is likely to interact with silver ions, and becomes an effective component as a dispersant and a protective agent when producing a nucleic acid / silver fine particle complex. At the same time, silver fine particles and nucleic acid alone do not exhibit fluorescence, but the combination of nucleic acid and silver fine particles provides strong luminescence (Raman scattering and charge transfer fluorescence) to provide highly biocompatible luminescent fine particles. be able to.

2. Silver fine particle and nucleic acid complex The first aspect of the present invention relates to a silver fine particle and nucleic acid complex having a diameter of 5 nm to 500 nm.
The complex of the present invention is a substantially spherical complex formed by complexing silver fine particles and nucleic acid, and has a diameter of 5 to 500 nm, preferably 5 to 50 nm, and more preferably 10 to 20 nm.
Here, the silver fine particles contained in the composite may be made of a single crystal lattice or may have a plurality of crystal structures. Also included are fine particles in which a plurality of crystals are fused in a mosaic pattern. Here, the mosaic shape is a state in which crystals are gathered without regularity, and partly refers to a state in which a part of nucleic acid enters between crystals.

Examples of the nucleic acid complexed with the silver particles provided by the present invention include DNA, RNA, phosphodiester derivatives thereof, PNA, 2-O-methyl RNA, oligonucleotide, peptide nucleic acid, and the like. Is not to be done.
The nucleic acid used in the present invention is preferably DNA or RNA, particularly 2 or more containing adenine and / or guanine, preferably 5 to 100, more preferably 5 to 50, and still more preferably 10 to 30. DNA or RNA consisting of a single base is preferred. The DNA or RNA used here may be single-stranded or double-stranded.
The nucleic acid used here is not limited to this, but preferably contains a large amount of adenine and / or guanine having a purine base structure. Therefore, the nucleic acid used here is preferably 50% or more, more preferably 60% or more, more preferably 70%, more preferably 80% or more (converted in terms of the number of nucleotides) of the constituting nucleotides. And / or guanine.
Further, from the viewpoint of preparing a more homogeneous complex, it is preferable that all constituent nucleotides are single nucleotides. Therefore, a nucleic acid in which all nucleotides are composed of any one of adenine, guanine, thymine and cytosine can be preferably used in the present invention.
The nucleic acid particularly preferably used in the present invention is selected from adenine or guanine 5-50 mer (preferably 10-30 mer, more preferably 10-20 mer).

When the complex of the present invention is used as a fluorescent stain, it is preferable to bind an antibody that recognizes a target protein. For this purpose, a functional group such as biotin or thiol is preferably bonded to the end of the nucleic acid in the complex.
The composite of the present invention is substantially spherical. The sphericity of this complex is not particularly limited, but is preferably 0.7 or more, more preferably 0.8 or more.
In the complex according to a preferred embodiment of the present invention, the silver particles and the nucleic acid are complexed in a state where the nucleic acid is present inside and / or outside the silver fine particles. However, in order to reduce cytotoxicity, it is desirable that the surface of the silver fine particles be covered with nucleic acid. The extent to which the nucleic acid covers the surface of the silver fine particles is not particularly limited as long as the cytotoxicity can be reduced to a desired level. Preferably, in the complex of the present invention, 30% or more, more preferably 50% or more, and further preferably 70% or more of the surface of the silver fine particles is covered with the nucleic acid.

3. Next, a method for producing the composite of the present invention will be described.
The present invention provides a method for producing the above-mentioned complex of silver fine particles and nucleic acid using a solution containing silver ions and nucleic acid. More specifically, in the production method of the present invention, a nucleic acid such as DNA or oligonucleotide is dissolved in water or a buffer solution and mixed with an aqueous solution containing silver ions such as an aqueous silver nitrate solution. Silver fine particles coated with nucleic acid and complexed with nucleic acid can be prepared by irradiating the mixed aqueous solution with light such as ultraviolet rays or reducing with a reducing agent.

As a compound which produces | generates silver ion required for preparation of the silver fine particle of this invention, silver chloride, silver sulfate, silver nitrate, silver phosphate, silver sulfite, silver nitrite, silver phosphite etc. can be mentioned. Of these, silver nitrate can be preferably used. The concentration of these aqueous solutions is preferably 10M to 10mM, but 10mM to 100mM is preferable in view of cost and the like.

  These compounds that produce silver ions are used as aqueous solutions. The compound is preferably a sulfuric acid aqueous solution, a hydrochloric acid aqueous solution, or a Tris-HCl buffer solution, and particularly preferably a Tris-HCl buffer solution.

  Various reducing agents for precipitating silver from silver ions can be used, but reducing agents that are stable in aqueous solution such as sodium borohydride, dimethylamine borane, hydrazine, sodium ascorbate, sodium citrate, etc. It is preferable to use an aqueous solution of The concentration of the reducing agent aqueous solution is not particularly limited, but is usually 100 to 100 mM.

Reduction by light for precipitating silver can be performed by ultraviolet rays, but as an ultraviolet irradiation method, it is preferable to irradiate ultraviolet rays having a wavelength in the region from 254 nm to 365 nm as a main wavelength. For example, a low pressure U V lamp having a dominant wavelength of 254 nm, a high pressure U V lamp having a dominant wavelength of 365 nm, or the like can be used. Although there is no restriction | limiting in particular in the irradiation amount and irradiation time of an ultraviolet-ray, less than 1 hour is preferable, More preferably, it is less than 10 minutes.
It is also possible to obtain silver fine particles by reduction with a nitrogen gas laser (337 nm, 10 ns pulse).

4). Fluorescent staining agent of the present invention The complex of the present invention having the structure as described above exhibits fluorescence (particularly Raman scattering) due to the interaction between nucleic acid and silver fine particles. Therefore, such a complex can be used as a fluorescent stain (or fluorescent labeling agent) used in the bio field. When used as a fluorescent stain, as described above, a functional group such as biotin or thiol is bound to the end of the nucleic acid in the complex, and an antibody that recognizes the target protein is bound to the functional group. Keep it. The fluorescent staining agent of the present invention can be used according to a known technique (nature biotechnology, 21 (1) 41-46 (2003).
The silver fine particle-nucleic acid complex prepared above is stably dispersed and dissolved in water, and can be safely taken up by cells to exhibit biocompatibility. When light irradiation is performed, the silver fine particles are colored even when taken up by cells. In particular, fine particles in which the structure of particles formed inside forms a mosaic shape have enhanced Raman scattering at hot spots sandwiched between the particles, and the Raman scattering does not quench even if the exposure is continued for a long time. Therefore, the fluorescent staining agent of the present invention can be used, for example, as an imaging reagent for cell staining, a staining reagent for gel electrophoresis, a reagent used in a fluorescence quantification method for proteins, and the like.

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

Example 1: Preparation of microparticles using dA20 (deoxyadenine 20-mer) Gel filtration-purified oligonucleotide dA20 (deoxyadenine 20-mer) Tris-HCl buffer aqueous solution so that the unit conversion (base number concentration) is 10 mM. (10 mM Tris-HCl (pH 7.8, 20 ° C.)). A silver nitrate aqueous solution AgNO ₃ (Wako Pure Chemical Industries) was used to prepare 10 mM.
DA20 and an aqueous silver nitrate solution were mixed at Nucleobase (base): Ag ⁺ ion = 1: 1 (molar ratio), and diluted with a 10 mM Tris-HCl buffer aqueous solution so that the final concentration was 100 μM. 1 ml of this mixed aqueous solution was placed in a 4 ml glass bottle. Ultraviolet rays were irradiated for 5 minutes from the top of the bottle using a UV (ultraviolet) spot light source ([LIGHTNINGCURE ^™ L8868, HAMAMATSU], 300 μW / cm ² , 360 nm).

Example 2 Structural Evaluation of Fine Particles After UV irradiation shown in Example 1, a UV-Vis absorption spectrum [UV-1650PC, SHIMADZU] of the solution was measured.
As a result of irradiating ultraviolet light into a buffer solution containing silver ions and dA20, new absorption was observed at around 410 nm (FIG. 1). Since this coincides with the already known plasmon absorption of silver nanoparticles, the formation of silver nanoparticles was confirmed from a buffer solution containing silver ions and dA20.
The shape was observed with a transmission electron microscope (TEM) [HD-2000, HITACHI]. (FIG. 2) As a result, it was revealed that nanoparticles having a particle size of about 20 nm were formed. .
Furthermore, as a result of elemental analysis of fine particles using EDAX (energy dispersive X-ray fluorescence spectrometer), a peak derived from Ag3d was observed. From this, it was revealed that the prepared fine particles were silver nano-particles.

Example 3: Internal structure of fine particles FIG. 3 shows an electron micrograph in which the internal structure and surface state of the silver nanoparticles formed in Example 1 were observed in more detail. A plurality of silver lattice images were confirmed in the silver nanoparticles shown in FIG. Furthermore, a diffraction image as shown in (c) was obtained from the silver nanoparticles shown in FIG. This confirmed the presence of silver crystals inside the fine particles. Usually, in silver nanoparticles prepared by a usual method such as citrate reduction method, the crystal lattice of the whole particle should be aligned in a certain direction, but the silver nanoparticles prepared by this method have a lattice orientation It depends on the location. That is, the fine particles produced this time are not particles composed of a single crystal, but are a plurality of silver nanocrystals inside the particles, which are mosaic-like particles that are aggregated to form a single particle.
Further, when the particle surface is seen, a protective layer having a thickness of 3-4 nm is present (FIG. 3 (b)). From this, it is considered that the oligonucleotide covers the surface of the silver nanoparticles.
In the shaded portion of the photograph, fluctuations due to charging caused by electron irradiation were observed, which revealed the presence of non-conductive components. That is, it was suggested that dA20 which is a non-conductive component is contained in the fine particles.
From the above results, it was suggested that the prepared fine particles were fine particles in which dA20 and a plurality of silver nanocrystals were combined.

Example 4: Measurement of surface state of fine particles The surface charge state of fine particles formed in Example 1 was measured using a laser zeta potentiometer [ELS-8000, Otsuka Electronics]. As a result, the zeta potential was -24.77 mV, indicating that the surface was negatively charged (Figure 4). This causes electrostatic repulsion between the particles, thereby maintaining the dispersed state without causing the fine particles to aggregate. The negative charge is thought to be due to the phosphate group of dA20.
In order to examine whether dA20 is present on the surface of the silver nanoparticles, the following experiment was conducted. Add 200 μl (100 μM) of dT20 or poly (U) with a base complementary to adenine and 200 μl (100 μM) of dG20, which is a non-complementary base, to 200 μl (100 μM) of a silver nanoparticle dispersion prepared using dA20 for 5 minutes at room temperature. Left alone. 10 μl of each solution was dropped onto a cleaned glass substrate and dried in a nitrogen gas atmosphere. The sample was observed with a fluorescence microscope [BX51, U-LH100HGAPO, Olympus] and a microscope camera [DP71, Olympus]. The objective lens was UplanAPO 100x / 1.35 oil Iris and observed with dark field illumination.
When the produced silver nanoparticles were observed (FIG. 5 (a)), dark field images of the individual silver nanoparticles were observed, and no aggregates were observed. Next, when dG20, which is a 20-mer of guanine that does not form a hydrogen bond with adenine, was added to silver nanoparticles, the same dispersion state was observed (FIG. 5 (b)). On the other hand, when dT20, poly (U), which is a continuous sequence of thymine which is a base complementary to adenine, was added, a large number of aggregates were observed as shown in FIGS. 5 (c) and 5 (d). This suggests that the nucleobase (adenine) of dA20 protrudes from the surface of the silver nanoparticle, and as a result of the formation of hydrogen bonds between thymine and adenine, the fine particles aggregated. This revealed that not only the phosphate group of dA20 but also a nucleobase was present on the outermost surface of the silver nanoparticle.

Example 5: Evaluation of luminous characteristics of fine particles In order to examine the luminous characteristics of the silver nanoparticles prepared in Example 1, the following experiment was conducted. 10 μl of 200 μl (100 μM) of a silver nanoparticle dispersion prepared using dA20 was dropped on a cleaned glass substrate and dried in a nitrogen gas atmosphere. The sample was observed by B excitation, G excitation, and U excitation with a fluorescence microscope [BX51, U-LH100HGAPO, Olympus] and a microscope camera [DP71, Olympus]. (The objective lens is UplanAPO 100x / 1.35 oil Iris) The results are shown in FIG. 6, and light emission from the fine particles was observed at any excitation light wavelength.
From this, it became clear that the microparticles produced this time emit light corresponding to a wide range of excitation wavelengths.

Example 6: Identification of emission origin of fine particles It is found from the UV absorption spectrum that the silver fine particles prepared in Example 1 have almost no absorption in the wavelength band of G excitation. Therefore, in order to identify the origin of luminescence, microspectroscopy of the emission spectrum from the fine particles was performed. The sample was measured with the glass substrate fixed. An argon ion laser (oscillation wavelength 514 nm) was used as an excitation light source. A laser was introduced into the microscope, and the sample was excited by focusing with an objective lens. The scattered light of the excitation light laser was cut with a filter, and the light emitted from the sample was collected with an optical fiber and then introduced into a spectrometer. The spectrally separated light was detected with a CCD camera to obtain a spectrum (FIG. 7).
In this spectrum, broad emission of 530 nm to 570 nm and sharp peaks of 534 nm (Raman shift 730 cm ⁻¹ ) and 552 nm (Raman shift 1320 cm ⁻¹ ) were observed. This sharp peak is attributed to Raman scattering of adenine.
From this, it became clear that the light emission from the fine particles was fluorescence and Raman scattering as a result of the interaction between adenine and silver fine particles. Since Raman scattering is observed at a wavelength shifted from the excitation light, it is observed at any excitation wavelength.

Example 7: Evaluation of fading property of fine particles Next, the fading property of silver fine particles prepared in Example 1 was examined.
Commercially available fluorescent semiconductor particles (EviDots ^TM CdSe / ZnS core-shell type nanoparticles)
And compared. A sample in which EviDots ^™ was dropped onto a glass substrate in the same manner as the silver fine particles was prepared. EviDots ^™ , a silver fine particle sample, was irradiated with B excitation (470-495 nm) for 10 minutes under a microscope. Thereafter, the emission intensity was examined using each excitation light. As a result, the fluorescence intensity of EviDots ^™ was remarkably attenuated after UV irradiation, whereas no decrease in fluorescence intensity was observed for the silver fine particles. This is thought to be due to the fact that the light emission of the silver nanoparticle contains a component due to Raman scattering, so that fading does not easily occur and is less likely to fade than the fluorescent semiconductor fine particle.

Example 8: Toxicity test to cells Next, a cytotoxicity test of the silver fine particles prepared in Example 1 will be described.
The prepared microparticles were dropped onto HeLa cells and cultured in a serum-free medium at 5% CO ₂ and 37 ° C. for 6 hours. Thereafter, the solution was washed with PBS to remove unnecessary fine particles, and observed with a confocal laser scanning microscope (FV300, Olympus).
The result is shown in FIG. Green luminescence was observed in the extended HeLa cells. From this, it was confirmed that the produced silver fine particles were taken into the cells and emitted light even inside the cells.
Further, after the silver fine particle solution was dropped, the culture was performed for 24 hours.
When the cells were observed after culturing for 24 hours, it was observed that the cells were dividing and proliferating (FIG. 10). From this, it was clarified that the silver fine particles are hardly cytotoxic because they are protected by a biopolymer called dA20.

Comparative Example 1: Silver fine particles prepared by adding citric acid
36 mg of silver nitrate was dissolved in 200 ml of ultrapure water and boiled with stirring. 4 ml of an aqueous solution of sodium citrate prepared to 1% by weight was added and stirred for 10 minutes while boiling to produce silver nanoparticles. An electron micrograph (TEM) of the silver nanoparticles thus produced is shown in FIG. As shown in FIG. 11, when produced by citric acid reduction, both the particle shape and shape were non-uniform. Moreover, the internal structure like the silver nanoparticle produced in Example 1 was not seen.

Example 9: Effect of solution on silver nanoparticle formation (1) Preparation of reagent and solution
Oligonucleotide dA20, which is an adenine 20mer, was prepared as DNA. Further, aqueous silver ion solution was prepared by Silver nitrate (AgNO ₃₎ so as to 10 mM using the Wako Pure Chemical] ultrapure water.
As a buffer solution, 10 mM Tris-HCl aqueous solution pH 7.8, 20 ° C, pH 7.0, pH 9.0, 20 ° C were prepared. Other solutions include 10 mM Bis-Tris buffer aqueous solution (Bis (2-hydroxoyethyl) iminotirs- (hydroxymethyl) methane, pH 5.8, 20 ° C), 10 mM phosphate buffer aqueous solution (Sodium dihydrogenphosphate dehydrate, Sodium phosphate dibasic basic dodecahydrate, pH 6.8, 20 ° C), 10 mM 2-aminoethanol (AE) aqueous solution (2-Aminoethanol, pH 8.1, 20 ° C), 10 mM 2-dimethylaminoethanol (DMAE) aqueous solution (N, N-Dimethylethanolamine, pH 8.0, 20 ° C) A trimethylol ethane (TME) aqueous solution (Trimethylol ethane, pH 5.8, 20 ° C.) was prepared. The pH of Bis-Tris buffered aqueous solution, AE aqueous solution, and DMAE aqueous solution was adjusted using hydrochloric acid. The structure of Tris, Bis-Tris, AE, DMAE, and TME is shown in FIG.

(2) UV irradiation experiment Prepare a mixed aqueous solution in which adenine: Ag ⁺ ion = 1: 1 (molar ratio) is diluted with ultrapure water or various buffered aqueous solutions to a final concentration of 100 μM, and 1 ml is put into a 4 ml glass bottle. I put it in. Each was irradiated with ultraviolet rays from the top of the bottle for 5 minutes using a UV spot light source ([LIGHTNINGCURE ^™ L8868, HAMAMATSU], 300 μW / cm ² , 360 nm).

(3) Results of photoreduction in photoreduced ultrapure water in various solutions are shown in FIGS. 13 (a) and 13 (b), and results of photoreduction in a 10 mM Tris-HCl buffer aqueous solution are shown in FIG. It is shown in c) and (d). FIGS. 13A and 13C show AgNO ₃ only, and FIGS. 13B and 13D show solutions in which AgNO ₃ and dA20 are added. In the solutions of FIGS. 13 (a) and (b), no change in absorption spectrum is observed even after irradiation with ultraviolet light, and it is clear that silver nanoparticles are not formed even in the presence of the oligonucleotide dA20 in ultrapure water. became.
In contrast, when photoreduction was performed in an aqueous Tris-HCl buffer solution, a peak derived from plasmon absorption was observed around 410 nm regardless of the presence or absence of dA20, so silver ions were reduced and silver nanoparticles were formed. it seems to do.
Next, the reason why the silver ion photoreduction occurs in the Tris-HCl buffer aqueous solution may be that the amine contained in the structure of Tris acted as a reducing agent by light irradiation. Therefore, it was examined whether photoreduction proceeds using various molecules having a structure similar to Tris shown in FIG.
The results are shown in FIG. Since AE, DMAE, and TME have no buffering capacity, experiments were conducted after adjusting the pH so that diammine silver (I) complex was not formed. When photoreduction is performed in 2-aminoethanol (AE) and 2-dimethylaminoethanol (DMAE) with amine (Fig. 14 (a), (b)), the difference between primary amine and tertiary amine is Regardless, silver nanoparticles were formed. On the other hand, in trimethylolethane (TME) containing no amine (FIG. 14 (c)) and in an aqueous phosphate buffer solution (FIG. 14 (d)), the absorption spectrum changes before and after UV irradiation. Since it is not seen, it is considered that photoreduction of silver ions has not progressed. From this, it was clarified that the amine contained in the solution acts as a reducing agent and can reduce silver ions by irradiating UV light as a mechanism of photoreduction.

(4) pH dependence in photoreduction Next, the effect of the pH of the solution was examined. The results of changing the pH of the Tris-HCl buffer aqueous solution are shown in FIG. Bis-Tris was substituted because it exceeded the buffer capacity of Tris-HCl at pH 5.8. When a compound containing an amine such as Tris (see FIG. 12) is present, the silver ion takes three states depending on pH.
1. Ag ⁺
2. Ag ₂ O 2Ag ⁺ + 2OH ^- _⇔ Ag ₂ O + 2H ₂ O
^{_{3. Ag (NH 3) + Ag}} 2 O + 4NH 3 (aq) + H 2 O ⇔ 2 [Ag (NH 3) 2] + + 2OH -
From FIG. 15 (b), strong plasmon absorption around 410 nm was observed under the conditions of pH 5.8 and pH 7.0 as in the case of pH 7.8. Moreover, although it was broad also in the solution of pH 9.0, 410 nm plasmon absorption was seen, and formation of the silver nanoparticle was confirmed.
From this, it became clear that the production of luminescent silver nanoparticles requires an aqueous solution containing an amine and a buffer solution, and does not depend on pH.

Example 10: Preparation of microparticles with various nucleobases (1) Preparation of reagents and solutions All oligonucleotides (dA5, dT5, dA20, dC20, dG20, dT20, dA50, dT50) were gel filtered as molecules containing nucleic acids. The 3′-terminal biotinylated oligonucleotide (3′-biotin-dA20) (FIG. 16) was prepared by HPLC purification. In addition, mononucleotide (AMP, CMP, GMP, TMP), polynucleotide (Poly (A), Poly (C), Poly (G), Poly (U)), double-stranded DNA (λDNA), circular DNA (M13mp8) RFIDNA) was used without purification. These were all prepared using a Tris-HCl buffer aqueous solution (10 mM Tris-HCl (pH 7.8, 20 ° C.)) so that the base concentration was 10 mM.
The silver ion aqueous solution was prepared with ultrapure water to 10 mM using silver nitrate.

(2) Preparation of metal nanoparticles
A mixed aqueous solution was prepared for each DNA by diluting DNA and an aqueous silver nitrate solution with a 10 mM Tris-HCl buffer aqueous solution so that the base number: Ag ⁺ ion = 1: 1 (molar ratio) and a final concentration of 100 μM.
1 ml of this solution was placed in a 4 ml glass bottle. Each was irradiated with ultraviolet rays from the top of the bottle for 5 minutes using a UV spot light source ([LIGHTNINGCURE ^™ L8868, HAMAMATSU], 300 μW / cm ² , 360 nm).

(3) Effects due to differences in nucleotide sequences
FIG. 17 shows the results of UV-Vis absorption spectra when oligonucleotides having the base sequences of dA20. dC20, dG20, and dT20 were used. When the spectra were compared, strong plasmon absorption near 410 nm was observed when dA20 and dG20 were used. On the other hand, plasmon absorption was also observed in dC20 and dT20, although they were weak. When these samples were observed by TEM, when dA20 / dG20 was used, relatively uniform silver nanoparticles with an average particle size of about 20 nm were observed, and with dC20 / dT20, a large number of particles of 50 nm or more were observed. (Figure 18).
From this, it was clarified that small nanoparticles were formed when DNA with purine base was used.

(4) Effects due to differences in DNA chain length In order to investigate how the oligonucleotide chain length affects the formation of silver nanoparticles, continuous sequences of adenine (AMP, dA5, dA20, dA50, poly (A)) was prepared. AMP is a monomer, and poly (A) is a polymer with an irregular length of about several thousand bases. The measurement results of UV-Vis absorption spectrum are shown in FIG. Only when AMP, which is a monomer among the five oligonucleotides, was used, plasmon absorption indicating silver nanoparticle formation was not observed after UV light irradiation. This is probably because one molecule of DNA cannot play the role of forming and assembling silver clusters in the monomer. For dA5, dA20, and dA50, there was no shift in plasmon absorption as the silver nanoparticle size increased, but the baseline of 500-700 nm increased when Poly (A) was used ( FIG. 20) revealed that silver nanoparticles with various particle sizes were formed. This is thought to be due to the fact that poly (A) chain lengths are not uniform, so silver nanoparticles also have various particle sizes.
From the above results, it was shown that fine particles can be produced by having a chain length of a dimer or more. Moreover, it is possible to change the size of the fine particles formed by changing the chain length.

(5) Preparation of microparticles with double-stranded DNA All the experiments so far have used single-stranded oligonucleotides. Therefore, λDNA (48,502bp, 16.5μm), which is a double-stranded long DNA, and M13mp8 RFIDNA (7,228), which is a circular DNA.
bp) and similar experiments were performed. FIG. 21 shows the change in absorption spectrum before and after UV irradiation when λDNA is used. When UV light was irradiated for 5 minutes in the same manner as in the previous experimental method, plasmon absorption centered at 405 nm was observed, confirming the production of silver nanoparticles even when λDNA was used. In addition, when circular DNA was used, nanoparticles were generated as in the case of using single-stranded DNA. In the case of circular DNA, the average particle size was about 40 nm.
In this way, nanoparticles can be formed not only in single-stranded DNA but also in linear double-stranded DNA or circular DNA.

(6) Preparation of silver nanoparticles presenting biotin In order to make the prepared silver microparticles have the ability to recognize molecules other than DNA nucleobases, the preparation of microparticles with DNA to which functional functional groups and dye molecules are added is examined. did.
Silver nanoparticles were prepared using the same biotinylated dA20 under the same conditions as the others. Changes in absorption spectra before and after UV irradiation and TEM images of the formed particles are shown in FIGS. As in the case of using dA20 with no terminal modification, plasmon absorption indicating silver nanoparticle formation was observed after UV irradiation, and silver nanoparticles having an average particle diameter of about 20 nm were formed from the TEM image. It was revealed that similar silver nanoparticles were formed even when DNA ends were modified.
Next, in order to examine whether biotin is present on the surface of the fine particles, streptavidin was added to confirm whether the fine particles were aggregated (FIG. 24). Streptavidin solution prepared to 0.2 mg / ml in 200 μl (100 μM) to the prepared silver nanoparticle dispersion solution (Streptavidin is Streptavidin, Type II [Wako Pure Chemicals] derived from Streptomyces avidinii, and TE buffer (1
(0 mM Tris-HCl, 1 mM EDTA, pH 7.8, 20 ° C.) was adjusted to 0.2 mg / ml) and mixed at 20 ° C. for 30 minutes. 10 μl of the solution was dropped on the washed glass substrate and dried in a nitrogen gas atmosphere. Thereafter, observation was performed by dark field illumination under a microscope.
The results are shown in FIG. As in the case of silver nanoparticles coated with dA20, it was observed that only silver nanoparticles prepared with terminal biotinylated dA20 were dispersed (FIG. 25 (a)). When streptavidin was added to the silver nanoparticles, it was found that the silver nanoparticles were cross-linked by streptavidin to form aggregates as shown in the schematic diagram of FIG. 24 (FIG. 25 (b)). This revealed that biotin was present on the surface of the silver nanoparticles. In this experiment, a biotin-avidin bond was used, but silver nanoparticles having molecular recognition ability can be prepared very simply by modifying various functional groups at the end of the oligonucleotide.

  As described above, the silver fine particle-nucleic acid complex of the present invention is usually coated with a nucleic acid, and thus is easily taken up by cells. In addition, the complex of the present invention has low cytotoxicity and exhibits stable luminescence for a long time in the cell. For example, an imaging reagent for cell staining, a staining reagent for gel electrophoresis, and a reagent used for fluorescence determination of protein Etc. are available.

The change of UV-Vis absorption spectrum before and after UV irradiation in the solution is shown. It is a transmission electron microscope (TEM) photograph of metal fine particles. 2 is a high-resolution TEM image (a, b) and diffraction image (c) of silver nanoparticles coated with dA20. The zeta potential measurement result of silver fine particles coated with dA20 is shown. This is a dark field image of silver nanoparticles prepared with dA20 ((a) Silver nanoparticles only, (b) Silver nanoparticles + dG20, (c) Silver nanoparticles + dT20 (d) Silver nanoparticles + poly (U) ). It is a fluorescence-microscope image of silver fine particles. (Left) B excitation (Excitation wavelength: 470-495 nm, Fluorescence wavelength: 510-nm) (Center) G excitation (Excitation wavelength: 530-550 nm, Fluorescence wavelength: 575-625 nm) (Right) U excitation (Excitation wavelength: 360 -370nm, fluorescence wavelength: 420 ~ 460nm) The spectrum of silver fine particles produced from dA20 is shown. The top row is a fluorescence micrograph of Evidots ^TM (fluorescence before left UV irradiation, right UV after 10 minutes irradiation), and the bottom row is a fluorescence micrograph of silver nanoparticles (fluorescence before left UV irradiation, after right UV irradiation for 10 minutes). . The microscopic image of the silver fine particle produced from dA20 taken in by the cell is shown. The microscopic image of the cell which added the silver fine particle produced from dA20 and was cultured for 24 hours is shown. The TEM image of the silver fine particle produced by adding a citric acid is shown. The structural formula of the substance used for the buffer solution is shown. The change of UV-Vis absorption spectrum before and after UV irradiation is shown. Photoreduction of silver ions in various solutions (conditions: dA20 100 μM, AgNO ₃ 100 μM) is shown. The pH dependence (conditions: dA20 100 μM, AgNO ₃ 100 μM, (a) before UV irradiation, (b) after UV irradiation) in the photoreduction of silver ions is shown. The structure of the terminal biotinylated oligonucleotide is shown. Changes in absorption spectrum when different base sequences are used are shown. The TEM image when a different base sequence is used is shown. The change (1) in the absorption spectrum when using adenine sequences with different chain lengths is shown. Changes in absorption spectrum (2) when using adenine sequences with different chain lengths are shown. Changes in absorption spectrum before and after UV irradiation when λDNA, which is a double-stranded DNA, is used. Changes in absorption spectrum before and after UV irradiation are shown. A TEM image of silver nanoparticles consisting of terminal biotinylated dA20 is shown. A schematic diagram of silver nanoparticle aggregation presenting biotin is shown. A dark field image of silver nanoparticles presenting biotin is shown. (a) silver nanoparticles presenting biotin, (b) silver nanoparticles presenting biotin + streptavidin.

Claims (16)

A complex of silver fine particles having a diameter of 5 nm to 500 nm and a nucleic acid , wherein the silver fine particles have a plurality of crystal structures . The complex according to claim 1, wherein the diameter is 5 nm to 50 nm. The composite according to claim 1 or 2, wherein the silver fine particles have a mosaic structure. The complex according to any one of claims 1 to 3 , wherein the nucleic acid is DNA or RNA consisting of 5 to 50 bases containing adenine and / or guanine. The complex according to claim 4 , wherein the nucleic acid is single-stranded DNA or RNA containing adenine or guanine. Said nucleic acid, is selected from 5-50 mer adenine, claim 5 complex according. Said nucleic acid, is selected from 5-50 mer guanine, claim 5 complex according. The complex according to any one of claims 4 to 7 , wherein the DNA or RNA has a functional group at its terminal. The complex according to claim 8 , wherein the functional group is biotin or thiol. The complex according to any one of claims 1 to 9 , wherein the silver particles and the nucleic acid are complexed in a state where the nucleic acid is present inside and / or outside the silver fine particles. The complex according to any one of claims 1 to 10 , wherein a surface is covered with the nucleic acid. The composite according to any one of claims 1 to 11 , which exhibits Raman scattering. The complex according to claim 12, which exhibits fluorescence due to an interaction between the nucleic acid and the silver fine particles. Fluorescent dye containing conjugate of any of claims 1-13. A method for producing a complex of silver fine particles and nucleic acid having a diameter of 5 nm to 500 nm using a solution containing silver ions and nucleic acids , wherein the silver ions are irradiated by ultraviolet irradiation in the solution containing silver ions and nucleic acids. How to reduce . A complex of silver particles and a nucleic acid obtained by the method according to claim 15 .

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