JP2008222502A - Composite particle with high dispersibility inhibiting nonspecific adsorption, composite particle colloid, analysis reagent using the same, particle surface modification method and method for manufacturing composite particle - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide composite particles and a composite particle colloid that prevents nonspecific adsorption except for intended adsorption upon specifically coupling biomolecules, shows high dispersibility and allows introduction of functional groups for coupling biomolecules, to provide a method for manufacturing the above particles and the colloid, and to provide an analysis reagent having excellent reproducibility of measurement results, high reliability and a high signal-noise ratio. <P>SOLUTION: The composite particles comprise silica particles and organic molecules, in which the organic molecules are adsorbed by electrostatic attractive force to the surfaces of the silica particles. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to highly dispersible composite particles, composite particle colloids, analytical reagents using the same, particle surface modification methods, and composite particle production methods, in which organic molecules are adsorbed onto the particle surfaces by electrostatic attraction. . Furthermore, the present invention provides composite particles, composite particle colloids, analytical reagents using the same, particle surface modification methods using the same, which prevent the occurrence of nonspecific adsorption other than intended when biomolecules are specifically bound. The present invention relates to a method for producing composite particles.

In recent years, fine particles of about several nm to 1 μm have been applied to various fields and attracted attention. For example, porous silica particles and zeolite particles used for adsorbents and catalysts, carbon black used for pigments, metal oxide particles, inorganic compound particles, metal nanoparticles used for conductive materials, and resin reinforcing agents. There are a wide variety of materials and applications of particles such as silica particles. Further, semiconductor nanoparticles and silica particles encapsulating a fluorescent substance are expected to be applied to fluorescent reagents as new fluorescent labeling agents, particularly in the bio field.
A fluorescent reagent is a protein or DNA bound to the surface of a fluorescent particle, and is used for detection, quantification, staining, etc. of a biomolecule by interacting with the specific biomolecule. is there. Since fluorescent particles such as semiconductor nanoparticles and fluorescent silica particles have high brightness and high light stability, the development of fluorescent reagents using fluorescent particles has attracted attention.
In order to use fluorescent particles as a fluorescent reagent, surface modification that satisfies the following problems is required.
(1) Introduction of a functional group for binding a biomolecule (2) Dispersion stabilization in a buffer solution (3) Prevention of non-specific adsorption Hereinafter, each of the above problems (1) to (3) is specifically described. State.
(1) Introduction of functional group for binding biomolecules In order to integrate silica particles and biomolecules to function stably, it is necessary to bind the silica particles and biomolecules irreversibly. For this purpose, it is necessary to covalently bond silica particles and biomolecules, not ionic bonds or physicochemical adsorption.
Protein has an amino group, a carboxyl group, a thiol group, and the like. In addition, DNA can be given an amino group or a thiol group by terminal modification. Therefore, if an amino group, a carboxyl group, a thiol group or the like is introduced into the silica particles, it is possible to covalently bond the silica particles and the biomolecules using a crosslinking agent or a condensing agent.
(2) Dispersion stabilization in a buffer solution The DLVO theory is known as a modeled representation of attractive and repulsive forces that act on the aggregation and dispersion of particles. This explains the agglomeration / dispersion of particles by electrostatic repulsive force and van der Waals force between particles (for example, see Non-Patent Document 1).
Biomolecules are often used in an aqueous solution having a high ion concentration such as physiological saline. In an aqueous solution with a high ion concentration, the ions weaken the electrostatic repulsion of the silica particles due to the shielding effect, so that the particles tend to aggregate. In order to stably disperse particles even in an aqueous solution having a high ion concentration, it is necessary to apply a repulsive force other than an electrostatic repulsive force.
There is a solid repulsive force that acts as a repulsive force between particles in addition to the electrostatic repulsive force. This works as a repulsive force between particles by repelling molecular chains with degrees of freedom. Therefore, the dispersibility of the particles can be improved by introducing a molecular chain having a high degree of freedom into the particle surface.
(3) Prevention of non-specific adsorption In order for the fluorescent particles to function as a fluorescent reagent, it is necessary that biomolecules bound to the fluorescent particles be selectively bound to the target by specific binding or the like. If non-specific binding to a biomolecule other than the target or adsorption to the substrate occurs, the sensitivity, accuracy, and reliability of the measurement will decrease accordingly.
In order to reduce non-specific adsorption, it is necessary to impart steric repulsion as well as improvement in dispersibility.

From the above viewpoints, surface modification is required for application of fluorescent particles. Particle surface modification methods include silane coating with a silane coupling agent, incorporation into polymer beads, entrapment in lipid bilayers, and attachment of small molecules via thiol groups. It is known (for example, see Patent Documents 1 to 3). However, the method of coating with a silane coupling agent has a problem that applicable particles are limited to inorganic particles, metal particles, silica particles, etc., and sufficient dispersibility cannot be obtained only by silane coating. is there. The method of incorporating the polymer beads is costly and has a problem that the particle size increases. The method of encapsulating in a lipid bilayer has a problem in terms of long-term dispersibility, although the dispersibility in water is high, but the lipid bilayer itself is unstable. Also regarding the method of binding a small molecule through a thiol group, applicable particles are limited to metals.
As described above, a surface modification method that can be applied to particles of various materials and is stable for a long time is not yet known.
Fumio Kitahara, Kunio Furusawa, Masataka Ozaki, Hiroyuki Oshima, “Zeta Potential Zeta Potential: Physical Chemistry of Fine Particle Interfaces”, Scientist, 1995 JP 2003-11505 A JP 2004-77389 A JP 2006-131771 A

In view of the above problems, the object of the present invention is to prevent the occurrence of non-specific adsorption other than intended when biomolecules are specifically bound, have high dispersibility, and It is an object of the present invention to provide composite particles and composite particle colloids capable of introducing functional groups for bonding.
Another object of the present invention is to provide an analytical reagent having excellent reproducibility of measurement results, high reliability, and a high signal / noise ratio. Another object of the present invention is to provide a particle surface modification method capable of adsorbing cationic organic molecules and anionic organic molecules on the particle surface in a superimposed manner and applicable to particles of various materials.
Furthermore, the object of the present invention is a composite that can be applied to particles of various materials, prevents the occurrence of non-specific adsorption, has high dispersibility, and can introduce functional groups for binding biomolecules. The object is to provide a method for producing particles.

The above problems have been achieved by the following means.
(1) Composite particles comprising silica particles and organic molecules, wherein organic molecules are adsorbed on the surface of the silica particles by electrostatic attraction.
(2) Silica particles / organic molecules / polyethylene glycol derivatives, wherein organic molecules are adsorbed on the surface of silica particles by electrostatic attraction, polyethylene glycol derivatives are covalently bonded, and located outside the organic molecules. Composite particles.
(3) An organic molecule is adsorbed to the surface of the silica particle by electrostatic attraction, and a polyethylene glycol derivative is covalently bonded to the organic molecule at one end of the derivative, located outside the organic molecule, and A composite particle of silica particles / organic molecule / polyethylene glycol derivative / biomolecule, wherein a biomolecule is covalently bonded to the other end of the polyethylene glycol derivative.
(4) The organic molecules adsorbed on the surface of the silica particles are cationic organic molecules and anionic organic molecules, the innermost cationic organic molecule is adsorbed on the silica particles, and the anionic organic molecules are A structure that is adsorbed to a cationic organic molecule and located outside the cationic organic molecule, or a structure that the anionic organic molecule is adsorbed to the cationic organic molecule and located outside the cationic organic molecule. The composite particle according to any one of (1) to (3), which is repeated a plurality of times and has a structure in which the anionic organic molecule is located on the outermost side.
(5) The composite particle according to (4), wherein the cationic organic molecule and the anionic organic molecule each form an organic molecular layer.
(6) The composite particle according to (4) or (5), wherein the polyethylene glycol derivative is covalently bonded to the outermost anionic organic molecule.
(7) The anionic organic molecule is a polypeptide containing at least one of polyacrylic acid, alginic acid, polystyrene sulfonic acid, citric acid, glutamic acid, aspartic acid, polyglutamic acid, polyaspartic acid, or glutamic acid or aspartic acid. The composite particle according to (6), wherein the cationic organic molecule is any of polyallylamine hydrochloride, chitosan, polylysine, or lysine.
(8) The anionic organic molecules are bonded via a covalent bond, the cationic organic molecules are bonded via a covalent bond, and / or the anionic organic molecule and the cationic organic The composite particle according to (6) or (7), wherein the molecule is partially bonded through a covalent bond.
(9) The polyethylene glycol derivative has a terminal functional group covalently bonded to the organic molecule, which is any one of a hydroxyl group, an amino group, a carboxyl group, a thiol group, a maleimide group, or a succinimidyl ester group, Any of (4) to (8), wherein the functional group at the other end of the glycol derivative is any one of a carboxyl group, a thiol group, a methoxy group, a hydroxyl group, an amino group, a maleimide group, or a succinimidyl ester group. 2. Composite particles according to item 1.
(10) Any one of (3) to (9), wherein the biomolecule is an antigen, antibody, DNA, RNA, sugar, sugar chain, ligand, receptor, peptide or chemical substance. The composite particles according to 1.
(11) The composite particle according to any one of (1) to (10), wherein the silica particles have an average particle diameter of 1 nm to 1 μm.

(12) The composite particle according to any one of (1) to (11), wherein the organic molecule has a molecular weight of 50 to 100,000.
(13) The composite particle according to any one of (1) to (12), wherein an absolute value of ζ potential in pure water at pH 7 is 1 to 60 mV.
(14) The composite particle according to any one of (1) to (13), wherein the absolute value of the ζ potential in pure water at pH 7 is 35 to 60 mV.
(15) The composite particle according to any one of (1) to (14), wherein the silica particle has an average particle diameter of 1 nm to 1 μm and a molecular weight of the organic molecule of 50 to 100,000.
(16) The composite particles according to (15), wherein the absolute value of the ζ potential in pure water at pH 7 is 1 to 60 mV.
(17) The composite particles according to (16), wherein the absolute value of the ζ potential in pure water at pH 7 is 35 to 60 mV.
(18) A composite particle colloid in which the composite particles according to any one of (1) to (17) are dispersed in a dispersion medium.
(19) The composite particle colloid according to (18), wherein the dispersion medium is a buffer solution.
(20) An analytical reagent using the composite particle colloid according to (18) or (19).
(21) The cationic organic molecule is adsorbed on the surface of the particle, and further, the anionic organic molecule is adsorbed, and the cationic organic molecule and the anionic organic molecule are alternately applied a plurality of times. A particle surface modification method, wherein a plurality of organic molecules are adsorbed on the particle surface in a superimposed manner by performing the adsorption treatment.
(22) The particle surface modification method according to (21), wherein the particles are silica particles.

(23) The cationic organic molecule and the anionic organic molecule are alternately applied a plurality of times by the procedure of performing adsorption treatment of the cationic organic molecule on the surface of the particle and further performing the adsorption treatment of the anionic organic molecule. A method for producing composite particles comprising particles and organic molecules, wherein the adsorption treatment is performed.
(24) The method for producing composite particles according to (23), wherein the colloid of the particles and a solution of organic molecules having a charge opposite to the surface charge of the particles are mixed.
(25) Further, the colloid of the composite particles composed of the particles and the organic molecules, and a solution of organic molecules having a charge opposite to the surface charge of the composite particles are mixed, (23) or (24) The method for producing composite particles according to (24).
(26) After producing the composite particles comprising the particles and the organic molecules by the production method according to any one of (23) to (25),
A method for producing particle / organic molecule / polyethylene glycol derivative composite particles, wherein a polyethylene glycol derivative is covalently bonded to an organic molecule located on the outermost side of the composite particles by a condensing agent or a crosslinking agent.
(27) The composite particle according to (26), wherein a colloid of the composite particle composed of the particle and an organic molecule, a solution of the polyethylene glycol derivative, and the condensing agent or a crosslinking agent are mixed. Production method.
(28) After producing the composite particles of the particles / organic molecules / polyethylene glycol derivative by the production method according to (26),
Furthermore, a method for producing a particle / organic molecule / polyethylene glycol derivative / biomolecule composite particle, wherein a biomolecule is covalently bonded to one end of the polyethylene glycol derivative.
(29) The covalent bond of the biomolecule to one end of the polyethylene glycol derivative is a colloid of the composite particle of the particle / organic molecule / polyethylene glycol derivative, a solution of the biomolecule, the condensing agent, or a cross-linking. The method for producing composite particles according to (28), which is performed by mixing with an agent.
(30) The method for producing composite particles according to any one of (23) to (29), wherein the particles are silica particles.

Since the composite particles of the present invention are formed by adsorbing organic molecules having a charge on the surface of the silica particles, the electrostatic repulsion between the composite particles can be increased and the dispersibility can be improved.
The composite particle of the present invention further increases the steric repulsion between the composite particles by covalently bonding a polyethylene glycol derivative to the adsorbed organic molecule, improves dispersibility, and specifically binds biomolecules. In this case, non-specific adsorption other than intended can be prevented. It is also possible to introduce functional groups for binding biomolecules.
Therefore, the composite particles of the present invention prevent nonspecific adsorption by a low molecular weight compound or a high molecular compound, nonspecific adsorption to a substrate or a container, and are excellent in dispersibility and have high ionic strength such as physiological saline. Aggregation hardly occurs even in dispersed colloids.
Here, “non-specific adsorption” refers to adsorption that does not follow rules other than specifically binding to a specific functional group or ligand, and refers to an adsorption phenomenon other than intended.

Since the composite particle colloid of the present invention uses the composite particle, when specifically binding a biomolecule, it prevents non-specific adsorption other than intended, has excellent dispersibility, and ions such as physiological saline. Aggregation is unlikely to occur even with water-dispersed colloids with high strength.
The analytical reagent of the present invention uses the composite particle colloid that prevents non-specific adsorption other than intended and has excellent dispersibility when specifically binding biomolecules, so that the reproducibility of measurement results is improved. Highly sensitive analysis of extremely small target samples with excellent and reliable signal / noise ratio is possible.

In the particle surface modification method of the present invention, the ζ potential of a particle is measured in advance to determine whether the particle is positively charged or negatively charged. When the particle is positively charged, an anionic organic molecule is negatively charged. When charged, the organic molecules can be adsorbed on the surface of the particles by adsorbing cationic organic molecules, and can be applied to particles of various materials regardless of the material of the particles.
The particle surface modification method of the present invention can adsorb cationic organic molecules and anionic organic molecules in a superimposed manner on the particle surface by alternately adsorbing cationic organic molecules and anionic organic molecules.
Similarly, in the method for producing the composite particles of the present invention, the ζ potential of the particles is measured in advance to determine whether the particles are positively charged or negatively charged. When negatively charged, organic molecules can be adsorbed on the particle surface by adsorbing cationic organic molecules, and can be applied to particles of various materials regardless of the material of the particles. Further, by alternately adsorbing the cationic organic molecules and the anionic organic molecules, the cationic organic molecules and the anionic organic molecules can be adsorbed on the particle surface in a superimposed manner.
The method for producing composite particles of the present invention can provide composite particles that hardly cause aggregation even in an aqueous dispersion having high ionic strength such as physiological saline. Moreover, the composite particle which can suppress the nonspecific adsorption | suction of the low molecular compound and high molecular compound with respect to particle | grains, and the nonspecific adsorption | suction with respect to the board | substrate and container of particle | grains can be provided. Furthermore, a functional group for binding biomolecules can also be introduced.

First, a first embodiment of the composite particle of the present invention will be described.
The composite particles of the present invention have a structure composed of silica particles and organic molecules, in which organic molecules are adsorbed on the surface of the silica particles by electrostatic attraction.
Here, “electrostatic attractive force” refers to Coulomb force acting between positive charge and negative charge. The “adsorption” refers to integration by electrostatic attraction, van der Waals force or hydrophobic interaction.
In the present invention, the silica particles used are not particularly limited, and may be silica particles obtained by any arbitrary preparation method. Examples thereof include silica particles prepared by the sol-gel method described in Journal of Colloid and Interface Science, 159, 150-157 (1993).
The present inventors have applied for a patent for a method for preparing fluorescent dye compound-containing colloidal silica particles (for example, Japanese Patent Application No. 2005-376401). It is particularly preferable to use silica particles containing a functional compound obtained according to the method.
Here, specific examples of the functional compound include fluorescent dye compounds, light absorbing compounds, magnetic compounds, radiolabeled compounds, pH sensitive dye compounds, and the like.
Specifically, the silica particles containing the functional compound are reacted with a product obtained by reacting the functional compound with a silane compound and covalently bonding, ionic bonding, or other chemical bonding or adsorption. Alternatively, it can be prepared by polymerizing two or more silane compounds.
Preferred embodiments of the method for preparing silica particles containing the functional compound include N-hydroxysuccinimide (NHS) ester group, maleimide group, isocyanate group, isothiocyanate group, aldehyde group, paranitrophenyl group, diethoxy. The functional compound having an active group such as a methyl group, an epoxy group, or a cyano group, and a silane coupling agent having a substituent (for example, an amino group, a hydroxyl group, or a thiol group) that reacts with the active group. The product obtained by reacting and covalently bonding can be prepared by polymerizing one or more silane compounds.

  Specific examples of the functional compound having the active group include fluorescent dyes having an NHS ester group such as DY550-NHS ester or DY630-NHS ester (both trade names, manufactured by Dynamics GmbH) represented by the following formulae, respectively. A compound can be mentioned.

  Specific examples of the silane coupling agent having a substituent include γ-aminopropyltriethoxysilane (APS), 3- [2- (2-aminoethylamino) ethylamino] propyl-triethoxysilane, N-2 ( Examples thereof include silane coupling agents having an amino group such as (aminoethyl) 3-aminopropylmethyldimethoxysilane and 3-aminopropyltrimethoxysilane. Of these, APS is preferable.

  The silane compound to be polymerized is not particularly limited, but tetraethoxysilane (TEOS), γ-mercaptopropyltrimethoxysilane (MPS), γ-mercaptopropyltriethoxysilane, γ-aminopropyltriethoxysilane (APS). ), 3-thiocyanatopropyltriethoxysilane, 3-glycidyloxypropyltriethoxysilane, 3-isocyanatopropyltriethoxysilane, and 3- [2- (2-aminoethylamino) ethylamino] propyl-triethoxy Mention may be made of silane. Among these, TEOS, MPS, or APS is preferable.

When prepared as described above, spherical or nearly spherical silica particles can be produced. The nearly spherical silica particles specifically have a shape in which the ratio of the major axis to the minor axis is 2 or less.
In order to obtain silica particles having a desired average particle size, ultrafiltration is performed using an ultrafiltration membrane such as YM-10, YM-100 (both trade names, manufactured by Millipore), and the particle size is large. It is possible to remove particles that are too small or too small, or to centrifuge at an appropriate gravitational acceleration and collect only the supernatant or precipitate.

  The present invention can also be applied to particles of any material that is not silica particles. Specifically, metal particles such as gold, silver and copper, oxide particles such as ferrite and titanium oxide, semiconductor nanoparticles such as cadmium selenide, and polymers such as polystyrene and polymethylmethacrylic acid may be used. It may be a particle.

In the present invention, the organic molecule to be used is preferably a cationic organic molecule or an anionic organic molecule from the viewpoint of adsorbing to the surface of the silica particles by electrostatic attraction.
In particular, the organic molecule that is directly adsorbed and formed on the surface of the silica particles is preferably the cationic organic molecule from the viewpoint of having a charge opposite to the surface charge of the silica particles.
Examples of the anionic organic molecule include polyacrylic acid, alginic acid, polystyrene sulfonic acid, citric acid, glutamic acid, aspartic acid, polyglutamic acid, polyaspartic acid, or a polypeptide containing at least one of glutamic acid or aspartic acid. And is preferably polyacrylic acid or alginic acid.
Examples of the cationic organic molecule include polyallylamine hydrochloride, chitosan, polylysine, or lysine, and polyallylamine hydrochloride or chitosan is preferable.
The molecular weight of these organic molecules is preferably 50 to 100,000, more preferably 2000 to 20,000 (in the present invention, the molecular weight means a weight average molecular weight unless otherwise specified). The compounds are polydisperse and do not necessarily have the same molecular weight or particle weight, so the value obtained by measuring the molecular weight is the average molecular weight averaged in some way, the main ones being There are three types: 1) number average molecular weight Mn, 2) weight average molecular weight Mw, 3) Z average molecular weight Mz, and a relationship of Mn <Mw <Mz is established. ).

In the composite particle of the present invention, it is preferable to form a structure in which the cationic organic molecule and the anionic organic molecule are alternately adsorbed, and the cationic organic molecule and the anionic organic molecule are each organic. It is more preferable to form a multilayer structure formed by forming a molecular layer.
For example, since the silica particle has a negative ζ potential, which will be described later, when processing four layers of organic molecules, the first layer from the inside is a cationic organic molecule, the second layer is an anionic organic molecule, 3 The layer can be a cationic organic molecule, and the outermost fourth layer can be an anionic organic molecule.
From the viewpoint of sufficiently reducing the exposure of the surface of the silica particles and suppressing nonspecific adsorption to the particle surface, the number of alternately adsorbed is preferably 2 to 10, and similarly when a layer is formed. 2-10 layers are preferred.
When the composite particles of the present invention are used in a buffer solution such as PBS (phosphate buffered saline), Tris buffer solution, HEPES buffer solution, the buffer solution contains a negative ionic species with high adsorptivity. From the viewpoint of preventing the ionic species from adsorbing to the composite particles by electrostatic attraction, weakening the electrostatic repulsion, and preventing the composite particles from aggregating, The organic molecule is preferably an anionic organic molecule.

The composite particle of the present invention has a structure in which the anionic organic molecule is adsorbed on the cationic organic molecule adsorbed on the surface of the silica particle and is located outside the cationic organic molecule, or the anionic organic molecule is A structure that adsorbs to the cationic organic molecule and is located outside the cationic organic molecule is repeated a plurality of times, preferably 2 to 10 times, and has an alternating structure in which the anionic organic molecule is located on the outermost side. Particularly preferred.
Moreover, it is preferable that the organic molecules forming the alternating structure are bonded to each other via a covalent bond by using a crosslinking agent or a condensing agent. The anionic organic molecules are bonded via a covalent bond, the cationic organic molecules are bonded via a covalent bond, and / or the anionic organic molecule and the cationic organic molecule are More preferably, they are partially bonded via a covalent bond. This stabilizes the adsorption between the organic molecules and can prevent the organic molecules from desorbing. As a result, long-term dispersion stabilization of the composite particles is possible.
Here, “partially bonded via a covalent bond” means that, in the present invention, the anionic organic molecule and the cationic organic molecule are bonded by adsorption, but are due to the covalent bond. It means that the bonds are mixed.
Specific examples of the crosslinking agent or condensing agent used include N- (6-maleimidocaproyloxy) succinimide (EMCS), glutaraldehyde, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and the like. Can be mentioned. The number of equivalents of the crosslinking agent or condensing agent used in the reaction, the type and volume of the dispersion medium or solvent, and the reaction conditions such as the reaction temperature are not particularly limited as long as the reaction proceeds.

Next, a second embodiment of the composite particle of the present invention will be described.
The composite particle of the present invention is a silica particle / organic molecule in which a polyethylene glycol derivative (hereinafter sometimes simply referred to as “PEG derivative”) is covalently bonded to the organic molecule and located outside the organic molecule. / PEG derivative is preferable, and the PEG derivative is more preferably covalently bonded to the outermost anionic organic molecule. Also in the second embodiment of the composite particle of the present invention, in the structure in which the cationic organic molecule and the anionic organic molecule are alternately adsorbed, the cationic organic molecule and the anionic organic molecule are each organic. More preferably, a molecular layer is formed.
In the present invention, the “PEG derivative” refers to a compound having PEG in the basic skeleton and having functional groups described below at both ends.
In the present invention, the PEG compound covalently bonded to the organic molecule can form a covalent bond with the organic molecule using a crosslinking agent or a condensing agent. The molecular weight of the PEG derivative is preferably 50 to 10000, more preferably 2000 to 8000.
Specific examples of the crosslinking agent or condensing agent include an aqueous solution having an arbitrary mixing ratio of 1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (Sulfo-NHS). Or a buffer solution etc. are mentioned. The number of equivalents of the crosslinking agent or condensing agent used in the reaction, the type and volume of the dispersion medium or solvent, and the reaction conditions such as the reaction temperature are not particularly limited as long as the reaction proceeds.
Regarding the terminal functional group of the PEG derivative, examples of the terminal covalently bonded to the organic molecule include a hydroxyl group, an amino group, a carboxyl group, a thiol group, a maleimide group, a succinimidyl ester group, and the like. Preferably there is.
Examples of the functional group at the other end opposite to the end covalently bonded to the organic molecule include a carboxyl group, a thiol group, a methoxy group, a hydroxyl group, an amino group, a maleimide group, and a succinimidyl ester group. Or it is preferable that it is a thiol group.

Next, a third embodiment of the composite particle of the present invention will be described.
The composite particle of the present invention is a silica particle in which the PEG derivative is covalently bonded at one end thereof, located outside the organic molecule, and a biomolecule is covalently bonded to the other end of the PEG derivative. It is particularly preferable to have a structure consisting of / organic molecule / PEG derivative / biomolecule. The PEG derivative is preferably covalently bonded to the outermost anionic organic molecule.
Also in the third embodiment of the composite particle of the present invention, in the structure in which the cationic organic molecule and the anionic organic molecule are alternately adsorbed, the cationic organic molecule and the anionic organic molecule are each organic. More preferably, a molecular layer is formed.
The biomolecule is preferably covalently bonded to the PEG derivative directly or via a crosslinking agent. Specific examples of the crosslinking agent include N- (6-maleimidocaproyloxy) succinimide (EMCS), glutaraldehyde and the like. There are no particular restrictions on the reaction conditions such as the number of equivalents of the crosslinking agent used in the reaction, the type / volume of the dispersion medium or solvent, and the reaction temperature, as long as the reaction proceeds.
In the third embodiment of the composite particle of the present invention, not all of the PEG derivative bound to the organic molecule may be bound to the biomolecule, that is, the PEG derivative bound to the biomolecule and the living body. The PEG derivative to which no molecule is bonded may be mixed. Such surface modification can be performed by mixing the PEG derivatives having different terminal functional groups with respect to the PEG derivative bonded to the organic molecule.
For example, a PEG derivative having an amino group at one end and a carboxyl group at the other end, and a PEG derivative having an amino group at one end and a methoxy group at the other end, preferably in a molar ratio of 1: 4. After mixing at a molar ratio and covalently bonding the amino group of the PEG derivative and the carboxyl group of the organic molecule using a condensing agent, the biomolecule is bound only with respect to the PEG derivative having a carboxyl group at the other end. Can be done by the method.
Examples of the biomolecule to be covalently bonded to the end of the PEG derivative moiety include antigens, antibodies, DNA, RNA, sugars, sugar chains, ligands, receptors, peptides or chemical substances.
Here, a ligand refers to a substance that specifically binds to a protein, such as a substrate that binds to an enzyme, a coenzyme, a regulatory factor, a hormone, or a neurotransmitter, as well as a low molecular weight molecule or ion. Also includes high molecular weight materials.
The chemical substances are not limited to natural organic compounds, but include artificially synthesized physiologically active compounds and environmental hormones.

In this invention, it is preferable that the average particle diameter of the said silica particle to be used is 1 nm-1 micrometer, and it is more preferable that it is 20 nm-500 nm.
The average particle size of the composite particles of the present invention is not particularly limited, but is preferably 1 nm to 1 μm.
In the present invention, the average particle size is calculated from the total projected area of 50 silica particles or composite particles randomly selected from an image such as a transmission electron microscope (TEM) or a scanning electron microscope (SEM). Alternatively, the area occupied by the composite particles is obtained by an image processing apparatus, and the average value of the diameters of the circles corresponding to the value obtained by dividing the total occupied area by the number of selected silica particles or composite particles (50 particles) (corresponding to the average circle) (Diameter).
The variation coefficient of the particle size distribution, the so-called CV value, is not particularly limited, but is preferably 10% or less, more preferably 8% or less.
In the present specification and claims, monodisperse means a particle group having a CV value of 15% or less.

Next, the ζ potential of the composite particles of the present invention will be described.
Charge separation occurs at the interface when the dispersion medium and colloidal particles moving relative to it come into contact. At this time, ions having a sign opposite to that of the particles gather on the particle surface to form a layer. This is called an electric double layer. In a region close enough to the particle surface, ions with opposite charges are strongly attracted to the surface and have no mobility, so they are called stationary phases. The ions outside it have mobility and are called diffusion layers. The contact surface between the fixed layer and the diffusion layer is called a sliding surface. The potential of the sliding surface when the potential in a region where the charge is sufficiently far away from the particle is defined as zero is ζ potential (hereinafter sometimes simply referred to as “zeta potential”). Electric potential.
The zeta potential is an index for evaluating the dispersibility, cohesiveness, surface modification and the like of colloidal particles. That is, since the colloidal particles are charged and the magnitude of the electrostatic repulsion due to the charging corresponds to the magnitude of the absolute value of the zeta potential, the magnitude of the absolute value of the zeta potential is It is an index of the dispersion stability of particles (see, for example, Fumio Kitahara, Kunio Furusawa, Masataka Ozaki, Hiroyuki Oshima, “Zeta Potential Zeta Potential: Physical Chemistry of Fine Particle Interface”, Scientist, 1995).
Further, when the colloidal particles are negatively charged, the zeta potential becomes a negative value, while when the colloidal particles are positively charged, the zeta potential becomes a positive value.
The composite particles of the present invention preferably have an absolute value of ζ potential in pure water of pH 7 of 1 to 60 mV, and more preferably 35 to 60 mV.
If the absolute value is too small, aggregates are easily formed. If the absolute value is too large, nonspecific adsorption increases due to electrostatic interaction.
As the zeta potential measuring device, Zetasizer Nano (trade name, manufactured by Malvern), ELS-Z1 (trade name, manufactured by Otsuka Electronics Co., Ltd.), NICOMP 380ZLS (trade name, manufactured by IBC) and the like can be used.

Next, the composite particle colloid of the present invention will be described.
The composite particle colloid of the present invention is obtained by dispersing the composite particles of the present invention in a dispersion medium.
The dispersion medium for the composite particle colloid is not particularly limited as long as it uniformly disperses the composite particles. For example, water, ethanol, PBS (phosphate buffered saline), Tris buffer, HEPES buffer And the like.

Next, the analysis reagent of the present invention will be described.
The analytical reagent of the present invention is achieved by using the composite particle colloid and imparting a label such as fluorescence, light absorption, magnetism, radiation, pH sensitivity to the composite particle contained in the composite particle colloid. . As a method for imparting the label to the composite particles, as described above, silica particles containing the functional compound such as a fluorescent dye compound, a light absorbing compound, a magnetic compound, a radiolabeled compound, and a pH sensitive dye compound are used. Examples include a method for producing the composite particles.
Specific examples of the analysis reagent of the present invention include a biomolecule detection reagent, a biomolecule quantification reagent, a biomolecule separation reagent, a biomolecule recovery reagent, or an immunostaining reagent.
When the composite particles contained in the composite particle colloid of the present invention have a structure composed of silica particles / organic molecules / polyethylene glycol derivatives / biomolecules, the biomolecules or bioactive substances that recognize the biomolecules are targeted. It can be used as an analytical reagent for detecting, quantifying, separating or recovering the target biomolecules or physiologically active substances. When the molecular recognition between the biomolecule and the target biomolecule or physiologically active substance is an antigen-antibody reaction, an immunostaining reagent using the composite particle colloid can be used.
Here, molecular recognition refers to (1) hybridization between DNA molecules or DNA-RNA molecules, (2) antigen-antibody reaction, (3) reaction between enzyme (receptor) and substrate (ligand), etc. A specific interaction between molecules.

Next, the particle surface modification method of the present invention will be described.
In the particle surface modification method of the present invention, the cationic organic molecule, the anionic organic molecule, and the anionic organic molecule are adsorbed on the surface of the particle, followed by the adsorption treatment of the anionic organic molecule. The cationic organic molecules and the anionic organic molecules are superposedly adsorbed on the particle surfaces by alternately performing the adsorption treatment a plurality of times, preferably 2 to 10 times.
In the particle surface modification method of the present invention, the material of the particles is not particularly limited, silica particles, metal particles such as gold, silver and copper, oxide particles such as ferrite and titanium oxide, and semiconductor nanoparticles such as cadmium selenide. Or may be polymer particles such as polystyrene and polymethyl methacrylic acid, but are preferably silica particles obtained by any preparation method such as the sol-gel method described above. Silica particles containing a functional compound obtained according to the method described in Japanese Patent Application No. 2005-376401 are particularly preferred.
The adsorption treatment with the organic molecules is preferably performed by adding and mixing the particle colloid to the organic molecule solution, or adding and mixing the organic molecule solution to the particle colloid. There is no restriction | limiting in particular about the solvent of the solution of the said organic molecule, What is necessary is just a solvent which melt | dissolves the said organic molecule. Further, the mixed dispersion obtained by mixing the organic molecule solution and the particle colloid needs to form a uniform phase.

Whether or not the particles can be adsorbed by the organic molecules can be confirmed by measuring the zeta potential of the particles before and after the treatment and evaluating whether the sign of the zeta potential is reversed.
When the zeta potential of the particle is negative, the first adsorption treatment can be performed by mixing the solution of the cationic organic molecule and the colloid of the particle. After the adsorption treatment, separation of the particles and organic molecules not adsorbed on the particles can be performed by centrifugation or ultrafiltration.
By measuring the zeta potential of the composite particles after washing and confirming that the zeta potential is positively reversed, it can be determined whether or not the organic molecules have been adsorbed. Similarly, in the process of multilayering the organic molecules, it can be confirmed whether or not the adsorption process can be performed based on whether the sign of the zeta potential is reversed.

Next, the manufacturing method of the composite particle of this invention is demonstrated.
The method for producing composite particles according to the present invention includes the step of subjecting the surface of the particles to adsorption treatment of the cationic organic molecules, and further subjecting the anionic organic molecules to adsorption treatment. It is characterized in that the adsorption treatment is alternately carried out a plurality of times, preferably 2 to 10 times, with organic organic molecules.
In the method for producing composite particles of the present invention, the material of the particles is not particularly limited, and silica nanoparticles, metal particles such as gold, silver, and copper, oxide particles such as ferrite and titanium oxide, and semiconductor nanoparticles such as cadmium selenide. Particles may be used, and polymer particles such as polystyrene and polymethyl methacrylic acid may be used, but silica particles obtained by any preparation method such as the sol-gel method described above are preferable, as described above. Silica particles containing a functional compound obtained according to the method described in Japanese Patent Application No. 2005-376401 are particularly preferable.
In the production method of the present invention, it is preferable to mix the colloid of the particles and a solution of organic molecules having a charge opposite to the surface charge of the particles. For example, when silica particles are used, since the surface charge is negative, the solution to be mixed can be a solution of the cationic organic molecule.
Further, the mixing of the colloid of the composite particles composed of the particles and the organic molecules obtained as described above and the organic molecule solution having a charge opposite to the surface charge of the composite particles is repeated one or more times. Thus, the desired composite particles can be produced.

In the method for producing composite particles of the present invention, in order to produce composite particles of particles / organic molecules / polyethylene glycol derivatives, as described above, after producing the composite particles composed of the particles and the organic molecules,
This can be achieved by covalently bonding a polyethylene glycol derivative to the organic molecule with a condensing agent or a crosslinking agent. The condensing agent or crosslinking agent is as described above.
As described above, in the method for producing the composite particle of particle / organic molecule / polyethylene glycol derivative, a colloid of the composite particle composed of the particle and the organic molecule, a solution of the polyethylene glycol derivative, the condensing agent or It is preferable to carry out by mixing with a crosslinking agent. There is no restriction | limiting in particular about the solvent of the solution of the said polyethyleneglycol derivative, What is necessary is just a solvent which melt | dissolves the said polyethyleneglycol derivative.

In the method for producing composite particles of the present invention, in order to produce composite particles of particles / organic molecules / polyethylene glycol derivatives / biomolecules, the composite particles of particles / organic molecules / polyethylene glycol derivatives are used as described above. After manufacturing
Furthermore, this can be achieved by covalently bonding the biomolecule to a functional group at one end of the polyethylene glycol derivative.
As described above, in the method for producing the composite particle of particles / organic molecule / polyethylene glycol derivative / biomolecule, the colloid of the composite particle of particle / organic molecule / polyethylene glycol derivative, the solution of the biomolecule, It is preferably carried out by mixing the condensing agent or the crosslinking agent. The solvent for the biomolecule solution is not particularly limited as long as it is a solvent that dissolves the biomolecule. The condensing agent or crosslinking agent is as described above.

Hereinafter, the present invention will be described in more detail based on examples. The present invention is not limited to these examples. Unless otherwise specified, in the following examples, “part” represents “part by mass”, “%” represents “mass%”, and “molecular weight” represents “weight average molecular weight”.
Reference Example (Preparation of silica particles used for production of composite particles of the present invention)
5.6 mg of DY550-NHS ester (trade name, manufactured by Dynamics GmbH) was dissolved in 1 ml of dimethyl sulfoxide (DMSO). 1.3 μl of APS was added thereto and reacted at room temperature (23 ° C.) for 1 hour.
To 100 μl of the obtained reaction solution, 32 ml of ethanol, 100 μl of TEOS, 7.2 ml of distilled water, and 100 μl of 28% by mass ammonia water were added and reacted at room temperature for 24 hours.
The reaction solution was centrifuged at a gravity acceleration of 22000 × g for 30 minutes, and the supernatant was removed. 1 ml of distilled water was added to and dispersed in the precipitated silica particles, and centrifuged again at a gravitational acceleration of 22000 × g for 30 minutes. This washing operation was further repeated twice to remove unreacted TEOS, ammonia and the like contained in the fluorescent silica particle dispersion, thereby obtaining 24.5 mg of silica particles having an average particle diameter of 64 nm. Yield about 91%.

Example 1
(Method for preparing composite silica particles modified with PAH / PAA)
9 ml of a PAH (polyallylamine hydrochloride, Fluorochem) aqueous solution (1 mg / ml) was placed in a 30 ml beaker. A stir bar was added and mixed well. To this, 1 ml of an aqueous dispersion colloid of silica particles (particle size: 64 nm, 10 mg / ml) prepared in the above Reference Example was slowly added while stirring over 3 minutes.
The obtained dispersion mixture was stirred at room temperature (23 ° C.) for 15 minutes, then centrifuged (20,000 g) for 30 minutes to settle the particles, and the supernatant was immediately removed. The obtained precipitate was re-dispersed in 5 ml of distilled water, and centrifuged (20,000 g) again for 30 minutes to settle the particles. The same operation was repeated three times to remove excess PAH.
Finally, the particles were dispersed in 1 ml of distilled water to obtain composite silica particles modified with PAH as a water-dispersed colloid (yield 10 mg / ml × 1 ml).
1000 μl of distilled water was added to 50 μl of the water-dispersed colloid, and the zeta potential of the composite silica particles modified with the PAH was measured with a zeta potential measuring device (Zeta Sizer Nano, manufactured by Malvern).
FIG. 1 is a graph showing changes in zeta potential due to surface modification.
In FIG. 1, a is a zeta potential of surface untreated silica particles not having PAH before PAH treatment, b is a zeta potential of composite silica particles modified with PAH after PAH treatment, and c is described below. Figure 2 shows the zeta potential of PAH and PAA modified composite silica particles further treated with PAA.
As is clear from FIG. 1, the zeta potential a of the surface untreated silica particles not having PAH before PAH treatment is negative, whereas the zeta potential b of the composite silica particles after PAH treatment is positive. Therefore, it was confirmed that the silica particles had PAH on the surface layer.

Next, a PAA (polyacrylic acid, average molecular weight of about 5,000, manufactured by Wako Pure Chemical Industries, Ltd.) aqueous solution (1 mg / ml) was placed in a 9 ml beaker. A stir bar was added and the aqueous solution was mixed well. Here, 1 ml of the aqueous dispersion colloid of composite silica particles modified with PAH obtained above was slowly added while stirring over 3 minutes.
After stirring the mixed dispersion for 15 minutes at room temperature, excess PAA was removed by centrifugation (20,000 g) in the same manner as in the PAH treatment.
Finally, the particles were dispersed in 500 μl of distilled water to obtain composite silica particles modified with PAH and PAA as a water-dispersed colloid (yield 19 mg / ml × 500 μl).
1000 μl of distilled water was added to 25 μl of the aqueous dispersion colloid, and the zeta potential of the composite silica particles was measured with the zeta potential measuring device.
As is clear from FIG. 1, since the zeta potential c of the composite silica particles further treated with PAA is negative, the composite silica particles are modified with PAH and PAA, which have PAA on the outermost side. It was confirmed to be composite silica particles.

Example 2
(Binding of PEG derivative and avidin)
200 μl of colloid (19 mg / ml) of composite silica particles modified with PAH and PAA treated with PAH and PAA was placed in a microtube. 600 μl of distilled water was added thereto.
200 μl of an aqueous solution containing 10 mM EDC (1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride) and 25 mM Sulfo-NHS (N-hydroxysulfosuccinimide) is added to the colloid of the composite silica particles for 15 minutes. And mixed at room temperature. After mixing for 15 minutes, a PEG derivative (molecular weight 2,000, SUNBRIGHT MEPA-20H, manufactured by NOF Corporation) having one amino group at one end and a methoxy group at the other end is at a concentration of 4 mM. 200 μl of an aqueous solution containing the above-mentioned two kinds of PEG derivatives in which a PEG derivative (molecular weight 2,000, SUNBRIGHT PA-020HC, manufactured by NOF Corporation), which is an amino group and a carboxyl group at the other end, is dissolved at a concentration of 1 mM In addition, mixing was performed at room temperature for 3 hours.
After mixing for 3 hours, centrifugation (20,000 g) was performed for 30 minutes to settle the particles, and the supernatant was immediately removed. The obtained precipitate was redispersed in 1 ml of distilled water, and centrifuged (20,000 g) again for 30 minutes to settle the particles. The same operation was repeated three times to remove excess PEG derivative, EDC and Sulfo-NHS. Finally, the precipitate was dispersed in 800 μl of distilled water.
To the obtained dispersion, 200 μl of an aqueous solution containing 10 mM EDC and 25 mM Sulfo-NHS was added to the composite silica particle colloid, and mixed for 15 minutes at room temperature. After mixing for 15 minutes, 50 μl of 2 mg / ml avidin (trade name, manufactured by Wako Pure Chemical Industries, Ltd.) solution was added and stirred for 3 hours.
After mixing for 3 hours, centrifugation (20,000 g) was performed for 30 minutes to settle the particles, and the supernatant was immediately removed. The obtained precipitate was redispersed in 1 ml of distilled water, and centrifuged (20,000 g) again for 30 minutes to settle the particles. The same operation was repeated 4 times to remove excess avidin. Finally, the particles were dispersed in 1 ml of distilled water to obtain composite silica particles modified with PAH / PAA / PEG derivative / avidin (yield 3.5 mg / ml × 1 ml).

Example 3
(Dispersibility evaluation)
950 μl each of the aqueous dispersion colloid (10 mg / ml) modified with PAH and PAA modified with PAH and PAA, and the untreated surface untreated silica particle (10 mg / ml) with no treatment. On the other hand, 50 μl of 1 mg / ml avidin aqueous solution was added and mixed for 30 minutes. 100 μl of the obtained two types of colloids are added to 900 μl each of different PBS (phosphate buffered saline), and the time change of the particle size is measured by dynamic light scattering method using Zetasizer Nano (trade name, manufactured by Malvern). Measured. The measurement results are shown in FIG.
As shown in FIG. 2, the surface untreated silica particles (e in the figure) that were not treated with PAH and PAA became cloudy immediately after addition to PBS, the particle size increased to about 800 nm, and aggregation occurred. It was confirmed that this was happening. On the other hand, PAH and PAA-modified composite silica particles treated with PAH and PAA (d in the figure) did not increase in particle size even after 3 hours and maintained high dispersibility.

Example 4
(Suppression of non-specific adsorption)
PAH and PAA-modified composite silica particles treated with PAH and PAA and PAH and PAA-treated composite silica particles were each dispersed in distilled water (particle concentration 0). .6 mg / ml). Biotin-4-fluorescein (trade name, manufactured by Ana Spec Inc.) was added to 1 ml of these colloidal silica particle colloids at 4 μg / ml and mixed at room temperature for 6 hours.
After mixing for 6 hours, centrifugation (20,000 g) was performed for 30 minutes to settle the particles, and the supernatant was immediately removed. The obtained precipitate was redispersed in 1 ml of distilled water, and centrifuged (20,000 g) again for 30 minutes to settle the particles. The same operation was repeated 4 times to remove excess biotin-4-fluorescein.
The fluorescence of the obtained particles was measured. The measurement was performed using a fluorescence spectrophotometer FP-6500 (manufactured by JASCO Corporation). The fluorescence intensity derived from silica particles, that is, the emission intensity at 578 nm in the excitation light at 557 nm and the emission intensity from biotin-4-fluorescein, that is, the emission intensity at 520 nm in the excitation light at 490 nm were measured.
In order to estimate the amount of biotin-4-fluorescein bound per unit particle, the emission intensity derived from biotin-4-fluorescein was divided by the fluorescence intensity derived from silica particles, and the obtained value was defined as a labeling parameter. The obtained results are shown in FIG.
As shown in FIG. 3, the labeling parameter of the composite silica particles bound with the PEG derivative is 1/5 of the labeling parameter of the composite silica particles not bound with the PEG derivative, and nonspecific adsorption is suppressed by the binding of the PEG derivative. Confirmed that it was.

FIG. 1 is a graph showing changes in zeta potential due to surface modification. FIG. 2 is a graph showing the change over time of the particle size of the particles in PBS. FIG. 3 is a graph showing the label parameter.

Claims (30)

  A composite particle composed of silica particles and organic molecules, in which organic molecules are adsorbed to the surface of the silica particles by electrostatic attraction.   Silica particles / organic molecules / polyethylene glycol derivatives in which organic molecules are adsorbed on the surface of silica particles by electrostatic attraction, and polyethylene glycol derivatives are covalently bonded to the organic molecules and located outside the organic molecules. Composite particles.   An organic molecule is adsorbed to the surface of the silica particle by electrostatic attraction, and a polyethylene glycol derivative is covalently bonded to the organic molecule at one end of the derivative, located outside the organic molecule, and the polyethylene glycol. A composite particle of silica particle / organic molecule / polyethylene glycol derivative / biomolecule, wherein a biomolecule is covalently bonded to the other end of the derivative.   The organic molecules adsorbed on the surface of the silica particles are a cationic organic molecule and an anionic organic molecule, the innermost cationic organic molecule is adsorbed on the silica particle, and the anionic organic molecule further is the cationic organic molecule. A structure that is adsorbed to a molecule and located outside the cationic organic molecule, or a structure that the anionic organic molecule is adsorbed to the cationic organic molecule and located outside the cationic organic molecule is repeated a plurality of times. The composite particle according to any one of claims 1 to 3, which has a structure in which the anionic organic molecule is located on the outermost side.   The composite particle according to claim 4, wherein the cationic organic molecule and the anionic organic molecule each form an organic molecular layer.   The composite particle according to claim 4 or 5, wherein the polyethylene glycol derivative is covalently bonded to the outermost anionic organic molecule.   The anionic organic molecule is any one of polyacrylic acid, alginic acid, polystyrene sulfonic acid, citric acid, glutamic acid, aspartic acid, polyglutamic acid, polyaspartic acid, or a polypeptide containing at least one of glutamic acid or aspartic acid. The composite particle according to claim 6, wherein the cationic organic molecule is any one of polyallylamine hydrochloride, chitosan, polylysine, and lysine.   The anionic organic molecules are bonded via a covalent bond, the cationic organic molecules are bonded via a covalent bond, and / or the anionic organic molecule and the cationic organic molecule are The composite particle according to claim 6 or 7, which is partially bonded through a covalent bond.   The polyethylene glycol derivative has a terminal functional group covalently bonded to the organic molecule, which is a hydroxyl group, an amino group, a carboxyl group, a thiol group, a maleimide group, or a succinimidyl ester group, and the polyethylene glycol derivative is The composite according to any one of claims 4 to 8, wherein the functional group at the other end has any of a carboxyl group, a thiol group, a methoxy group, a hydroxyl group, an amino group, a maleimide group, or a succinimidyl ester group. particle.   The composite particle according to any one of claims 3 to 9, wherein the biomolecule is an antigen, antibody, DNA, RNA, sugar, sugar chain, ligand, receptor, peptide, or chemical substance. .   The composite particle according to any one of claims 1 to 10, wherein an average particle diameter of the silica particles is 1 nm to 1 µm.   The composite particle according to any one of claims 1 to 11, wherein the molecular weight of the organic molecule is 50 to 100,000.   The composite particle according to any one of claims 1 to 12, wherein an absolute value of ζ potential in pure water at pH 7 is 1 to 60 mV.   The composite particle according to any one of claims 1 to 13, wherein an absolute value of ζ potential in pure water having a pH of 7 is 35 to 60 mV.   The composite particle according to any one of claims 1 to 14, wherein an average particle diameter of the silica particles is 1 nm to 1 µm, and a molecular weight of the organic molecule is 50 to 100,000.   Furthermore, the composite particle of Claim 15 whose absolute value of ζ potential in the pure water of pH 7 is 1-60 mV.   Furthermore, the composite particle of Claim 16 whose absolute value of zeta potential in the pure water of pH7 is 35-60mV.   A composite particle colloid in which the composite particles according to any one of claims 1 to 17 are dispersed in a dispersion medium.   The composite particle colloid according to claim 18, wherein the dispersion medium is a buffer solution.   An analytical reagent comprising the composite particle colloid according to claim 18 or 19.   The adsorption treatment of the cationic organic molecules on the surface of the particles, followed by the adsorption treatment of the anionic organic molecules, and the adsorption treatment of the cationic organic molecules and the anionic organic molecules alternately, a plurality of times. A particle surface modification method characterized in that a plurality of organic molecules are superposedly adsorbed on the particle surface by performing the step.   The particle surface modification method according to claim 21, wherein the particles are silica particles.   The adsorption treatment of the cationic organic molecules on the surface of the particles, followed by the adsorption treatment of the anionic organic molecules, and the adsorption treatment of the cationic organic molecules and the anionic organic molecules alternately, a plurality of times. A method for producing composite particles composed of particles and organic molecules.   The method for producing composite particles according to claim 23, wherein a colloid of the particles and a solution of organic molecules having a charge opposite to the surface charge of the particles are mixed.   25. The colloid of the composite particles comprising the particles and the organic molecules, and a solution of organic molecules having a charge opposite to the surface charge of the composite particles are mixed. A method for producing composite particles. After producing the composite particles composed of the particles and the organic molecules by the production method according to any one of claims 23 to 25,
A method for producing particle / organic molecule / polyethylene glycol derivative composite particles, wherein a polyethylene glycol derivative is covalently bonded to an organic molecule located on the outermost side of the composite particles by a condensing agent or a crosslinking agent.   27. The method for producing composite particles according to claim 26, wherein a colloid of the composite particles composed of the particles and the organic molecules, a solution of the polyethylene glycol derivative, and the condensing agent or a crosslinking agent are mixed. . After producing the composite particles of the particles / organic molecules / polyethylene glycol derivative by the production method according to claim 26,
Furthermore, a method for producing a particle / organic molecule / polyethylene glycol derivative / biomolecule composite particle, wherein a biomolecule is covalently bonded to one end of the polyethylene glycol derivative.   The covalent bond of the biomolecule to one end of the polyethylene glycol derivative comprises a colloid of the composite particle of the particle / organic molecule / polyethylene glycol derivative, a solution of the biomolecule, and the condensing agent or cross-linking agent. It is performed by mixing, The manufacturing method of the composite particle of Claim 28 characterized by the above-mentioned.   The method for producing composite particles according to any one of claims 23 to 29, wherein the particles are silica particles.

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