JP4665161B2 - Method for measuring Raman spectrum, noble metal particles used in the method, and method for producing the noble metal particles - Google Patents

Description

  The present invention relates to a method for measuring a Raman spectrum, a noble metal particle used in the method, and a method for producing the noble metal particle.

  A living body has a complicated mechanism for maintaining homeostasis against changes in the external environment and the internal environment, and when such a mechanism is disturbed, it appears as a symptom such as a disease. Therefore, there is a possibility that it can be applied to diagnosis of diseases by grasping the state of existence of information transmission substances (for example, hormones and otachoids) related to maintenance of homeostasis in the living body. For example, in a cancer cell, a specific gene is excessively expressed or the expression is suppressed. Therefore, cancer is diagnosed by measuring the presence of a compound (RNA, signal transmitter, etc.) corresponding to the gene. It is thought that there is a possibility that the presence or absence of a sign can be determined.

  Thus, various methods have been studied in which a target compound is labeled with a fluorescent group or the like through a group (such as an antibody) that specifically binds to the target compound, and the target compound is quantitatively analyzed based on the intensity of fluorescence development. In recent years, not only a conventional quantitative analysis method using fluorescence but also a method using a Raman spectrum has been studied.

The Raman spectrum is a spectrum of scattered light (Raman scattered light) other than the same frequency (ν ₀ ± ν _i ) in the scattered light generated by irradiating a substance with monochromatic light having a constant frequency ν ₀ . This Raman frequency ν _i is equal to the vibration frequency between the vibrational and rotational energy levels of the molecules and crystals that make up the material, so it is an information source for determining, identifying, and quantifying the energy level of the material. (See "Chemical Dictionary", Tokyo Chemical Doujin). However, since the Raman scattered light is very weak, it is measured after being enhanced using the surface plasmon resonance effect. This surface plasmon resonance effect is based on the phenomenon that light does not generally couple with electron waves (plasmons) but causes coupling on the surface of metal particles. For example, when gold particles are used, A strong absorption peak appears for light near 520 nm. As an application of such a technique, there are inventions disclosed in Patent Documents 1 and 2. In these inventions, the dispersion stability of particles in a solution is improved by coating particles such as gold with polyethylene glycol having a terminal reactive group for binding to an antibody, etc., and then surface plasmon resonance effect is applied. The Raman scattered light of the target compound is enhanced.

  However, in the art, detailed information such as chemical structure to achieve specific binding to a target compound is required, and a compound for labeling the compound must be prepared.

  On the other hand, if information on not only a specific compound but also compounds existing in the entire sample can be obtained by a Raman spectrum, a comprehensive judgment on the sample can be made. For example, with respect to biological samples, it is possible to grasp disease diagnosis and signs thereof, and it is possible to determine what harmful substances are contained in the wastewater. Further, according to the Raman spectrum, information on the structure of an unknown compound can also be obtained, so that it can be used for searching for a new useful compound.

  However, in the case of a biological sample containing various compounds, it is necessary to further increase the sensitivity of Raman spectrum measurement. Therefore, it is conceivable to increase the surface area of the metal particles in order to improve the surface plasmon resonance effect. However, the dispersion stability naturally decreases as the particles become larger.

  Conventionally, in order to ensure dispersion stability in a sample, metal fine particles used in Raman spectrum measurement have an ionic group such as citric acid supported on the surface and have used ion repulsion between particles. However, when an ionic group is present on the metal surface, there is a problem that particles cannot be measured and aggregated very easily when the ionic strength of the sample is high.

  On the other hand, it is also conceivable to apply the techniques described in Patent Documents 1 and 2 to suppress aggregation and sedimentation of particles by supporting polyethylene glycol on the metal surface. However, the purpose of enhancing Raman scattered light cannot be achieved. That is, since the surface plasmon resonance effect increases as the surface area of the metal particles increases, it is effective to increase the apparent particle surface area by bringing the metal particles closer to each other without causing aggregation and sedimentation. However, if the surface of the particle is covered with a polymer such as polyethylene glycol, there is a limit to the proximity of the metal particles, so the surface plasmon resonance effect must remain unique to a single particle. Absent.

  As described above, in order to enhance the weak Raman scattered light in the Raman spectrum measurement, it is possible to increase the apparent particle surface area by appropriately agglomerating metal particles at the time of measurement and effectively use the surface plasmon resonance effect. Conceivable. However, when such aggregation is achieved, the dispersion stability of the particles decreases. As described above, the conventional techniques cannot simultaneously satisfy the mutually contradicting characteristics of the dispersion stability of the metal particles and the effective use of the surface plasmon resonance effect.

  Further, in the production methods described in Patent Documents 1 and 2, metal particles are produced mainly by reducing chloroauric acid with a reducing agent such as sodium borohydride, but the metal obtained by such a method is used. The particles have a small particle size and cannot fully exhibit the surface plasmon resonance effect.

On the other hand, in patent document 3, the compound which gold | metal | money and saccharides couple | bonded through the sulfur atom by the reducing power of saccharide itself without using a reducing agent especially is manufactured. However, considering the purpose of use (synthesis intermediates for photographic materials and photographic additives) and the fact that chloroauric acid and saccharide are mixed with ice cooling, such compounds are present on the metal particles. It is unlikely that the sugar is substituted. In fact, the general formula described in the literature and the chemical formula of the actually synthesized compound (C ₆ H ₁₁ SAu = 392.17) indicate that the gold atom (Au) and the saccharide are bonded one-to-one. Considering the bulkiness of saccharides, it is unlikely that this compound is aggregated to form particles. In other words, it can be said that the compound is not a saccharide substituted on the surface of the gold particle, based on the common general knowledge.
Japanese Patent Laid-Open No. 13-200050 (Claim 1, paragraphs [0002] and [0003]) Japanese Patent Laid-Open No. 14-80903 (Claims 1 to 4, paragraphs [0002] and [0003]) JP 2003-113194 A (Claim 1, paragraphs [0001] and [0060])

  Under the circumstances described above, the problem to be solved by the present invention is a method capable of obtaining a comprehensive Raman spectrum of not only a specific compound but also a compound contained in a sample, and the dispersion stability of the additive It is to provide a method that has succeeded in satisfying both conflicting characteristics of enhancement of Raman-dispersed light by the surface plasmon resonance effect.

  Another object of the present invention is to provide noble metal particles that are excellent in dispersion stability, can effectively exhibit the surface plasmon resonance effect, and can enhance Raman-dispersed light, and a method for producing the same.

  In order to solve the above-mentioned problems, the present inventor has conducted intensive research in order to search for noble metal particles that are excellent in dispersion stability and can be appropriately aggregated under a certain condition to effectively exhibit the surface plasmon resonance effect. Proceeded. As a result, it was found that if a saccharide having a hydroxyl group as a substituent is supported on the surface of the noble metal particle, the dispersion stability is improved, and it is appropriately aggregated by the presence of a cation to further enhance the Raman scattered light of the sample. The present invention has been completed.

  That is, the Raman spectrum measurement method according to the present invention is characterized in that noble metal particles having a substituent represented by the following formula (I) on the surface are added to a sample, and the noble metal particles of the present invention have the following formula (I It is characterized by having a substituent represented by

[In the formula, (Saccharide) represents a monosaccharide or disaccharide having no ionic group in the side chain. ].

  In the Raman spectrum measurement method and the noble metal particles, the average particle size of the particles is preferably 30 to 50 nm. This is because the noble metal particles having an average particle diameter in the above range are more excellent in the dispersion stability in the sample and the effect of enhancing Raman dispersion light.

  Furthermore, in the method for producing noble metal particles according to the present invention, a solution of a compound represented by the following formula (II) is added dropwise to a halogenated noble metal acid or a salt thereof or a solution of a noble metal salt of an inorganic acid while heating. Features.

[In the above formula, (Saccharide) indicates a monosaccharide or disaccharide having no ionic group in the side chain. ].

  According to the production method, not only has an appropriate particle size, but also the particle sizes of individual particles are relatively uniform, the particle size distribution range is narrow, and noble metal particles having an excellent Raman dispersion light enhancement effect can be easily obtained. be able to.

  Since the Raman spectrum measurement method of the present invention can effectively enhance weak Raman dispersion light, the sensitivity is extremely high as compared with the conventional measurement method. Moreover, since the noble metal particles used in the measurement method of the present invention are extremely excellent in dispersion stability, they can easily aggregate and settle even when measuring a sample containing a large amount of electrolyte such as a biological sample. It can be measured stably. Therefore, the Raman spectrum measurement method of the present invention is very useful in that it can be applied to diagnosis of diseases and analysis of waste liquids that are expected to contain a large number of compounds.

  Furthermore, the noble metal particles of the present invention are industrially useful as an additive used in the above-described measurement method, and the production method of the present invention is capable of producing the noble metal particles in a large amount and at a low cost.

  The method for measuring a Raman spectrum according to the present invention is summarized in that noble metal particles having a substituent represented by the following formula (I) on the surface are added to a sample.

[In the formula, (Saccharide) represents a monosaccharide or disaccharide having no ionic group in the side chain. ].

  “Noble metal” refers to gold, silver, platinum, rhodium, ruthenium, palladium, osmium, and iridium. In the present invention, any of gold, silver, platinum, or rhodium is preferably used, and gold or silver is particularly preferable. Can be used for This is because gold and silver have been demonstrated to exhibit the surface plasmon resonance effect well.

  In the present invention, the term “particle” refers to a particle in which the noble metal atoms are bonded to each other by a metal bond, and therefore, the noble metal ions are not aggregated by an ionic bond with a counter ion.

  The noble metal fine particles of the present invention have a substituent represented by the formula (I) on the surface. The type of chemical bond between the substituent and the noble metal is not necessarily clear, but it is not an ionic bond between at least the noble metal ion and the sulfur ion group. This is because no surface plasmon resonance effect is observed in the aggregate of noble metal ions.

  In the noble metal particles of the present invention, the noble metal atom and the substituent (I) are not bonded one to one. Substituent (I) is present on the particle surface, and only noble metal atoms should be present inside the particle, and from the bulk of the saccharide, one saccharide per noble metal atom 1 on the particle surface It is difficult to think that is bound. In this respect, it can be clearly distinguished from the compound described in Patent Document 3 in which a gold atom and a saccharide are bonded one to one.

  In the present invention, the “ionic group” refers to a group that can have a charge in its structure even under relatively mild conditions, and that causes noble metal particles to easily aggregate and settle due to the presence of counter ions. Examples of such groups include amino groups, carboxyl groups, phosphoric acid groups, and lower alkyl groups substituted therewith. In addition, although a hydroxyl group can become an ion by presence of a strong base, since the state in which a strong base exists is not a relatively mild condition, a hydroxyl group is not an ionic group.

  The “monosaccharide” in the present invention refers to a monovalent group in which one hydroxyl group is eliminated from a general carbohydrate that cannot be hydrolyzed any more, and two “disaccharides” are the same or different from each other. A monovalent group to which a monosaccharide is bonded.

  Accordingly, examples of “(Saccharide)” (monosaccharide or disaccharide having no ionic group in the side chain) of the present invention include tricarbon sugar (glyceraldehyde); tetracarbon sugar (erythrose or throse); A pentose such as ribose or xylose; a hexose such as glucose or fructose; and a disaccharide in which two saccharides selected from these are combined, and a monovalent group in which one hydroxyl group is eliminated. And “(Saccharide)” present on the particle surface has at least one hydroxyl group while having no ionic group. Since the high surface plasmon resonance effect can be exhibited by aggregating the light, it is possible to effectively enhance the weak Raman dispersion light.

  However, when producing noble metal particles by the method of the present invention described later, a part of hydroxyl groups present in (Saccharide) may be oxidized to form aldehyde groups. The definition of “(Saccharide)” in the present invention includes monosaccharides or disaccharides having an aldehyde group as described above.

  When an aldehyde group is present at (Saccharide) in a substituent, it can react with an amino group to form an imino group. In the case of (Saccharide) which is a substituent of the noble metal particles produced by the method of the present invention described later, some hydroxyl groups may be oxidized to form aldehyde groups. Therefore, the noble metal particles of the present invention may be particularly able to enhance the Raman scattered light by binding to a protein having an amino group in the structure. In order to obtain the same effect with the prior art, for example, a functional group having reactivity with the target compound must be introduced into the terminal of polyethylene glycol in advance.

  The average particle diameter of the noble metal particles of the present invention is preferably in the range of 30 to 50 nm. This is because if it is less than 30 nm, the effect of enhancing Raman scattered light may not be sufficient, and if it exceeds 50 nm, particles may aggregate excessively during measurement. Furthermore, the noble metal particles within the above range can form an aggregate having an appropriate particle size to such an extent that aggregation and sedimentation is not caused by addition of an appropriate amount of salt to the measurement system, and Raman scattering light can be further enhanced. However, since the range has an average particle size, even when the average particle size is in the range, particles having a particle size of about 20 nm or about 60 nm may exist. If the average particle size is within the range, there may be a case where a more excellent effect is exhibited by the presence of particles having a particle size not within the range of 30 to 50 nm. That is, particles having a particle size of about 20 nm are excellent in dispersion stability, and particles having a particle size of about 60 nm have a large surface area, so that a good surface plasmon resonance effect can be exhibited.

  The method for measuring the average particle size is not particularly limited, but it is considered that the particle size to be measured is in the range of several nanometers to several tens of nanometers, so that a laser scattering method suitable for particle size measurement of fine particles can be applied ( The average particle size may be calculated after measuring the particle size distribution with a laser scattering photometer.

  The shape of the noble metal particles is considered to be almost spherical, but those having a polyhedral structure are suitable. This is because a polyhedral noble metal particle can be expected to have a high surface plasmon resonance effect at its apex.

  The Raman spectrum measuring method of the present invention can use a known measuring method or measuring apparatus. However, a liquid sample is used, and if necessary, a salt is added during measurement.

  Specifically, a liquid sample or a sample in which a sample is dissolved or dispersed in a liquid is used. The solvent used for preparing the sample is preferably water; or a mixed solvent of water and a water-soluble solvent such as a lower alcohol. This is because the noble metal particles of the present invention are highly hydrophilic and can be easily dissolved or uniformly dispersed in an aqueous solvent.

Further, at the time of measurement, a salt is added as necessary. Since the noble metal particles of the present invention do not have an ionic group, they will not easily aggregate and settle even if ions are present in the sample. On the other hand, since it has at least one hydroxyl group, it can be appropriately aggregated by the presence of a cation, and can exhibit a higher surface plasmon resonance effect than when noble metal particles are present alone. , The intensity of Raman dispersed light can be increased. Therefore, when a proper cation is contained in the sample, it may be measured as it is, but when the precious metal particles do not aggregate appropriately, an appropriate amount of salt (electrolyte) is added. As such a salt, common sodium salts such as NaCl, NaH ₂ PO ₄ , NaBr, NaI, CH ₃ CO ₂ Na, NaNO ₃ can be used. Further, when the ionic strength of the sample is too high, the sample may be diluted with the above-described solvent. These specific conditions can be determined by preliminary experiments.

  As described above, the presence of cations in the measurement system and the addition of an appropriate amount of salt allow the noble metal particles of the present invention to form aggregates with an appropriate particle size without agglomeration and sedimentation. If the particle size of such an aggregate is about 0.6 to 1.5 μm, it does not aggregate and settle, and the apparent surface area increases, so that it shows a more excellent Raman dispersion light enhancement effect. This is demonstrated in the examples described later.

  The noble metal particles of the present invention can be synthesized by dropping a solution of the following compound (II) into a corresponding halogenated noble metal acid or a salt thereof or a solution of a noble metal salt of an inorganic acid while heating.

[In the above formula, (Saccharide) has the same meaning as described above. ].

  The “halogenated noble metal acid” may be any one depending on the noble metal constituting the target noble metal particle. For example, a halogenated platinic acid such as chloroauric acid or a haloplatinic acid such as chloroplatinic acid. Can be used as a raw material. Further, as the “halogenated noble metal acid salt”, an alkali metal salt or an alkaline earth metal salt of a metal halide acid can be used. The “noble metal salt of an inorganic acid” may be a common noble metal salt of an inorganic acid. For example, a noble metal salt of nitric acid such as silver nitrate can be used. However, since the halogenated noble metal acid and its salt and the noble metal salt of an inorganic acid need to be in solution, it must be soluble in the solvent used.

  Compound (II) is a monosaccharide or disaccharide having one hydroxyl group substituted with a thiol group. If this compound (II) is marketed, it may be used, and even if it is not commercially available, it can be easily synthesized from the corresponding monosaccharide or disaccharide.

  The solvent used in the solution of the noble metal particles and the compound (II) is not particularly limited as long as these compounds can be appropriately dissolved or uniformly dispersed. For example, water (pure water, distilled water, or the like containing water as a main component) As long as it is a thing, the name will not ask | require in particular); Or the mixed solvent of water and water-soluble solvents, such as a lower alcohol, ie, an aqueous solvent, can be used conveniently. Further, different solvents may be used for these solutions, but the same is preferable.

  In the method for producing noble metal particles according to the present invention, the solution of the compound (II) is dropped while heating the halogenated noble metal acid solution. At this time, since the particle diameter of the noble metal particles obtained tends to increase as the temperature increases, it is preferably heated to reflux. The heating reflux temperature depends on the solvent to be used, but in the method of the present invention, water or a mixed solvent containing water (aqueous solvent) is mainly used as the solvent, and therefore the temperature is in the range of about 80 to about 110 ° C. In addition, since the particle diameter tends to increase as the dropping speed decreases, the dropping speed may be adjusted according to the desired particle diameter. Furthermore, since the distribution range of the particle diameter is considered to be narrower as the temperature change of the reaction solution is smaller, the dropping speed is preferably as slow as possible within the range in which a desired particle diameter can be obtained.

  As described above, there is a close relationship between the dropping rate of the solution of compound (II) and the particle size of the generated particles. Therefore, it is necessary to control the dropping speed. However, by reducing the concentration of the halogenated noble metal acid solution and the compound (II) solution, good noble metal particles can be produced without particularly controlling the dropping rate. For example, by adjusting the concentration of the compound (II) solution to about 0.1 g / L to 1 g / L and the concentration of the halogenated noble metal acid solution to about 0.05 g / L to 0.4 g / L, the compound (II) Even if the dropping rate of the solution of II) is not strictly controlled, noble metal particles having a relatively narrow particle size distribution and an appropriate average particle size can be obtained without aggregation and sedimentation.

  After completion of the reaction, if purification is performed using a membrane filter having a pore size corresponding to a desired particle size, coarse particles having poor dispersion stability can be excluded. Further, excessively small particles can be eliminated by further using membrane filters having different pore sizes. Incidentally, according to the production method of the present invention, noble metal particles having an appropriate particle size can be produced efficiently, so that they can be sufficiently put into practical use without eliminating unnecessary particles. Further, since the production method of the present invention is very simple, the target noble metal particles can be produced at low cost and in large quantities. Furthermore, as demonstrated in the examples described later, the noble metal particles produced by the method of the present invention have a polyhedral structure having a particularly high surface plasmon resonance effect.

  Hereinafter, the present invention will be described in more detail by way of examples, but the scope of the present invention is not limited thereto.

  Production Example 1 Production of thioglucose-modified gold nanoparticles

[In the above formula, the product compound is schematically represented, and the shape of the particles and the number of sugars present on the surface are not limited to this. ]
Thioglucose (42.3 mg, 0.194 mmol) was dissolved in water (5 mL) to prepare a 38.8 mM thioglucose aqueous solution. Separately, chloroauric acid (HAuCl ₄ , 24.7 mg, 0.12 mmol) and water (50 mL) were placed in a reaction vessel and heated to reflux. First, 26% (1.3 mL) of the total amount of thioglucose aqueous solution prepared in advance was dropped at a rate of 80 to 95 μL / second, and then the remaining amount at a rate of 60 to 70 μL / second. When dropped, the color of the reaction solution quickly changed from yellow to reddish purple indicating the formation of gold particles. Even after the completion of the dropwise addition, heating under reflux was continued for another 10 minutes.

  Then, pure thioglucose modified gold nanoparticles were obtained by filtration using a membrane filter having a pore size of 50 nm (manufactured by whatman, Nycrypore Polycarbonate Track Etch Membrane).

Production Example 2 Production of Thioglucose-Modified Gold Nanoparticles Nanogold particles were produced using a thioglucose solution and a gold ion solution having a concentration lower than that of Production Example 1.

  Thioglucose (2.73 mg 0.0125 mmol) was dissolved in water (5 ml) to prepare a 2.5 mM thioglucose aqueous solution. Separately, chloroauric acid (10.3 mg, 0.025 mmol) and water (50 ml) were added to the reaction vessel and heated to reflux. Here, when the thioglucose aqueous solution prepared in advance was dropped without particularly controlling the dropping rate, the reaction solution rapidly changed from yellow to reddish purple indicating the formation of gold particles. After completion of the dropwise addition, the mixture was further heated to reflux for 10 minutes. 20 ml of the reaction solution was put in a dialysis tube (Spectra / Por cellulose ester MWCO: 5000) manufactured by Spectrum and dialyzed for 24 hours in 1 L of ultrapure water adjusted to pH 10 to remove unreacted raw materials and purify.

Comparative Production Example 1 Production of Citric Acid-Modified Gold Nanoparticles A 0.01% by mass chloroauric acid aqueous solution (50 mL) was placed in a reaction vessel and heated to reflux, and 1.0% by mass sodium citrate separately prepared here Aqueous solution (0.5 mL) was added dropwise. After the dropwise addition, the mixture was further heated and refluxed for 10 minutes. As a result, the reaction solution turned blue and eventually turned red.

  After completion of the reaction, the product was purified by the same method as in Production Example 1 to obtain citric acid-modified gold nanoparticles.

Test Example 1 Measurement of optical properties of thioglucose-modified gold nanoparticles The optical properties of various gold nanoparticles were measured using an ultraviolet-visible spectrophotometer (manufactured by JASCO Corporation, U-560). The results are shown in FIG.

Here, in general, the larger the particle size, the higher the absorption intensity (Iλ _max ) at the maximum wavelength, but as the particle aggregation proceeds in the solution, Iλ _max decreases, and in the near infrared region near 800 nm. It can be said that the absorption strength (I ₈₀₀ ) of the resin increases. That is, as an indicator of the particle size and dispersion state of the synthesized gold nanoparticles, can be used Airamuda _max and I _800. Therefore, as shown in Table 1, the relative intensity of Airamuda _max and I ₈₀₀ for I _350, for convenience categorized gold nanoparticles into four classes. That is, with respect to Iλ _max / I ₃₅₀ = 0.8, those having a relatively small particle size are class I and II, those having a relatively large particle size are class III and IV, and based on I ₈₀₀ / I ₃₅₀ = 0.2. Those having a relatively tendency to aggregate were designated as classes II and IV, and those having a relatively low tendency to aggregate were designated as classes I and III.

  According to FIG. 1, the absorption spectrum of the thioglucose modified gold nanoparticles showed strong absorption around 520 nm. This is considered to be derived from the surface plasmon resonance effect of gold. It can also be seen that class IV gold nanoparticles have the strongest absorption at the maximum wavelength and are large in particle size. However, since there is an absorption peak due to particle aggregation at 600 nm or more, it is considered that the dispersion stability is somewhat low. On the other hand, no absorption peak indicating aggregation of particles was observed in the absorption spectra of class I and III gold nanoparticles, indicating that the dispersion stability was extremely excellent. When the optical properties of the gold nanoparticles produced in Production Example 1 were measured, they could be classified into Class I. Therefore, it was revealed that the gold nanoparticles obtained in Production Example 1 are excellent in dispersion stability.

Test Example 2 Measurement of Average Particle Size and Particle Size Distribution of Thioglucose-modified Gold Nanoparticles The average particle size and particle size distribution of the gold nanoparticles produced in Production Example 1 were measured using a dynamic light scattering photometer (Otsuka Electronics Co., Ltd. , DLS-6000DLX). The results are shown in FIG. According to the result, the average particle size of the gold nanoparticles of Production Example 1 was about 33.8 nm, the particle size distribution was relatively narrow, and formation of aggregates was not confirmed. Therefore, the gold nanoparticles can be expected to be applied as an excellent Raman sensitizing reagent with excellent dispersion stability.

Test Example 3 Shape of Thioglucose Modified Gold Nanoparticles The gold nanoparticles produced in Production Example 1 were observed with a transmission electron microscope (JEM-100CX type, manufactured by JEOL Ltd.). The electron micrograph is shown in FIG. According to the result, polyhedral particles were confirmed. Polyhedral metal particles have been reported to have a high electric field enhancement effect at the apex (see KLKelly et al., Computing in Science & Engineering, Vol. 3, 67-73 (2001)). The gold nanoparticle of Example 1 has a high electric field enhancing effect and was proved to be excellent as a reagent for Raman sensitization.

Test Example 4 Evaluation of Dispersion Stability of Gold Nanoparticles The gold nanoparticles produced in Production Example 1 and Comparative Production Example 1 were redispersed in a 100 mM NaCl aqueous solution, and the intensity change of the absorption spectrum was observed. The results are shown in FIG. According to the result, in the citric acid-modified gold nanoparticles having an ionic group (carboxyl group) on the surface, the aggregation of the particles proceeded immediately after redispersion, and precipitated after 3 hours. On the other hand, with the thioglucose-modified gold nanoparticles of the present invention, it was confirmed that there was almost no change in absorption intensity even after 24 hours and that the thioglucose-modified gold nanoparticles were stable even under high ionic strength.

  Furthermore, it was redispersed in a HEPES buffer solution (pH = 7.4) having various ionic strengths (10 to 200 mM NaCl), and changes in visible transmittance were observed. The results are shown in FIGS. According to the results, the citrate-modified gold nanoparticles aggregated immediately after being redispersed and were so unstable that they cannot be evaluated in a buffer solution. On the other hand, with thioglucose-modified gold nanoparticles, although a slight decrease in dispersion stability was observed under an ionic strength of 200 mM, there was almost no change in the absorption strength of the gold nanoparticles after 24 hours under an ionic strength of 100 mM or less. It was confirmed that it was stable.

Test Example 5 Particle Size Distribution and Shape of Thioglucose-Modified Gold Nanoparticles For the gold nanoparticles produced in Production Example 2, the average particle size and particle size distribution were measured in the same manner as in Test Example 2. The results are shown in FIG. According to the results, the average particle size of the gold nanoparticles of Production Example 1 was about 34.7 ± 9.7 nm, the particle size distribution was relatively narrow, and formation of aggregates was not confirmed. Moreover, it observed with the transmission electron microscope similarly to the said Experimental example 3. The electron micrograph is shown in FIG. According to the result, polyhedral particles were confirmed.

  From these results, when using a low concentration thioglucose solution and a gold ion solution as raw materials, nanogold particles having excellent properties can be produced without strictly controlling the dropping rate of the thioglucose solution. Proven.

Test Example 6 Raman spectrum measurement Thioglucose-modified gold nanoparticles were produced in the same manner as in Production Example 2 above. When the particle size distribution and average particle size of the gold nanoparticles were measured by the same method as in Test Example 2, it was as shown in FIG. The gold nanoparticles were dissolved in 10 mM HEPES buffer (pH 7.4), and the concentration of the solution was adjusted so that the intensity of the visible absorption band observed near 500 to 600 nm was 1.0. 1.0 ml of this solution was taken and NaCl was added to a concentration of 65 mM to form aggregates having a size of about 0.9 to 1.3 μm (FIG. 9B). The aggregate was dispersed in the buffer without aggregation and sedimentation.

  The obtained aggregate dispersion was mixed well with 10 μL of 1 mM pyridine aqueous solution or 10 μM mercaptophenol aqueous solution, and then wavelength: 785 nm using a microscopic laser Raman spectrometer (LabRam HR-800, manufactured by Horiba Jobibon). Output: A semiconductor laser beam of 30 mW was irradiated, and a Raman spectrum was measured. For comparison, measurement was also performed when no gold nanoparticles were added. The results are shown in FIGS. 10 (a) and 10 (b).

Also, a dispersion of gold nanoparticle aggregates was prepared by the same method as above except that various inorganic salts (NaF, NaBr, NaI, NaOAc, NaNO ₃ or NaH ₂ PO ₄ ) were added instead of NaCl. And 10 μL of 1 μM rhodamine 6G (fluorescent dye) aqueous solution, and Raman Raman spectrum was measured. The results are shown in FIG.

  From the above results, by adding the thioglucose-modified gold nanoparticles according to the present invention and measuring the Raman spectrum, a ring concentration vibration of a low concentration compound that cannot be measured normally was strongly observed (FIG. 10 (a)). And (b)). As described above, the effect that the Raman scattered light intensity can be remarkably enhanced was observed in gold nanoparticles aggregated with any inorganic salt, but the most remarkable enhancement effect was observed particularly when NaBr was used as a flocculant. (See FIG. 10 (c)).

  Further, according to the method of the present invention, it has been proved that Raman spectrum measurement is possible even when near infrared light of 785 nm is used as excitation light. This indicates that Raman spectrum measurement can be performed without using light having a short wavelength that is considered to be harmful to a biological sample.

Test Example 7
The thioglucose-modified gold nanoparticles produced in Production Example 2 above are dissolved in a 10 mM HEPES buffer (pH 7.4), and the intensity of the visible absorption band observed at around 500 to 600 nm is 1.0. The concentration was adjusted. Ethylenediamine (1.2 mg, 0.02 mmol) was added to 20 mL of the solution so that the concentration became 10 mM, and the mixture was stirred at room temperature for 30 minutes. The reaction solution changed from red to blue with the passage of time, and it was found that the gold nanoparticles aggregated, but they did not aggregate and settle. This reaction solution was put into a dialysis tube (Spectra / Por cellulose ester MWCO: 5000), dialyzed for 24 hours in 1 L of ultrapure water adjusted to pH 10, and purified by removing unreacted ethylenediamine. After dialysis, the solvent was removed, the residue was dried under reduced pressure, and the IR spectrum of the gold nanoparticles was measured. For comparison, IR spectra were similarly measured for gold nanoparticles purified without being treated with ethylenediamine. The results are shown in FIG.

According to the result, an untreated gold nanoparticle showed an absorption peak at 1731 cm ⁻¹ derived from an aldehyde group. This is considered that a part of the hydroxyl group of glucose substituted on the gold nanoparticle surface is oxidized. Further, in the IR spectrum of gold nanoparticles treated with ethylenediamine, the absorption peak at 1731 cm ⁻¹ disappeared, and the absorption peak at 1635 cm ⁻¹ derived from the imino group appeared instead.

  From the above results, the aldehyde group is introduced into the substituent of the gold nanoparticle according to the present invention, and since it has the ability to react with an amino group, it binds to a compound having an amino group and is attributed to the compound. It is considered that the intensity of Raman scattered light can be enhanced. Therefore, the gold nanoparticles according to the present invention can be used for detection of biomolecules containing amino groups such as proteins.

FIG. 3 is a diagram showing a light absorption spectrum of thioglucose-modified gold nanoparticles produced in Production Example 1. It is a figure which shows the result of having measured the particle size distribution of the thioglucose modification gold | metal | money nanoparticle manufactured in manufacture example 1 by the laser scattering method. It is a transmission electron micrograph of the thioglucose-modified gold nanoparticles produced in Production Example 1, and it can be observed that it has a polyhedral structure. It is a figure which shows the dispersion stability of the thioglucose modification gold nanoparticle manufactured in manufacture example 1, and the citric acid modification gold nanoparticle concerning a prior art. It is a figure which shows the absorption spectrum in the 50 mM NaCl HEPES buffer solution (pH = 7.4) of the thioglucose modification gold nanoparticle manufactured by manufacture example 1, Comprising: It is a figure which shows the dispersion stability of the said particle | grain. It is a figure which shows the absorption spectrum in 10-200 mM NaCl HEPES buffer (pH = 7.4) of the thioglucose modification gold nanoparticle manufactured by manufacture example 1, Comprising: It is a figure which shows the dispersion stability of the said particle | grain. . It is a figure which shows the result of having measured the particle size distribution of the thioglucose modification gold | metal | money nanoparticle manufactured in manufacture example 2 by the laser scattering method. It is a transmission electron micrograph of the thioglucose-modified gold nanoparticles produced in Production Example 2, and it can be observed that it has a polyhedral structure. (A) Particle size distribution and average particle size of the gold nanoparticles produced in Test Example 6, and (b) Particle size distribution and average particle size of the gold nanoparticle solution obtained by adding a salt to aggregate It is. The dispersion of the gold nanoparticle aggregate according to the present invention was added, (a) the result of Raman spectrum measurement for a low concentration solution of pyridine, (b) the result of measurement for a low concentration solution of mercaptophenol, (c ) It is a figure which shows the result measured about the low concentration solution of rhodamine 6G, changing the kind of inorganic salt to add. It is IR spectrum of the thing which processed the gold nanoparticle concerning this invention with ethylenediamine, and an unprocessed thing.

Claims (6)

A method for measuring a Raman spectrum, comprising adding a noble metal nanoparticle having a substituent represented by the following formula (I) on the surface and a salt to a sample, and setting a cation concentration in the sample to 10 to 100 mM.
[In the above formula, (Saccharide) indicates a monosaccharide or disaccharide having no ionic group in the side chain. ] The method for measuring a Raman spectrum according to claim 1, wherein the noble metal nanoparticles have an average particle size of 30 to 50 nm. 2. The method of Raman spectrum measurement according to claim 1, wherein the noble metal nanoparticles are aggregated to a particle size of 0.6 to 1.5 [mu] m. A substance for enhancing the surface plasmon resonance effect, comprising a noble metal nanoparticle having a substituent represented by the following formula (I) on its surface .
[In the above formula, (Saccharide) indicates a monosaccharide or disaccharide having no ionic group in the side chain. ] 5. The substance for enhancing the surface plasmon resonance effect according to claim 4, wherein the noble metal nanoparticles have an average particle size of 30 to 50 nm. 6. The noble metal nanoparticle according to claim 4 or 5, wherein a solution of a compound represented by the following formula (II) is dropped into a solution of a halogenated noble metal acid or a salt thereof or a noble metal salt of an inorganic acid while heating. Particle production method.
[In the above formula, (Saccharide) indicates a monosaccharide or disaccharide having no ionic group in the side chain. ]