JP2005328809A - Method for producing particle for nucleic acid detection and nucleic acid transportation and use thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a bioprobe for highly sensitive detection of a nucleic acid while of suppressing nonspecific adsorption and the use of the bioprobe, and to provide a metal particle for supporting a nucleic acid enabling efficient nucleic acid transportation and the use of the metal particle. <P>SOLUTION: The metal particle supporting the nucleic acid is produced by protecting the surface of the particle with a polyethylene glycol-polycation copolymer to stabilize the dispersion of the particle and chemically modifying the nucleic acid to a part of a polyamine segment through mercapto group, dithiol group or sulfide group or directly supporting the nucleic acid to the surface of the metal particle. This invention further provides a high-sensitivity sensing material, a material usable as a carrier for gene therapy and a method for using the material. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a nucleic acid / microparticle complex that can be used in a bioprobe or biocarrier, and particularly relates to a microparticle for nucleic acid detection and nucleic acid transport.

  The bioprobe is suitable for detection of a specific test substance using, for example, a biological sample as a specimen, and various bioprobes are currently proposed. Typical examples of bioprobes for detecting nucleic acids include those that greatly amplify and detect the signal of a sensor that senses hybridization or dissociation of nucleic acids carried on the surface of a sensor or fine particles. .

  As one of main biosensors that can be suitably used as a nucleic acid detection sensor, a biosensor using surface plasmon resonance (SPR) (hereinafter also referred to as an SPR sensor) is provided. In this SPR sensor, a metal thin film such as gold or silver is formed on the bottom surface of a prism, and laser light or the like is incident from the back surface of the metal thin film while changing the incident angle, and the reflected light is measured. Is a biosensor capable of detecting a reaction such as nucleic acid hybridization occurring on the metal surface with high sensitivity without labeling, using light of a specific incident angle absorbed as a marker, It is known to be sensitive to changes in the refractive index at and near the surface of a metal thin film (eg, A. Szabo et al., Curr. Opin. Strength. Biol. 5 (1995) 699-705).

  Currently, fluorescent labels, radioisotopes, and the like are used as bioprobes for the purpose of improving the detection sensitivity of a small amount of specimen. Since the SPR sensor is a device that measures the change in the refractive index at and near the surface of the metal thin film as the change in surface plasmon resonance, the metal fine particles that increase the refractive index and affect the surface plasmon resonance are highly sensitive. The detection sensitivity of the SPR sensor, which is assumed to be present, can be further greatly amplified, and is suitable for detecting a sample containing a very small amount in a sample with high sensitivity.

  In particular, in recent years, the decoding of genes has progressed, and it has been required to detect subtle mutations of nucleic acids such as single nucleotide substitution mutations with high sensitivity, non-specific reactions are suppressed, simple and sensitive, The provision of a nucleic acid detection bioprobe capable of detecting a desired nucleic acid mutation is highly desired.

  Biocarriers are intended to deliver drugs intended for treatment to the target site in the body. Typical examples include biocarriers in which drugs are combined with liposomes, polymer micelles, polycations, dendrimers, etc. .

As prior literature relating to the present invention to be described later, for example, the following literatures are listed. JP-A-7-316285 (Patent Document 1)
Patent Document 1 describes a method for producing a polyalkylene oxide derivative that can be used on the surface of a chip of an SPR sensor, but this document itself does not disclose the application of the polyalkylene oxide derivative to a biosensor. Not

2. Japanese Patent Laid-Open No. 11-322916 (Patent Document 2)
Patent Document 2 discloses a polyoxyethylene derivative having a biotin residue at the ω-terminal, which can be more suitably used on the surface of an SPR sensor. However, this document itself describes this heteropolyoxyethylene derivative. There is no disclosure regarding application to biosensors.

3. JP 2001-200050 A (Patent Document 3)
Patent Document 3 describes that metal fine particles such as gold loaded with a polyethylene glycol derivative have good dispersibility, etc., but this document itself describes the application of the metal fine particles to biosensors. No disclosure has been made.

4). JP 2002-80903 A (Patent Document 4)
Patent Document 4 discloses metal fine particles such as gold having polyethylene glycol as a block and having a specific methacrylic acid polymer as another block and carrying a block polymer derivative. As such, there is no disclosure regarding the application of this metal fine particle to a biosensor. In addition, the block polymer of this patent document 4 is a block polymer which can be used also in this invention.

5). Japanese Patent No. 2815120 (Patent Document 5)
In Patent Document 5, HS-R-Y (R is a hydrocarbon group having more than 10 carbon atoms, which may be interrupted by a hetero atom, and Y shares a ligand or a biocompatible porous matrix. This organic molecule is packed tightly through the thiol group and bound to the surface of a free metal such as gold or silver. A surface is described in which a single layer is provided and then a hydrogel composed of agarose, dextran, polyethylene glycol or the like is covalently bonded as a biocompatible porous matrix.

6). Japanese Patent No. 3071823 (Patent Document 6)
Patent Document 6 describes a surface in which a hydrophilic linker portion and a solid-phase reactant of a biotin residue are covalently bonded in order to a spacer molecule bonded to a holding substrate via a sulfur atom of a thiol group.

7). JP 2001-178442 A (Patent Document 7)
In Patent Document 7, a DNA fragment having a thiol group at a terminal portion and a chain molecule having a reactive substituent capable of reacting with the thiol group to form a covalent bond are immobilized on the surface at one end. A method for immobilizing a DNA fragment on the surface of a solid phase carrier is described, wherein a covalent bond is formed between the DNA fragment and a chain molecule by contacting the solid phase carrier in a liquid phase. Yes.

8). Japanese translation of PCT publication No. 2004-501340 (patent document 8)
In Patent Document 8, an oligonucleic acid modified with a thiol at one end is immobilized on the surface of a gold fine particle to stabilize the dispersion of the gold fine particle, and a complementary oligonucleic acid is recognized by a combination of sequences of the immobilized oligonucleic acid. Although it is shown that it can be applied to various detection methods, there is no description that the water-soluble polymer and the nucleic acid are immobilized simultaneously. In addition, it has been shown that it is possible to remove nucleic acid once immobilized on the surface of metal fine particles by using a low molecular weight compound such as mercaptoethanol. It is not described that it can be applied as fine particles for transporting nucleic acids.

9. Japanese translation of PCT publication No. 2003-514224 (Patent Document 9)
Patent Document 9 shows that a metal fine particle probe made of gold, silver, copper, aluminum, or the like whose surface is modified with oligonucleic acid or protein can dramatically improve the measurement sensitivity when combined with an SPR sensor. However, there is no description that the nucleic acid or protein and the water-soluble polymer are immobilized on the surface at the same time.

10. “Roberts et al, J. Am. Chem. Soc. 1998, 120, 6548-6555” (Non-patent Document 1)
Non-Patent Document 1 describes a charcoal layer self-assembled on a gold surface through a thiol group using a compound based on an HS-spacer molecule-hydrophilic linker. In addition, the surface in which the hydrophilic linker part is formed from a mixture of a compound of 3 ethylene oxide units and a compound of 6 ethylene oxide units promotes ligand-specific binding of cells, but the volume of protein by the attached cells. Is also taught to reduce.

11. "Pavey et al., Biomaterials 20 (1999) 885-890" (Non-patent Document 2)
Non-Patent Document 2 describes a surface in which various combinations of two kinds of polyethylene oxide-polypropylene oxide-polyethylene oxide triblock polymers are deposited on a metal thin film for SPR detection. In addition, it has been suggested that polyethylene oxide can be stretched in a solution on the surface to form a brush-like structure.

12 JP 2001-56311 A (Patent Document 10)
In Patent Document 10, an oligonucleic acid is immobilized on a conductive substrate via a thiol group, and a low molecular thiol compound such as 2-mercaptoethanol or 6-mercaptohexanol is immobilized outside the immobilization region. Although it has been shown that nucleic acid can be quantified with high sensitivity, there is no description of the surface of metal fine particles, and there is no description of a water-soluble polymer in a substance immobilized outside the oligonucleic acid immobilization region.

13. “B. Sihgh et al., J. Pharmacy and Pharmacology, 55 (2003) 1631640” (Non-patent Document 3)
In Non-Patent Document 3, nucleic acid is electrostatically adsorbed on a gold colloid coated with polycation, but there is no description about coexistence with a water-soluble block polymer on the surface, and it is loose in a high ionic strength aqueous solution. It is described as forming an aggregate. Further, there is no description that the nucleic acid supported on the surface of the gold fine particles is released by a competitive reaction with the low molecular weight compound.
JP-A-7-316285 JP 11-322916 A JP 2001-200050 A JP 2002-80903 A Japanese Patent No. 2815120 Japanese Patent No. 3071823 JP 2001-178442 A JP-T-2004-501340 Special table 2003-514224 gazette JP 2001-56311 A Roberts et al. , J .; Am. Chem. Soc. 1998, 120, 6548-6555 Pavey et al. , Biomaterials 20 (1999) 885-890. B. Sihgh et al. , J .; Pharmacy and Pharmacology, 55 (2003) 1631640

Problems to be solved by the invention

Although microparticles carrying nucleic acids are expected as a core technology for sensors and gene therapy, they have problems such as nonspecific adsorption in the living environment, low dispersion stability, and insufficient gene hybridization ability. there were.
The inventors previously bonded a polycation with a water-soluble polymer such as PEG and electrostatically bonded it to a negatively charged surface so that the polycation is the outermost surface and the water-soluble polymer is in a brush shape. After forming the constructed surface, it was shown that a surface having high recognition ability can be constructed by electrostatically adsorbing and binding oligonucleic acid to the surface and used as a sensor (Japanese Patent Application No. 2003-18081, Application 2003-18085). By applying such a surface construction method to the fine particle surface, we succeeded in preparing highly functional fine particles that have not only high recognition ability but also dispersion stability. It is an object of the present invention to provide a base material that can be used not only as a base particle for high-sensitivity sensing but also as a carrier for gene therapy.

Means for solving the problem

    In order to solve this problem, the present inventor further studied the surface structure of a bioprobe for nucleic acid detection that can suppress non-specific reactions. As a result, a hydrophilic polymer-polycation copolymer is electrostatically or covalently bonded to the surface of the fine particles to provide a hydrophilic polymer brush-like structure, and nucleic acid is placed in the gap between the brush-like structures. By supporting it, not only the dispersion stability of the metal fine particles in water is improved, but also the nucleic acid can be easily supported on the surface of the metal fine particles, and the nonspecific reaction when hybridizing the nucleic acid is remarkably suppressed. It was found that the hybridization detection sensitivity of the nucleic acid on the surface of the SPR sensor is remarkably improved. Furthermore, it has been found that this nucleic acid / fine particle complex releases a nucleic acid carried in the body environment. These can be used as high-sensitivity sensors and basic materials for gene therapy.

  The fine particles in the present invention are not particularly limited, but latex such as polystyrene and polybradiene, inorganic particles such as silica and clay, gold, silver, copper, aluminum having strong plasmon absorption, alloys thereof, and / or Those containing these metals in a part of the structure of the fine particles are preferable. In particular, gold, silver, and copper are the most suitable metals because they bind to mercapto groups, disulfide groups, or amino groups very stably.

  The method of supporting a nucleic acid having one end thiol-modified on the surface of the fine particle allows a large amount of nucleic acid to be supported on the surface of the fine particle at one time, and that the supported nucleic acid peels off the surface of the metal fine particle under normal body environment. In addition, since the metal fine particles have a certain size, it is possible to avoid extracorporeal discharge in the kidney or the like. In addition, the nucleic acid-carrying metal microparticles taken up into the cell are competitive with the terminal thiol of the nucleic acid carried on the surface of the metal microparticle by a low-molecular thiol compound typified by glutathione present in large quantities in the cell. It has been found that it can be released into cells by being exchanged to act as nucleic acid transport microparticles. The point to be emphasized is that even after all the nucleic acid has been peeled off, the hydrophilic polymer remains on the surface of the metal fine particles, which can prevent the metal fine particles from aggregating with each other until they are discharged out of the cell and show cytotoxicity. It is characterized by not.

That is, the present invention relates to polyethylene glycol / polycation copolymer R1-PEG-R2 (R1 is acetal, aldehyde, hydroxyl group, amino group, carboxyl group, active ester azide group, biotin group, monosaccharide, oligosaccharide, amino acid, A functional group selected from the group consisting of a nucleic acid, an allyl group, a vinylbenzyl group, a methacryloyl group and an acryloyl group, PEG is — (CH ₂ CH ₂ O) n—, and R2 is a polycation (for example, tetraethylenepentapentane). Min, pentaethylenehexamine, poly (methacrylic acid, 2-N, N-dimethylaminoethyl), poly (lysine), chitin, chitosan, poly (N, N-dimethylaminomethylstyrene) are fixed on the surface of fine particles. , Directly or indirectly carrying a thiol-modified oligonucleic acid at one end Metal nanoparticles that perform efficient sequence recognition when performing reactions such as hybridization of nucleic acids, and sensing of complementary nucleic acid strands that suppress nonspecific reactions as much as possible, or efficient nucleic acid transport , Also referred to as the present fine particles) and a method for using the metal fine particles (hereinafter also referred to as the present method of use).

  In the above-mentioned patent documents (Japanese Patent Application Nos. 2003-18081 and 2003-18085), as in the present invention, a nucleic acid is supported on the surface of a metal fine particle provided with a hydrophilic polymer brush-like structure. Is not described or suggested.

      This metal fine particle is preferably used for detection in a biosensor using surface plasmon resonance, but is not limited to this, and a combination of a ligand and a receptor, for example, an antigen or a hapten and an antibody, a sugar and a lectin Any sensor that can detect any change on the chip surface and amplify the signal with this microparticle by forming a binding pair in a set of substrate and enzyme, hormone and its receptor, oligonucleotide and its complementary strand, etc. For example, it can be used in any category of sensors. Examples of the change to be detected include radioactivity, contact angle, sedimentation, aggregation, ultraviolet-visible light spectroscopy, fluorescence, chemiluminescence, electrochemiluminescence, and the like in addition to the above surface plasmon resonance.

  As described above, the present invention uses the fine metal particles as a bioprobe, and uses the fine metal particles to notify that the test substance in the test object is bound to the sensor surface with high sensitivity. As a preferred embodiment of the present method for using the present microparticles, detection by an instrument capable of detecting the state of the present microparticles using surface plasmon resonance (SPR) can be mentioned. The present invention also provides a method for using fine particles that can be stably dispersed even in a living environment and release nucleic acids capable of si-RNA and antisense treatment in cells by the method for producing metal fine particles of the present invention.

  The amount of cation in the PEG / polycation copolymer is in the range of 1 to 100,000 per molecule. Preferably, it is in the range of 3 to 10,000, more preferably in the range of 4 to 1,000.

The invention's effect

The fine particle dispersion obtained in the present invention is stable at a high concentration, and is stable without aggregation even under high ionic strength. In addition, compared with metal fine particles whose surface is modified only with oligonucleic acid, the metal fine particles have no problem in dispersion stability even if the negative charge density on the particle surface is lowered, but rather improve the ability to recognize nucleic acid complementary strands. It is possible. By using this fine particle, the surface plasmon wave on the surface of the SPR sensor interacts with the plasmon absorption of the metal fine particle, so that the detection sensitivity in the SPR sensor can be remarkably improved. It can be suitably used for diagnosis.
In addition, since the nucleic acid fixed on the particle surface is released by the influence of the surrounding environment, the fine metal particle after the nucleic acid release can maintain the dispersion stability by the PEG derivative. It can be preferably used.

    Examples and the like specifically showing the configuration and effects of the present invention will be described below.

Example 1
Preparation of colloidal gold solution (metal fine particles with PEG / PAMA on the particle surface)
Gold fine particles in which 30 mg of aceta-PEG / PAMA (5700/3800) is dissolved in 5 ml of ultrapure water, 1 ml of 2.5 mg / ml chloroauric acid is added and stirred vigorously overnight, and the surface is modified with aceta-PEG / PAMA Got. Since free block polymer and DNA were complexed, centrifugal purification (65,000 × g, 30 min, 20 ° C. × 3) was performed using sterilized water. The average particle size of the obtained particles was determined from a TEM image. The concentration of gold atoms was determined by atomic absorption measurement, and the concentration of gold fine particles in the solution was determined using the average particle size determined from the TEM image.
5′-terminal thiol-modified nucleic acid (hereinafter also referred to as SH-DNA) is dissolved in sterilized water to a concentration of 100 μM, subjected to a predetermined SH treatment, added 50 times the amount of gold fine particles, and overnight. Shake. Purification was carried out by ultracentrifugation (80,000 × g, 15 min, 15 ° C. × 5), and the solvent was replaced with 30 mM sodium citrate (1M NaCl, 1 mM EDTA, pH 7) solution at the same time. The particle concentration was determined by atomic absorption measurement. UV-visible light absorption was measured before and after addition of SH-DNA, and the dispersion stability of the particles and the stability against ionic strength were evaluated. (Fig. 1) This clearly shows that gold particles stabilized only with conventional terminal thiolated oligonucleic acids or those having dispersion stability equivalent to gold particles stabilized only with PEG / PAMA were prepared. It was done.

Example 2
Evaluation of surface structure of fine particles after nucleic acid support 1
After preparing PEG / PAMA-modified gold fine particles by the method of Example 1, SH-DNA-FITC fluorescently labeled at the 3 ′ end was supported and purified by the same method as in Example 1, and then the solvent was added to 10 mM pH 8 phosphorous. After substituting with acid buffer, 450 μl was taken out, 50 μl of 1.0M NaCN adjusted to pH 8 was added, and gold was completely dissolved by pipetting, and the fluorescence spectrum was measured. As a result, SH-DNA-FITC was obtained. The derived fluorescence and spectrum were observed. (FIG. 2) This revealed that SH-DNA was carried on the surface of gold fine particles coated with PEG / PAMA.

Example 3
Evaluation of surface structure of fine particles after loading with nucleic acid 2
After using fluorescein-PEG / PAMA chemically modified with a fluorescent group FITC at the end in advance, PEG / PAMA-modified gold fine particles were prepared by the method of Example 1, and then SH-DNA was supported and purified in the same manner as in Example 1. Then, after replacing the solvent with 10 mM pH 8 phosphate buffer, 450 μl was collected, 50 μl of 1.0 M NaCN adjusted to pH 8 was added, and gold was completely dissolved by pipetting. When measured, fluorescence and spectrum derived from fluorescein-PEG / PAMA were observed. (FIG. 3) This revealed that PEG / PAMA was still present on the surface of the gold fine particles even after SH-DNA was supported.

Example 4
Hybridization test using nucleic acid-carrying gold microparticles SH-DNA (SH 5′-dT (20) GCCA C CAGC-3 ′, SH5′-dT (20) GCTG G TGGC-) containing a complementary base sequence in the structure 3 ') or SH-DNA containing a non-complementary base sequence in the structure (SH-DNA carried on the surface of SH-coated gold fine particles was found to have a hybridizing ability.

Example 5
About 10,000-fold molar amount (4.08 mg) of MeO-PEG-b-PAMA (molecular weight: 12,000) was added to 5 ml of a commercially available 10 nm colloidal gold solution, stirred for one day, and then purified by centrifugation using sterile water (60 , 000 × g, 20 ° C., 15 min × 3), and the absorbance (λ = 520 nm) was measured. The particle concentration of the prepared PEGylated gold colloid solution was determined by atomic absorption, and 4.6 ml of this PEGylated gold colloid solution was collected, and after that, a 95-fold amount of DNA was added to the gold nanoparticles, Stir overnight and protected from light. Thereafter, centrifugal purification (80,000 × g, 20 ° C., 20 min, × 5) was performed using 10 mM tris-HCl buffer with 0.15M NaCl, and the fluorescence intensity was measured by measuring the total supernatant of 5 times to 20 ml. (Calculation of DNA introduction rate for one gold nanoparticle by quantification of free DNA based on fluorescence intensity). Also, the gold particle concentration was determined by measuring the UV-vis spectrum of the prepared SH-DNA-FITC / PEG-b-PAMA co-immobilized gold nanoparticles and comparing the absorbance at 520 nm with the absorbance of commercially available colloidal gold. did. Then, 50 μl of 1M DTT solution was added to 450 μl of 2.00 nM SH-DNA / PEGylated gold colloid solution (DNA: 0.984 nmol), the concentration was adjusted to 100 mM, and the mixture was shaken at 37 ° C. for 6, 12, 20, 40 h and then centrifuged. (80,000 × g, 20 ° C., 20 min) was performed, and 200 μl of the supernatant was collected, and the fluorescence intensity was measured to observe the release of DNA from the gold nanoparticle surface. (Fig. 5)

ＳＨ−ＤＮＡのみで安定化した金コロイドの調製
市販金コロイド（１０ｎｍ ｐｏｌｙ ｓｃｉｅｎｃｅ社製）３ｍｌにＳＨ化処理を施したＳＨ−ＤＮＡｓｅｑｕｅｎｃｅＩ_２０を２００倍モル量加え、２４時間攪拌した。時間後、２０ｍＭリン酸バッファー（０．２ＭＮａＣｌ，ｐＨ７）２倍量にメスアップし、さらに４０時間攪拌した。遠心精製（１２００００ｘｇ，４℃，１５ｍｉｎ）で精製し、同時に２×ＳＳＣ １ｍＭ ＥＤＴＡに置換した。粒子濃度は原子吸光ＴＥＭから求めた。Comparative Example 1
Comparison of hybridization ability with gold microparticles stabilized only with nucleic acid

Preparation of gold colloid stabilized with only SH-DNA A 200-fold molar amount of SH-DNA sequence I ₂₀ subjected to SH treatment was added to 3 ml of commercially available gold colloid (manufactured by 10 nm polyscience) and stirred for 24 hours. After the time, the volume was increased to 2 volumes of 20 mM phosphate buffer (0.2 M NaCl, pH 7), and the mixture was further stirred for 40 hours. Purification was performed by centrifugal purification (120,000 × g, 4 ° C., 15 min), and simultaneously replaced with 2 × SSC 1 mM EDTA. The particle concentration was determined from atomic absorption TEM.

PEG / PAMA, SH-DNA co-immobilized gold substrate A few drops of PEG / PAMA (5660/2780) aqueous solution adjusted to 1.6 mg / ml was placed on a gold substrate that had been subjected to ozone cleaning, and then at 40 ° C. (sealed) for 1 hour. Then, SH-DNA (5 μM, sequence II ₂₀ ) was allowed to flow at 5 μl / min for 10 min on a chip that was allowed to stand and further flowed with PEG / PAMA in the flow path system. At this time, the sample was adjusted so that I = 0.3. (37 ° C, Running buffer: MilliQ)

SH-DNA single immobilization chip SH-DNA (5 μM, sequence II ₂₀ 5 μl / min was allowed to flow for 30 min on a gold substrate subjected to ozone cleaning.
At this time, the sample was adjusted so that I = 0.3. (37 ° C., Running buffcr: 2 × SSC 1 mM EDTA)

Hybridization and adsorption of each gold colloid under each chip condition PEG / PAMA, SH-DNA co-immobilized gold colloid, and gold colloid stabilized only with SH-DNA were flowed to each sensor chip described above. The DNA sequence on the gold colloid side was sequence I ₂₀ , which was complementary to the sensor chip side. (Conditions: 25 ° C., 5 μl / min, 10 min, Running buffer:
Hybridization and adsorption of each gold colloid under each chip condition PEG / PAMA, SH-DNA co-immobilized gold colloid, and gold colloid stabilized only with SH-DNA were flowed to each sensor chip described above. The DNA sequence on the gold colloid side was sequence I ₂₀ , which was complementary to the sensor chip side. (Conditions: 25 ° C., 5 μl / min, 10 min, Running buffer: 2 × SSC 1 mM EDTA)

  When the colloidalized gold colloid was allowed to flow on each of the above gold substrates, either the gold colloid stabilized with SH-DNA alone or the amount of hybridization on the gold substrate modified with only SH-DNA was either Was smaller than that obtained on the PEG / PAMA, SH-DNA co-immobilized surface. This shows that since the introduction density of SH-DNA is too high, electrostatic repulsion between the DNA strands decreases the hybridizing ability. By making the surface co-immobilized with PEG / PAMA, It shows that the hybridizing ability of is improved.

The invention's effect

  According to the present invention, a metal fine particle capable of detecting a nucleic acid with higher sensitivity than conventional techniques and also functioning for nucleic acid transport is provided.

  FIG. 1 shows the spectrum before and after nucleic acid loading of the gold fine particles described in Example 1.

  FIG. 2 shows a fluorescence spectrum derived from SH-DNA-FITC carried on the surface of the gold fine particles described in Example 2.

  FIG. 3 shows a fluorescence spectrum derived from fluorescein-PEG / PAMA immobilized on the surface of the gold fine particles described in Example 3.

  FIG. 4 shows a TEM observation image of the gold fine particles described in Example 4.

  FIG. 5 is a fluorescence spectrum showing that SH-DNA-FITC supported on the PEG / PAMA-modified gold colloid surface described in Example 5 was released into a solution by adding an excess of a thiol compound (dithiothreitol). It is.

  It is a figure which shows the result of having compared the hybridizing ability about the gold substrate surface created using PEG / PAMA and SH-DNA suitably, and the gold fine particle surface.

Claims (17)

  The surface of the fine particle is coated with a hydrophilic polymer having a water-soluble polycation as a block segment, and the oligonucleic acid is directly or indirectly supported on the surface ensuring dispersion stability in water, thereby sensing a nucleic acid complementary strand. Alternatively, fine particles that perform nucleic acid transport.   The fine particle according to claim 1, wherein the fine particle is a metal.   The nucleic acid-carrying fine particles according to claim 1 or 2, wherein the hydrophilic polymer is a PEG / polycation copolymer which is polyethylene glycol having a polymerization degree of 2 to 10,000. The hydrophilic polymer is a hydrophilic polymer segment constituting the first block of the block polymer having the following characteristics (a) and (b), and the polymer segment constituting the second block is on the surface of the fine particles. The nucleic acid-carrying metal fine particles according to any one of claims 1 to 3, wherein the following block polymer is fixed to the surface of the fine particles through a functional group having coordination ability on the support.
(A) A segment of the hydrophilic polymer as a first block component (b) A monomer having a monomer having a functional group having a coordination ability on a support on the surface of a metal fine particle, the degree of polymerization being 1 to 200 Let the polymer segment be the second block. The nucleic acid-carrying metal microparticle according to claim 1, wherein the functional group having a coordination ability on the support on the surface of the microparticle is a mercapto group, a dithiol group, a sulfide group, or an amino group. The second block of the block polymer is of formula (1)

[Wherein, m represents an integer of 1 to 10, n represents an integer of 1 to 100, and R1 and R2 each independently represent an alkyl group having 1 to 5 carbon atoms, an amino group, a mercapto group, or The metal fine particle according to any one of claims 1 to 5, which is a segment of a methacrylic acid polymer or a pentaethylenehexamine group represented by [representing a sulfide group]. The nucleic acid is chemically modified to a part of the polyamine segment or the surface of the fine particle via a mercapto group, a dithiol group, or a sulfide group, and is supported on the surface of the gold fine particle. Nucleic acid-supported metal fine particles. The nucleic acid-carrying metal fine particles according to any one of claims 1 to 7, wherein the fine particles are formed of a material selected from at least one metal selected from gold, silver, copper, platinum, palladium, and aluminum. The nucleic acid-carrying metal fine particles according to claim 1, wherein the metal fine particles have a particle size of 1 nm or more and a size of less than 100 nm. The nucleic acid-carrying metal fine particles according to any one of claims 1 to 9, wherein the nucleic acid is selected from single-stranded DNA, double-stranded DNA, siRNA, and PNA. 12. The fine metal particle for nucleic acid transport according to any one of claims 1 to 11, wherein the nucleic acid supported directly or indirectly on the surface of the fine metal particle is released in a living environment. A metal fine particle for nucleic acid detection or transport having the surface according to any one of claims 1 to 11. A method for using the metal fine particles for nucleic acid transport according to claim 12. The method for using the metal fine particles for nucleic acid detection according to claim 13, wherein the metal fine particles for nucleic acid detection and a nucleic acid sample are brought into contact with each other to detect hybridization of the metal fine particles with the immobilized nucleic acid. The method for using the metal fine particles for nucleic acid detection according to claim 14, wherein the detection of hybridization between nucleic acids is performed by a sensor utilizing surface plasmon resonance. 15. The method for using metal fine particles for nucleic acid detection according to claim 14, wherein the sensor chip of the surface plasmon sensor uses a sensor chip having the surface according to claims 1 to 11. The method for using nucleic acid-detecting metal fine particles according to claim 15, wherein the detection of hybridization between nucleic acids is performed by an optical technique.

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