JP2006271244A - Functional particle and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide functional particles capable of highly efficiently immobilizing a target physiologically active substance and capable of highly efficiently recovering the target physiologically active substance which is immobilized. <P>SOLUTION: The functional particles comprise particles of FePt, FePd, and a CoPt alloy, have a gold or gold alloy film which are formed on the surfaces of the particles of the FePt, the FePd, and the CoPt alloy, and are magnetized. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a functional particle for binding a specific physiologically active substance and a method for producing the same. More specifically, the present invention relates to a functional particle useful for use as a bacterial separation carrier, a nucleic acid purification carrier, a protein purification carrier, an immobilized enzyme carrier, an immobilized antibody carrier, and a method for producing the same.

  Many physiologically active substances such as enzymes and antibodies have a high selectivity for a specific substance. Conventionally, the properties of such a physiologically active substance have a high accuracy in the components of a specific biological substance. Is used to detect in. In particular, after immobilizing a physiologically active substance that reacts with a target substance on a carrier, the specimen is brought into contact, and further reacted with a labeled protein that specifically binds to the target substance (immunoassay method). It is often used in immunological tests.

  As a method for immobilizing a specific physiologically active substance on a carrier, a method utilizing specific affinity between molecules is used. Moreover, as a material for immobilizing a physiologically active substance, a material composed of a silanol derivative is known (for example, see Patent Document 1). In order to immobilize the physiologically active substance on the carrier, first, the immobilization material is introduced into the base material by the silane coupling agent, then activated, and then the activated substance is brought into contact with the activated immobilization material. . This immobilization material has the advantage that an arbitrary physiologically active substance can be immobilized on the substrate by arbitrarily changing the chain length, but contains an easily decomposed bond, and the immobilization material itself is stable. There is a problem that is bad.

  Examples of the physiologically active substance include proteins having functions of enzymes, antibodies, coenzymes, glycoproteins, saccharides, etc. Among them, carriers for immobilizing enzymes are known (for example, patent documents). 2 and 3). The former is an inorganic or organic carrier for immobilizing an enzyme to which a coupling agent having a functional group having affinity for an enzyme and a hydrophobic functional group is bound. On the other hand, the latter is an inorganic carrier treated with a silane coupling agent having a special functional group. The enzyme is immobilized thereon, washed and dried, and then impregnated with a fatty acid to obtain an immobilized enzyme carrier.

  Gold is known to have an affinity for a thiol group. By forming a gold film on a carrier, a physiologically active substance can be immobilized via the thiol group. For example, in applications such as biosensors, those in which a metal film is formed on a flat substrate are used (see, for example, Patent Documents 4 and 5). In the former, a metal film such as gold, silver, or platinum is directly formed on a substrate such as glass or polyethylene terephthalate, and the physiologically active substance is immobilized on the metal film via a thiol group. The binding property of this physiologically active substance is detected by surface plasmon resonance analysis. In the latter, fine particles made of plastic are arranged on a substrate, and gold is evaporated from above. Part of the surface of the fine particles is coated with gold, and the physiologically active substance is immobilized on the gold surface. However, these are suitable for measuring the binding properties of physiologically active substances, but are not suitable for purification of physiologically active substances because they easily bind to substances other than the target substance.

  As a use for purifying a physiologically active substance, particles having a size of 0.1 μm to 10 μm obtained by coating the surface of a polymer with gold are known (for example, see Patent Document 6). These functional particles are formed by coating the surface of polymer particles with gold having affinity for thiol groups, so that physiologically active substances can be immobilized via thiol groups and substances other than the target substance can be bound. Suitable for purification of physiologically active substances.

  However, since the functional particles for use in purifying a physiologically active substance described in Patent Document 6 do not have a configuration capable of aggregating as needed, the functional particles are recovered with high efficiency from the state dispersed in the specimen. Therefore, there is a problem that the recovery efficiency of the target physiologically active substance is low.

Japanese Patent Application Laid-Open No. 5-344485 JP-A-5-219952 JP-A-9-257 JP 2003-194820 A Japanese Patent Laid-Open No. 11-326193 JP 2004-150841 A

  Accordingly, an object of the present invention is to provide functional particles capable of highly efficiently immobilizing a target physiologically active substance and recovering the immobilized target physiologically active substance with high efficiency, and a method for producing the same. It is.

  The invention according to claim 1 as means for solving the problems includes FePt, FePd and CoPt alloy particles, and a gold or gold alloy film formed on the surface of the alloy particles, and is magnetic. It is the functional particle characterized by this.

  As a means for solving the above-mentioned problem, the invention according to claim 2 is the functional particle according to claim 1, wherein the FePt, FePd, and CoPt alloy particles are selected from B, Bi, Cu, Sn, Sb, and Pb. The functional particle according to claim 1, comprising at least one element.

  As a means for solving the above-mentioned problem, the invention according to claim 3 is that the average particle size of the alloy particles is in the range of 3 nm to 50 nm, and the thickness of the gold or gold alloy film is in the range of 3 nm to 100 nm. It is a functional particle in any one of Claims 1-2 characterized by the above-mentioned.

  The invention according to claim 4 as means for solving the problem further comprises a spacer layer formed on a surface of the gold or gold alloy film. Functional particles.

  As a means for solving the above-mentioned problems, the invention according to claim 5 is characterized in that the spacer layer has a general formula, RSH, RSR, RSSR (wherein R is an amino group having 20 or less carbon atoms, Having at least one functional group selected from the group consisting of carboxyl group, hydroxyl group, halogen, thiol group, nitro group, aldehyde group, carbonyl group, sulfonyl group, sulfonic acid group, nitroso group, amide group and azide group, A linear or branched alkyl group, a linear or branched alkenyl group, a linear or branched alkynyl group, an alicyclic group, an aromatic group, a condensed cyclic group or a heterocyclic group; 5) The compound is formed by bonding to the lower noble metal film through a compound containing a thiol group, sulfide group or disulfide group represented by It is a function of particle.

  The invention according to claim 6 as means for solving the problem comprises at least the functional particle according to any one of claims 1 to 5, wherein a physiologically active substance is bound to the surface of the functional particle. A bioactive substance capture / recovery / purification kit characterized by capturing, recovering and purifying a target bioactive substance.

  Since the functional particle of the present invention has a gold film or a gold alloy film on the outermost surface, a physiological activity containing a thiol group, sulfide group or disulfide group having affinity for the gold or gold alloy film in the molecule. The substance can be captured. In addition, since the functional particles of the present invention are magnetic, the physiologically active substance captured by the functional particles can be efficiently recovered and purified by applying a magnetic field.

  Physiologically active substances that do not contain thiol groups, sulfide groups or disulfide groups in the molecule do not bind to the gold film or gold alloy film, but by providing a specific spacer layer on the outermost layer of the functional particles, Alternatively, a physiologically active substance having no disulfide group can also be captured. For example, a mercaptan compound having a thiol group is first bonded to gold or a gold alloy film to provide a spacer layer, and various physiologically active substances can be captured through the functional group of the mercaptan compound forming the spacer layer. . A specific physiologically active substance can be selectively captured by appropriately selecting a functional group in the mercaptan compound. Since the functional particles having the spacer layer are also magnetized, physiologically active substances such as proteins captured by the functional particles can be effectively recovered and purified by applying a magnetic field.

  Therefore, the functional particles of the present invention can efficiently capture a specific physiologically active substance, recover or purify it with or without utilizing magnetism.

  Hereinafter, the functional particles of the present invention will be specifically described with reference to the drawings. FIG. 1 is a schematic sectional view of an example of the functional particles of the present invention. The functional particle 1A of the present invention basically has FePt, FePd, and CoPt particles 5 as nuclei and has a gold film 7 on the outer surface of the combined FePt, FePd, and CoPt particles 5.

  FIG. 2 is a schematic cross-sectional view of another example of the functional particles of the present invention. The functional particle 1B shown in FIG. 2 has FePt, FePd, and CoPt particles 5 as nuclei and a gold film 7 on the outer surface of the nickel particles 5 as in the functional particle 1A shown in FIG. Unlike the functional particles 1A, the gold film 7 further has a spacer layer 9 bonded to a specific physiologically active substance on the outer surface.

Since FePt, FePd, and CoPt are superparamagnetic materials after synthesis, when functional particles 1A and 1B use FePt, FePd, and CoPt particles 5, functional particles 1A and 1B are responsive to magnetic fields. By operating the magnetic field, dispersion into the specimen and aggregation in the specimen can be performed at any time, so that functional particles can be easily collected from the specimen. Further, FePt, B in FePd and CoPt particles, Bi, Cu, Sn, Sb , by the inclusion of at least one element selected from Pb, the L1 ₀ ordered phase that exhibits a high magnetic anisotropy The phase transition temperature can be reduced below 300 ° C. For example, when refluxing at 300 ° C. using an alcohol reduction method, FePtBi particles have an L10 ordered phase structure after synthesis. Therefore, it has high magnetic anisotropy and can be easily collected by a magnet.

  On the other hand, gold or a gold alloy is a metal material having a high affinity with a thiol group, sulfide group or disulfide group. By forming a gold film or a gold alloy film on the outermost surface of the functional particle, a thiol group is formed on the functional particle. A physiologically active substance can be immobilized via a compound containing a sulfide group or a disulfide group. Therefore, the functional particle 1A having the FePt, FePd, and CoPt alloy particles 5 and the gold film or the gold alloy film 7 formed on the surface of the FePt, FePd, and CoPt alloy particles has the recovery efficiency of a desired physiologically active substance. Can be increased.

  Even if the gold film or gold alloy film 7 is present on the outermost surface of the functional particle 1A, the physiologically active substance that does not contain a thiol group, sulfide group, or disulfide group in the molecule is bonded to the gold film or gold alloy film 7. I can't. Therefore, in the functional particle 1B of the present invention, by providing the specific spacer layer 9 in the outermost layer of the functional particle, it is possible to capture a physiologically active substance having no thiol group, sulfide group or disulfide group. For example, a mercaptan compound having a thiol group is first bonded to a gold film or a gold alloy film 7 to provide a spacer layer 9, and various physiologically active substances are captured through the functional group of the mercaptan compound forming the spacer layer. be able to. A specific physiologically active substance can be selectively captured by appropriately selecting a functional group in the mercaptan compound.

  In the functional particles 1A and 1B of the present invention, the average particle size of the FePt, FePd, and CoPt alloy particles 5 is preferably in the range of 3 nm to 50 nm. If the average particle size of the FePt, FePd, and CoPt alloy particles 5 is too small, the particles tend to aggregate even when no magnetic field is applied, resulting in poor dispersibility in the specimen (sample). On the other hand, if the average particle size of the FePt, FePd, and CoPt alloy particles 5 is too large, the specific surface area decreases, and the immobilization efficiency of the physiologically active substance decreases. According to experiments by the present inventors, by making the average particle size of FePt, FePd and CoPt alloy particles 5 in the range of 3 nm to 50 nm, the dispersibility in the specimen (sample) is good and the physiologically active substance Functional particles having high immobilization efficiency can be obtained.

  In the functional particle 1A of the present invention, the thickness of the gold film or the gold alloy film 7 is preferably in the range of 3 nm to 100 nm. The immobilization efficiency of the physiologically active substance in the functional particles varies depending on the thickness of the noble metal film 7. That is, when the thickness of the noble metal film 7 is less than 3 nm, the immobilization efficiency of the physiologically active substance in the functional particles decreases. In addition, since the surface of the underlying FePt, FePd, and CoPt alloy particles cannot be sufficiently coated, Fe and Co ions flow out, Fe and Co oxides are formed on the particle surface, and peptide bonds in the specimen are cleaved. This is not preferable because it may occur. When the thickness of the noble metal film 7 exceeds 100 nm, the immobilization efficiency of the physiologically active substance is saturated. Therefore, by setting the thickness of the noble metal film 7 in the range of 3 nm to 100 nm, the physiologically active substance can be immobilized with high efficiency and economical functional particles. The gold alloy film is formed using a gold alloy having an affinity for a thiol group, sulfide group or disulfide group, such as an AuPd alloy.

  In the functional particle 1B of the present invention, the spacer layer 9 is bonded to the noble metal film 7 through a thiol group (—SH), a sulfide group (—S—) or a disulfide group (—S—S—). A thiol group, a sulfide group or a disulfide group has a high affinity with gold or a gold alloy and has a characteristic that it can be easily bonded. Therefore, the spacer layer 9 has a general formula, RSH, RSR, RSSR (wherein R is a carbon atom number of 20 or less, amino group, carboxyl group, hydroxyl group, halogen, thiol group, nitro group, aldehyde) A linear or branched alkyl group having at least one functional group selected from the group consisting of a group, a carbonyl group, a sulfonyl group, a sulfonic acid group, a nitroso group, an amide group and an azide group, a linear group Or a branched alkenyl group, a linear or branched alkynyl group, an alicyclic group, an aromatic group, a condensed cyclic group or a heterocyclic group)), a thiol group, a sulfide group or a disulfide group. Can be used. Therefore, the spacer layer 9 is bonded to the noble metal film 7 as, for example, Au—S—R. Since the physiologically active substance that can be recovered can be appropriately changed according to the type of the compound containing the thiol group, sulfide group, or disulfide group constituting the spacer layer 9, it is possible to make the functional particle 1B versatile. it can.

  Since the functional particle 1A of the present invention has the gold film or the gold alloy film 7 on the outermost surface, the thiol group, sulfide group or disulfide group having affinity for the gold or gold alloy film 7 is contained in the molecule. A physiologically active substance (eg, glutathione) can be captured. Moreover, since the functional particles 1A are magnetized, the physiologically active substance captured by the functional particles 1A can be effectively recovered and purified by applying a magnetic field.

  The functional particle 1A can capture a physiologically active substance having a thiol group, sulfide group or disulfide group in the molecule, but cannot capture a physiologically active substance having no thiol group, sulfide group or disulfide group. In contrast, the functional particle 1B can capture various physiologically active substances by various functional groups in the compound containing the thiol group, sulfide group or disulfide group constituting the spacer layer 9. In addition, a specific physiologically active substance can be selectively captured by appropriately selecting this functional group. Similar to the functional particle 1A, the functional particle 1B is also magnetized, so that the physiologically active substance captured by the functional particle 1B can be effectively recovered and purified by applying a magnetic field.

  In the functional particles of the present invention, the gold film or gold alloy film is preferably formed by an electroless plating method. According to the electroless plating method, a gold film or a gold alloy film can be uniformly and highly efficiently formed on the surface of FePt, FePd and CoPt alloy particles, so that high-performance functional particles can be produced at low cost. it can.

  The method for producing the functional particle 1B of the present invention includes the following steps (1) to (3).

(1) a step of activating the outer surface of FePt, FePd and CoPt alloy particles with an acid;
(2) a step of immersing the acid-activated FePt, FePd and CoPt alloy particles in a substitutional electroless noble metal plating bath to form a gold film or a gold alloy film on the outer surface of the FePt, FePd and CoPt alloy particles; And (3) a compound containing the gold or gold alloy film and a thiol group, sulfide group or disulfide group represented by the general formulas RSH, RSR, RSSR (wherein R is as defined above) To form a spacer layer.

  By immersing FePt, FePd, and CoPt alloy particles in acid, ultrathin rust and smut on the metal surface are removed, and a fresh metal surface appears. As the acid to be used, dilute hydrochloric acid, dilute sulfuric acid, dilute nitric acid and the like can be used. Among them, it is preferable to use dilute hydrochloric acid that has a high ability to remove the surface oxide passive film and has no oxidizing property. Subsequent to the acid activation treatment, when the FePt, FePd, and CoPt alloy particles are immersed in an electroless noble metal plating bath, a gold film or a gold alloy film can be formed on the outer surface of the FePt, FePd, and CoPt alloy particles. It is preferable to use a substitution type precious metal plating bath for the precious metal plating. For example, substitutional gold plating utilizes the difference in ionization tendency between iron and gold, and gold is deposited on the outer surface of nickel particles according to the following reaction formula (3).

3Fe + 2Au ³⁺ = 3Fe ²⁺ + 2Au (1)
In substitutional precious metal plating, the reaction stops basically when the surface of FePt, FePd, and CoPt particles is covered with gold or a gold alloy.
In a general substitution type noble metal plating bath, the deposition rate is 0.1 nm / second to 0.2 nm / second. Based on this deposition rate, the film thickness of the gold or gold alloy can be controlled.

Metal Fe, metal Co, and iron oxides such as Fe ₃ O ₄ and γ-Fe ₂ O ₃ also exhibit magnetism, but are inappropriate for use in the present invention. Metal Fe has a higher ionization tendency than metal Ni and is easily eluted as a cation. Metal Co is higher in cost than metal Ni and cannot be manufactured at low cost. On the other hand, the outer surface of Fe ₃ O ₄ or γ-Fe ₂ O ₃ iron oxide particles cannot be completely covered by the substitutional electroless gold plating method. γ-Fe ₂ O ₃ flows out as ions when used under acidic conditions. Although Fe ₃ O ₄ has acid resistance, the particle surface cannot be completely covered with gold, and thus the peptide bond of the physiologically active substance may be cleaved on the exposed Fe ₃ O ₄ surface. Therefore, metal Fe, metal Co, and iron oxide are magnetic even when recovering and purifying the physiologically active substance due to high ionization tendency, high cost, incomplete coating with a gold film, and cleavage and decomposition of the physiologically active substance. Even a body cannot be used in place of FePt, FePd, and CoPt alloy particles coated with gold or a gold alloy film.

  FIG. 3 is a conceptual diagram showing an example of a method for using the functional particles of the present invention. In step (1), an analysis sample 13 such as blood of a laboratory animal is filled in a suitable container such as a beaker 11. The analysis sample 13 contains various proteins 15-17. The functional particle 1A or 1B of the present invention is put into the analysis sample 13 and sufficiently mixed with stirring to bind the specific protein 15 contained in the analysis sample 13. Next, in step (2), the analytical sample 13 of the beaker 11 is transferred to, for example, a separatory funnel 19 or the like, and a magnetic field is applied from the outside of the separatory funnel 19 by a magnet 21 or the like, so that the functional particles 1A or 1B is collected around the magnetic field, and the remaining analysis sample 13 in the funnel 19 is discarded. Thereafter, in step (3), the recovered functional particles 1A or 1B are transferred to another container 23, and a separation liquid (for example, physiological saline) 24 is added to the functional particles 1A or 1B. Since the protein 15 is separated, only the specific protein 15 can be efficiently separated by collecting the supernatant.

  FIG. 4 is a conceptual diagram showing another example of the method for using the functional particles of the present invention. Since the functional particle of the present invention has magnetism, it is preferable to use it by a magnetic sorting method as shown in FIG. 3, but it can also be used by other than the magnetic sorting method. For example, as shown in step (1), the functional particles 1A or 1B of the present invention are confined in a microchannel 27 of a known microchemical chip 25, and the analysis sample 13 is allowed to flow from the upstream side of the microchannel 27. . The analysis sample 13 contains various proteins 15-17. Next, in step (2), the analysis sample 13 and the functional particles 1A or 1B are sufficiently brought into contact with each other to bind the specific protein 15 to the functional particles 1A or 1B, and the unnecessary analysis sample 13 is microchanneled. 27 washed away. Thereafter, a separation liquid (for example, physiological saline) 24 is flowed from the upstream side of the microchannel 27 to separate the protein 15 from the functional particles 1A or 1B, and the separated specific protein 15 is recovered on the downstream side.

  FIG. 5 is a conceptual diagram showing another example of the method for using the functional particles of the present invention. The functional particles of the present invention can be suitably used as a carrier for an enzyme immunoassay (ELISA) method. FIG. 5 is a conceptual diagram illustrating the direct adsorption method of the ELISA method. In step (1), an antigen (for example, HIV antigen) 29 is bound to the solid phase surface (that is, the carrier surface) of the functional particle 1A or 1B of the present invention. In step (2), a test sample (eg, serum) is added. If there is an antibody (for example, an anti-HIV antibody (produced in an average of about 12 weeks when infected with HIV and specifically binds to the HIV antigen)) 30 in the sample, it reacts with and binds to the antigen 29. Next, in step (3), the enzyme-attached secondary antibody 32 is added as a labeling substance. The enzyme-attached secondary antibody 32 can specifically bind to the antibody 30 in the test sample. Thereafter, in step (4), a substance 34 that reacts with the enzyme of the antibody with antibody 32 and develops color is added. In step (5A), the color development state of the reaction product 36 is measured. As shown in step (5B), if there is no antibody (for example, anti-HIV antibody) 30 in the test sample, no color is developed because no reaction occurs even if the coloring substance 34 is added. Such a series of reactions can be carried out in the microchannel 27 of the microchemical chip 25 as shown in FIG. Therefore, if an antigen capable of binding to the solid phase surface (that is, the carrier surface) of the functional particle 1A or 1B of the present invention is previously identified, an antibody that reacts specifically with this antigen can be easily detected by ELISA. can do.

1.69 mmol of Pt (acac) ₂ and 1.69 mmol of Fe (acac) ₃ were dissolved in 100 ml of tetraethylene glycol, respectively. 0.338 mmol of Bi (CH ₃ COO) ₃ was dissolved in 100 ml of tetraethylene glycol and added to the above solution. Thereafter, 1.69 mmol of oleylamine and 1.69 mmol of oleic acid were added to the above solution. Under a nitrogen stream, the solution was refluxed at 200 ° C. for 30 minutes, and then refluxed at 298 ° C. for 30 minutes. After completion of the reaction, tetraethylene glycol was distilled off, and the produced fine particles were centrifuged. The supernatant was removed, ethanol was added and the mixture was ultrasonically dispersed, and centrifuged to obtain FePtBi particles. FePtBi particles were immersed in 0.1N aqueous hydrochloric acid for 1 minute. The FePtBi particles were washed by filtration, immersed in a substitutional electroless gold plating bath for 5 minutes, and gold was plated on the surface of the FePtBi particles. The synthesized particles were hardened with an epoxy resin, and thin pieces were produced with a microtome. Observation with a transmission electron microscope revealed that the obtained functional particles were spherical particles, and the average particle diameter was 5 nm. Moreover, it turned out that the film thickness of an outer periphery gold film | membrane is 10 nm. Furthermore, it was found that the gold film completely covered the FePtBi particle surface. Moreover, as a result of measuring the magnetic characteristics using a vibrating sample magnetometer, the coercive force was 2,500 Oe. Furthermore, after the functional particles were dispersed in water, it was confirmed that there was sufficient magnetism collection with a magnet.

1.69 mmol Pt (acac) ₂ and 3.38 mmol Co (acac) ₃ were each dissolved in 100 ml tetraethylene glycol. 0.338 mmol of Bi (CH ₃ COO) ₃ was dissolved in 100 ml of tetraethylene glycol and added to the above solution. Thereafter, 1.69 mmol of oleylamine and 1.69 mmol of oleic acid were added to the above solution. Under a nitrogen stream, the solution was refluxed at 200 ° C. for 30 minutes, and then refluxed at 298 ° C. for 30 minutes. After completion of the reaction, tetraethylene glycol was distilled off, and the produced fine particles were centrifuged. The supernatant was removed, ethanol was added and ultrasonically dispersed, and centrifuged to obtain CoPtBi particles. FePtBi particles were immersed in 0.1N aqueous hydrochloric acid for 1 minute. The CoPtBi particles were separated by filtration and immersed in a substitutional electroless gold plating bath for 5 minutes, and gold was plated on the CoPtBi particles. The synthesized particles were hardened with an epoxy resin, and thin pieces were produced with a microtome. Observation with a transmission electron microscope revealed that the obtained functional particles were spherical particles, and the average particle diameter was 5 nm. Moreover, it turned out that the film thickness of an outer periphery gold film | membrane is 10 nm. Furthermore, it was found that the gold film completely covered the CoPtBi particle surface. Moreover, as a result of measuring the magnetic characteristics using a vibrating sample magnetometer, the coercive force was 2,200 Oe. Furthermore, after the functional particles were dispersed in water, it was confirmed that there was sufficient magnetism collection with a magnet.
(Comparative Example 1)
2 g of silica particles having an average particle size of 5 μm were dispersed in 100 ml of pure water, and 1 ml of 1% chloroauric acid aqueous solution and 5 ml of 1% sodium citrate aqueous solution were added and stirred. Sodium citrate was added to this mixture and further stirred. After heating to 80 ° C. and stirring for 30 minutes, it was filtered and dried. Observation with a scanning electron microscope revealed that the obtained functional particles were spherical particles. As a result of exposing the cross-section of this functional particle by focused ion beam (FIB) processing and observing with a scanning electron microscope, the film thickness of the gold film is 30 nm, and the entire silica surface is covered with the gold film. I found out that it was not.

  The functional particles obtained in Examples 1 and 2 and Comparative Example 1 were subjected to a bioactive substance adsorption experiment. As the physiologically active substance, glutathione, which is a tripeptide having a thiol group having affinity for gold, was used. A certain amount of functional particles and a glutathione standard solution were mixed and stirred for several minutes. The concentration of glutathione in the supernatant was colorimetrically determined, and the amount of glutathione adsorbed on the functional particles was calculated. The results are shown in Table 1 below.

As is clear from the results shown in Table 1, the functional particles of Examples 1 and 2 are excellent in glutathione adsorption efficiency. Furthermore, since the functional particles of Examples 1 and 2 have magnetism, they can be recovered with a magnet, and the adsorbed glutathione can be recovered with high efficiency. On the other hand, the functional particles of Comparative Example 1 have low glutathione adsorption efficiency and are not magnetic, so that the adsorbed glutathione could not be recovered with high efficiency.

The example which provided the spacer layer in the functional particle obtained in Example 1 and 2 and capture | acquired specific protein is shown.
First, the functional particles obtained in Examples 1 and 2 and 8-amino-1-octanethiol, which is a mercaptan compound having an amino group at the terminal, were mixed to introduce amino groups on the surface of the functional particles. Next, functional particles into which amino groups were introduced were dispersed in Tris-HCl buffer (pH 7.5), peroxidase was added as a specific protein, and peroxidase was captured by the functional particles. As a method for measuring the amount and activity of the captured peroxidase, a coloring reaction of TOOS-4-AA system was used. This color reaction involves oxygen produced by the reduction of hydrogen peroxide catalyzed by peroxidase, TOOS [N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline] and 4-AA ( 4-aminoantipyrine) is used to produce a dye having absorption at a wavelength of 546 nm. As a result, it was found that the peroxidase capture rates for the functional particles were 83.5% and 82.9%.

The functional particles obtained in Examples 1 and 2 were mixed with 10-carboxy-1-decanethiol, which is a mercaptan compound having a carboxyl group at the terminal, to introduce carboxyl groups on the surface of the functional particles. Next, functional particles into which carboxyl groups were introduced were dispersed in a phosphate buffer (pH 7.0), carbodiimide hydrochloride was added, rabbit antibody IgG was added as a specific protein, and the antibodies were captured on the functional particles. The amount of antibody captured was determined from the absorbance at a wavelength of 280 nm. As a result, it was found that the rabbit antibody capture rates for the functional particles were 81.3% and 82.0%.

  The functional particle of the present invention has a magnetic property and has a gold film or a gold alloy film having a high affinity with a compound containing a thiol group, sulfide group or disulfide group on the outermost surface. The recovery efficiency is high, and it can be applied to the purification of physiologically active substances such as proteins using a magnetic field.

  In the development of new drugs, proteins related to diseases are separated from analytical samples such as body fluids (for example, blood) of laboratory animals, and their properties are investigated in detail, or new drug candidate substances such as compounds that suppress their action are searched. However, the functional particles of the present invention can be suitably used for such purposes. It can also be used for research to find proteins that bind strongly to specific drug candidates.

  Furthermore, the functional particles of the present invention can be used for the purpose of examination or diagnosis of diseases associated with specific proteins such as BSE and Alzheimer's disease.

It is an outline sectional view of an example of functional particles of the present invention. It is an outline sectional view of another example of functional particles of the present invention. It is a conceptual diagram of an example of the usage method of the functional particle of this invention. It is a conceptual diagram of another example of the usage method of the functional particle of this invention. It is a conceptual diagram of the other example of the usage method of the functional particle | grains of this invention.

Explanation of symbols

1A, 1B Functional particles of the present invention 5 FePt alloy particles 7 Gold film or gold alloy film 9 Spacer layer 11 Container 13 Analytical sample 15, 16, 17 Protein 19 Separation funnel 21 Magnet 23 Container 24 Liquid 25 for separation Microchemical chip 27 Microchannel 29 Antigen 30 Antibody 32 Secondary antibody with enzyme 34 Coloring reagent 36 Reaction product

Claims (6)

A functional particle having FePt, FePd, and CoPt alloy particles and gold or a gold alloy film formed on the surface of the alloy particles and having magnetism. 2. The functional particle according to claim 1, wherein the FePt, FePd, and CoPt alloy particles contain at least one element selected from B, Bi, Cu, Sn, Sb, and Pb. Functional particles. The average particle size of the alloy particles is in the range of 3 nm to 50 nm, and the thickness of the gold or gold alloy film is in the range of 3 nm to 100 nm. particle. The functional particle according to claim 1, further comprising a spacer layer formed on a surface of the gold or gold alloy film. The spacer layer has a general formula, RSH, RSR, RSSR (wherein R is a carbon atom number of 20 or less, amino group, carboxyl group, hydroxyl group, halogen, thiol group, nitro group, aldehyde group, carbonyl group) A linear or branched alkyl group, a linear or branched chain having at least one functional group selected from the group consisting of a group, a sulfonyl group, a sulfonic acid group, a nitroso group, an amide group and an azide group A thiol group, a sulfide group, or a disulfide group represented by a alkenyl group, a linear or branched alkynyl group, an alicyclic group, an aromatic group, a condensed cyclic group, or a heterocyclic group. The functional particle according to claim 4, wherein the functional particle is formed by bonding to a lower noble metal film via a metal. It consists of at least the functional particles according to any one of claims 1 to 5, and captures, recovers and purifies the target physiologically active substance by binding the physiologically active substance to the surface of the functional particle. A bioactive substance capture / recovery / purification kit.

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