Nucleic acid-immobilized gold nanoparticles, and method for manufacturing the same

Abstract

To provide nucleic acid-immobilized gold nanoparticles that can deliver nucleic acid from an intracellular endosome to a cytoplasm highly efficiently, and a method for manufacturing the nucleic acid-immobilized gold nanoparticles.SOLUTION: A compound expressed by the following formula (1): (HS)n-R1-R2-R3-O-P(=O)O--O-Nuc...(1) (in the formula (1), n denotes 1 or 2 integers, R1 denotes a divalent or trivalent linking group, R2 denotes an acid-cleavable group cleavable by a change from neutral to pH 4 to 5 of acid, R3 denotes a divalent linking group, Nuc denotes a residue in which a group expressed by -O-P(=O)O--OH of 5' terminal is removed from a nucleic acid molecule, or a residue in which hydrogen atoms in a hydroxy group of 3' terminal is removed from a nucleic acid molecule) is allowed to bond to surfaces of gold nanoparticles as a ligand.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to nucleic acid-immobilized gold nanoparticles and a method for producing the same. [Background technology]

[0002] Gold nanoparticles (Nuc-AuNPs) bearing a dense layer of nucleic acid on their surface are composite nanomaterials that combine the high molecular recognition ability of nucleic acids with the vivid coloring properties of gold nanoparticles. Recently, it has been revealed that such Nuc-AuNPs exhibit high stability in blood and high cellular uptake efficiency. Therefore, the application of Nuc-AuNPs as drug delivery vehicles for nucleic acid medicines has been investigated.

[0003] One of the studies related to the application of Nuc-AuNPs as drug carriers is a study on specifically enhanced Raman imaging and photothermal therapy of cancer cells using gold nanoparticles with Cy3-modified single-stranded DNA attached to their surface (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Mohzibudin Z. Quazi,Ukjae Lee,Sanghak Park,Seonhye Shin, Eunseop Sim,Hyugbin Son,and Nokyoung Park,ACS Appl. Bio Mater. 2021,4,8337-8385 Summary of the Invention [Problem to be solved by the invention]

[0005] However, to achieve highly efficient in vivo nucleic acid drug delivery using Nuc-AuNPs, it is necessary to improve the efficiency of nucleic acid drug transfer from intracellular endosomes to the cytoplasm.

[0006] The present invention has been made in view of the above-mentioned problems, and aims to provide nucleic acid-immobilized gold nanoparticles that can transfer nucleic acids from intracellular endosomes to the cytoplasm with high efficiency, and a method for producing such nucleic acid-immobilized gold nanoparticles. [Means for solving the problem]

[0007] The present inventors have applied a compound represented by the following formula (1): (HS) n -R 1 -R 2 -R 3 -OP(=O)O - -O-Nuc···(1) (In formula (1), n ​​is an integer of 1 or 2, and R 1 is a divalent or trivalent linking group, and R 2 is an acid-cleavable group that can be cleaved by changing from neutral to acidic at pH 4 to 5, and R 3 is a divalent linking group, and Nuc is -OP(=O)O at the 5' end of the nucleic acid molecule. - It is a residue in which a group represented by -OH has been removed, or a residue in which the hydrogen atom of the 3'-terminal hydroxyl group has been removed from a nucleic acid molecule. The present invention has been accomplished based on the discovery that the above-mentioned problems can be solved by binding a compound represented by the following formula (I):

[0008] (1) A ligand having a nucleic acid-derived residue is bound to the surface of a gold nanoparticle; The ligand has the following formula (1): (HS) n -R 1 -R 2 -R 3 -OP(=O)O - -O-Nuc···(1) (In formula (1), n ​​is an integer of 1 or 2, and R 1 is a divalent or trivalent linking group, and R 2 is an acid-cleavable group that can be cleaved by changing from neutral to acidic at pH 4 to 5, and R 3is a divalent linking group, and Nuc is -OP(=O)O at the 5' end of the nucleic acid molecule. - It is a residue in which a group represented by -OH has been removed, or a residue in which the hydrogen atom of the 3'-terminal hydroxyl group has been removed from a nucleic acid molecule. A nucleic acid-immobilized gold nanoparticle, which is a compound represented by the formula:

[0009] (2) The acid-cleavable group is -CH=N-, -CO-O-, -C(COOH)=CH-CO-NH-, or -CR 01 =N-NH-, R 01 is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.

[0010] (3) The nucleic acid-immobilized gold nanoparticle according to (2), wherein the acid-cleavable group is -CH=N-.

[0011] (4) R 1 The nucleic acid-immobilized gold nanoparticle according to (3), wherein the divalent linking group is a phenylene group.

[0012] (5) The nucleic acid-immobilized gold nanoparticles according to any one of (1) to (4), wherein bis(p-sulfonatophenyl)phenylphosphine is bound to the surface of the gold nanoparticles.

[0013] (6) The surface of gold nanoparticles is coated with the following formula (11): (HS) n -R 1 -R 4 ···(11) (In Equation (11), R 4 is a group capable of generating an acid-cleavable group that can be cleaved by changing from neutral to acidic at pH 4 to 5, and n and R 1 is R in Eq. (1) 1 is the same as and binding a thiol compound represented by the formula: R in thiol compounds bound to the surface of gold nanoparticles 4 and the following equation (12): R 5 -R3 -OP(=O)O - -O-Nuc···(12) (In equation (12), R 5 is the R in the formula (11). 4 is a group capable of reacting with to generate the acid-cleavable group, R 3 is R in Eq. (1) 3 is the same as R in the nucleic acid derivative represented by 5 to form an acid-cleavable group; (1) A method for producing a nucleic acid-immobilized gold nanoparticle, comprising:

[0014] (7) R 4 is a formyl group, and R 5 is an amino group, and R 2 (6) The method for producing nucleic acid-immobilized gold nanoparticles according to (6), wherein is -CH=N-.

[0015] (8) The method for producing nucleic acid-immobilized gold nanoparticles according to (6) or (7), wherein bis(p-sulfonatophenyl)phenylphosphine is bound to the surface of the gold nanoparticles before the thiol compound is bound to the surface of the gold nanoparticles. [Effects of the Invention]

[0016] According to the present invention, it is possible to provide nucleic acid-immobilized gold nanoparticles that can transfer nucleic acids from intracellular endosomes to the cytoplasm with high efficiency, and a method for producing the nucleic acid-immobilized gold nanoparticles. DETAILED DESCRIPTION OF THE INVENTION

[0017] <Nucleic acid-immobilized gold nanoparticles> In nucleic acid-immobilized gold nanoparticles, a ligand having a residue derived from a nucleic acid is bound to the surface of the gold nanoparticle. The ligand has the following formula (1): (HS) n -R 1 -R 2 -R 3 -OP(=O)O --O-Nuc···(1) (In formula (1), n ​​is an integer of 1 or 2, and R 1 is a divalent or trivalent linking group, and R 2 is an acid-cleavable group that can be cleaved by changing from neutral to acidic at pH 4 to 5, and R 3 is a divalent linking group, and Nuc is -OP(=O)O at the 5' end of the nucleic acid molecule. - It is a residue in which a group represented by -OH has been removed, or a residue in which the hydrogen atom of the 3'-terminal hydroxyl group has been removed from a nucleic acid molecule. It is a compound represented by the formula:

[0018] The inventors focused on the fact that intracellular endosomes are an environment of pH 4 to 5, and discovered that by immobilizing the above-mentioned ligand, which contains a thiol group capable of coordinating with gold, an acid-cleavable group that can be cleaved upon a change from neutral to an acidic pH of 4 to 5, and a nucleic acid residue, on the surface of a gold nanoparticle, molecules containing nucleic acid residues can be released from the surface of the gold nanoparticle with high efficiency in intracellular endosomes, thereby enabling highly efficient transfer of nucleic acids from intracellular endosomes to the cytoplasm.

[0019] <Gold nanoparticles> As the gold nanoparticles, any gold fine particles having a nanoscale particle size produced by a conventionally known production method can be used without any particular limitation. The particle size of the gold nanoparticles is preferably 2 nm or more and 100 nm or less, and more preferably 5 nm or more and 40 nm or less, as an average particle size measured by dynamic light scattering.

[0020] <Ligand> The ligand has the following formula (1): (HS) n -R 1 -R 2 -R 3 -OP(=O)O - -O-Nuc···(1) (In formula (1), n ​​is an integer of 1 or 2, and R 1 is a divalent or trivalent linking group, and R 2is an acid-cleavable group that can be cleaved by changing from neutral to acidic at pH 4 to 5, and R 3 is a divalent linking group, and Nuc is -OP(=O)O at the 5' end of the nucleic acid molecule. - It is a residue in which a group represented by -OH has been removed, or a residue in which the hydrogen atom of the 3'-terminal hydroxyl group has been removed from a nucleic acid molecule. It is a compound represented by the formula:

[0021] R in Equation (1) 1 is a divalent or trivalent linking group. The divalent or trivalent linking group is not particularly limited as long as the desired effect is not impaired. The divalent or trivalent linking group is typically an organic group, and is preferably a hydrocarbon group. In terms of the ease with which many ligands can be bound to the surface of gold nanoparticles, n in formula (1) is 1 and R 1 is preferably a divalent linking group.

[0022] The number of carbon atoms in the organic group as a divalent or trivalent linking group is not particularly limited, and the number of carbon atoms in the organic group is preferably 1 to 20, more preferably 1 to 12, and even more preferably 1 to 6.

[0023] R 1 The divalent or trivalent linking group is preferably a hydrocarbon group. The hydrocarbon group may be an aliphatic hydrocarbon group, an aromatic hydrocarbon group, or a combination of an aliphatic hydrocarbon group and an aromatic hydrocarbon group.

[0024] Specific preferred examples of the divalent hydrocarbon group include alkylene groups such as methylene, ethane-1,2-diyl (ethylene), propane-1,3-diyl, propane-1,2-diyl, butane-1,4-diyl, pentane-1,5-diyl, hexane-1,6-diyl, heptane-1,7-diyl, octane-1,8-diyl, nonane-1,9-diyl, decane-1,10-diyl, undecane-1,11-diyl, and dodecane-1,12-diyl. phenylene groups such as p-phenylene, m-phenylene, and o-phenylene; naphthalenediyl groups such as naphthalene-2,6-diyl, naphthalene-2,7-diyl, naphthalene-1,4-diyl, naphthalene-1,2-diyl, naphthalene-1,3-diyl, and naphthalene-2,3-diyl; and biphenylene groups such as biphenyl-4,4'-diyl, biphenyl-3,3'-diyl, and biphenyl-3,4'-diyl.

[0025] R 2 is an acid-cleavable group that can be cleaved by changing from neutral to acidic at pH 4 to 5. As the acid-cleavable group, any well-known functional group that can be cleaved at pH 4 to 5 can be used without any particular limitation.

[0026] The acid cleavable group is -CH=N-, -CO-O-, -C(COOH)=CH-CO-NH-, or -CR 01 Preferably =N-NH-. 01 is a hydrogen atom or an alkyl group having from 1 to 4 carbon atoms. From the viewpoints of ease of synthesis of the ligand and rapid cleavage at pH 4 to 5, the acid-cleavable group is more preferably —CH═N—.

[0027] R 3 is R 2 and a residue derived from a nucleic acid molecule as Nuc. R 3 The structure of the linking group is not particularly limited, and structures derived from various terminal modification structures that can be introduced into nucleic acid molecules by known methods can be used without particular limitation. For example, the ligand represented by formula (1) is preferably a compound represented by the following formula (1A): 1 , R 2 , Nuc, and n are R in formula (1). 1 , R 2 , Nuc, and n. Alk is an alkylene group which may have a branch. (HS) n -R 1 -R 2 -Alk-OP(=O)O - -O-Nuc···(1A) The number of carbon atoms in the alkylene group represented by Alk is not particularly limited. The number of carbon atoms in the alkylene group is, for example, preferably 1 or more and 20 or less, and more preferably 2 or more and 16 or less. The alkylene group for Alk includes a trimethylene group (-(CH2)3-), a hexamethylene group (-(CH2)6-), and a dodecamethylene group (-(CH2) 12 -) are mentioned.

[0028] Nuc is the -OP(=O)O at the 5' end of the nucleic acid molecule. - It is a residue in which a group represented by -OH has been removed, or a residue in which the hydrogen atom of the 3'-terminal hydroxyl group has been removed from a nucleic acid molecule.

[0029] R of nucleic acid-derived residues as Nuc 3 The end not bound to the linking group may be labeled with various conventionally known fluorescent dyes, such as cyanine dyes such as Cy3 (registered trademark), Cy5 (registered trademark), and Cy7 (registered trademark), coumarin dyes, rhodamine dyes, and fluorescein dyes.

[0030] The nucleic acid molecule that provides Nuc is not particularly limited, and is preferably a nucleic acid molecule that is generally recognized as a nucleic acid drug. The nucleic acid molecule that provides Nuc may be DNA or RNA. Furthermore, the nucleic acid molecule that provides Nuc may be single-stranded or double-stranded. Suitable examples of nucleic acid molecules that provide Nuc include siRNA, shRNA, dsRNA, miRNA, antisense DNA, antisense RNA, ribozymes, aptamers, and decoy nucleic acids. The phosphodiester bonds in the nucleic acid molecules may be replaced with phosphotriester, phosphorothioate, phosphorodithioate, H-phosphonate, or alkylphosphonate bonds, etc., as needed. The base sequence of the nucleic acid molecule that provides Nuc is appropriately selected from the sequences of known nucleic acid drugs depending on the type of disease to be treated. The number of bases when the nucleic acid drug is single-stranded and the number of base pairs when the nucleic acid drug is double-stranded are not particularly limited, but are preferably 5 to 200, more preferably 10 to 50.

[0031] <Bis(p-sulfonatophenyl)phenylphosphine> In the nucleic acid-immobilized gold nanoparticles, the ligand represented by formula (1) is preferably bound to the surface of the gold nanoparticles to which bis(p-sulfonatophenyl)phenylphosphine is bound. By bonding bis(p-sulfonatophenyl)phenylphosphine to the surface of gold nanoparticles, the dispersibility of the gold nanoparticles is improved.

[0032] <Method for producing nucleic acid-immobilized gold nanoparticles> There are no particular limitations on the method for producing nucleic acid-immobilized gold nanoparticles, as long as the method can bind the ligand represented by the above formula (1) to the surface of the gold nanoparticles. The ligand represented by formula (1) may be directly bound to the surface of the gold nanoparticle, or the ligand represented by formula (1) may be synthesized on the surface of the gold nanoparticle.

[0033] Suitable examples of the method for producing nucleic acid-immobilized gold nanoparticles include: The surface of the gold nanoparticles is coated with the following formula (11): (HS) n -R 1 -R 4 ···(11) (In formula (11), R 4 is a group capable of generating an acid-cleavable group that can be cleaved by changing from neutral to acidic at pH 4 to 5, and n and R 1 is R in Equation (1). 1 is the same as and binding a thiol compound represented by the formula: R in thiol compounds bound to the surface of gold nanoparticles 4 and the following equation (12): R 5 -R 3 -OP(=O)O - -O-Nuc···(12) (In equation (12), R 5 is R in Equation (11). 4 is a group capable of reacting with to form an acid-cleavable group, R 3 is R in Eq. (1) 3 is the same as In the nucleic acid derivative represented by 5 and reacting the compound with the compound to form an acid-cleavable group.

[0034] When the acid cleavable group is -CH=N-, R 4 is a formyl group, and R 5 is an amino group or R 4 is an amino group, and R 5 is a formyl group. The reaction between the formyl group and the amino group proceeds, for example, by mixing gold particles bound to a thiol compound represented by formula (11) with a nucleic acid derivative represented by formula (12) at a temperature of 0°C or higher and 80°C or lower, preferably 10°C or higher and 60°C or lower. In formula (11), when n is 2, a cyclic disulfide compound in which two thiol groups in the thiol compound represented by formula (11) are oxidatively condensed to form a disulfide bond can be used as a precursor of the thiol compound represented by formula (11). In the presence of gold nanoparticles, the cyclic disulfide compound can be reacted with a reducing agent such as tris(2-carboxyethyl)phosphine to reductively cleave the disulfide bond, producing a thiol compound represented by formula (11). The resulting thiol compound can be coordinated to the surface of gold nanoparticles. The reaction solvent is not particularly limited as long as it does not inhibit the progress of the reaction, and may be water, an organic solvent, or an aqueous solution of an organic solvent. Suitable reaction solvents include, for example, dimethyl sulfoxide and an aqueous solution of dimethyl sulfoxide.

[0035] When the acid cleavable group is -CO-O-, R 4 , and R 5 The combination of is not particularly limited as long as it is a combination of functional groups that produces a carboxylic acid ester. R 4 , and R 5 When the combination of is a combination of a carboxy group and a hydroxyl group, -CO-O- can be produced using a known condensing agent. R 4 , and R 5 When the combination of is a combination of a methoxycarbonyl group and a hydroxyl group, -CO-O- can be produced by a so-called transesterification reaction. R 4 , and R 5 When the combination is a combination of a halogenocarbonyl group such as a chlorocarbonyl group and a hydroxyl group, -CO-O- can be generated by mixing gold particles bound to a thiol compound represented by formula (11) with a nucleic acid derivative represented by formula (12).

[0036] When the acid cleavable group is -C(COOH)=CH-CO-NH-, R 4 is a 2,5-dioxofuran-1-yl group (maleic anhydride group), and R 5 is an amino group or R 4 is an amino group, and R 5 is a 2,5-dioxofuran-1-yl group.

[0037] The acid cleavable group is -CR 01 =N-NH-, R 4 But, -CO-R 01 is a group represented by R 5 is a group represented by -NH-NH2, or R 4 is a group represented by -NH-NH2, and R 5 Ga-CO-R 01 It is a group represented by the formula:

[0038] As described above, the acid-cleavable group is preferably —CH═N— in view of ease of synthesis of the ligand and rapid cleavage at pH 4 to 5. R 4 is a formyl group, suitable specific examples of the thiol compound represented by formula (11) include 2-formylethyl mercaptan, 3-formylpropyl mercaptan, 2-formylpropyl mercaptan, 4-formylbutyl mercaptan, 5-formylpentyl mercaptan, 6-formylhexyl mercaptan, 4-mercaptobenzaldehyde, 3-mercaptobenzaldehyde, and 2-mercaptobenzaldehyde. R 4 is an amino group, specific preferred examples of the thiol compound represented by formula (11) include 2-aminoethyl mercaptan, 3-aminopropyl mercaptan, 2-aminopropyl mercaptan, 4-aminobutyl mercaptan, 5-aminopentyl mercaptan, 6-aminohexyl mercaptan, 4-mercaptoaniline, 3-mercaptoaniline, and 2-mercaptoaniline.

[0039] In terms of dispersibility of gold nanoparticles, it is preferable to bind bis(p-sulfonatophenyl)phenylphosphine (BSPP) to the surface of the gold nanoparticles before binding a thiol compound to the surface of the gold nanoparticles. The amount of BSPP used is not particularly limited as long as the dispersibility of gold nanoparticles is improved to the desired extent. Gold nanoparticles and BSPP are mixed in a dispersion medium such as dimethyl sulfoxide at a temperature of, for example, 0°C or higher and 80°C or lower, preferably 10°C or higher and 60°C or lower, whereby BSPP binds to the surface of the gold nanoparticles. When BSPP is bound to gold nanoparticles, the concentration of BSPP in the dispersion containing gold nanoparticles is preferably 10 mg / mL or less.

[0040] The nucleic acid-immobilized gold nanoparticles produced as described above are typically used in a dispersion medium for various purposes. [Example]

[0041] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. It is not something that is done.

[0042] Example 1 According to the following scheme, nucleic acid-immobilized gold nanoparticles were produced in which a ligand (Cy5-DNA-Shiff) having a structure corresponding to the structure of the aforementioned formula (1) was bound to the surface of gold nanoparticles (AuNPs). [ka]

[0043] (Preparation of BSPP-bound gold nanoparticles (BSPP-AuNPs)) To a dispersion of gold nanoparticles (average particle size 15 nm) in dimethyl sulfoxide (DMSO) at a concentration of 1 nmol / L, BSPP was added to a concentration of 1 mg / 1 mL. The dispersion of gold nanoparticles containing BSPP was then left overnight at room temperature (20-25°C) to obtain gold nanoparticles with BSPP bound to their surface (BSPP-AuNP). The dispersion containing BSPP-AuNP was concentrated by centrifugation (20,000 G, 20 minutes). The supernatant was then removed from the concentrated dispersion, and 250 μL of water was added to the residue. The resulting aqueous dispersion of BSPP-AuNP was again concentrated by centrifugation (20,000 G, 20 minutes). 220 μL of the supernatant was then removed to obtain 30 μL of an aqueous dispersion of BSPP-AuNP. 30 μL of DMSO was added to 30 μL of the aqueous dispersion of BSPP-AuNP, yielding 60 μL of a dispersion of BSPP-AuNP in a DMSO aqueous solution (water:DMSO (volume ratio): approximately 1:1).

[0044] (Preparation of gold nanoparticles (CHO-AuNPs) with 4-mercaptobenzaldehyde bound to the surface of BSPP-AuNPs) 2 μL of a DMSO solution containing 4-mercaptobenzaldehyde at a concentration of 0.5 mol / L was added to 60 μL of a dispersion of BSPP-AuNP in a DMSO aqueous solution (water:DMSO (volume ratio): approximately 1:1). Next, the dispersion of BSPP-AuNP containing 4-mercaptobenzaldehyde was left to stand at 50 °C for 1 hour. Thereafter, the dispersion was left to stand at room temperature (20-25 °C) for 24 hours. After standing, the dispersion was concentrated by centrifugation (20,000 G, 20 minutes), the supernatant was removed, and 60 μL of DMSO aqueous solution (water:DMSO (volume ratio): approximately 1:1) was added to the residue. This procedure was repeated twice to remove excess 4-mercaptobenzaldehyde, yielding 60 μL of a dispersion in which CHO-AuNPs were dispersed in DMSO aqueous solution (water:DMSO (volume ratio): approximately 1:1).

[0045] (Preparation of nucleic acid-immobilized gold nanoparticles (Cy5-DNA-Shiff-AuNP) in which a ligand (Cy5-DNA-Shiff) having a structure corresponding to the structure of formula (1) above is bound to the surface of BSPP-AuNP) 20 μL of an oligonucleotide aqueous solution containing a 100 μmol / L single-stranded oligo DNA with 15 bases, the 5' (3') end of which was modified with an amino group and the 3' (5') end with a fluorescent dye (Cy5), was added to 60 μL of a dispersion of CHO-AuNPs in a DMSO aqueous solution (water:DMSO (volume ratio): approximately 1:1). The dispersion containing the oligonucleotides and CHO-AuNPs was left standing overnight at 50°C. Then, 0.5 μL of 0.5 mol / L phosphate buffer solution (PBS) was added to the dispersion. The dispersion with the added phosphate buffer was left standing at 50°C for 48 hours. After standing, the dispersion was concentrated by centrifugation (20,000 G, 20 minutes), the supernatant was removed, and 60 μL of TE buffer (pH 8) was added to the residue. This process was repeated twice to remove excess oligo-DNA, yielding 60 μL of a dispersion of Cy5-DNA-Shiff-AuNPs dispersed in TE buffer (pH 8).

[0046] The average diameter of the CHO-AuNPs obtained by the above method was measured by dynamic light scattering and was found to be 18.6 nm, and that of the Cy5-DNA-Shiff-AuNPs was measured by dynamic light scattering and was found to be 26.4 nm. This difference in average particle size indicates that oligo DNA is bound to CHO-AuNP. Furthermore, when a dispersion of Cy5-DNA-Shiff-AuNPs at pH 7.4 was kept at room temperature for 30 minutes, the dispersion after 30 minutes showed red fluorescence, while a dispersion of Cy5-DNA-Shiff-AuNPs adjusted to pH 5.5 was kept at room temperature for 30 minutes, the dispersion after 30 minutes showed blue fluorescence. The change from red to blue fluorescence is due to the aggregation of gold nanoparticles. This indicates that the acid-cleavable group -CH=N- in the ligand (Cy5-DNA-Shiff) is cleaved at pH 5.5. The cleavage of the acid-cleavable group causes fragments containing nucleic acid molecules to be released from Cy5-DNA-Shiff-AuNP, resulting in the aggregation of gold nanoparticles due to the reduction of steric hindrance on the surface of the gold nanoparticles.

Claims

1. a ligand having a nucleic acid-derived residue is attached to the surface of the gold nanoparticle; The ligand is represented by the following formula (1): (HS) ｎ -R １ -R ２ -R ３ -O-P(=O)O － -O-Nuc・・・(1) (In formula (1), n ​​is an integer of 1 or 2, and R １ is a divalent linking group, and R ２ is an acid-cleavable group that can be cleaved by changing from neutral to acidic at pH 4-5, and R ３ is a divalent linking group, and Nuc is —O—P(═O)O at the 5′ end of the nucleic acid molecule. － It is a residue in which a group represented by -OH has been removed, or a residue in which the hydrogen atom of the 3'-terminal hydroxyl group has been removed from a nucleic acid molecule. A nucleic acid-immobilized gold nanoparticle, which is a compound represented by the formula:

2. The acid cleavable group is -CH=N-, -CO-O-, -C(COOH)=CH-CO-NH-, or -CR ０１ =N-NH-, The R ０１ The nucleic acid-immobilized gold nanoparticle according to claim 1, wherein is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.

3. The nucleic acid-immobilized gold nanoparticle of claim 2, wherein the acid-cleavable group is -CH=N-.

4. The R １ The nucleic acid-immobilized gold nanoparticle according to claim 3, wherein the divalent linking group is a phenylene group.

5. The nucleic acid-immobilized gold nanoparticle according to any one of claims 1 to 4, wherein bis(p-sulfonatophenyl)phenylphosphine is bound to the surface of the gold nanoparticle.

6. The surface of the gold nanoparticles is coated with the following formula (11): (HS) ｎ -R １ -R ４ ・・・(11-1) (In formula (11), R ４ is a group capable of generating an acid-cleavable group that can be cleaved by changing from neutral to acidic at pH 4 to 5, n, and R １ is R in formula (1). １ (Similar to the above.) and binding a thiol compound represented by the formula: The R in the thiol compound bound to the surface of the gold nanoparticle ４ and the following formula (12): R ５ -R ３ -O-P(=O)O － -O-Nuc・・・(12) (In formula (12), R ５ is the R in the formula (11). ４ is a group capable of reacting with to generate the acid-cleavable group, and R ３ is R in formula (1). ３ (Similar to the above.) In the nucleic acid derivative represented by ５ to generate the acid-cleavable group; The method for producing nucleic acid-immobilized gold nanoparticles according to claim 1, comprising:

7. The R ４ is a formyl group, and the R ５ is an amino group, and the R ２ The method for producing nucleic acid-immobilized gold nanoparticles according to claim 6, wherein is -CH=N-.

8. 8. The method for producing nucleic acid-immobilized gold nanoparticles according to claim 6, wherein bis(p-sulfonatophenyl)phenylphosphine is bound to the surface of the gold nanoparticles before the thiol compound is bound to the surface of the gold nanoparticles.

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