JP4667798B2 - Method for producing metal composite - Google Patents

Description

  The present invention relates to a method for producing a metal composite comprising a metal and a target compound obtained by reacting metal fine particles with a target compound.

Coordination compounds of metals, e.g., d ⁸ coordination compounds gold or platinum, are attracting attention as a drug such as an anticancer agent. Why d ⁸ coordination compound of the gold or platinum express anticancer effect, by coordination compound gold or platinum to form a coordination complex with a base portion of the DNA, the activity of DNA-dependent enzymes It is thought to be for suppression. That is, when a metal ion such as gold or platinum is bound to the base portion of DNA, the secondary structure or tertiary structure of DNA is changed in addition to the chemical structure of DNA, and polymerase, nuclease, esterase, kinase, other nucleotides or Affects the activity of polynucleotide-dependent enzymes. As a result, the metal coordination compound can be used as an anticancer agent, a cytotoxic agent, an anti-rheumatic agent, and the like.

  In order to make a metal coordination compound function as an anticancer agent or the like, for example, an amine derivative of gold chloride or platinum chloride is synthesized organically in advance, and this is donated to cells. This derivative then reacts with DNA to form a complex of its base moiety and metal chloride. As a result, the enzyme activity is affected.

  As a method for obtaining such a metal complex, for example, in Patent Document 1, a metal complex of a metal coordination compound and a nucleic acid is prepared by reacting a nucleic acid-specific metal coordination compound with a nucleic acid, Metal coordination compounds and / or byproducts that have not formed a complex are removed, and a reducing agent is allowed to act on the metal complex of the metal coordination compound and the nucleic acid to produce a metal complex of the metal fine particles and the nucleic acid. Nucleic acid metallization methods are described.

  In addition, as a method for obtaining a metal complex, for example, Patent Document 2 describes a method of producing nanoparticles to which oligonucleotides are attached by using charged particles and contacting with a salt solution in water. ing. In this method, metal particles are reduced with citric acid to prepare a gold colloid, and then oligonucleotides are attached.

JP 2002-371094 A JP-T-2004-501340

  However, in order to use a metal coordination compound as an anticancer agent or the like as in the prior art, it is necessary to synthesize the gold chloride or platinum chloride amine derivative, etc., and it takes time and effort. In addition, it is necessary to administer this derivative into the living body, and it is difficult to control the time space of the reaction.

  Even in the methods of Patent Documents 1 and 2, a plurality of processes are performed to obtain a metal composite, and for example, it is difficult to generate a metal composite in vivo. Therefore, development of a method for easily producing a metal composite is desired.

  The present invention is a method for producing a metal complex for easily obtaining a metal complex containing a metal and a target compound obtained by reacting metal fine particles with a target compound.

  The present invention relates to a method for producing a metal complex containing a metal and a target compound, which is obtained by reacting metal fine particles with a target compound, wherein a reaction liquid containing the metal fine particles and the target compound is irradiated with a laser. To produce the metal composite.

  In the method for producing a metal composite, the reaction solution preferably further contains an amine compound and a reaction accelerator.

  In the method for producing the metal complex, the target compound is preferably a biopolymer.

  In the method for producing a metal complex, the biopolymer is preferably a nucleic acid.

  In the method for producing a metal complex, the biopolymer is preferably a protein.

  In the method for producing a metal composite, the metal fine particles include Au, Ag, Ru, Rh, Pd, Os, Ir, Pt, Cu, Hg, Re, Fe, Ni, Co, Cr, Mn, Mo, It is preferable to include at least one metal selected from W, Ta and Nb.

  In the method for producing a metal composite, the amine compound may be Au, Ag, Ru, Rh, Pd, Os, Ir, Pt, Cu, Hg, Re, Fe, Ni, Co, Cr, Mn, Mo, It is preferable to include an amine compound that easily forms a coordination bond with at least one metal selected from W, Ta, and Nb.

  In the method for producing a metal complex, the amine compound may be proline, trishydroxymethylaminomethane (Tris), histidine, 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES). ), At least one amine compound selected from triethylamine acetate, urea, asparagine, arginine, tryptophan and spermidine.

  In the method for producing a metal composite, the reaction accelerator is preferably a cation.

  In the method for producing a metal complex, the cation is preferably a divalent or higher polyvalent cation.

  In the method for producing a metal complex, the metal complex is preferably a metal coordination compound.

  In the metal composite manufacturing method, the laser is preferably a pulse laser.

  In the method for producing a metal composite, the wavelength of the laser is preferably near the surface plasmon resonance wavelength of the metal fine particles.

  In the method for producing a metal composite, the wavelength of the laser is preferably a wavelength at which the metal fine particles have a large absorption coefficient.

  In the method for producing a metal composite, the wavelength of the laser is preferably in the vicinity of an interband transition of the metal fine particles.

  Hereinafter, embodiments of the present invention will be described.

  In the method for producing a metal complex according to an embodiment of the present invention, a reaction solution containing metal fine particles and a target compound is preferably a reaction containing metal fine particles, a target compound, an amine compound, and a reaction accelerator. By irradiating the liquid with a laser, a metal composite containing the metal and the target compound is produced. The metal complex is, for example, a coordination compound between a metal and a target compound.

  The metal composite manufacturing method according to the present embodiment is performed using, for example, a metal composite manufacturing apparatus shown in FIG. The metal complex manufacturing apparatus 1 is configured to include a laser generator 10, a condensing unit such as a lens 12, a stirring unit such as a cell 14 and a stirring bar 16, and the like. Specifically, the manufacturing method of the metal composite according to the present embodiment is performed according to the following procedure.

  First, a reaction liquid containing metal fine particles, a target compound, an amine compound, and a reaction accelerator is prepared. At this time, the metal fine particles are dispersed in the solution, but the target compound, the amine compound and the reaction accelerator are dissolved in the reaction solution from the viewpoint of improving the reaction efficiency. preferable.

  The metal of the metal fine particles used in the present embodiment is not particularly limited as long as it is a typical metal or a transition metal, but Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Hg, Re, Y, It is preferably a transition metal such as Zr, Nb, Mo, W, Ta, Nb, Ru, La, Ce, Pr, Nd, Au, Ag, Ru, Rh, Pd, Os, Ir, and Pt. Among transition metals, Au, Ag, Ru, Rh, Pd, Os, Ir, Pt, Cu, Hg, Re, Fe, Ni, Co, Cr, Mn, Mo, W, Ta, and Nb are more preferable. Preferably, noble metals such as Au, Ag, and platinum group (Ru, Rh, Pd, Os, Ir, Pt) are more preferable from the viewpoint of being hardly oxidized, and Au and Pt are particularly preferable.

  The particle size of the metal fine particles is preferably 1 μm or less, more preferably 1 nm to 100 nm, and more preferably 5 nm to 20 nm in view of high dispersibility in a metal solution. Is more preferable. If the particle size of the metal fine particles is smaller than 1 nm, the wavelength of the irradiation laser tends to be short, and the operation may be complicated.

  Examples of the method for producing metal fine particles include SF-LAS (Surfactant-free laser ablation in solution) in which the surface of a metal plate is ablated by laser irradiation or microwave irradiation in a liquid such as water, and water added with a surfactant. Specific examples include SC-LAS (Surfactant-controlled laser ablation in solution), ablation of metal plate surface in liquid by laser irradiation or microwave irradiation, chemical reduction method, discharge method in solution, etc. There is no. The addition of a surfactant to the metal fine particles is preferable because the metal fine particles can be stabilized and the operation becomes easy in production.

  As the surfactant, anionic, cationic, nonionic or amphoteric surfactants can be used. Usually, sodium dodecyl sulfate (SDS) is used from the viewpoint of the solubility of the surfactant, the stabilizing power of the metal fine particles in the solvent, and the like.

  In the present embodiment, the target compound that forms the metal complex with the metal fine particle is not particularly limited as long as it is a compound that forms the metal complex with the metal fine particle, but a biopolymer; a polymer having charge mobility, etc. Among them, a biopolymer is preferable from the viewpoint of easy coordination with a metal. Here, the biopolymer is a polymer compound synthesized in vivo, and examples thereof include proteins; nucleic acids such as DNA and RNA; polysaccharides and the like. Of these, proteins or nucleic acids are preferable, and nucleic acids such as DNA and RNA are nitrogen-containing compounds, and are more preferable from the viewpoint that they are easily coordinated with metals. Further, DNA and RNA may be single-stranded or double-stranded.

  In this embodiment, the presence of an amine compound is necessary in order to form a metal complex including a metal and a target compound. The amine compound is not particularly limited as long as it is a primary amine, a secondary amine, a tertiary amine, a compound containing a quaternary amine, for example, an aliphatic amine or an aromatic amine. For example, Proline , Trishydroxymethylaminomethane (Tris), histidine, HEPES, triethylamine acetate, urea, asparagine, arginine, tryptophan, spermidine and the like. Examples of amine compounds include metal ions such as Au, Ag, Ru, Rh, Pd, Os, Ir, Pt, Cu, Hg, Re, Fe, Ni, Co, Cr, Mn, Mo, W, Ta, and Nb. A compound that easily forms a coordination bond with at least one selected metal is preferable. Examples of such a compound include a compound having a low ionization energy. This is presumably because the lower the ionization energy, the easier the electron is transferred to the metal ion, and the metal is more easily coordinated to the amine compound. As a tendency, the ionization energy is preferably 8 eV or less.

In this embodiment, in order to form a metal complex containing a metal and a target compound, a reaction accelerator is required in addition to the amine compound. Here, examples of the reaction accelerator include a cation and a polyamine compound. The cation is not particularly limited, for ^example, Na +, ^{K +,} periodic table 1A metals ions ^etc; Mg 2+, periodic table Group 2A metal ion ^{Ca 2+} and the like; other ^{Mn 2+,} Metal ions of groups 3A to 7A, 8 and 1B to 4B of the periodic table such as Fe ²⁺ , Co ²⁺ , Ni ²⁺ , Cu ²⁺ , Zn ²⁺ , Fe ³⁺ , Co ³⁺ , Al ³⁺ , Sn ⁴⁺ , etc .; Etc. Among these, in order to increase the reaction efficiency for forming a metal complex including a metal and a target compound, it is preferably a divalent or higher polyvalent metal ion such as Mg ²⁺ or Ca ²⁺ , and has a high valence. More preferred. There is no restriction | limiting in particular as a salt which generate | occur | produces these cations, For example, halogen salts, such as chlorine, a bromine, an iodine; Sulfate; Carbonate; Nitrate; Ammonium salt;

  Examples of the polyamine compound include putrescine, spermidine, spermine and the like.

  As a solvent used in the reaction solution, water or a general organic solvent can be used. Water is not particularly limited and includes, for example, tap water, ground water, ion exchange water, pure water, ultrapure water, etc. In order to improve the reaction efficiency, it is better that there are fewer impurities, usually ion Exchange water, pure water, and ultrapure water are used. Examples of the organic solvent include alcohol solvents such as methanol, ethanol and isopropyl alcohol; aromatic solvents such as benzene and toluene; halogen solvents such as methylene chloride, chloroform and carbon tetrachloride; n-hexane, n-heptane and the like. Linear saturated hydrocarbon solvents; cyclic saturated hydrocarbon solvents such as cyclohexane; acetonitrile and the like can be used. Among these, water and alcohol-based solvents are preferable because of wide application range, and water is more preferable.

The concentration of the metal fine particles in the reaction solution is not particularly limited as long as the metal fine particles can be suspended, but is usually in the range of 0.1 μg / mL to 1000 μg / mL. The amount of the target compound in the reaction solution is usually in the range of 10 ⁴ mol% to 10 ⁸ mol% with respect to the amount of metal fine particles. Moreover, the density | concentration of the amine compound in a reaction liquid is the range of 0.001M-5M normally. The concentration of the reaction accelerator in the reaction solution is preferably in the range of 0.001M to 10M. If the cation concentration is less than 0.1M, the formation of the metal composite may not proceed sufficiently, and if the cation concentration exceeds 10M, the formation of the metal composite may become saturated. It is not necessary to add to

  The reaction liquid containing the metal fine particles, the target compound, the amine compound, and the reaction accelerator is put into the cell 14. The cell 14 is usually made of a material such as quartz or glass.

The reaction liquid put in the cell 14 is irradiated with a laser emitted from the laser generator 10. When the reaction solution is irradiated with a laser at a predetermined intensity and for a predetermined time, a metal atom or metal ion or metal cluster or metal cluster ion generated from the metal fine particles reacts with the target compound to obtain a metal complex. It is done. When irradiating the reaction liquid with a laser, the laser is preferably condensed in the reaction liquid by a condensing means such as a lens 12. The condensing region of the laser in the reaction solution is preferably in the range of (1 μm) ³ to (1 mm) ³ . Due to restrictions on the apparatus, it is difficult to narrow the condensing region to be smaller than (1 μm) ^{3. When the} condensing region is larger than (1 mm) ³ , reaction efficiency may be lowered. The laser is preferably a pulse laser.

  The intensity of the laser applied to the reaction solution is not particularly limited as long as the metal fine particles are efficiently ablated, but is preferably in the range of 100 μJ / pulse to 100 mJ / pulse, and 5 mJ / pulse to 20 mJ / pulse. A range of pulses is more preferable. When the laser irradiation intensity is lower than 100 μJ / pulse, the reaction may not proceed. When the laser irradiation intensity is higher than 100 mJ / pulse, the target compound or reaction product may be decomposed.

  The time for irradiating the reaction solution with the laser may be determined according to the reactivity between the metal fine particles to be used and the target compound, and is not particularly limited, but is usually in the range of 1 minute to 1 hour, preferably 5 minutes to The range is 10 minutes.

The wavelength of the laser irradiated to the reaction solution may be a wavelength at which the metal fine particles are efficiently ablated, and is not particularly limited, but is a wavelength near the surface plasmon resonance wavelength of the metal fine particles, or metal It is preferable that the particle has a wavelength having a large absorption coefficient, or a wavelength near the interband transition of the metal particle. Here, it is preferable that a big absorption coefficient is absorption of the intensity | strength of 100 M ^{< -1} > cm < ^-1 > or more. For example, in the case of gold fine particles, it is preferable to use a wavelength of 532 nm near the surface plasmon resonance wavelength. When there is no particularly strong absorption in the visible region as in the case of platinum fine particles, a laser with any wavelength may be used.

  The type of laser to be used is not particularly limited as long as it is selected according to the wavelength of the laser to be irradiated. For example, a semiconductor laser, solid laser, gas laser, dye laser, excimer laser, or the like can be used. .

  In addition, it is preferable that the laser irradiated to the reaction liquid is irradiated through the open part of the cell 14. When irradiated through the cell 14, depending on the intensity of the laser, the cell 14 itself may be sputtered by the laser and damaged. For this reason, the cell 14 is preferably made of a transparent material. For example, as shown in FIG. 1, the laser is irradiated from the upper surface opening of the cell 14.

  The temperature of the reaction solution is not particularly limited as long as the target compound such as a biopolymer to be used is not decomposed, but is in the range of 0 ° C to 100 ° C, usually 10 ° C to 30 ° C.

  The reaction liquid placed in the cell 14 is preferably stirred by stirring means such as a stirring bar 16 or a stirring blade. By stirring, the entire reaction solution can be irradiated with a laser uniformly. When stirring means such as the stirrer 16 is not used, scanning may be performed so that the entire reaction liquid is irradiated with a laser.

Formation of a metal complex by reaction of metal fine particles with a target compound can be achieved, for example, by transferring the reaction solution after laser irradiation to an absorption spectrum measurement cell and measuring the ultraviolet-visible absorption spectrum using an ultraviolet-visible spectrometer. It is confirmed. A reaction solution prepared by dispersing and dissolving gold fine particles prepared by the SF-LAS method, λDNA, trishydroxymethylaminomethane (Tris), and Ca ²⁺ (calcium chloride) in water in a glass cell, Nd: YAG FIG. 2 shows the measurement results of the ultraviolet-visible absorption spectrum of the reaction solution irradiated with the laser at the second harmonic of 532 nm while stirring the solution (see Example 1 for detailed experimental conditions). In FIG. 2, the dotted line shows the absorption spectrum of the gold fine particle dispersion solution, and the thick solid line shows the absorption spectrum of the reaction solution irradiated with laser after adding the gold fine particles, λDNA, trishydroxymethylaminomethane (Tris), and CaCl ₂ . The peak intensity near 360 nm is the integral value of the shaded portion of the 360 nm peak. The intensities of the absorption peak at 517 nm derived from the gold fine particles and the absorption peak at 260 nm derived from the DNA base were reduced as compared with those before laser irradiation, and a new absorption peak at 360 nm was observed. This 360 nm peak is considered to be an absorption peak of a new compound generated by chemical bonding between the base portion of DNA and gold ions.

  This absorption peak at 360 nm is considered to be due to charge transfer absorption due to the movement of electrons from the lone electrons on the nitrogen portion of the DNA or the nitrogen atom of the amine to the d orbital of the gold ion. (Many compounds in which four amines are coordinated to gold or platinum have a charge transfer absorption band around 360 nm.)

  In addition, it is considered that the gold ion generated in the solution forms a complex with the amine and stabilizes it, and at the same time forms a complex with the base portion of DNA.

  From the above, the reaction mechanism was estimated as shown in FIG. First, when gold fine particles are ablated by laser irradiation, gold ions are released. This ion is considered to react with the nitrogen atom of the added amine compound to generate a reactive molecular intermediate. Furthermore, it is considered that this intermediate forms a complex of the nitrogen moiety of the DNA base moiety and the gold-amine. The 360 nm peak in the visible ultraviolet absorption spectrum is considered to be derived from the charge transfer absorption band of this compound. Furthermore, it is considered that the solvated electrons released from the gold fine particles simultaneously with the gold ions are recombined with the gold ions in the coordination complex, reduced to gold atoms, and taken into the gold fine particles.

In this embodiment, by irradiating a reaction liquid containing metal fine particles, a target compound such as a biopolymer, an amine compound, and a reaction accelerator with a laser, the metal fine particles in the reaction liquid react with the target compound. Thus, a metal complex containing a metal and a target compound can be easily obtained. As a feature of the method for producing a metal composite according to the present embodiment, for example,
(A) Control of reaction time, space and reaction amount by laser (b) Utilization of inhibitory effect of nucleic acid-gold or nucleic acid-platinum chloride complex formation reaction due to steric hindrance of nucleic acid binding factor. The following examples can be given as application examples of the method for producing a metal composite according to the present embodiment using these characteristics.

(1) Anticancer agent (using inactivation of DNA reaction product)
When a metal ion binds to a base moiety, it changes the secondary or tertiary structure of DNA, affecting the activity of polymerases, nucleases, esterases, kinases, other nucleotide- or polynucleotide-dependent enzymes. As a result, it can be used as a cytotoxic agent, an anticancer agent, an anti-rheumatic agent or the like. Conventionally, when an amine derivative of gold chloride or platinum chloride is organically synthesized in advance and donated to a cell, this derivative reacts with DNA to form a complex of its base moiety and metal chloride. Arise. As a result, the enzyme activity is affected and a cytotoxic effect is exhibited. In the method for producing a metal complex according to this embodiment, it is not necessary to synthesize a derivative, and it is only necessary to irradiate a laser in the presence of a reaction essential factor. Moreover, since the reaction is induced only at the site irradiated with the laser, the control of the time space of the reaction is far superior to the case of administration of the derivative.

Specifically, in the conventional method, for example, a gold complex (complex) such as AuCl ₃ (Hpm), AuCl ₂ (pm), AuCl (Ph), Et ₃ AsAuCl, Ph (PhCh ₂ ) ₂ PAuCl, etc. Alternatively, platinum complexes such as cisplatin and cis-EE are synthesized organically in advance, and injected into the peritoneum to cancer cells cultured for 20 days under the subcutaneous membrane. Ten days later, the peritoneum is cleaved and the weight of the cancer cells is measured. By such a method, the function of these substances as an anticancer agent was examined.

  The factors that cause these substances to act as anticancer agents are that these complex compounds react with DNA to form a complex of DNA or the base or gold or platinum, and the activity of various enzymes that work on DNA is suppressed. Is thought to be killed.

  However, in the conventional method, there are various difficulties in accurately supplying these compounds to cancer cells and preventing other healthy cells from acting, and there has been a problem of drug delivery. In addition, it was difficult to control the action in the time and space most suitable as an anticancer agent. However, in the method for producing a metal complex according to this embodiment, DNA and gold or platinum form a complex only at the portion irradiated with the laser, so that there is an advantage that time and space can be easily controlled by laser irradiation.

(2) Biochemical analysis method Inhibition effect of nucleic acid-gold or platinum chloride complex formation reaction due to steric hindrance of nucleic acid binding factor, that is, when a nucleic acid and a nucleic acid binding factor interact, a metal irradiated with laser Since the metal compound produced from the fine particles cannot access the nucleic acid, the reaction at the base portion of the nucleic acid does not occur. Thereafter, in order to identify a site where the nucleic acid-gold chloride complex formation reaction has not occurred, for example, a DNase I partial digestion method in the DNase I footprinting method is used. DNase I activity inhibition occurs at the place where the complex formation reaction has occurred, and partial digestion with DNase I occurs at the place where the complex formation reaction has not occurred. When a site where DNase I activity inhibition has occurred is identified using analytical means such as electrophoresis, the DNA portion where the nucleic acid and the nucleic acid binding factor interacted can be identified.

  In the conventional method, the DNase I footprint method was used (conventional method 1). The DNase I footprinting method is performed to identify a factor that recognizes and binds to a specific base sequence and to determine the region of the specific base sequence. After reacting a DNA fragment labeled only on one end with a DNA binding factor, the DNA fragment is partially digested with DNase I under the condition of cutting about one site per DNA molecule. This DNA fragment is electrophoresed on urea polyacrylamide gel for base sequence determination, and autoradiography is performed. The resulting band is in the form of a ladder for each base, but since the region to which the DNA binding factor has been bound is protected from DNase I digestion, the corresponding band becomes thinner. Therefore, it is possible to determine the coupling area.

  In the conventional method 1, measurement is performed with time and space controlled. When the process of complex formation when the DNA binding factor is a complex or when the DNA binding factor tracks the process of searching for a DNA-specific binding sequence is time-resolved, the time-resolved DNase I footprint method is used. There is a need to do. However, in the conventional method 1, the digestion time of DNase I determines the upper limit of time resolution. In the case of DNase I, it takes several minutes.

  The method for producing a metal composite according to this embodiment has an advantage of having a time resolution of several microseconds because the laser irradiation time is the reaction time.

  Further, methylation interferometry has been known as the conventional method 2. When a DNA fragment in which guanine nucleotides are partially methylated is reacted with a DNA binding factor, the methylation inhibits the factor's DNA binding ability. As a result, bound guanine nucleotides are identified. Specifically, a gel shift method is performed by reacting a DNA probe partially methylated with guanine nucleotides and a DNA binding factor, and bound and unbound DNA probes are extracted from the gel. Polyacrylamide gel electrophoresis for base sequencing is performed to identify bound guanine nucleotides.

  However, in the conventional method 2, the upper limit of the time resolution is several tens of minutes or more of the time for extracting the bound and unbound DNA probes by the gel shift method. In addition, there is a problem that the base for determining binding / non-binding is limited to guanine nucleotides.

  In the metal composite manufacturing method according to the present embodiment, since the laser irradiation time is the reaction time, it has a time resolution of several microseconds. Further, the reaction shown in the method for producing a metal complex according to this embodiment has an advantage that it does not depend on the type of base.

(3) Binding activity (rapid measurement of binding dissociation constant)
This also utilizes the inhibitory effect of the nucleic acid-metal chloride complex formation reaction due to steric hindrance of the nucleic acid binding factor as described above. Specifically, the binding activity can be measured simply by mixing a nucleic acid and a nucleic acid binding factor, mixing the factor necessary for the reaction, irradiating the laser for about 10 minutes, and measuring the visible ultraviolet spectrum.

  In the conventional method, while the amount of DNA binding factor is changed, the amount of binding between DNA and DNA binding factor is measured using a gel shift method or a membrane immobilization method.

  However, the conventional method has a problem that operations such as gel shift method and membrane immobilization are complicated.

  The method for producing a metal complex according to the present embodiment has an advantage that the binding activity can be measured only by measuring the visible ultraviolet spectrum, which is very simple.

(4) Higher order structure of nucleic acid, difference in reactivity between single strand and double strand Similar to the above principle, metal irradiated by laser depending on higher order structure of nucleic acid or whether nucleic acid is single strand or double strand Differences in the reaction at the base portion of the nucleic acid occur due to the accessibility of the reactive molecular intermediate generated from the microparticles to the nucleic acid. This difference can be detected by the absorption intensity of the visible ultraviolet spectrum, and knowledge about the form of the nucleic acid can be obtained.

  As conventional methods, there are many methods for detecting higher-order structures of nucleic acids. For example, DNA morphology detection by light scattering or electrophoresis. In addition, for detecting whether a nucleic acid is single-stranded or double-stranded, there is a method using a difference in the ultraviolet absorption cross section of a base.

  However, the conventional method has a problem that time-resolved measurement of DNA structure is difficult.

  When the method for producing a metal complex according to the present embodiment is used, DNA higher-order structure measurement with a time resolution of several microseconds at the time of laser irradiation is possible. That is, there is an advantage that the structure of DNA at the time of laser irradiation can be fixed.

  Thus, the method for producing a metal composite according to the present embodiment has the advantages described above, and has advantages over conventional methods in use as an anticancer agent, use as a biochemical analysis, and the like. Various applications are possible.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail more concretely, this invention is not limited to a following example.

<Formation of metal complex of gold fine particles and λDNA>
Example 1
Gold fine particles not containing a surfactant are treated with SF-LAS (surfactant-free laser ablation in solution) method (for example, JP-A-2003-286509 and F. Mafune, J. Kohno, Y. Takeda, T. Kondow and H. Sawabe: J. Phys. Chem. B, 105, (2001), 5114-5120 etc.). 100 mL of a 3 mM aqueous solution of λDNA (manufactured by TOYOBO), 100 mL of a 0.5 M aqueous solution of calcium chloride, and 100 mL of a solution in which 1.2 mg of fine gold particles not containing the above surfactant are dispersed are mixed and reacted. A liquid was prepared. 0.3 mL of this reaction solution was placed in a glass cell having a bottom surface of 1 cm × 1 cm. In this example, λDNA was used as it was with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA added. This was irradiated with a Nd: YAG laser double wave of 532 nm and a pulse laser of 17 mJ / pulse for 10 minutes from the opening of the cell. The laser beam was condensed in the reaction solution using a lens so as to be about (0.1 mm) ³ . During this time, a stirring bar having a length of 10 mm and a width of 1 mm was placed at the bottom of the cell, and the solution was stirred with a magnetic stirrer. Thereafter, the solution was transferred to a quartz cell for measuring an absorption spectrum, and an ultraviolet-visible absorption spectrum was measured using an ultraviolet-visible spectrometer (manufactured by Shimadzu Corporation, UV-1200 SPECTROTOPOMETER). The result is shown in FIG. In FIG. 2, the dotted line shows the absorption spectrum of the gold fine particle dispersion solution, and the thick solid line shows the absorption spectrum of the reaction solution irradiated with laser after adding the gold fine particles, λDNA, trishydroxymethylaminomethane (Tris), and CaCl ₂ . The intensities of the absorption peak at 517 nm derived from gold fine particles and the absorption peak at 260 nm derived from the DNA base were decreased compared with those before laser irradiation, and a new absorption peak at 360 nm was observed. This peak at 360 nm was found to be an absorption peak of a new compound generated by chemical bonding between the base portion of DNA and gold chloride. Note that the peak intensity near 360 nm indicates the integral of the shaded portion of the 360 nm peak in FIG.

  Next, the factors necessary for forming a complex of the target compound and gold by laser irradiation of the gold fine particles were searched.

<Finding factors necessary for complex formation>
(Comparative Example 1)
Using the reaction solution of Example 1, the reaction solution was mixed for 10 minutes without laser irradiation. After stirring for 10 minutes, an ultraviolet-visible absorption spectrum of the reaction solution was measured, but an absorption peak near 360 nm was not observed, and the reaction hardly occurred. From this, it was confirmed that laser irradiation at 532 nm was necessary for the appearance of an absorption peak at 360 nm.

(Comparative Example 2)
In Example 1, laser irradiation was performed in the same manner as in Example 1 except that λDNA was not added to the reaction solution, and a visible ultraviolet absorption spectrum was measured. An absorption peak near 360 nm was not observed, and the reaction hardly occurred. From this, it was confirmed that DNA was necessary for the appearance of an absorption peak at 360 nm.

(Comparative Example 3)
In Example 1, laser irradiation was performed in the same manner as in Example 1 except that no gold fine particles were added to the reaction solution, and a visible ultraviolet absorption spectrum was measured. An absorption peak near 360 nm was not observed, and the reaction hardly occurred. As a result, it was confirmed that gold fine particles were necessary for the appearance of an absorption peak at 360 nm.

(Comparative Example 4)
In Example 1, laser irradiation was performed in the same manner as in Example 1 except that a polyvalent cation (Ca ²⁺ ) was not added to the reaction solution, and a visible ultraviolet absorption spectrum was measured. An absorption peak near 360 nm was not observed, and the reaction hardly occurred. This confirmed that a cation was required for the appearance of an absorption peak at 360 nm.

(Comparative Example 5)
In Example 1, instead of gold fine particles, a gold (Au) plate (size 10 mm × 10 mm × thickness 3 mm) was placed at the bottom of the reaction solution, and the fundamental wave of the Nd: YAG laser was 1064 nm and the second harmonic wave was 532 nm. Laser irradiation was performed in the same manner as in Example 1 except that a laser beam of 100 mJ / pulse was focused on the surface of the gold plate and irradiated for 10 minutes, and a visible ultraviolet absorption spectrum was measured. An absorption peak near 360 nm was not observed, and the reaction hardly occurred. As a result, it was confirmed that the gold fine particles were required to be dispersed in the solution for the appearance of an absorption peak at 360 nm.

(Comparative Example 6)
In Example 1, λDNA was not added, gold fine particles and cations were mixed to obtain a reaction solution, and a Nd: YAG laser double wave of 532 nm, 17 mJ / pulse pulse laser was used in the same manner as in Example 1. Was irradiated for 10 minutes. Immediately thereafter, λDNA (manufactured by TOYOBO) from which the buffer was removed was added, and when a visible ultraviolet absorption spectrum was measured, an absorption peak near 360 nm was not observed. This indicates that the gold fine particles, cations, amine compound and DNA coexist at the time of laser irradiation and the reaction proceeds.

(Comparative Example 7)
In Example 1, λDNA (TOYOBO) supplemented with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA was used as it was. However, in this comparative example, λDNA (manufactured by TOYOBO) was used as a reaction solution. In place of), laser irradiation was performed in the same manner as in Example 1 except that λDNA (manufactured by TOYOBO) was dialyzed to remove the buffer, and a visible ultraviolet absorption spectrum was measured. An absorption peak near 360 nm was not observed, and the reaction hardly occurred. This revealed that the appearance of an absorption peak at 360 nm has a reaction essential factor in addition to DNA, gold fine particles, and polyvalent cations.

<Search for reaction essential factors>
(Example 2)
In order to clarify the reaction essential factor, in Comparative Example 6, λDNA (manufactured by TOYOBO) was dialyzed and the buffer was removed, and further, proline (Proline), trishydroxymethylaminomethane (Tris), histidine (Histidine). ), 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (2- [4- (2-Hydroxyethyl) -1-piperazinyl] ethanesulfonic acid: HEPES), triethylamine acetate, urea, asparagine ( Aspergine, Arginine, Tryptophan, Spermidine, Ethylenediamine, Formamide, Hydrazine, Aniline, Nicotinic acid, Lysozyme, Dimethylsulfoxide (DMSO), Acetonitrile , EDTA, glycine, lysine, ammonium nitrate, dATP, dAMP, adenine, thymine, guanine, cytosine at a concentration of 1M, respectively Laser irradiation was performed in the same manner as in Example 5, and a visible ultraviolet absorption spectrum was measured. An absorption peak around 360 nm was observed in the solutions to which proline, trishydroxymethylaminomethane (Tris), histidine, HEPES, triethylamine acetate, urea, asparagine, arginine, tryptophan, and spermidine were added. On the other hand, an absorption peak near 360 nm is obtained in a solution to which ethylenediamine, formamide, hydrazine, aniline, nicotinic acid, Lysozyme, DMSO, acetonitrile, EDTA, glycine, lysine, ammonium nitrate, dATP, dAMP, adenine, thymine, guanine, and cytosine are added. Only a few were observed. From these results, it was found that the reaction requires certain amine compounds.

<Influence of laser wavelength>
(Example 3)
In Example 1, instead of the Nd: YAG laser double wave 532 nm, 17 mJ / pulse pulse laser, the Nd: YAG laser fundamental wave 1064 nm, 17 mJ / pulse pulse laser was irradiated for 10 minutes. In the same manner as above, laser irradiation was performed, and a visible ultraviolet absorption spectrum was measured. Only a few absorption peaks around 360 nm were observed. As a result, it was confirmed that irradiation of the surface plasmon resonance wavelength of the gold fine particles was effective for the appearance of an absorption peak at 360 nm.

<Cation concentration dependence>
Example 4
In Example 1, except that the concentration of added calcium chloride (Ca ²⁺ ions) was changed, laser irradiation was performed in the same manner as in Example 1, and a visible ultraviolet absorption spectrum was measured. FIG. 4 shows the Ca ²⁺ ion concentration dependence of the absorption peak intensity at a concentration of 360 nm. The horizontal axis represents the concentration of Ca ²⁺ , and the vertical axis represents the intensity of absorption near 360 nm. The reaction hardly occurs when the Ca ²⁺ ion concentration is lower than 0.001M, and the reaction amount is saturated to a constant value when the Ca ²⁺ ion concentration is higher than 0.1M. Furthermore, a rapid increase in the reaction amount is observed when the Ca ²⁺ ion concentration is around 0.005M. Cations such as Ca ²⁺ ions bind to the phosphate portion of the DNA molecule, and have the function of neutralizing the negative charge of the DNA and neutralizing the DNA in the solution. It is considered that the neutralized DNA is adsorbed on the surface of the gold fine particles, and the reaction probability with gold ions generated by laser irradiation is increased. FIG. 4 shows that the DNA molecule and Ca ²⁺ ion binding reaction is an equilibrium reaction having an equilibrium constant of 7.6 × 10 ⁻³ M. The equilibrium constant 7.6 × 10 ⁻³ M determined here is the dissociation constant 3.3 × 10 ⁻⁵ M between the DNA molecule and the Ca ²⁺ ion, or the DNA phosphate group concentration 4.0 × 10 ^{−4 in the} solution. Greater than any of M. It is considered that a higher concentration of Ca ²⁺ ions is necessary to neutralize λDNA in which huge negative charges are present at high density.

<Formation of metal complex of gold fine particles and polynucleotide>
(Example 5)
The λDNA used in the above examples and comparative examples is a double-stranded DNA having 48502 base pairs. Therefore, the reaction was performed in the same manner as in Example 1 for DNA having a small number of base pairs and single-stranded DNA. The length of the implemented DNA was 2mer (single-stranded DNA is hereinafter referred to as ssDNA), 4mer (ssDNA), 56mer (ssDNA), and 18mer (ssDNA) all produced peaks near 360 nm, indicating that the reaction occurred. confirmed. Furthermore, similar reactions were carried out for monomeric dATP, dAMP, adenine, thymine, guanine, and cytosine, but no reaction occurred. From the above, since a reaction occurs with a polynucleotide of 2 mer or more, it is considered that the gold ion reacts across two base parts.

(Example 6)
<Product hydrolysis>
After the metal complex was formed, it was confirmed whether the product decomposed with time. After the laser irradiation, a visible ultraviolet absorption spectrum was measured after a certain time. The main cause of the decomposition is considered to be that solvated electrons released from the gold fine particles simultaneously with the gold ions in the solution recombine with the gold ions in the coordination complex and are reduced to gold atoms. In fact, the peak near 360 nm that appeared by laser irradiation attenuated with time after laser irradiation. The half-life was 36 minutes at 25 ° C. The results are shown in FIG. The result of fitting the measured value with an exponential function is shown by a solid line.

(Example 7)
<Detection of interaction between DNA and DNA binding factor>
The inhibitory effect of complex formation reaction on DNA due to steric hindrance of DNA binding factor was examined. That is, when DNA and a DNA binding factor interact, it is considered that the reaction at the base portion of DNA does not occur because the reactive molecular intermediate generated from the gold fine particles irradiated with the laser cannot approach DNA. Specifically, the interaction between ssDNA (Seq: ggatcctaatgaccaagg) and T4 page 32 protein (hereinafter referred to as p32 protein) was detected. The p32 protein is a protein encoded by T4 phage, which binds to ssDNA non-specifically during DNA replication and contributes to stabilization of ssDNA. The reaction was carried out using a 1.0 μM ssDNA aqueous solution, a 10 mM Tris aqueous solution, a 1 M CaCl ₂ aqueous solution, A: 0 M, B: 0.3 × 10 ⁻⁶ M, and C: 1.0 ×. A laser beam of 532 nm was irradiated in the same manner as in Example 1 to a reaction solution containing an aqueous solution of p32 protein having three concentrations of 10 ⁻⁶ M. Thereafter, a visible ultraviolet absorption spectrum was measured, and an absorption peak intensity at 360 nm was measured. When the concentration of p32 protein was high, the absorption peak area at 360 nm decreased. The relationship between the concentration of p32 protein and the absorption peak at 360 nm is shown in FIG. The arrow indicates the position of the peak at 360 nm. This shows that the dissociation constant of the p32 protein is 10 ⁻⁶ or less. According to literature values, the dissociation constant of p32 protein for ssDNA is 10 ⁻⁸ M.

<Formation of metal complex of gold fine particles prepared by SC-LAS method and λDNA>
(Example 8)
SC-LAS (surfactant-controlled laser ablation in solution) method (a method for producing gold fine particles in a liquid to which SDS is added. The size of gold fine particles generated can be controlled by changing the concentration of SDS, for example. JP, 2003-286509, and F. Mafune, J. Kohno, Y. Takeda, T. Kondow and H. Sawabe: J. Phys. Chem. B, 105, (2001), 5114-5120, etc. The reaction was carried out in the same manner as in Example 1 using the gold fine particles produced by the above method. This is advantageous because the gold fine particles produced by the SC-LAS method are more stable in the solution than the gold fine particles produced by the SF-LAS (surfactant-free laser ablation in solution) method. When SDS was contained in an amount of 0.01 M or more, a precipitate was formed, making it difficult to measure the absorption spectrum. However, when the SDS is 0.001 or less, an absorption spectrum can be measured, and a peak at 360 nm is generated, confirming that the reaction proceeds even in the presence of SDS.

Example 9
Platinum fine particles containing no surfactant were produced by the SF-LAS method. In the same manner as in Example 1, a reaction solution in which λDNA (manufactured by TOYOBO), calcium chloride, and platinum fine particles not containing a surfactant were dispersed and dissolved was placed in a glass cell having a bottom surface of 1 cm × 1 cm. This was irradiated with a Nd: YAG laser double wave of 532 nm, 17 mJ / pulse pulse laser for 10 minutes from the opening of the cell, and an ultraviolet-visible absorption spectrum was measured. The result is shown in FIG. In addition to the 260 nm absorption peak derived from the DNA base, a new absorption peak at 360 nm was observed. This 360 nm peak is an absorption peak of a new compound produced by the reaction between the base portion of DNA and a platinum compound. In addition, the fact that calcium chloride and trishydroxymethylaminomethane (Tris) are required for the reaction, the reaction does not occur with monomeric ATP, and the reaction also occurs with ssDNA showed the same tendency as in the case of gold fine particles. . In FIG. 7, A: UV-visible absorption spectrum when reacting by adding λDNA and calcium chloride to a platinum fine particle dispersion, B: Ultraviolet when reacting using a reaction solution obtained by removing calcium chloride from A Visible absorption spectrum, C: A λDNA is changed to ATP, and reaction is carried out using a reaction solution to which trishydroxymethylaminomethane (Tris) is added, D: A λDNA is converted into 4mer DNA In addition, UV-visible absorption spectrum when reaction is performed using a reaction solution to which trishydroxymethylaminomethane (Tris) is added, and reaction is performed using a reaction solution from which E: D trishydroxymethylaminomethane (Tris) is removed. Shows the UV-visible absorption spectrum. The arrow indicates the position of the peak at 360 nm.

It is a figure which shows an example of a structure of the metal complex manufacturing apparatus which concerns on embodiment of this invention. In the manufacturing method of the metal composite_body | complex which concerns on embodiment of this invention, it is a figure which shows the measurement result of the visible absorption spectrum of the reaction liquid before and behind reaction. It is a figure which shows the reaction mechanism which the metal complex produces | generates estimated in the manufacturing method of the metal complex which concerns on embodiment of this invention. In the manufacturing method of the metal composite_body | complex which concerns on embodiment of this invention, it is a figure which shows the Ca2 ⁺ ion concentration dependence of the absorption peak intensity | strength of 360-nm density | concentration in the reaction liquid after reaction. In the manufacturing method of the metal complex which concerns on embodiment of this invention, it is a figure which shows the change accompanying the time passage of the 360-nm absorption peak intensity | strength of the reaction liquid immediately after laser irradiation. It is a figure which shows the relationship between the density | concentration of p32 protein, and the absorption peak of 360 nm in the manufacturing method of the metal complex which concerns on embodiment of this invention. It is a figure which shows the measurement result of the visible absorption spectrum of the reaction liquid after reaction when making it react using the platinum microparticles in the manufacturing method of the metal composite_body | complex which concerns on embodiment of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Metal complex manufacturing apparatus, 10 Laser generator, 12 Lens, 14 cells, 16 Stirrer.

Claims (15)

A method for producing a metal complex comprising a metal and a target compound, wherein metal fine particles and a target compound are reacted.
A method for producing a metal complex, characterized in that the metal complex is produced by irradiating a reaction liquid containing the metal fine particles and the target compound with a laser. It is a manufacturing method of the metal composite according to claim 1,
The method for producing a metal complex, wherein the reaction solution further contains an amine compound and a reaction accelerator. It is a manufacturing method of the metal composite according to claim 1 or 2,
The method for producing a metal complex, wherein the target compound is a biopolymer. It is a manufacturing method of the metal composite according to claim 3,
The method for producing a metal complex, wherein the biopolymer is a nucleic acid. It is a manufacturing method of the metal composite according to claim 3,
The method for producing a metal complex, wherein the biopolymer is a protein. It is a manufacturing method of the metal complex according to any one of claims 1 to 5,
The metal fine particles are at least one selected from Au, Ag, Ru, Rh, Pd, Os, Ir, Pt, Cu, Hg, Re, Fe, Ni, Co, Cr, Mn, Mo, W, Ta, and Nb. A method for producing a metal composite comprising two metals. It is a manufacturing method of the metal composite according to any one of claims 1 to 6,
The amine compound is at least one selected from Au, Ag, Ru, Rh, Pd, Os, Ir, Pt, Cu, Hg, Re, Fe, Ni, Co, Cr, Mn, Mo, W, Ta, and Nb. A method for producing a metal composite comprising an amine compound that easily forms a coordination bond with two metals. It is a manufacturing method of the metal complex according to any one of claims 1 to 7,
The amine compound includes proline, trishydroxymethylaminomethane (Tris), histidine, 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES), triethylamine acetate, urea, asparagine, arginine, A method for producing a metal complex, comprising at least one amine compound selected from tryptophan and spermidine. It is a manufacturing method of the metal composite according to any one of claims 1 to 8,
The method for producing a metal complex, wherein the reaction accelerator is a cation. It is a manufacturing method of the metal composite according to claim 9,
The method for producing a metal complex, wherein the cation is a divalent or higher valent cation. It is a manufacturing method of the metal composite according to any one of claims 1 to 10,
The method for producing a metal complex, wherein the metal complex is a metal coordination compound. It is a manufacturing method of the metal composite according to any one of claims 1 to 11,
The method of manufacturing a metal composite, wherein the laser is a pulse laser. It is a manufacturing method of the metal composite according to any one of claims 1 to 12,
The method of producing a metal composite, wherein the laser has a wavelength in the vicinity of a surface plasmon resonance wavelength of the metal fine particles. It is a manufacturing method of the metal composite according to any one of claims 1 to 12,
The method of manufacturing a metal composite, wherein the laser has a wavelength at which the metal fine particles have a large absorption coefficient. It is a manufacturing method of the metal composite according to any one of claims 1 to 12,
The method of manufacturing a metal composite, wherein the laser has a wavelength in the vicinity of an interband transition of the metal fine particles.

Images