JP2011242387A - Composite fine particle for capturing or separating biological substance - Google Patents

Abstract

An object of the present invention is to provide capture microbeads that are inexpensive and can be used for analysis / separation with improved mechanical resistance to centrifugation and the like.
A gold layer-coated fine particle in which a gold layer adheres directly to a fine particle having a resin surface or via an organic binder layer, and a ligand that is linked to the gold layer and can specifically bind to a biological substance. The above-mentioned problems are solved by composite microparticles for capturing or separating a biological substance characterized by comprising:
[Selection figure] None

Description

  The present invention relates to composite microparticles for capturing or separating biological substances and a separation or detection method using the same.

Currently, micro-sized resin beads are widely used for analysis and separation, but most of them are functionalized on the resin surface with carboxyl groups or amino groups.
In order to modify the resin surface with a ligand such as an antibody, as shown in FIG. 1, the carboxyl group on the particle surface is converted to an activated ester with a carbodiimide coupling agent (1) and a succinimide derivative (2); hydrazine or a derivative thereof Via a multi-step chemical reaction such as amidation (hydrazide) by (3); and reductive amination in the presence of a reducing agent (5) to bind to the ligand (4) introduced with an aldehyde group. There is a need to.
In such a method, there are many problems in the introduction efficiency and introduction density of the ligand.

Gold microparticles can be used as the microbeads, but the ligand can be bound to the gold surface in a single step by thiolation of the ligand.
However, the gold fine particles themselves are very expensive.

  In addition, microbeads whose particle surfaces are gold-plated are known in the field of electronic materials. However, since the gold-plated microbead has a nickel base plating, the plating layer is rigid, and the plating layer is peeled off by centrifugation frequently used for analysis and separation (FIG. 5). For this reason, it is difficult to use gold-plated microbeads having a nickel plating layer as a base for analysis and separation.

  Accordingly, there has been a demand for the development of capture or separation microbeads that are inexpensive and can be used for analysis / separation with improved mechanical resistance to centrifugation and the like.

  According to the present invention, a gold layer-coated fine particle in which a gold layer is adhered to a fine particle having a resin surface directly or via an organic binder layer, and can be specifically bonded to a biological substance connected to the gold layer. Provided is a composite microparticle for capturing or separating a biological substance comprising a ligand.

  According to the present invention, the biological material in the sample is captured by the composite fine particles, and the composite fine particles are separated from the sample by centrifugation or magnetic separation. A separation method is provided.

  Furthermore, according to the present invention, the biological substance in the sample is captured by the composite microparticle, and the presence of the biological substance captured by the composite microparticle is specifically bound to the biological substance. There is provided a method for detecting a biological substance in a sample, characterized in that the detection is performed using a ligand.

  According to the present invention, the biological material in the sample is captured in the composite fine particles, the composite fine particles are accumulated on the working electrode by magnetic force, and the presence of the biological material captured in the composite fine particles is electrochemically detected. A method for detecting a biological substance in a sample is provided.

According to the composite particles for capture or separation of the present invention, it is possible to greatly simplify the introduction of the ligand, increase the efficiency, increase the density, and reduce the cost.
In addition, the composite fine particles for capture or separation of the present invention have improved mechanical resistance than conventional gold-plated resin particles and can be used in the same manner as gold fine particles, but can be manufactured at a lower cost than gold fine particles.

A conventional method for introducing a ligand onto the surface of a resin particle will be described. It is a scanning electron microscope (SEM) photograph of the resin particle used for preparation of gold layer coating resin fine particles. It is a SEM photograph after coat | covering the gold nanoparticle on the resin particle of FIG. It is a SEM photograph of gold layer coating resin fine particles. It is a microscope picture which shows the state after centrifugation of the gold plating micro bead which has a nickel base plating layer. It is a microscope picture which shows the state after centrifugation of gold layer coating | cover resin fine particle. 2 is an SEM photograph of magnetic beads used for the production of gold layer-coated magnetic resin fine particles 1. It is a SEM photograph after coat | covering a gold nanoparticle on the magnetic bead of FIG. It is a SEM photograph of gold layer covering magnetic resin fine particles. The general procedure of a DNA capture experiment using the composite microparticles of the present invention is shown. The result of the DNA capture experiment using the composite microparticles of the present invention is shown. The horizontal axis represents wavelength (nm) and the vertical axis represents absorbance. 2 is a particle size distribution (a) and a TEM photograph (b) of positively charged gold nanoparticles used in Example 2. FIG. In the graph of (a), the horizontal axis represents the particle diameter (nm), and the vertical axis represents the number of particles having the corresponding particle diameter as a ratio (%) to the total number of particles. 4 is a SEM photograph of gold nanoparticle-coated magnetic resin particles (a) and gold layer-coated magnetic resin particles (b) produced in Example 2. 4 is a cross-sectional TEM photograph of gold nanoparticle-coated magnetic resin particles (a) and gold layer-coated magnetic resin fine particles (b) produced in Example 2. FIG. “AuNP” represents gold nanoparticles, and “plastic” represents resin fine particles. 4 is a fluorescence micrograph of a dispersion of biotin-introduced gold layer-coated magnetic resin fine particles prepared in Example 2. FIG. a and b are a bright field image and a fluorescence field image, respectively, when the dispersion is observed alone. c and d are a bright field image and a fluorescence field image, respectively, when the dispersion liquid to which fluorescein-labeled streptavidin is added is observed. An outline procedure for producing one embodiment of a magnetic electrode that can be used in the detection method of the present invention will be described. It is the cyclic voltammogram in the sulfuric acid solution of the magnetic electrode (Au / Fe electrode) and gold electrode (Au electrode) which were produced in the Example. The horizontal axis represents the potential (V) with respect to the reference electrode (Ag | AgCl), and the vertical axis represents the current density (μA / mm ² ). It is the cyclic voltammogram of potassium ferrocyanide in the PBS buffer solution measured with the magnetic electrode (Au / Fe electrode) produced in the Example, and the gold electrode (Au electrode). The horizontal axis represents the potential (V) with respect to the reference electrode (Ag | AgCl), and the vertical axis represents the current density (μA / mm ² ). The increase in the electrode area due to the accumulation of the gold layer-coated magnetic resin fine particles on the electrode surface (and the periphery thereof) of the magnetic electrode (a) will be described (b). (a) Cyclic voltammogram of potassium ferrocyanide in a PBS buffer solution measured with a magnetic electrode in which gold layer-coated magnetic resin particles are accumulated on the electrode surface by an external magnetic force. The horizontal axis represents the potential (V) with respect to the reference electrode (Ag | AgCl), and the vertical axis represents the current (μA). (b) It is a figure which shows the relationship between gold layer coating | coated magnetic resin particle fixed amount, and oxidation current peak value ((DELTA) _Ipeak ). The horizontal axis represents the fixed amount of particles in μg, and the vertical axis represents the oxidation current peak value in μA. It is a cyclic voltammogram of fluorescein in a PBS buffer (pH 7.4) solution measured by (a) a magnetic electrode and (b) a magnetic electrode in which gold layer-coated magnetic resin particles are accumulated on the electrode surface by an external magnetic force. In either case, the horizontal axis represents the potential (V) with respect to the reference electrode (Ag | AgCl), and the vertical axis represents the current (μA). It is the schematic explaining the procedure of the detection method of this invention. It is a figure explaining the reduction reaction of hydrogen peroxide catalyzed by HRP. It is a cyclic voltammogram in a 250 μM H ₂ O ₂ / PBS buffer (pH 7.4) solution measured with a magnetic electrode (a) and a magnetic electrode (b, c) having biotin immobilized on the surface. The biotin-fixed magnetic electrode was used by binding HRP-labeled streptavidin to biotin (b) or without binding (c). The horizontal axis represents the potential (V) with respect to the reference electrode (Ag | AgCl), and the vertical axis represents the current (nA). It is a cyclic voltammogram in a 250 μM H ₂ O ₂ / PBS buffer (pH 7.4) solution measured with a magnetic electrode in which gold layer-coated magnetic resin fine particles are accumulated on the electrode surface by an external magnetic force. (a) Gold layer-coated magnetic resin particles in which biotin was introduced into the gold layer were used as the gold layer-coated magnetic resin particles, and measurement was performed by binding HRP-labeled streptavidin to biotin. (b) Measurement was performed using biotin-introduced gold layer-coated magnetic resin particles without binding HRP-labeled streptavidin to biotin. (c) Biotin non-introduced gold layer coated magnetic resin particles were used. The horizontal axis represents the potential (V) with respect to the reference electrode (Ag | AgCl), and the vertical axis represents the current (μA). Biotin-introduced gold layer-coated magnetic resin particles in which biotin is bound with streptavidin labeled with fluorescein (a), rhodamine (b), and HRP (c), respectively, and biotin-introduced gold layer-coated magnetic resin in which none is bound FIG. 5 is a cyclic voltammogram in 250 μM H ₂ O ₂ / PBS buffer (pH 7.4) measured with a magnetic electrode in which fine particles (d) are accumulated by an external magnetic force.

<Composite fine particles of the present invention>
The capture or separation composite fine particles according to the present invention (hereinafter, also simply referred to as “composite fine particles”) include a gold layer-coated fine particle in which a gold layer is adhered to fine particles having a resin surface directly or via an organic binder layer, It consists of a ligand that is linked to the layer and can specifically bind to the capture or separation object.
In other words, the gold-coated fine particles include fine particles having a resin surface and a gold layer that covers the resin surface directly or via an organic binder layer.

  The resin surface may be the surface of resin fine particles obtained by atomizing a resin, or the surface of the resin layer of resin-coated fine particles in which core fine particles are coated with a resin layer.

  As the resin constituting the resin surface, any resin that can be used for resin particles or resin-coated particles in this field can be used. Examples include (co) polymers of styrene, (meth) acrylic acid, olefins, amides, imides and derivatives thereof, and crosslinked products thereof. More specific examples include polystyrene (PS), polypeptide methyl methacrylate (PMMA), polypentaerythritol tetraacrylate, polytrimethylolpropane triacrylate, nylon, polyolefin and copolymers and cross-linked products thereof, acrylic resin And phenol resin. In addition, examples of the resin containing a “site capable of interacting with gold” described later include polylysine, polyallylamine, polyacrylic acid, polyacrylamine, polyamide, polyimide, polyvinylpyridine, urea resin, polyurethane, polysulfide, and the like. It is done.

  Any method that can be used in the art, such as monomer casting, suspension polymerization, melt spin coating, ultracentrifugation, and ultrasonic waves, can be used as a method for making the resin into fine particles, and is appropriately selected depending on the type of the resin.

As the core fine particles, any known fine particles that can be used as core particles of resin-coated particles in the art can be used. Examples include those made of magnetic materials (for example, various ferrites such as magnetite, iron, etc.).
The resin layer on the core fine particles can be formed by a known method. For example, it can be formed by applying a coating solution in which a resin is dissolved or dispersed in an appropriate solvent to the surface of the core fine particles (by a dipping method or the like), followed by drying and curing.

  The resin fine particles or the resin layer (when the core fine particles are not composed of a magnetic material) may contain the magnetic material as described above so that it is preferably uniformly distributed.

  The average particle diameter (volume average particle diameter) of the fine particles having a resin surface can be nano-order to micro-order, for example, 1 nm to 100 μm, preferably 10 nm to 100 μm, more preferably 1 μm to 10 μm.

The gold layer is provided directly covering the resin surface. Therefore, there is no other layer between the resin surface and the gold layer, except for an “organic binder” layer described later.
The gold layer covering the resin surface is preferably formed on the resin surface by first fixing gold nanoparticles and then precipitating gold.

  The gold nanoparticles can be fixed on the resin surface by mixing fine particles having the resin surface in a gold nanoparticle dispersion (gold colloid solution) and stirring or standing. The gold nanoparticle dispersion may optionally contain an organic binder (see below). The fixed reaction temperature may be any temperature as long as the dispersion does not completely freeze or evaporate during the reaction period, but is preferably near room temperature (for example, 10 to 35 ° C.). The fixation reaction time depends on the temperature, but can be, for example, 2 hours to 48 hours (2 days) at room temperature.

The number average particle diameter of the gold nanoparticles is sub-nano order to nano order (about 0.1 to 1000 nm), for example, 0.1 to 500 nm, preferably 0.1 to 100 nm, more preferably 1 to 100 nm, and more preferably Can be 5-50 nm.
The average particle diameter of the gold nanoparticles is, for example, 1 / 100,000 to 1/10, preferably 5 / 100,000 to 1/10, more preferably 5/10000 to 5/100, of the average particle diameter of the fine particles having a resin surface. possible.

As the gold nanoparticle dispersion liquid, a commercially available product may be used, but it can be easily produced by an in-solution reduction reaction using a gold ion (gold complex ion) -containing solution and a reducing agent. For example, it can be obtained by adding citric acid to a chloroauric acid solution and heating.
When electrostatic interactions are used to immobilize gold nanoparticles on the resin surface, the gold nanoparticles are manufactured to have a positive or negative charge. In this case, an organic binder described later having a group capable of electrostatically interacting with the resin surface (group) is added to the gold ion (gold complex ion) -containing solution and stirred, and then a reducing agent is added and further stirred. Thus, a dispersion of gold nanoparticles having a positive charge or a negative charge can be obtained.
The zeta potential of the gold nanoparticles having a positive charge or a negative charge is arbitrary, but may be, for example, +/− 5 to +/− 50 mV.

  The fixation of the gold nanoparticles on the resin surface can be via a site that can interact with the gold present on the resin surface. Here, “interaction” refers to chemical bond, van der Waals force, electrostatic interaction, hydrophobic interaction, adsorption force and the like.

  Examples of the site (group) (X) that can interact with gold include, for example, a thiol group, a sulfide group (—S—), a disulfide group (—S—S—), an amino group, an imino group (—N═), Examples thereof include a carboxyl group, a carbonyl group, a sulfonyl group, and a phosphoryl group. Among these, a thiol group, a disulfide group, an amino group, and an imino group are preferable, and a thiol group and a disulfide group are more preferable.

A part capable of interacting with gold may be present on the surface of the resin as a part of the resin, or may be included in the organic binder.
Here, the “organic binder” is an organic compound having a part capable of interacting with gold together with a part capable of interacting with a resin constituting the resin surface (or a group present on the resin surface).

Examples of the site (Y) that can interact with the resin include an alkyl group (for example, 1 to 10, preferably 2 to 8 carbon atoms), an aromatic ring (for example, a benzene ring), a heterocyclic ring, or a derivative thereof (phenol). , Amino group, carboxyl group, sulfo group and hydroxyl group.
Specific examples of the alkyl group and derivatives thereof are — (CH ₂ ) _n CH ₃ , — (CH ₂ ) _m (CH) _n CH ₃ , — (CH ₂ ) _n COOH, — (CH ₂ ) _n OH ( here, the hydrogen on the carbon may be substituted by C ₁ -C ₄ alkyl group).
Specific examples of the aromatic ring and derivatives thereof include a benzene ring, a 5- to 7-membered heteroaromatic ring (1 or 2 heteroatoms are selected from N, S, and O) and 2 or 3 condensed rings thereof. it is (wherein, hydrogen rings C ₁ -C ₄ alkyl group, a carboxyl group, a hydroxyl group, may be substituted with an amino group).

In the organic binder, the part capable of interacting with gold and the part capable of interacting with the resin constituting the resin surface (or a group present on the resin surface) may be directly bonded or a saturated hydrocarbon chain. (— (CH ₂ ) _p —; p may be an integer, for example, 0 to 20, more preferably 2 to 10) may be bonded via a spacer (Z).
That is, if X is a monovalent group represented by a thiol group, an organic binder has the general formula X-Z _q -Y _(q is 0 or 1) can expressed as.

Organic binders include, for example, alkanethiol (CH ₃ — (CH ₂ ) _n —SH), carboxyalkanethiol (HOOC— (CH ₂ ) _{n + 1} —SH), hydroxyalkanethiol (HO— (CH ₂ ) _{n + 1} -SH), which may be an amino alkanethiol _{(H 3 N- (CH 2)} n + 1 -SH) ( wherein, n represents an integer, preferably 0 to 20, more preferably 1 to 10, more preferably is 2-5. hydrogen on carbon may be substituted by C ₁ -C ₄ alkyl group).
The organic binder can also be, for example, Y ¹ — (CH ₂ ) _p —SH (where Y ¹ is an aromatic ring and derivatives thereof, p is an integer, preferably 1-8).
The organic binder is also, for example, (—S— (CH ₂ ) _m —OH) ₂ , (—S— (CH ₂ ) _m —COOH) ₂ (where n is an integer, preferably 0 to 20, more preferably 2-10. hydrogen on carbon may be substituted by C ₁ -C ₄ alkyl group).
Organic binders also include, for example, dithia (C ₄ -C ₈ ) cycloalkane- (CH ₂ ) _n -CH ₃ , dithia (C ₄ -C ₈ ) cycloalkane- (CH ₂ ) _{n + 1} -COOH, dithia ( C ₄ -C ₈₎ cycloalkane _{- (CH 2) n + 1} -OH, dithia (C ₄ ~C ₈₎ cycloalkane _{- (CH 2) n + 1} -NH 2, dithia (C ₄ ~C ₈₎ cycloalkyl Alkane- (CH ₂ ) _q -Y ¹ (wherein two S in the cycloalkane form a disulfide, n is an integer, preferably 0-20, more preferably 1-10, more preferably is 2 to 5, Y ¹ is an aromatic ring and derivatives thereof, p is an integer, preferably 1 to 10).

Specific examples of the organic binder include thioctic acid, aminophenyl disulfide, allyl mercaptan, propanethiol, butanethiol, p-aminothiophenol, amyl mercaptan, 2-aminoethanethiol and the like.
The organic binder may be present in the gold nanoparticle dispersion in a free state during the fixing reaction, or may be previously attached to the gold nanoparticles via a group capable of interacting with gold.
The resin surface may be activated with, for example, a carboxyl group, an amino group, an epoxy group, or a tosyl group before the fixing reaction.

The electrical resistance of the fine particles to which the gold nanoparticles are fixed may be, for example, 10 ³ to 10 ⁹ Ω, more preferably 10 ³ to 10 ⁵ Ω.
In the present invention, the electric resistance of the fine particles (gold nanoparticle fixed or metal-coated) is about 1 × 1 cm between a platinum plate and a tungsten needle (tip diameter 5 μm; manufactured by Suruga Seiki Co., Ltd., M904-0017). The electrical resistance is measured with a digital multimeter (model 34970A, manufactured by Agilent, applied current: 1 mA) in a compressed state of 20%. The electric resistance value is obtained as an average value of measured values of 10 fine particles.

  Gold can be deposited on fine particles on which gold nanoparticles are fixed by adding the fine particles and a reducing agent to a solution of a gold compound or a gold complex compound and stirring or allowing to stand. The precipitation reaction temperature may be any temperature as long as the dispersion does not completely freeze or evaporate during the reaction period, and may be, for example, 10 to 80 ° C. The deposition reaction time depends on the thickness and temperature of the gold layer to be obtained, but can be, for example, 30 minutes to 24 hours.

Examples of the gold compound or the gold complex compound include chloroauric acid (tetrachloroauric acid), sodium chloroaurate, diethylamineauric acid trichloride, gold bromide, gold hydroxide, and gold oxide.
The reducing agent is not particularly limited as long as it is a substance that can reduce gold ions or gold complex ions to simple gold. For example, citric acid, sodium citrate, sodium ascorbate, sodium borohydride, methanol, hydrogen peroxide water and the like can be used.

  In order to prevent aggregation between the fine particles, it is preferable to add a dispersant to the solution of the gold compound or the gold complex compound. Examples of the dispersant include alcohols such as methanol, ethanol, and polyvinyl alcohol, polyvinyl pyrrolidone, and polyethylene glycol.

The thickness of the gold layer may be any thickness, but may be, for example, about 3 times the average particle diameter of the gold nanoparticles used.
The electric resistance of the obtained gold layer-coated fine particles can be, for example, 0.01 to 100Ω, preferably 0.01 to 10Ω.

“Ligand” refers to one of a pair of substances that specifically bind to each other under a specific condition (“specific binding pair”), and is also referred to as “specific binding partner”.
Specific binding pairs include antibodies and antigens, complementary nucleic acid molecules, enzymes and substrates, aptamers and their target proteins or polypeptides, lectins and sugar chains, biotin and avidin, metal ions and proteins or peptides (e.g., copper ions This bond is used for protein quantification by the Biuret method, and nickel ions are used for purification of fusion proteins having a histidine tag such as (His) ₆ . It has been known.

The ligand used in the present invention can be appropriately selected from known specific binding pairs according to the target to be captured or separated using the composite microparticles, such as antibodies, antigens, nucleic acid probes, Aptamers, lectins (eg, concanavalin A, kidney bean lectin (PHA)), glutathione, avidin, streptavidin, biotin. The ligand can be fixed to the gold layer-coated fine particles through an adsorption reaction of the composite fine particles on the coated gold layer or through thiolation of the ligand.
The capture or separation object may consist of biological material, for example. “Biological substances” include proteins, nucleic acids, lipids (including phospholipids), and carbohydrates (sugar chains). “Nucleic acid” includes artificial nucleic acids (eg, PNA, LNA) having specific binding properties to DNA or RNA having complementary base sequences in addition to DNA and RNA.

  Metal ions are immobilized on the surface of the gold layer-coated fine particles by thiolated metal ligands, and can be used for separation and purification through binding of the metal and sample protein or peptide. On the other hand, the capture material further includes obtaining a different capture function using other specific reactions. As one example, DNA and RNA are biotinylated and captured by avidin / thiolated biotin-modified composite microparticles, and used for applications such as purification of DNA and RNA binding proteins. Similarly, it is also included that the antigen or antibody is biotinylated and captured in composite microparticles and used for applications such as separation of microorganisms such as fungi, cell separation, protein purification, immunoassay, and immunoprecipitation.

  For example, when the capture or separation target is a protein or polypeptide, the ligand used in the composite microparticle of the present invention can be an antibody or an aptamer. Further, when the capture or separation target is a nucleic acid, the ligand can be a nucleic acid probe, and when the capture or separation target is a sugar, the ligand can be a lectin. Furthermore, when the capture or separation object is bound to glutathione S-transferase (GST), biotin or (strept) avidin, the ligand can be glutathione, (strept) avidin or biotin, respectively.

In the present invention, antibodies include polyclonal antibodies, monoclonal antibodies, and fragments thereof that retain specific binding activity (for example, single-chain antibodies, F (ab ′) ₂ , Fab, Fab ′, or Fv). To do. The antibody or fragment thereof is preferably monoclonal.

  In the present invention, the nucleic acid probe has a form as long as a single-stranded region (for example, 10 bases or more, preferably 15 bases or more, more preferably 20 bases or more) that can specifically hybridize with a complementary strand exists. Regardless, a part may be double-stranded.

Any known method that can be used in the art can be used for linking the ligand to the surface of the gold layer.
For example, by immersing the gold layer-coated fine particles in a solution containing a ligand (for example, a buffer solution), the two can be linked.

  In addition, for example, a method may be used in which molecules having a thiol group, a sulfide group, or a disulfide group are linked using the property of forming a self-assembled monolayer (SAM) on the gold surface. This method can be easily carried out by immersing the gold layer-coated fine particles in a solution containing a ligand into which the above group has been introduced in advance. Methods for introducing thiol groups, sulfide groups, or disulfide groups into polypeptides, proteins, or nucleic acid probes are known in the art, and for example, alkane thiols and commercially available reagents (eg, SAM reagents) can be used.

The composite microparticles of the present invention may be treated with any blocking agent capable of blocking non-specific binding such as bovine serum albumin (BSA) or skim milk powder after ligation of the ligand.
Methods for masking non-specific binding regions with blocking agents are known in the art. For example, it can be performed by incubating the composite microparticles of the present invention in a buffer containing a blocking agent.

<Separation method of the present invention>
The method for separating an object in a sample according to the present invention is characterized in that the object in the sample is captured by the composite fine particles according to the present invention described above, and the composite fine particles are separated from the sample by centrifugation or magnetic separation. And

  The sample that can be used in the separation method of the present invention is not particularly limited as long as it is a sample that may contain an object to be separated. The sample can be, for example, a biological sample from animals including humans and livestock animals such as cows, horses, pigs, goats, sheep, chickens, rats, mice. Examples of biological samples include blood (including serum and plasma), lymph, cerebrospinal fluid, ascites, tissue exudate or secretion (including tears, sweat, saliva), body fluids such as sputum and urine; brain And tissues such as spinal cord, heart, liver, mucous membrane (homogenates, lysates or extracts thereof); cells (including lysates and extracts thereof); culture supernatants of cells or tissues, and the like. In the present invention, the “sample” includes the diluted product.

  The sample may be pretreated for removal of components other than the object (including decomposition) or amplification of the object, if necessary. Examples of the pretreatment include filtration, enzyme treatment, and PCR amplification.

  As the composite fine particles that can be used in the separation method of the present invention, those having a substance that specifically binds to the separation target as a ligand are selected.

  Capture of an object that may be present in a sample with the composite microparticles is performed by combining the composite microparticles with the sample or a dilution thereof under conditions that allow the ligand in the composite microparticles to specifically bind to the target. It can be easily achieved by mixing. Methods for determining specific binding conditions are known in the art.

  Separation of the composite fine particles from the sample can be performed, for example, by centrifugation. When a magnetic material is used in the composite fine particles (for example, as the core fine particles or in the resin layer), the separation can also be performed by magnetic force.

  The separation method of the present invention can be used for purification of an object. For example, when a recombinant polypeptide or protein is expressed as a GST-fusion, the recombinant expression product can be purified from the culture supernatant or cell lysate using composite microparticles containing glutathione as a ligand. Also, for example, when PCR amplification is performed on a target nucleic acid using a biotin or (strept) avidin-bound primer, the target nucleic acid is purified using a composite microparticle containing (strept) avidin or biotin as a ligand. Can do.

  The separation method of the present invention can also be implemented as part of a method for detecting or measuring an object.

<First Detection Method of the Present Invention>
In the method for detecting an object in a sample according to the present invention, the object in the sample is captured by the composite microparticle according to the present invention described above, and the presence of the object captured by the composite microparticle is detected in the object. Detection is performed using another ligand that specifically binds.

  The sample that can be used in the detection method of the present invention is not particularly limited as long as it is a sample that may contain a detection target, and is the same as that described for the separation method of the present invention.

  In the detection method of the present invention, the composite fine particles are appropriately selected to have a substance that specifically binds to the detection target as a ligand (hereinafter also referred to as “first ligand” for convenience). Preferably, the first ligand can be an antibody, an antigen, a nucleic acid probe, or an aptamer.

Capturing the detection target that may be present in the sample with the composite microparticle includes, for example, the sample and the composite microparticle under conditions sufficient to capture the detection target that may be present in the sample by the composite microparticle (for example, time and It can be carried out by contact under temperature). Such a method of setting conditions is known in the art, and can be performed, for example, by a preliminary reaction test.
This contact may be performed in a stationary state or in a shaking state.

  After contact, if necessary, the composite fine particles may be separated from the sample by the separation method according to the present invention described above (or may be concentrated without being completely separated), and / or washed. May be. By performing separation or concentration and / or washing, the S / N ratio at the time of detection is increased, and the detection accuracy (detection sensitivity) can be improved.

  Another ligand (hereinafter also referred to as “second ligand” for convenience) is the same type of ligand as the first ligand in the composite microparticle according to the present invention as long as it specifically binds to the detection (capture) target. Alternatively, it may be a heterogeneous ligand. That is, when the first ligand is an antibody, the second ligand may be an antibody or an aptamer (which can of course be a ligand other than an aptamer). However, the first ligand and the second ligand must not compete for binding to the detection (capture) object.

The second ligand can be appropriately selected from known specific binding pairs according to the detection (capture) target and the first ligand.
Such a method of using two ligands for one detection (capture) object is known in the art as a sandwich method.

  In the sandwich method, preferably the second ligand has a label for detection. The label is known in the art and is appropriately selected depending on the detection method used. The label can be detected optically, chemically, radioactively, magnetically or electrically. The label is selected from the group consisting of, for example, a fluorescent dye, an enzyme, an absorbing dye, a chemiluminescent group, a radioisotope, a spin label, and an electrochemical label.

Detection of the presence of an object captured in a composite, microparticle using a second ligand, for example,
The second ligand and the composite microparticle are bound to the second ligand to be detected (which is captured by the composite microparticle (the first ligand therein) if present in the sample) Under sufficient conditions,
The unbound second ligand is removed, for example by washing,
It can be done by detecting the presence of the second ligand.

A method for setting contact conditions (for example, time and temperature) is known in the art, and can be performed, for example, by a preliminary test. The contact may be performed in a stationary state or in a shaking state.
The presence of the second ligand can be detected, for example, by detecting a signal from the label. For detection, the signal from the label may be measured quantitatively.

  The detection method of the present invention can be used in combination with an immunoassay (enzyme-linked immunosorbent assay (ELISA) method, fluorescent immunoassay method, radioimmunoassay method, etc.).

<Second Detection Method of the Present Invention>
In the second detection method of the present invention, a detection target (biological substance) in a sample is captured by the composite fine particles according to the present invention described above, which have magnetism, and the composite fine particles act by magnetic force. The method is characterized by electrochemically detecting the presence of an object that is accumulated on an electrode and captured by the accumulated composite fine particles.

  Samples, composite microparticles and ligands that can be used in the second detection method of the present invention are as described in the “Separation method of the present invention” and “First detection method of the present invention” section. In addition, the capture of the detection target that may be present in the sample by the composite fine particles is also as described above. However, composite fine particles having magnetic properties are used.

After being captured by the composite fine particles, the composite fine particles are accumulated on the electrode (working electrode in the electrochemical detection method) by an external magnetic force. The magnetic force used for the accumulation can be determined as appropriate so as not to affect the redox current obtained during the electrochemical measurement, while sufficient to maintain the accumulation state of the composite fine particles.
Since the composite fine particles according to the present invention are conductive (the surface is covered with a gold layer), they may be integrated without being limited by the electrode area of the working electrode itself (that is, three-dimensionally).

In order to ensure that substances that may be present in the sample and have unintended effects on the electrochemical detection are reliably excluded before the accumulation, preferably the composite microparticles are separated by the separation method according to the invention described above. Separate and collect from the sample (may be concentrated without being completely separated).
When it is clear that the sample used does not contain such a substance, the separation / recovery step can be omitted.

As the working electrode, a magnetic electrode capable of accumulating magnetic particles on the surface (particularly the electrode surface) by applying an external magnetic force is used. The magnetic electrode body is made of, for example, a rod-like magnetic / conductive material (e.g., a magnetic metal such as iron), but a non-magnetic / non-conductive material (e.g., leaving a predetermined region to define an effective area as an electrode) , Teflon (registered trademark), PEEK resin, epoxy resin). The coating layer may be formed with a thickness that does not affect the external magnetic force. Further, the electrode surface may be covered with a noble metal (for example, gold) in order to suppress corrosion and elution of metal ions into the electrolyte (including acid, base, and supporting salt). The magnetic electrode whose electrode surface is coated with a noble metal includes an electrode made of a noble metal used (for example, a gold (Au) electrode) and electrochemical characteristics (for example, a potential at which a peak associated with the formation / reduction of an oxide film is observed). Are preferably the same.
The source of the external magnetic force can be a magnet such as a permanent magnet or an electromagnet. The application of an external magnetic force to the working electrode can be performed by any means and mode that can apply a magnetic force necessary to accumulate magnetic particles on the working electrode, for example, by bringing a magnet into contact with the magnetic electrode body. .

  For the counter electrode (and reference electrode if necessary), an electrode usually used in electrochemical detection can be used, for example, a noble metal electrode such as platinum or platinum black, an oxide electrode such as titanium oxide or manganese oxide, A silver chloride electrode and a carbon electrode are mentioned. Preferably, a platinum electrode is used as the counter electrode and a silver / silver chloride electrode is used as the reference electrode.

When the detection target itself is a substance suitable for electrochemical detection (for example, oxidase / reductase), the presence of the detection target itself can be detected electrochemically.
When the detection target itself is not suitable for electrochemical detection, it is another ligand that specifically binds to the detection target and has a label suitable for electrochemical detection (hereinafter referred to as “secondary” for convenience. The presence of the detection target can be detected electrochemically by applying an electrochemical label to the detection target captured on the electrode in a sandwich method using a ligand). The second ligand is as described for the first detection method, except that the label is suitable for electrochemical detection. Suitable labels for electrochemical detection include oxidizing / reducing enzymes such as (per) oxidase and dehydrogenase (e.g. horseradish peroxidase (HRP), alkaline phosphatase, urease, catalase, glucose oxidase, etc.) or oxidizing / reducing (For example, fluorescein).

As a specific method of electrochemical detection, a known method can be used depending on the detection target or the second ligand.
The oxidation-reduction reaction used for electrochemical detection can be appropriately selected depending on the detection target or the label used. For example, when the label is HRP, an oxidation reaction of H ₂ O ₂ + 2e ⁻ + 2H ⁺ → 2H ₂ O can be used. In this case, hydrogen peroxide can be used as a substrate.
For electrochemical detection, a mediator such as potassium ferrocyanide / potassium ferricyanide may be used.

For the second method of the present invention, any electrochemical detection method known in the art can be used. Electrochemical detection may be performed by cyclic voltammetry, potential step chronoamperometry, coulometry, convective voltammetry, differential pulse voltammetry, square wave voltammetry, or AC impedance measurement.
The electrochemical measurement system includes a working electrode, a counter electrode, a reference electrode, and a potentiostat.

  According to the second detection method of the present invention, the detection target is accumulated on the electrode via the composite fine particles, so that even when the detection target has a low concentration in the sample or when the sample itself is a small amount. Detection with high sensitivity is possible.

(1) Preparation of gold layer-coated resin fine particles 156 mL of ultrapure water, 24 mL of 1% aqueous solution of tetrachlorogold (III) acid tetrahydrate (Wako Pure Chemical Industries) and 3% aqueous solution of sodium citrate (Katayama Chemical) 20 mL was added and stirred with a stirrer at 80 ° C. for 20 minutes to obtain a dispersion of gold nanoparticles (30 ± 5 nm).

  To 17 mL of the obtained gold nanoparticle dispersion liquid, 30 mg of acrylic resin particles (Hayakawa Rubber Co., Ltd .; Haya beads M-11R; particle size 5 μm; acrylic resin; FIG. 2) and 240 μL of 10 mM thioctic acid / ethanol solution are added to room temperature. For 4 hours using a stirrer. Next, the mixture was filtered through an omnipore membrane filter (manufactured by Nihon Millipore; pore size 0.2 μm), and washed with ultrapure water to obtain gold nanoparticle-coated resin particles (FIG. 3).

  Next, the obtained gold nanoparticle-coated resin particles are added to 20 mL of ultrapure water, and further 0.5% polyvinyl alcohol (Wako Pure Chemical Industries, Ltd .; degree of polymerization: about 500) 300 μL, 1% tetrachlorogold ( III) 4 mL of an acid tetrahydrate aqueous solution and 270 μL of 30% hydrogen peroxide water were added, and the mixture was stirred at 60 ° C. for 3 hours using a stirrer. Subsequently, it filtered with the omnipore membrane filter and wash | cleaned with the ultrapure water, and the gold-layer coating | cover resin fine particle was obtained (FIG. 4).

The gold-plated microbeads (micropearl AU) having the obtained gold layer-coated resin fine particles and nickel base plating layer were each subjected to centrifugation (6037 × g, 30 seconds), and the state after centrifugation was observed.
In the gold-plated microbeads having the underlying nickel plating layer, peeling of the plating layer was observed after centrifugation (FIG. 5).
On the other hand, in the gold-coated resin fine particles, no peeling of the gold layer due to centrifugation was observed (FIG. 6).

(2) Preparation of gold layer-coated magnetic resin fine particles 18 mL of a gold nanoparticle dispersion obtained in the same manner as above was added to a polymer polymer magnetic bead (Dynabeads (registered trademark) M-270 Carboxylic Acid (Invitrogen); Diameter: 3.0 μm; Fig. 7) 750 μL and 100 mM aminophenyl disulfide / ethanol solution 27 μL were added and stirred for 4 hours at room temperature using an omnipore membrane filter (Nippon Millipore Corp .; pore size 0.2 μm). By filtration and washing with ultrapure water, gold nanoparticle-coated magnetic resin particles were obtained (FIG. 8).

  Next, the obtained gold nanoparticle-coated magnetic resin particles were added to 60 mL of ultrapure water, and further, 0.5% polyvinyl alcohol (manufactured by Wako Pure Chemical Industries, Ltd .; degree of polymerization: about 500) 960 μL, 1% tetrachlorogold ( III) 13 mL of an acid tetrahydrate aqueous solution and 880 μL of 30% aqueous hydrogen peroxide were added, and the mixture was stirred at 60 ° C. for 3 hours using a stirrer. Next, the solution was filtered through an omnipore membrane filter and washed with ultrapure water to obtain gold layer-coated magnetic resin fine particles (FIG. 9).

(3) Confirmation of DNA capture using composite fine particles of the present invention A 12-base thiolated DNA probe (probe A: 3 ′ HS- (T) ₁₂ ; or probe B: (T) ₁₂ -SH 5 ′) The gold layer-coated resin fine particles (4 mg) prepared as described above were dispersed in a solution (1 mM, TE buffer (pH 7)) and stirred at room temperature for 1 hour.
Gold layer-coated resin fine particles were precipitated by centrifugation (6037 × g, 30 seconds), the supernatant was removed, and fresh TE buffer (1.0 mL) was added. Centrifugation and resuspension were repeated two more times to remove excess DNA.

To a target DNA (poly (A) ₂₄ ) solution (1 mM), composite microparticles containing probe A as a ligand and composite microparticles containing probe B as a ligand were added and stirred at room temperature for 1 hour. The composite fine particles were precipitated by centrifugation (6037 × g, 30 seconds), and UV measurement (absorbance measurement) of the supernatant was performed (UV-2400-PC UV-visible spectrophotometer; manufactured by Shimadzu Corporation; sweep range 240 to 300 nm) . The UV measurement was also performed before adding the composite microparticles (FIG. 10).

The results are shown in FIG.
Note that destruction of the composite fine particles by centrifugation (particularly, peeling of the gold layer) was not observed.

As is clear from FIG. 11, the absorbance at 260 nm (maximum absorption wavelength of nucleic acid) is reduced by the addition of the composite fine particles of the present invention. This decrease in absorbance corresponds to a decrease in nucleic acid of about 8 pmol. From this, the DNA density on the gold-coated fine particles was calculated to be 5.1 pmol cm ^-2, and this value is different from the DNA modification density of 17 pmol cm ^-2 on the 12 nm diameter gold nanoparticles due to the difference in particle diameter. It was a comparable value when considered.
That is, FIG. 11 clearly shows that a part of the nucleic acid in the solution was captured by the composite microparticles of the present invention.

(4) Capture of streptavidin using the composite fine particles of the present invention
(4-1) Preparation of composite fine particles of the present invention having biotin as a ligand
(4-1-1) Preparation of positively charged gold nanoparticles To 37.66 mL of ultrapure water, 2.34 mL of a 1 wt% aqueous solution of tetrachloroauric (III) acid tetrahydrate (Wako Pure Chemical Industries) and 2-aminoethane 189 μL of a 450 mM aqueous solution of thiol hydrochloride (Wako Pure Chemical Industries) was added and stirred at 25 ° C. for 20 minutes. A suspension of gold nanoparticles was obtained by adding 10 μL of a 10 mM aqueous solution of sodium borohydride (Wako Pure Chemical Industries) and stirring vigorously at room temperature for 10 minutes in a dark room.

  2.0 μL of the resulting suspension was dropped onto a TEM grid (Nisshin EM Co., Ltd .; 200 mesh; No. 6511), vacuum-dried at room temperature for 24 hours, and then transmission electron microscope (TEM, JEOL Ltd.) Observed by JEM-2000FXII). Formation of gold nanoparticles with a particle size of about 40 nm was confirmed (FIG. 12b).

The zeta potential and particle size distribution of the gold nanoparticles were measured by a zeta potential / particle size measurement system (manufactured by Otsuka Electronics Co., Ltd .; ELSZ-2). The average particle size was 42.5 ± 6.8 nm (FIG. 12a). The zeta potential was +36.47 mV, confirming a positive charge. This positive charge is considered to be due to the amino group (—NH ₃ ⁺ ) of aminoethanethiol.

(4-1-2) Preparation of gold nanoparticle-coated magnetic resin particles Dynabeads (registered trademark) M-270 Carboxylic Acid (manufactured by Invitrogen; particle size: 3.0 μm) was used as magnetic beads (magnetic resin fine particles). This magnetic bead has a carboxylic acid group on its surface. The commercially available product was suction filtered, washed thoroughly with 100 mL of ultrapure water, and then vacuum-dried at room temperature for 24 hours before use.
30 mg of the dried magnetic beads were added to 18 mL of the positively charged gold nanoparticle suspension obtained above and dispersed by sonication for 3 minutes, and then mixed rotor (AS ONE; MIX-ROTAR VMR-5) was stirred at room temperature for 2 hours. Next, the beads were recovered by an external magnetic force, dispersed in 500 μL of ultrapure water, and recovered again by an external magnetic force. After performing this purification operation three times, it was vacuum-dried at room temperature for 24 hours to obtain gold nanoparticle-coated magnetic resin particles (FIG. 13a).

Here, the adsorption between magnetic beads and gold nanoparticles is due to electrostatic interaction due to the negative charge of the carboxylic acid group present on the magnetic bead surface and the positive charge of the amino group of aminoethanethiol on the gold nanoparticle:
^{Magnetic bead (-COO -) + AuNP} (-NH 3 +) → Magnetic bead (-COO - ... + H 3 N-) AuNP
It is thought that.

(4-1-3) Preparation of gold layer-coated magnetic resin fine particles 30 mg of gold nanoparticle-coated magnetic resin particles were added to 60 mL of ultrapure water and dispersed by sonication for 3 minutes. To the resulting dispersion, 1.05 mL of a 0.5 wt% aqueous solution of polyvinyl alcohol (Wako Pure Chemical Industries), 12.8 mL of an aqueous solution of 1% tetrachloroauric (III) acid tetrahydrate and 0.88 mL of 30 wt% hydrogen peroxide water And stirred for 12 hours at room temperature using a mix rotor (manufactured by AS ONE; MIX-ROTAR VMR-5). Next, the beads were recovered by an external magnetic force, dispersed in 500 μL of ultrapure water, and recovered again by an external magnetic force. After performing this purification operation three times, it was vacuum dried at room temperature for 24 hours to obtain gold layer-coated magnetic resin fine particles (FIG. 13b).

  Gold nanoparticle-coated magnetic resin particles and gold layer-coated magnetic resin fine particles were each dispersed in an epoxy resin (Oken Shoji Co., Ltd .; Epok 812) and allowed to stand at 60 ° C. overnight to be cured. The surface of the cured resin is polished with a polishing film (Sumitomo 3M; # 4000), then cut thinly with a diamond knife (Nisshin EM Co., Ltd .; size 2.0 mm, 45 °; DiATOME), and a TEM grid (Nisshin EM) 200 mesh; No. 6511) and observed with a transmission electron microscope (manufactured by JEOL; TEM; JEM-2000FXII). The film thicknesses of the gold nanoparticle-coated magnetic resin particles and the gold layer-coated magnetic resin fine particles were estimated to be about 50 nm and 100 nm, respectively (FIGS. 14a and 14b).

The electrical resistances of the gold nanoparticle-coated magnetic resin particles and the gold layer-coated magnetic resin fine particles were 8.0 MΩ and 1.3 Ω, respectively, and the conductivity of the gold layer-coated magnetic resin fine particles was dramatically improved (Table 1). This is because, as is apparent from the cross-sectional TEM image (FIG. 14), a continuous gold layer was formed as a result of gold being deposited in the voids between the gold nanoparticles seen in the gold nanoparticle-coated magnetic resin particles. .

(4-1-4) Introduction of biotin into the surface of the gold layer-coated magnetic resin fine particles 1 mM biotin (manufactured by Thermo scientific; Sulfosuccinimidyl 2- (Biotinamido) ethyl-1,3'Dithiopropionate) 4 mg was mixed, dispersed by sonication for 3 minutes, and stirred at room temperature for 1 hour using a rotary mixer (FINEPCR; ROTARER; AG). Subsequently, the fine particles were collected by an external magnetic force, dispersed in 1 mL of PBS buffer, and collected again by an external magnetic force. This purification operation was performed three times, and then redispersed in 1 mL PBS buffer to obtain a dispersion of biotin-introduced gold layer-coated magnetic resin fine particles (composite fine particles of the present invention).

(4-2) Capture of streptavidin Add 5 μL of 4 mg / mL fluorescein-labeled streptavidin (Thermo scientific) aqueous solution as a target substance to 1 mL of biotin-introduced gold layer-coated magnetic resin particle dispersion and use a rotary mixer. And stirred at room temperature for 1 hour. Thereafter, the fine particles were collected by external magnetic force, dispersed in 1 mL of PBS buffer, and collected again by external magnetic force. This purification operation was performed three times, and then redispersed in 500 μL PBS buffer.

3 μL of biotin-introduced gold layer-coated magnetic resin particle dispersion alone and biotin-introduced gold layer-coated magnetic resin particle dispersion to which fluorescein-labeled streptavidin has been added are each dropped onto a slide glass, covered with a cover glass, and fluorescent microscope (OLYMPUS) Manufactured by BX51). Image capture was performed with a digital camera (manufactured by OLYMPUS; DP72) (FIG. 15).
In the biotin-introduced gold layer-coated magnetic resin fine particle dispersion (alone), a resin fine particle image was observed in the bright field (FIG. 15a), but no image was observed in the fluorescent field (FIG. 15c). On the other hand, in the biotin-introduced gold layer-coated magnetic resin fine particle dispersion to which fluorescein-labeled streptavidin is added, a fluorescent image by fluorescein is present at a location corresponding to the resin fine particle image observed in the bright field (Fig. 15b; bright field image). Was observed (FIG. 15d). This revealed that the biotin-introduced gold layer-coated magnetic resin fine particles can selectively capture the target (strept) avidin.

(4-3) Electrochemical detection using magnetic electrodes
(4-3-1) Magnetic electrode Production of magnetic electrode A 5 mm long Teflon rod (Fig. 16a) was drilled with a 1 mm diameter hole (Fig. 16b). Epoxy resin (manufactured by Oken; Epok 812) was applied to one end. Insert an iron wire (made by Niraco; FE-221480; φ1.0 mm; 99.995%) about 7 cm in length, and let the epoxy resin stand at 60 ° C overnight to cure it. It was fixed to a (registered trademark) rod (FIG. 16c). Thereafter, one end serving as the electrode surface was polished with a precision finishing polishing film (Sumitomo 3M; 2000 mesh), and the epoxy resin adhering to the electrode surface side was scraped off. An iron wire was pulled out from the opposite side of the electrode surface by about 2 mm to form a 2 mm deep gap on the electrode surface side (FIG. 16d). The gap was filled with gold leaf without any gaps. The surface of the electrode embedded with gold foil is made of a precision finishing polishing film (Sumitomo 3M; 2000 mesh), precision finishing polishing film (Sumitomo 3M; 4000 mesh), and liquid compound (Soft 99, 9800 mesh). They were used in order to give a mirror finish (Fig. 16e). In this way, an electrode having a structure that responds to an external magnetic force was produced using a magnetic / conductive material (FIG. 16f).

Characteristic evaluation of magnetic electrode Using magnetic or gold electrode (φ1.0 mm) prepared above as working electrode, platinum electrode (1 cm × 2 cm) as counter electrode, Ag | AgCl electrode as reference electrode, 0.1 M Cyclic voltammetry was performed in a sulfuric acid (Wako Pure Chemical Industries) solution with a sweep rate of 100 mV s ⁻¹ and a sweep range of −0.25 to +1.5 V, and the two were compared (FIG. 17).
Regardless of which working electrode was used, peaks associated with the formation and reduction of a gold oxide film were observed near 1.5 V on the anode side and 0.9 V on the cathode side. Therefore, it was confirmed that the gold-coated magnetic electrode produced above has the same electrochemical characteristics as the gold electrode.

Preparation of phosphate buffered saline (PBS buffer) Sodium chloride (Wako Pure Chemical Industries) 80 g, Potassium chloride (Wako Pure Chemical Industries) 2 g, Disodium hydrogen phosphate dodecahydrate (Wako Pure Chemical Industries) 36.3 g, Sodium dihydrogen phosphate (Wako Pure Chemical Industries) 2.4 g was dissolved in about 900 mL of ultrapure water, adjusted to pH 7.4 with 35% hydrochloric acid (Katayama Chemical Co., Ltd.), and ultrapure water was added to add 1 L I made a female up.

Electrochemical measurement of potassium ferrocyanide 5 mM potassium ferrocyanide (K ₄ [Fe (CN) ₆ ]) using the above magnetic electrode as working electrode, platinum electrode (1 cm × 2 cm) as counter electrode, and Ag | AgCl electrode as reference electrode Cyclic voltammetry was performed in a 3H ₂ O (Wako Pure Chemical Industries) / PBS buffer solution at a sweep rate of 10 mV s ⁻¹ and a sweep range of 0 to +0.5 V (FIG. 18).
A pair of redox peaks was obtained near 0.32 V and 0.25 V. This is a peak due to redox of ferrocyanide ions in the electrolytic solution, and a typical curve obtained with a gold electrode was obtained.

(4-3-2) Accumulation of gold resin coated magnetic resin particles on magnetic electrode and electrochemical measurement
The gold layer-coated magnetic resin fine particles prepared in (4-1) can be integrated by magnetic force and have high conductivity because they are coated with the gold layer. Utilizing these properties, gold layer-coated magnetic resin particles were accumulated on the surface of the magnetic electrode by an external magnetic force, thereby increasing the electrode area and increasing the sensitivity of the electrochemical response (FIG. 19).

Disperse 4 mg of gold layer coated magnetic resin particles in 1 mL of PBS buffer, drop 4 μL each from 4 to 32 μL onto the magnetic electrode, and collect and fix the magnetic resin particles on the electrode surface with an external magnet. This was the working electrode. A platinum electrode (1 cm x 2 cm) is used for the counter electrode, and Ag | AgCl is used for the reference electrode, and cyclic in a 5 mM potassium ferrocyanide / PBS buffer solution with a sweep rate of 10 mVs ^-1 and a sweep range of 0 to +0.5 V. Voltammetry was performed (Figure 20).
As a result of cyclic voltammetry, the current response accompanying the oxidation-reduction of ferrocyan increased as the fixed amount of the gold layer-coated magnetic resin particles on the surface of the magnetic electrode increased (FIG. 20a). The difference (ΔI _peak ) between the oxidation peak current value and the base current value was plotted (FIG. 20b). ΔI _peak increased proportionally when the amount of beads dispersed droplets was 24 μL or less, but ΔI _peak reached saturation when the amount was 24 μL or more. This is considered to be because the amount of gold-coated magnetic resin particles that can be fixed with respect to the electrode area of the magnetic electrode was saturated. Under this experimental condition, ΔI _peak increased to about 3 times compared to the case of a drop volume of 0 μL. Since the current value was proportional to the electrode area, it was suggested that the electrode area increased about three times.

Electrochemical measurement of fluorescein Cyclic voltammetry of fluorescein, which is a kind of fluorescent dye used for microscopic observation, was performed. Fluorescein has a pKa of 6.4 in the ionization equilibrium and exhibits pH-dependent absorption and fluorescence emission in the pH range of 5-9. Fluorescein is considered to have a structure represented by the following formula (I) depending on the pH of the solution.

When dissolved in PBS buffer (pH 7.4), fluorescein takes the structure of (B), the solution exhibits a yellowish green color, and in 0.1 M sulfuric acid solution (pH 1.0), it takes the structure of (A). Shows orange.
A magnetic electrode with magnetic electrodes and magnetic resin particles coated with gold layer coated on the working electrode by external magnetic force, a platinum electrode (1 cm × 2 cm) as the counter electrode, and Ag | AgCl as the reference electrode, 1 mM fluorescein (Wako Pure) Yakuhin Kogyo Co., Ltd.) Cyclic voltammetry was performed in a PBS buffer solution at a sweep rate of 10 mV s ⁻¹ and a sweep range of 0 to 0.8 V (FIG. 21).
As a result, an oxidation peak was observed around a potential of 0.7 V in PBS buffer. This is considered to be a peak due to the oxidation reaction represented by the following formula (II). Accumulation of gold layer-coated magnetic resin particles increased ΔI _peak by 11 times and increased the sensitivity of electrochemical signals.

Electrochemical measurement of HRP-labeled streptavidin with (4-3-3) magnetic electrode
Streptavidin was electrochemically detected using the biotin-introduced gold layer-coated magnetic resin fine particles prepared in (4-1) (FIG. 22).

HRP (Horseradish peroxidase) is known to exhibit a high catalytic function for the reduction reaction of hydrogen peroxide and to exhibit a high electrical response even without an electron mediator. Therefore, it is frequently used as a labeling substance in immunochemical staining and analytical chemistry experiments such as ELIZA. The enzyme-catalyzed reaction of HRP is represented by the following formulas () to (3).
HRP (Fe ³⁺ ) + H ₂ O ₂ → Compound-I (Fe ⁴⁺ = O, P ⁺ ) + H ₂ O (1)
^{Compound-I (Fe 4+ = O} , P +) + e - + H + → Compound-II (Fe 4+ = O) (2)
^{Compound-II (Fe 4+ = O} ) + e - + H + → HRP (Fe 3+) + H 2 O (3)
First, two-electron oxidation of HRP prosthesis heme by hydrogen peroxide occurs, and as a result, a reaction intermediate with an oxidation number of +5 consisting of oxyferryl iron (Fe ⁴⁺ = O) and porphyrin π-cation radical (P ⁺ ) Compound- I (Fe ⁴⁺ = O, P ⁺ ) is generated (1). Next, Compound-I (Fe ⁴⁺ = O, P ⁺ ) receives electrons from the electrode, the porphyrin π-cation radical is reduced by one electron, and the reaction intermediate Compound-II (Fe ⁴⁺ = O) with oxidation number +4 (2) Finally, Compound-II (Fe ⁴⁺ = O) receives the electrons and returns to the original state. Therefore, this reaction system is generally shown in the following formula (4) and FIG.
^{_{H 2 O 2 + 2e - +}} 2H + → 2H 2 O (4)

3 μL of 1 mM biotin (Thermo Scientific, EZ-Link ™ Sulfo-NHS-SS-Biotin) solution is dropped onto the surface of the magnetic electrode prepared in (4-3-1) and left at 4 ° C. for 30 minutes. Thus, biotin was modified on the electrode surface as a ligand. 3 μL of a 1 mg / mL HRP-labeled streptavidin (Ana Spec, Inc., Streptavidin, HRP Conjugated) solution was dropped onto the biotin-modified magnetic electrode, and the plate was allowed to stand in a dark room at 4 ° C. for 30 minutes. After rinsing with ultrapure water, this is the working electrode, the counter electrode is a platinum electrode (1 cm x 2 cm), the reference electrode is Ag | AgCl, and the sweep rate is 10 mVs in a 250 μM H ₂ O ₂ / PBS buffer solution. ^-1 and cyclic voltammetry was performed in a sweep range of -0.2 to +0.3 V (FIG. 24).
Peaks were observed at 0.19 V and 0.10 V at the anode and cathode, respectively. This is considered to be a peak due to direct electron transfer reaction with the electrode surface in the enzyme-catalyzed reaction of hydrogen peroxide by HRP. This suggested that HRP-labeled streptavidin exhibits electrochemical activity.

Electrochemical measurement of HRP-labeled streptavidin using gold layer coated magnetic resin particles
The biotin-introduced gold layer coated magnetic resin fine particles prepared in (1) capture HRP-labeled streptavidin in the sample solution, accumulate the gold layer coated magnetic resin particles on the electrode by an external magnetic force, and absorb hydrogen peroxide. Cyclic voltammetry was performed in the PBS buffer containing (FIG. 25). Peaks were observed at 0 V and 0.06 V at the anode and cathode, respectively. This is considered to be a peak due to direct electron transfer reaction with the electrode surface in the enzyme-catalyzed reaction of hydrogen peroxide by HRP. However, when the magnetic electrode is used (see above) and the gold layer coated magnetic resin particles are compared, the peak current value ΔIpc at the cathode (the peak current minus the base current) is 20 nA each. Amplification of the peak current more than 130 times was achieved. From this, it became possible to perform highly sensitive electrochemical measurement by using gold layer-coated magnetic resin particles.

Detection of target molecules using biotin-introduced gold layer-coated magnetic resin particles 20 μL of biotin-modified gold layer-coated magnetic resin particles dispersed in each labeled target are dropped onto the electrode, and gold is applied to the electrode surface by an external magnetic force. Layer-coated magnetic resin particles were accumulated. An electrode on which gold layer-coated magnetic resin particles were integrated was used as a working electrode, a counter electrode was a platinum electrode (1 cm × 2 cm), and a reference electrode was Ag | AgCl. In the case of HRP-labeled streptavidin, cyclic voltammetry was performed in a 250 μM H ₂ O ₂ / PBS buffer solution at a sweep rate of 10 mV s ⁻¹ and a sweep range of −0.2 to +0.3 V (FIG. 26).
Only when the gold layer-coated magnetic resin particles from which HRP-labeled streptavidin was recovered were measured, a pair of prominent redox peaks were observed. This is considered to be a redox peak of hydrogen peroxide by HRP, and only HRP-labeled streptavidin can be detected in this potential scanning range. Therefore, from this result, it was suggested that the measurement by the multi-array method in which a plurality of types of labeled antigens are collectively measured and only a specific antigen is detected is possible.

  From the above results, it can be understood that high sensitivity / high accuracy detection of a detection target can be achieved by accumulating gold layer-coated magnetic resin fine particles on a magnetic electrode by an external magnetic force.

  The above embodiments and examples are described as examples for facilitating the understanding of the present invention, and the present invention is limited only to the specific configurations described in this specification or the accompanying drawings. It should be noted that they are not intended. The specific configurations, means, and methods described in this specification can be replaced with many others known in the art without departing from the basic idea of the present invention understood based on this specification. This should be understood and can be easily recognized by those skilled in the art.

Claims (10)

A gold layer-coated fine particle in which a gold layer is attached to a fine particle having a resin surface directly or via an organic binder layer, and a ligand that is linked to the gold layer and can specifically bind to a biological substance. Composite fine particles for capturing or separating biological material. The gold layer-coated fine particles are obtained by mixing the fine particles having the resin surface with a gold nanoparticle dispersion liquid containing or not containing an organic binder to obtain fine particles having gold nanoparticles fixed on the resin surface. The composite fine particles according to claim 1, obtained by mixing the fine particles in a solution containing gold ions or gold complex ions in the presence of a reducing agent. The composite microparticle according to claim 1 or 2, wherein the ligand is selected from the group consisting of an antibody, an antigen, a nucleic acid probe, an aptamer, a lectin, glutathione, avidin, streptavidin, biotin, and a metal ion. The composite fine particles according to claim 1, wherein the fine particles having a resin surface are resin fine particles. The composite fine particles according to any one of claims 1 to 3, wherein the fine particles having a resin surface are magnetic fine particles. A biological substance in a sample is captured by the composite microparticle according to any one of claims 1 to 5, and the composite microparticle is separated from the sample by centrifugation. Method for separating substances. A method for separating a biological material in a sample, wherein the biological material in the sample is captured by the composite microparticle according to claim 5, and the composite microparticle is separated from the sample by magnetic separation. A biological substance in a sample is captured by the composite microparticle according to any one of claims 1 to 5, and the presence of the biological substance captured by the composite microparticle is specific to the biological substance. A method for detecting a biological substance in a sample, wherein the detection is performed using another ligand that binds to. The biological material in the sample is captured by the composite microparticle according to claim 5, the composite microparticle is accumulated on the working electrode by magnetic force, and the presence of the biological material captured by the composite microparticle is electrochemically detected. A method for detecting a biological substance in a sample, characterized by detecting. The method according to claim 9, wherein the electrochemical detection is performed by cyclic voltammetry.