JP2020025536A - Glycan immobilized magnetic metal nanoparticles, virus capture, concentration, purification and detection - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide sugar chain-immobilized magnetic metal nanoparticles in which a particle size does not change due to a delicate difference in preparation and a virus can be separated, concentrated and detected with good reproducibility without using an ultracentrifuge. .. SOLUTION: It has a linker compound, a sugar chain, and magnetic nanoparticles, an amino group of the linker compound is bonded to the sugar chain, and a sulfur atom is bonded to the magnetic nanoparticles, and the magnetic nanoparticles are gold or the like. Sugar chain-immobilized magnetic metal nanoparticles containing, and the surface of which is coated with gold or the like. [Selection diagram] Fig. 2

Description

  The present invention provides a sugar chain-immobilized magnetic metal nanoparticle having a uniform particle diameter, a virus capture by the sugar chain-immobilized magnetic metal nanoparticle, a method of concentrating and purifying the captured virus, and a real-time quantitative reverse transcription polymerase chain. This is a technique relating to a highly sensitive virus detection method by a reaction (RT-PCR) or a real-time quantitative polymerase chain reaction (PCR).

  Sugar chains are the third life chains after nucleic acids and proteins, and are important molecules for living organisms like nucleic acids and proteins. In particular, viruses are known to recognize sugar chains on cell surfaces and infect them. For example, influenza virus is known to recognize a sialic acid structure having a specific binding mode on the surface of a target cell serving as a host, and to propagate by inserting RNA having its own genetic information into the host cell. .

  On the other hand, in recent years, there has been concern about the emergence of highly pathogenic viruses and mutant viruses that cause a global pandemic, as well as the production of livestock and livestock products such as avian influenza and swine breeding and respiratory disorders. Influencing viral diseases have also been reported. Therefore, development of techniques for capturing viruses for virus research and techniques for early diagnosis of viral diseases for treatment are expected.

  Nanoparticles are materials applied in various fields such as life science, medical diagnosis, and biotechnology. Nanoparticles on which proteins such as antibodies are immobilized are useful for immunostaining by antigen-antibody reaction, antigen detection, separation / purification, and the like. Also, a drug delivery system (DDS) using nanoparticles in which a drug or a gene is immobilized, an analysis marker using labeled nanoparticles, and an analysis reagent such as a tracer are widely used.

  Magnetic nanoparticles can be used for cell purification, biomarkers, and the like because the state of the particles can be controlled by an appropriate external magnetic field due to their magnetic properties. Further, magnetic particles are highly practical elements having excellent characteristics such as being able to image living bodies and cells when used as a sensitizer for nuclear magnetic resonance imaging in the medical field. . In addition, silver can bond to a sulfur atom like metals such as gold, and is a highly versatile metal that can be complexed with biomolecules and organic compounds.

  For example, Patent Document 1 discloses a method of separating an antigen protein on the surface of a virus particle specifically binding to a sugar chain by using a sugar chain-immobilized gold nanoparticle, and obtaining an antibody using the antigen protein as an immunizing antigen. Has been described. Patent Literature 2 describes a virus concentration method using sugar chain-immobilized magnetic metal nanoparticles and a predetermined second magnetic substance.

JP 2013-159567 A JP 2011-45358 A

  The present invention solves the problems in sugar chain-immobilized gold nanoparticles and sugar chain-immobilized magnetic gold nanoparticles that have been developed so far for the detection, isolation, purification, and the like of sugar chain-binding proteins. .

  Sugar chain-immobilized gold nanoparticles (for example, Patent Document 1) are particles in which gold nanoparticles and sugar chains are immobilized. It has a maximum absorption wavelength peculiar to gold at about 530 nm and exhibits a purple-red color. Therefore, the activity of binding to a sugar chain-binding protein such as lectin can be visually observed by using a change in color, an agglutination reaction or the like as an index. Can be analyzed.

  However, isolation and purification of the components bound to the sugar chain-immobilized gold nanoparticles require large-sized equipment such as an ultracentrifuge.

  When a sugar chain-immobilized magnetic gold nanoparticle (for example, Patent Literature 2) binds to a target, a large-scale apparatus is not required, and a binding component such as a protein or a virus is isolated and purified only by magnetic separation. be able to. This technology has been applied as a simple test / diagnosis tool for bacteria and viruses in bedside clinics, farms, livestock farms, and the like, in addition to research.

  The sugar chain-immobilized magnetic gold nanoparticles have been found to be able to efficiently concentrate influenza virus and the like by immobilizing sulfated polysaccharides, and are currently being developed for clinical applications such as early diagnosis.

  However, the conventional sugar chain-immobilized magnetic gold nanoparticles have problems such as a change in particle size and dispersion stability after production depending on the type of sugar chain to be immobilized.

  To date, production methods for “sugar chain-immobilized gold nanoparticles” and “sugar chain-immobilized magnetic gold nanoparticles” using gold as a constituent atom have already been established, and their use in research and medical institutions has been expanded. I have. Among them, sugar chain-immobilized magnetic gold nanoparticles have high versatility, but have problems such as a change in particle size and dispersion stability depending on the type of sugar chain to be immobilized, manufacturing technique, and the like. . That is, there is a problem in controlling the particle size.

  Therefore, in the present specification, it is possible to immobilize various sugar chains, has high dispersion stability without depending on the type of sugar chains, and utilizes the magnetism of sugar chain-immobilized magnetic metal nanoparticles. A novel sugar-immobilized magnetic metal nanoparticle capable of concentrating a virus and a method for preparing the same, a method for capturing, concentrating, and purifying a binding component using the novel sugar-chain-immobilized magnetic metal nanoparticle and a method for detecting a virus are described.

  That is, in one embodiment of the present invention, the surface of a magnetic nanoparticle is coated with gold and / or silver capable of immobilizing a sugar chain via a thiol similar to gold, and a metal having magnetism is used to maintain magnetism. The present invention was carried out with respect to an intrinsic gold- and / or silver-containing sugar chain-immobilized magnetic metal nanoparticle, a method for preparing the sugar chain-immobilized magnetic metal nanoparticle, and a technique for capturing and separating a virus in a sample.

  The present invention includes the following embodiments.

<1> having an amino group at one end of a hydrocarbon chain or a hydrocarbon derived chain as a main chain, a linker compound having a sulfur atom at the other end, a sugar chain, and a magnetic nanoparticle;
The amino group is bonded to the reducing end of the sugar chain, the sulfur atom is bonded to the magnetic nanoparticle,
The magnetic nanoparticles contain gold and / or silver, and
A sugar chain-immobilized magnetic metal nanoparticle, wherein the surface of the magnetic nanoparticle is coated with gold and / or silver.

  <2> The sugar chain-immobilized magnetic metal nanoparticle according to <1>, wherein the sugar chain binds to a protein on the surface of the virus particle to capture the virus.

  <3> a step of applying a magnetic force to a mixture obtained by mixing the sugar chain-immobilized magnetic metal nanoparticles according to <1> or <2> with a specimen containing a virus, How to concentrate the virus.

  <4> The method for concentrating virus according to <3>, wherein the admixture contains a second magnetic substance having an average particle diameter larger than the sugar chain-immobilized magnetic metal nanoparticles.

  <5> The method according to <4>, wherein the surface of the second magnetic material is coated with silica.

  <6> Amplifying the virus RNA concentrated by the virus concentration method according to any one of <3> to <5> by real-time quantitative RT-PCR, or <3> to <5> A method for detecting a virus, comprising: amplifying, by real-time quantitative PCR, the DNA of the virus concentrated by the method for concentrating a virus according to any one of <1> to <3>.

  The sugar chain-immobilized magnetic metal nanoparticles used in the present invention can be prepared by simply mixing the sugar chain ligand complex treated with the reducing agent and the magnetic nanoparticles. Thus, sugar chains having high binding properties to various viruses can be respectively immobilized, so that sugar chain-immobilized magnetic metal nanoparticles capable of capturing a wide variety of viruses can be provided.

  In addition, by using the sugar chain-immobilized magnetic metal nanoparticles to capture and crush the virus particles, it is possible to purify and extract RNA in the virus in a short time and easily, Even a low concentration virus solution can be easily prepared into a high concentration virus solution.

  The prepared high-concentration virus solution can be expected to be detected in body fluids such as saliva and diluted virus solutions that are difficult to detect by conventional methods, by RNA amplification by real-time quantitative RT-PCR or DNA amplification by real-time quantitative PCR. .

  The sugar chain-immobilized magnetic metal nanoparticles according to one embodiment of the present invention do not change the particle size and dispersion stability depending on the type of the sugar chain to be immobilized, and rapidly detect and identify viruses, The effect is that it can be performed easily and with high sensitivity.

Sugar containing α2-3-linked sialyl lactose (2,3SL), dextran sulfate 2500 (DS25), β1-3-linked N-acetylgalactosamine-galactose, or α1-6-linked mannose-glucose, and a linker compound FIG. 3 is a view showing a structure of a chain ligand complex. It is a figure which shows the transmission electron microscope image of 2,3-sialyl lactose immobilized magnetic silver nanoparticle, and an average particle diameter. It is a figure which shows the dynamic light-scattering measurement value of 2,3-sialyl lactose immobilized magnetic silver nanoparticle, and an average particle diameter. FIG. 3 is a view showing an ultraviolet-visible absorption spectrum of 2,3-sialyl lactose-immobilized magnetic silver nanoparticles. It is a figure which shows the transmission electron microscope photograph of the virus particle which 2,3-sialyl lactose fixed magnetic silver nanoparticle captured. It is a figure which shows the amplification curve of the real-time quantitative RT-PCR of the RNA which the virus captured by the 2,3-sialyl lactose immobilized magnetic silver nanoparticle has. It is a figure which shows the transmission electron microscope photograph of DS25 immobilized magnetic gold silver nanoparticle, and an average particle diameter. It is a figure which shows the dynamic light-scattering measurement value of DS25 immobilized magnetic gold silver nanoparticles, and an average particle diameter. It is a figure which shows the ultraviolet-visible absorption spectrum of DS25 immobilized magnetic gold silver nanoparticles.

  Embodiments of the present invention will be described below, but the present invention is not limited thereto. Hereinafter, the “sugar chain-immobilized magnetic metal nanoparticles”, “the virus concentration method using sugar chain-immobilized magnetic metal nanoparticles”, and “real-time quantitative RT-PCR or real-time quantitative PCR” according to one embodiment of the present invention. The virus detection method will be described in detail.

[1. Sugar chain-immobilized magnetic metal nanoparticles]
The sugar chain-immobilized magnetic metal nanoparticles according to one embodiment of the present invention include a linker compound having an amino group at one end of a hydrocarbon chain or a hydrocarbon derivative chain as a main chain and a sulfur atom at the other end, and a sugar chain. And the magnetic nanoparticle, wherein the amino group is bonded to the reducing end of the sugar chain, the sulfur atom is bonded to the magnetic nanoparticle, and the magnetic nanoparticle contains gold and / or silver. Further, the magnetic nanoparticles are characterized in that the surface is coated with gold and / or silver.

  In the present specification, the “sugar chain-immobilized magnetic metal nanoparticle” is intended to mean a sugar chain ligand complex described in detail below and a magnetic nanoparticle bonded to each other. The “sugar chain ligand complex” refers to a linker compound capable of binding to magnetic nanoparticles containing gold and / or silver and a sugar capable of specifically interacting with a surface protein of a virus to be captured. And a chain. Therefore, the sugar chain ligand complex is required not to form a nonspecific interaction based on hydrophobicity with a substance such as a protein.

  The sugar chain-immobilized magnetic metal nanoparticles according to one embodiment of the present invention are produced by adding a water-soluble sugar chain ligand complex treated with a reducing agent to the surface of the nanoparticles, and have high dispersibility in an aqueous solution. Show. The sugar chain-immobilized magnetic metal nanoparticles have a surface modified with a sugar chain ligand complex, and preferably have an average particle diameter of 1 to 200 nm, more preferably about 1 nm to 50 nm.

  In addition, the sugar chain-immobilized magnetic metal nanoparticles according to one embodiment of the present invention include the sugar chain ligand complex, that is, the amino group present at one end of a linker compound having a hydrocarbon chain or a hydrocarbon derivative chain. A sugar chain-immobilized magnetic metal nanoparticle that is bonded to a sugar chain having a reducing end and is bonded to silver, gold, or another metal in a hydrocarbon structure containing a sulfur atom present at the other end of the linker compound It is.

  The sugar chain-immobilized magnetic metal nanoparticles according to one embodiment of the present invention can capture a virus by binding the sugar chain to a protein on the surface of a virus particle.

[2. Linker compound, sugar chain ligand complex]
The linker compound in one embodiment of the present invention has an amino group at one end of a hydrocarbon chain or a hydrocarbon-derived chain as a main chain, and has a sulfur atom at the other end.

  The “hydrocarbon-derived chain” means that a part of a carbon-carbon bond (CC bond), which is a main chain structure of the hydrocarbon chain, has a carbon-nitrogen bond (CN bond) or a carbon-oxygen bond ( (CO bond) and an amide bond (CO-NH bond).

  Preferably, the linker compound has a carbon-nitrogen bond in the hydrocarbon derived chain.

  Since the linker compound has an amino group at the terminal of a hydrocarbon chain or a hydrocarbon derivative chain, a sugar chain molecule can be easily introduced. The amino group may be a modified amino group (for example, an amino group modified with an acetyl group, a methyl group, a formyl group, or the like), an aromatic amino group, or an unmodified amino group. Good.

  Among them, the amino group is preferably an aromatic amino group. Under conditions of pH 3-4, which are the optimal conditions for the reductive amination reaction, it is necessary that the amino group is not protonated. Therefore, it is preferable that the lone pair is an aromatic amino group that can exist on a nitrogen atom even under conditions of pH 3 to 4 due to conjugation with an aromatic compound.

  In the sugar chain ligand complex, which is a complex of the linker compound and a sugar chain, a sugar chain having a reducing end at the amino group of the linker compound is introduced. That is, the sugar chain ligand complex has a structure in which the linker compound and the sugar chain having a reducing end are bonded via an amino group.

  This sugar chain can be introduced by a reductive amination reaction between the amino group of the linker compound and the sugar chain. That is, the aldehyde group (—CHO group) or ketone group (—CRO group, R is a hydrocarbon group) in the sugar chain generated by the equilibrium reacts with the amino group of the linker compound. Subsequent reduction of the Schiff base formed by this reaction makes it possible to easily introduce a sugar chain into the amino group.

  The “sugar chain having a reducing terminal” is a saccharide that can form a ketone or aldehyde having a reducing property at the molecular terminal. That is, the sugar chain having a reducing end is a monosaccharide chain, an oligosaccharide chain, or a polysaccharide chain in which the anomeric carbon atom is not substituted. That is, the sugar chain having a reducing end is a reduced sugar chain. As the sugar chain having a reducing end, a commercially available product or a natural product may be used.A product prepared by synthesis or a product prepared by decomposing a commercially available or natural polysaccharide may be used. it can.

  As a sugar chain having a reducing end, for example, glucose, galactose, mannose, maltose, isomaltose, lactose, panose, cellobiose, melibiose, mannooligosaccharide, chitooligo, etc., laminarioligosaccharide, glucosamine, N-acetylglucosamine, glucuronic acid, heparin, Heparan sulfate, chondroitin sulfate, dermatan sulfate, N-acetylgalactosamine, fucose and the like.

  More preferably, the sugar chain having a reducing end is α2-3 linked sialyl lactose (2,3SL), dextran sulfate (DS25) having a molecular weight of about 2500, β1-3 linked N-acetylgalactosamine-galactose, α1-6 Bound mannose-glucose.

  For example, 2,3SL is a sugar chain required for avian influenza infection, and DS25 is highly negatively charged due to a large number of sulfate groups and shows strong binding to the surface of virus particles such as influenza and herpes. . N-acetylglucosamine and fucose show binding to norovirus, and lactose shows binding to rotavirus. It is considered that these viruses can be captured via sugar chains by the sugar chain-immobilized magnetic metal nanoparticles.

  Dextran sulfate is a polyanionic dextran derivative mainly composed of a glucose skeleton linked by an α1-6 bond, and is synthesized from various high-purity dextran fractions. By adjusting the molecular weight of the dextran sulfate to about 2500, DS25 retains a large number of sulfate groups necessary for binding to the virus and controls the particle size of the product nanoparticles to about 10 nm. It is possible. The “product nanoparticle” is a sugar chain-immobilized magnetic metal nanoparticle in which the ligand complex is bonded to the magnetic nanoparticle. In addition, what was prepared by decomposing | disassembling a commercially available or natural polysaccharide chain can be used for said DS25.

  In addition, the above-mentioned linker compound has a metal atom with a gold-sulfur (Au-S) bond, a silver-sulfur (Ag-S) bond, or another metal by a sulfur atom provided at the other end of the hydrocarbon chain or the hydrocarbon derivative chain. A sulfur (MS) bond can be formed; More specifically, the linker compound has a hydrocarbon structure containing a disulfide bond (SS bond) or an SH group at the other end of the main chain.

  Sulfur atoms form metal-sulfur bonds (e.g., Au-S bonds, Ag-S bonds) with surface metals (gold and / or silver) in the magnetic nanoparticles, and can strengthen the bond with the metal. it can. Thus, in the linker compound, a sugar chain is bonded to the amino group and a sulfur atom is bonded to the magnetic nanoparticle, so that sugar chain molecules can be assembled and arranged on the magnetic nanoparticle.

  The sugar chain ligand complex in which the linker compound and the sugar chain are bonded can be firmly and easily bonded to the magnetic nanoparticles by the SH group sulfur. Therefore, sugar chains can be easily immobilized on the surface of the magnetic nanoparticles. Furthermore, the high hydration of the sugar chains bound on the magnetic nanoparticles makes it possible to stabilize the magnetic nanoparticles in a colloidal state.

  As the linker compound, a conventionally known linker compound, for example, a linker compound developed by the present inventors so far, WO2005 / 077965, a linker compound described in U.S. Patent No. 7320867B2, and the like can be used. It is not limited to.

  As the linker compound, a compound having a structure represented by the following chemical formula (1) can be preferably used:

(In the chemical formula (1), p and q are each independently an integer of 0 or more and 6 or less, X has an amino group or an aromatic amino group at a terminal, and has a carbon-nitrogen bond in a main chain. Is a structure comprising one, two or three chains of a hydrocarbon-derived chain which may be substituted, Y is a sulfur atom or a hydrocarbon structure containing a sulfur atom, and Z is a carbon-carbon bond or a carbon atom. A straight-chain or branched structure having an oxygen bond).

  The aromatic ring of the aromatic amino group that can be contained in X may be composed of only a hydrocarbon, or may be a heterocyclic ring containing an element other than carbon. X preferably has a structure represented by the following chemical formula (2), chemical formula (3), chemical formula (4), chemical formula (5), chemical formula (6) or chemical formula (7):

(In the chemical formulas (2) and (3), m ^{1 to} m ⁵ are each independently an integer of 0 or more and 6 or less.)

(In the chemical formula (6), ^{n 3} is an integer between 1 and 100, inclusive.)

  The Z may be represented by the following formula (8) or (9):

(In the chemical formulas (8) and (9), n ¹ and n ² are each an integer of 1 or more and 6 or less.)

  The linker compound is represented by the following chemical formula (10), chemical formula (11), chemical formula (12), chemical formula (13), chemical formula (14), chemical formula (15), chemical formula (16), or chemical formula (17). Compounds having the structure represented may be more preferably used:

(In the chemical formula (10), the chemical formula (11), the chemical formula (12), the chemical formula (13), and the chemical formula (15), m ^{1 to} m ⁵ are each independently an integer of 0 to 6, and n ¹ and n ² is 1 to 6 each an integer independently, ^{n 3} is an integer between 1 and 100, inclusive, q is 0 to 6. the integers.).

  The linker compound is produced, for example, by performing a condensation reaction between thioctic acid and an aromatic amino group terminal. At this time, the condensation reaction between the thioctic acid and the aromatic amino group condenses the carboxyl group and the aromatic amino group of the thioctic acid to form an amide bond, whereby the linker compound can be obtained. The sugar chain ligand complex is obtained by introducing a sugar chain having a reducing end into the linker compound thus prepared.

  The compound having the structure represented by the above chemical formula (10), chemical formula (11), chemical formula (12), chemical formula (13) or chemical formula (14) is, for example, thioctic acid (also referred to as α-lipoic acid) ) And an amine compound in which the terminal of the aromatic amino group is protected by a protecting group, and deprotecting the protecting group at the terminal of the aromatic amino group.

  The compound represented by the above formula (15), (16) or (17) can be produced, for example, by performing a condensation reaction between thioctic acid and one terminal amino group of a diamino compound.

  Specifically, the carboxy group of the thioctic acid and the amino group at one end of the diamino compound are condensed by the condensation reaction between the thioctic acid and the diamino compound, and an amide bond is formed. The compound represented by the chemical formula (16) or (17) can be obtained. Before subjecting the diamino compound to the condensation reaction between the thioctic acid and the diamino compound, the amino group at one terminal of the diamino compound may be protected by a protecting group. In this case, after the condensation reaction, the compound represented by the above formula (15), (16) or (17) is obtained by deprotecting the protecting group of the amino group protected by the protecting group. Can be.

  The thioctic acid is

Is provided. The amine compound is not particularly limited as long as it has an aromatic amino terminal protected by a protecting group.

The protecting group is a substituent introduced so that an amino group (for example, an amino group of an aromatic amino group or an amino group at one end of a diamino compound) is not reacted by the condensation reaction. Such a protective group is not particularly limited, but for example, a t-butoxycarbonyl group (—COOC (CH ₃ ) ₃ ; hereinafter also referred to as a Boc group), a benzyl group, an allyl carbamate group (− COOCH ₂ CH ＝ CH ₂ , Alloc group) and the like.

  A sugar chain ligand complex can be produced using the linker compound as described above. For example, by introducing a sugar chain into the linker compound by a reductive amination reaction between an amino group or an aromatic amino group contained in the linker compound (for example, contained in the above X of the chemical formula (1)) and the sugar chain, The sugar chain ligand complex can be obtained.

  FIG. 1 shows α2-3-linked sialyl lactose (2,3SL), dextran sulfate 2500 (DS25), β1-3-linked N-acetylgalactosamine-galactose, or α1-6-linked mannose-glucose, and a linker compound. It is a figure which shows the structure of the sugar chain ligand complex containing these. However, the sugar chain ligand complex used in one embodiment of the present invention is not limited to that shown in FIG.

  The sugar chain ligand complex obtained in this manner contains sulfur (S) contained in the linker compound (for example, contained in the above-mentioned Y in the chemical formula (1)) and gold and / or silver contained in the magnetic nanoparticles. It can be firmly and simply immobilized on the magnetic nanoparticles via the metal-sulfur bond formed between them. Therefore, the sugar chain ligand complex can be easily immobilized on the surface of the magnetic nanoparticle.

[3. Magnetic nanoparticles]
Since the magnetic nanoparticles used in the present invention have magnetism, the state of the particles can be controlled by an appropriate external magnetic field. Therefore, it can be used as cell purification, biomarker, and the like. Further, when magnetic nanoparticles are used as a sensitizer such as nuclear magnetic resonance imaging in the medical field, they can be used to image living organisms or cells, etc. It is.

  The magnetic nanoparticles are metal nanoparticles having magnetism, and are intended to be dispersed in an aqueous solution to form a colloid solution. Therefore, the average particle diameter of the magnetic nanoparticles is preferably in the range of 1 to 200 nm, more preferably in the range of 1 nm to 50 nm.

  When the average particle diameter of the magnetic nanoparticles is in the range of 1 to 200 nm, there is an advantage that the dispersion stability of the particles does not easily change with time. In addition, when nanoparticles having a size smaller than the target virus particles are used, the trapping efficiency is dramatically increased. Therefore, the average particle diameter of the magnetic nanoparticles is more preferably 1 nm to 50 nm.

  In the present specification, the “particle diameter” means the diameter of the largest inscribed circle with respect to the two-dimensional shape of the particle when the particle is observed with a transmission electron microscope. For example, when the two-dimensional shape of the particle is substantially circular, the diameter of the circle is intended, and when the particle is substantially elliptical, the minor axis of the ellipse is intended, and the particle is substantially square In this case, the length of the side of the square is intended, and when it is substantially rectangular, the length of the short side of the rectangle is intended.

  “Average particle size” refers to the average value of the above particle sizes of a plurality of particles. In the present specification, whether or not the average particle diameter has a value in a predetermined range is determined by observing 20 particles with a transmission electron microscope, measuring the particle diameter of each particle, and measuring the particle diameter of the 20 particles. The average value of the above-mentioned particle diameters was determined or confirmed by a dynamic light scattering method. Furthermore, it was confirmed by dynamic light scattering method that the sugar chain-immobilized magnetic metal nanoparticles in which sugar chains were immobilized on the magnetic nanoparticles had a larger particle diameter by the amount of the sugar chains.

  The magnetic substance (hereinafter, referred to as “first magnetic substance”) contained in the magnetic nanoparticles is not particularly limited, and a conventionally known magnetic substance can be used. For example, iron oxide, magnetite, chromium oxide, cobalt, ferrite, nickel, gadolinium, and the like can be given.

Among them, the first magnetic substance is preferably iron oxide and cobalt because of its strong magnetism. Note that, in this specification, iron oxide refers to trivalent iron oxide. Magnetite is an oxide of divalent and trivalent iron (Fe ²⁺ Fe ³⁺ ₂ O ₄ ).

  The magnetic nanoparticles contain gold and / or silver. Silver is a highly versatile metal that, like metals such as gold, can bind to a sulfur atom and can be complexed with biomolecules or organic compounds.

  In the above magnetic nanoparticles, gold and / or silver may be contained as atoms. Further, silver may be contained as an oxide.

  From the viewpoints of stability and magnetism of the immobilized sugar chain, the content of gold and / or silver in the magnetic nanoparticles is preferably from 20 to 70% by weight, and more preferably from 30 to 60% by weight based on the weight of the magnetic nanoparticles. Is more preferred.

In addition, the magnetic nanoparticles may contain other metals in addition to gold and / or silver. Examples of other metals include copper, aluminum, platinum, aluminum oxide, SrTiO ₃ , LaAlO ₃ , and ZrO ₂ .

  The surface of the magnetic nanoparticles is coated with gold and / or silver. For example, the magnetic nanoparticles have a core-shell-shell structure such that gold and / or silver, the first magnetic substance, and then gold and / or silver from the center.

  For example, JP-A-2006-153519 discloses a metal composite having a metal core such as gold, a first shell that is a magnetic material that covers the metal core, and a second shell such as gold that covers the first shell. It describes that the particles can be used for selective separation and collection of antigens, antibodies, and the like. In addition, Takahashi M. et al, ACS Omega, 2 (8): 4929-4937., Etc., describe magnetic separation of autophagosomes using the above metal complex particles.

  However, these documents do not disclose or suggest that a sugar chain is bound to a linker compound provided with the metal composite particles. In addition, there is no suggestion about the relationship between the metal composite particles and the above-described problem of the present invention. That is, by using the above magnetic nanoparticles, the conventional sugar chain-immobilized gold nanoparticle and the sugar chain in which the particle size, dispersion stability, and the like change depending on the type of sugar chain to be immobilized, manufacturing technique, and the like. The fact that the problem of the chain-immobilized magnetic gold nanoparticles can be solved is a unique finding found by the present inventors.

  The magnetic nanoparticles are heated, for example, by adding a solution of cobalt (II) acetylacetonate and iron (III) acetylacetonate to a solution of silver nitrate, and further adding sodium tetrachloroaurate (III) dihydrate or silver nitrate. Can be prepared as magnetic nanoparticles having a core-shell-shell structure.

[4. Method for Preparing Sugar Chain-Immobilized Magnetic Metal Nanoparticles]
The sugar chain-immobilized magnetic metal nanoparticles according to one embodiment of the present invention are obtained by mixing a solution containing the above magnetic nanoparticles with a linker compound (sugar chain ligand complex) bonded to a sugar chain by treatment with a reducing agent. be able to. Each S atom of the SS bond of the sugar chain ligand complex is bonded to gold, silver, or another metal on the magnetic nanoparticle by a metal-sulfur bond, whereby the sugar chain-immobilized magnetic metal nanoparticle is formed. It is formed.

  The sugar chain-immobilized magnetic metal nanoparticles exhibit high dispersibility in an aqueous solution. The average particle size of the sugar chain-immobilized magnetic metal nanoparticles is preferably in the range of 1 to 200 nm, and more preferably in the range of 1 nm to 50 nm.

  When the average particle diameter is 1 nm or more, it can be manufactured at low cost and is practical. In addition, it is preferable that the average particle diameter is 200 nm or less, because the change over time in the dispersion stability of the sugar chain-immobilized magnetic metal nanoparticles is easily suppressed.

  In addition, when the average particle diameter is 1 nm to 50 nm, nanoparticles having a size smaller than the target virus particles are used, and the virus capturing efficiency is significantly increased, which is preferable.

  As a method of bonding the linker compound to the sugar chain, it is preferable to mix the linker compound and the sugar chain at a molar ratio of 1: 1 to 50: 1.

  As a method for preparing the sugar chain-immobilized magnetic metal nanoparticles, specifically, for example, a solution containing a sugar chain ligand complex in a solution of silver-containing magnetic nanoparticles (hereinafter also referred to as magnetic silver nanoparticles) is used. May be added. Examples of the sugar chain ligand complex include, for example, a sugar chain ligand complex in which 2,3-sialyl lactose and a linker compound are bound by treatment with a reducing agent. Thus, the SS bond of the sugar chain ligand complex can be converted into a metal-sulfur bond on the magnetic nanoparticle, and the sugar chain-immobilized magnetic metal nanoparticle can be obtained.

  The reducing agent used in the reducing agent treatment is not particularly limited, and examples thereof include salts such as sodium borohydride and sodium cyanoborohydride. In addition, for example, as in the case of using potassium instead of the above-mentioned sodium, salts having a different cation component from the above-mentioned sodium may be used.

  The solvent used for the solution containing the magnetic nanoparticles and the solution containing the sugar chain ligand complex is not particularly limited, but includes, for example, ultrapure water, methanol, ethanol, propanol, and a mixed solvent thereof. Can be.

  In addition, the sugar chain-immobilized magnetic metal nanoparticles obtained by the above-mentioned mixing are subjected to washing by ultrafiltration, centrifugation, etc., and components such as low-molecular salts are removed, so that sugar chains immobilized in a solution state are stable. Magnetic metal nanoparticles can be obtained.

  The mixing ratio of gold, silver, the sugar chain ligand complex, and the reducing agent used to prepare the sugar chain-immobilized magnetic metal nanoparticles is not particularly limited, but the sugar chain-immobilized magnetic metal nanoparticles may be used. It is preferable that the final concentration of gold and / or silver in the contained solution is 0.1 to 1 mM, respectively.

  The concentration of the sugar chain ligand complex is preferably 0.1 to 10 mM as the final concentration in the solution containing the sugar chain-immobilized magnetic metal nanoparticles. The concentration of the reducing agent used is desirably 1 to 10 mM as the final concentration in the solution containing the sugar chain-immobilized magnetic metal nanoparticles.

  The method for preparing the sugar chain-immobilized magnetic metal nanoparticles described above utilizes the above-described method for producing magnetic nanoparticles and the method for producing a sugar chain ligand complex. The sugar chain-immobilized magnetic metal nanoparticles produced based on this method are characterized in that they are dispersed in an aqueous solution. That is, this production method is also a method of preparing a colloid solution of the sugar chain-immobilized magnetic metal nanoparticles.

[5. Capture and Concentration Method of Virus with Sugar Chain-Immobilized Magnetic Metal Nanoparticles]
According to one embodiment of the present invention, a method for capturing and concentrating a virus using a sugar chain-immobilized magnetic metal nanoparticle involves interacting a solution containing a sugar chain-immobilized magnetic metal nanoparticle with a solution containing a virus particle. After that, the conjugate and the non-conjugate are separated by magnetism.

  That is, the virus concentration method according to one embodiment of the present invention includes a step of applying a magnetic force to a mixture obtained by mixing a sugar chain-immobilized magnetic metal nanoparticle with a virus-containing specimen. The sugar chain-immobilized magnetic metal nanoparticles according to one embodiment of the present invention that have captured the virus are subjected to magnetic separation, and the supernatant is removed, whereby a high-concentration virus solution can be obtained.

  The specimen is not particularly limited as long as it can be brought into contact with the sugar chain-immobilized magnetic metal nanoparticles. Examples include saliva, nasal mucosa, bodily fluids of plants and animals, and the like.

  The above-mentioned saliva or the like may be used as a sample itself, or may be used as a liquid prepared by adding it to, for example, a MEM medium or physiological saline. By mixing such a sample with, for example, a solution prepared by adding sugar chain-immobilized magnetic metal nanoparticles to water, saline, or a phosphate buffer, the virus in the sample is immobilized on the sugar chain. Contact can be made with the magnetic metal nanoparticles.

  The “solution containing the sugar chain-immobilized magnetic metal nanoparticles” means a colloid solution in which the sugar chain-immobilized magnetic metal nanoparticles according to one embodiment of the present invention are dispersed in a liquid. As long as the solution contains sugar chain-immobilized magnetic metal nanoparticles, the solution may further contain a salt or the like. As the liquid, for example, ultrapure water or a buffer solution can be used.

  In the present invention, the “solution containing virus particles” (virus-containing specimen) is intended to mean a colloid solution in which the virus is dispersed in a liquid in the present invention. Also, a virus is intended to mean a virus that exhibits binding properties to the sugar chain ligand complex of the sugar chain-immobilized magnetic metal nanoparticles according to one embodiment of the present invention. The virus particles preferably have a particle size of 10 to 200 nm.

  As a liquid of the solution in which the virus particles are dispersed, ultrapure water, a buffer, or the like can be used as long as the solution does not contain a substance that may inhibit the binding between the sugar chain ligand complex and the virus particles. Further, the concentration of virus particles in the solution is preferably 1-10000 HAU (hemagglutination unit) in the case of a virus that induces hemagglutination.

  In the admixture obtained by mixing the sugar chain-immobilized magnetic metal nanoparticles and the sample containing the virus, the interaction between the sugar chain-immobilized magnetic metal nanoparticles and the virus causes the sugar chain-immobilized magnetic metal to be mixed. There are a conjugate which is a component to which the sugar chain of the nanoparticle and the virus are bound, and a non-conjugate which is a component which is not bound to the sugar chain-immobilized magnetic metal nanoparticle. By applying a magnetic force to the mixture, the bound body can be separated from the unbound body. As a result, the virus can be concentrated. The component separated and concentrated in the above step is defined as a nanoparticle-virus conjugate.

  Further, the admixture may contain a second magnetic substance having a larger average particle diameter than the sugar chain-immobilized magnetic metal nanoparticles. The second magnetic body assists the magnetism of the first magnetic body.

  The second magnetic material is not particularly limited, but is preferably magnetite or ferrate, or triiron tetroxide.

  As described above, the average particle size of the magnetic nanoparticles is preferably in the range of 1 to 200 nm, and more preferably in the range of 1 nm to 50 nm. The average particle diameter of the sugar chain-immobilized magnetic metal nanoparticles having the sugar chains immobilized on the magnetic nanoparticles is preferably in the range of 1 to 200 nm, and more preferably in the range of 1 nm to 50 nm.

  The range of the average particle diameter of the second magnetic material is not particularly limited as long as the upper limit of the average particle diameter is 100 μm and is larger than the average particle diameter of the sugar chain-immobilized magnetic metal nanoparticles. However, the thickness is preferably 100 nm or more and 100 μm or less, more preferably 100 nm or more and 50000 nm or less, and still more preferably 1000 nm or more and 10000 nm or less.

  By using the second magnetic material, a concentration effect comparable to that using centrifugation can be obtained more efficiently than when only the first magnetic material is used. Then, the average particle diameter of the sugar chain-immobilized magnetic metal nanoparticles is in the above range, and by adjusting the average particle diameter of the second magnetic substance to the above range, the average particle diameter of both is optimized. Guessed. As a result, by applying magnetic force to the mixture containing the sugar chain-immobilized magnetic nanoparticles, the second magnetic substance, and the sample containing the virus, a trace amount of virus in the sample is used by centrifugation. , And can be concentrated more efficiently, simply and safely.

The weight ratio of the sugar chain-immobilized magnetic metal nanoparticles to the second magnetic substance is preferably 1: 1 × 10 ³ to 1: 1 × 10 ¹¹ . By adjusting the ratio of the amount of the sugar chain-immobilized magnetic metal nanoparticles having the above-described average particle diameter to the ratio of the amount of the second magnetic substance used to the above range, the concentration efficiency can be further improved. .

  Regarding the above “applying a magnetic force”, the method of applying the magnetic force is not particularly limited. For example, a magnetic force can be applied to the mixture by bringing a conventionally known permanent magnet such as an electromagnet or a bar magnet into contact with the outer wall of the container containing the mixture. The strength of the magnetic force is preferably 100 to 500 millistellas in order to obtain the same concentration efficiency as in the case of using centrifugation.

  When the second magnetic substance is used, the coloration exhibited by the sugar chain-immobilized magnetic metal nanoparticles being dispersed in the liquid is the time when the step of applying a magnetic force to the above-mentioned mixture is performed, and the sugar chain is immobilized. The determination may be made based on a phenomenon in which the magnetic metal nanoparticles are thinned by being adsorbed by the second magnetic substance.

  When the second magnetic substance is not used, the end time of the step of applying a magnetic force to the above mixture is based on the phenomenon that the sugar chains immobilized magnetic metal nanoparticles to which the virus is bound are accumulated due to the magnetic force and the coloring becomes thin. May be visually determined.

  By applying a magnetic force, the sugar chain-immobilized magnetic metal nanoparticle to which the virus is bound, or the sugar chain-immobilized magnetic metal nanoparticle to which the virus is bound, and the second magnetic material are accumulated by the magnetic force. The method for recovering the virus gene from this aggregate is not particularly limited, and may be a conventionally known method. For example, after washing the aggregate with sterilized water or distilled water, pipetting is appropriately performed, and sterile water or distilled water containing the aggregate is heated at 100 ° C., whereby the viral gene can be collected in the supernatant. it can.

  The virus to be concentrated is not particularly limited. For example, influenza virus, herpes simplex virus, herpes simplex virus, AIDS virus, hepatitis B virus, hepatitis C virus, chickenpox virus (VZV), cytomegalovirus (CMV), adult T cell leukemia virus (HTLV-1), Examples include lentivirus, carp herpes virus, adenovirus, norovirus, rotavirus and the like.

  The virus binds to the sugar chain of the sugar chain-immobilized magnetic metal nanoparticle by the sugar binding site (sugar chain recognition site) of the virus surface protein in the molecule. Examples of the above bond include a hydrogen bond, an ionic bond, a bond by electrostatic interaction, a bond by Van der Waals force, and the like.

  In the virus concentration method according to one embodiment of the present invention, the surface of the second magnetic body may be coated with silica. As an embodiment of the coating, there can be mentioned, for example, an embodiment in which a silica film is formed on the surface of a second magnetic substance containing iron oxide as a component. According to this configuration, since the nucleic acid can be prevented from adsorbing to the second magnetic substance, the recovery rate of the virus-derived nucleic acid can be significantly improved.

As a method of coating the surface of the second magnetic substance with silica, for example, the surface of iron oxide (Fe ₂ O ₃ ) particles prepared by adding ammonia to ferrous chloride and ferric chloride is used. A method of treating with an excess amount of tetraethyl orthosilicate and ammonia in ethanol may be used. In addition, as a method for confirming that the surface of the second magnetic material is coated with silica, there can be mentioned observation by a composition analysis (EPMA) and a scanning electron microscope (SEM).

[6. Highly Sensitive Virus Detection Method by Real-Time Quantitative RT-PCR Using Nanoparticle-Virus Conjugate or Real-Time Quantitative PCR]
In the virus detection method according to one embodiment of the present invention, the virus RNA concentrated by the above-described virus concentration method may be amplified by real-time quantitative RT-PCR, or may be concentrated by the above-described virus concentration method. The DNA of the virus obtained is amplified by real-time quantitative PCR.

  The method for detecting a virus with high sensitivity by real-time PCR using the above-described nanoparticle-virus conjugate is characterized in that the nanoparticle-virus conjugate separated and concentrated by the above-described steps is crushed and RNA or DNA contained in the virus is subjected to real-time quantitative RT-PCR. To amplify.

  The sugar chain-immobilized magnetic metal nanoparticles capturing the virus are particularly bound to proteins on the surface of the virus particle. Therefore, it is assumed that a large amount of molecules derived from viruses, such as nucleic acids and proteins, are present in the solution after crushing of the virus particles. The virus RNA bound to the sugar chain-immobilized magnetic metal nanoparticles is amplified by real-time quantitative RT-PCR, or the virus DNA bound to the sugar chain-immobilized magnetic metal nanoparticles is subjected to real-time quantitative PCR. By the amplification, the presence or absence of a specific virus, the copy number of the virus, and the like can be determined.

  In the real-time quantitative RT-PCR and the real-time quantitative PCR, a Ct (Threshold Cycle) value when no sugar chain-immobilized magnetic metal nanoparticles are used is determined, and from the Ct value, a method according to an embodiment of the present invention is used. The difference obtained by subtracting the Ct value in the case of concentration was determined to evaluate the degree of virus concentration.

  The present invention also includes the following aspects (1) to (4).

  (1) The amino group present at one end of a linker compound having a hydrocarbon-derived chain binds to a sugar chain having a reducing end, and the sulfur atom present at the other end of the linker compound has silver as a surface structure. Sugar chain-immobilized magnetic silver nanoparticles, which are bound to particles.

  (2) The sugar chain-immobilized magnetic silver nanoparticles according to (1), wherein the sugar chain binds to a protein on the surface of the virus particle to capture the virus.

  (3) The sugar chain-immobilized magnetic silver nanoparticles according to (1), wherein the captured virus according to (2) is concentrated by magnetic separation.

  (4) Virus capture, concentration, purification, and virus amplification, wherein the RNA of the virus captured and concentrated by the sugar chain-immobilized magnetic silver nanoparticles according to (1) is amplified by real-time quantitative RT-PCR. How to detect.

  The present invention will be described more specifically based on examples, but the present invention is not limited thereto. Examples include “Preparation of sugar chain-immobilized magnetic silver nanoparticles”, “Preparation of sugar chain-immobilized magnetic silver nanoparticles”, “Influenza by sugar chain-immobilized magnetic silver nanoparticles or sugar chain-immobilized magnetic gold-silver nanoparticles” Capture of virus and concentration by magnetic separation ”,“ Capture of pig reproductive and respiratory disorder syndrome virus in pig saliva by sugar-immobilized magnetic gold-silver nanoparticles and magnetic separation ”,“ Magnetic silver nanoparticles with sugar chain immobilized Crushing of virus conjugate and detection of virus by real-time quantitative RT-PCR ".

[Example 1]
[Preparation of sugar-immobilized magnetic silver nanoparticles]
Silver nitrate (17.0 mg, 0.1 mmol, Wako Pure Chemical Industries) and 1,2-hexadecanediol (260 mg, 1.0 mmol, Tokyo Chemical Industry) were converted into oleic acid (2.3 ml, 8.0 mmol, Aldrich), oleylamine (3.2 ml, 10.0 mmol, Tokyo Chemical Industry) and tetraethylene glycol (10 ml, Tokyo Chemical Industry).

  The obtained solution was stirred while being heated with a mantle heater under an argon atmosphere, and after the temperature of the solution reached 170 ° C., a mixed solution of oleylamine and toluene (3.0 ml, 2: 1) was added, and the solution was further heated for 250 minutes. Heated to ° C. The mixed solution contains cobalt (II) acetylacetonate (70.6 mg, 0.2 mmol, Aldrich) and iron (III) acetylacetonate (53.4 mg, 0.2 mmol, Aldrich).

  Next, hydrophobic magnetic silver nanoparticles were synthesized by adding a mixed solution (2.0 ml, 1: 1) of oleylamine and toluene in which silver nitrate (17.0 mg, 0.1 mmol) was dissolved.

  The obtained hydrophobic magnetic silver nanoparticles are washed by adding excess acetone and then centrifuging (3,700 g, 5 min) twice to remove the remaining organic components, and concentrating under reduced pressure. The solvent was removed. Thereafter, the obtained hydrophobic magnetic silver nanoparticles were dispersed in a hexane solution (200 μl) so as to have a concentration of 17.0 mg / ml.

  The hydrophobic magnetic silver nanoparticles dispersed in the hexane solution were prepared by adding a 2,3-sialyl lactose-immobilized sugar chain ligand complex and an aqueous sodium borohydride solution (final concentration 2.5 mM, 1.0 ml, respectively) to 10 mM. Ultrasonic waves were applied to an aqueous solution adjusted to pH 12 with an aqueous sodium hydroxide solution.

  After adding 2.0 ml of ultrapure water while irradiating ultrasonic waves for 30 minutes, the reaction solution is centrifuged (3,700 g, 5 min), and the aqueous layer is subjected to ultrafiltration (3 k, 13,000 g, 15 min, 3 times) ) To remove unreacted reagents, salts, and the like, to synthesize 2,3-sialyl lactose-immobilized magnetic silver nanoparticles (2,3SL-MSNP).

  The sugar chain ligand complex is a reducing agent in a mixed solvent of water, dimethylacetamide and acetic acid, with 1.5 equivalents of a linker compound represented by the chemical formula (14) with respect to 2,3-sialyl lactose. It was prepared by adding 12 equivalents of sodium cyanoborohydride and performing a reductive amination reaction at 40 ° C. in a dark place for 3 days.

  FIG. 2 is a transmission electron microscope image of 2,3-sialyl lactose-immobilized magnetic silver nanoparticles (2,3SL-MSNP), and an average particle diameter obtained by observing 20 particles with a transmission electron microscope ( 9.24 nm).

  FIG. 3 is a diagram showing the particle size distribution of 2,3SL-MSNP and the average particle size (14.61 nm ± 2.67 nm) measured by the dynamic light scattering method.

  The degree of dispersion of the average particle diameter of 2,3SL-MSNP measured by the dynamic light scattering method is less than 30%. Although no data is shown, the degree of dispersion is lower than the sugar chain-immobilized gold nanoparticles described in Patent Document 1 and the sugar chain-immobilized magnetic gold nanoparticles described in Patent Document 2. That is, 2,3SL-MSNP has a more uniform particle diameter than conventional sugar chain-immobilized gold nanoparticles and sugar chain-immobilized magnetic gold nanoparticles, and has excellent particle diameter and dispersion stability after production. I can say.

  FIG. 4 is a diagram showing an ultraviolet-visible absorption spectrum of 2,3-sialyl lactose-immobilized magnetic silver nanoparticles. The vertical axis represents the absorbance, and the horizontal axis represents the wavelength.

[Capture of influenza virus by sugar chain-immobilized magnetic silver nanoparticles and concentration by magnetic separation]
Virus particles were captured using the 2,3SL-MSNP prepared as described above among the magnetic silver nanoparticles having sugar chains immobilized thereon. A 2,3SL-MSNP solution (10 μl) was added to an influenza virus dilution (500 μl) obtained by diluting a culture supernatant of avian influenza A (DUCK A / spot-billed duck / kagoshima / KU57 / 2015 (H11N9)) with PBS. Mixed.

  The mixed solution was transferred to an Eppendorf tube containing about 10 mg of the second magnetic material, mixed well, and allowed to stand for 1 minute with a magnet stand (TAKARA Magnetic Stand (6 tubes)) to perform magnetic separation. After the magnetic separation, the supernatant of the mixed solution was removed, and the precipitate subjected to magnetic separation using a 2,3SL-MSNP and a magnet was collected. As the second magnetic material, magnetite having a particle size of 20 to 100 μm was used.

  FIG. 5 is a diagram showing a transmission electron micrograph of virus particles captured by magnetic silver nanoparticles immobilized on 2,3-sialyl lactose. The right diagram of FIG. 5 is an enlarged view of a portion surrounded by a frame in the left diagram of FIG.

[Disruption of Sugar Chain-Immobilized Magnetic Silver Nanoparticle-Virus Conjugate and Virus Detection by Real-Time Quantitative RT-PCR]
To the precipitate obtained by the magnetic separation, 20 μl of a 0.1% SDS solution was added to crush the virus particles to obtain a precipitate fraction. As a control, 10 μl of each of the virus solution in which the sugar chain-immobilized magnetic silver nanoparticles were not used and the supernatant fraction after the magnetic separation were collected, and 10 μl of a 0.1% SDS solution was added thereto. A minute was prepared.

  For the precipitated fraction and the control fraction, 2 μl of each of the obtained solutions was added to 23 μl of a reagent for PCR, and subjected to real-time quantitative RT-PCR. As a reagent, One Step SYBR PrimeScript RT-PCR Kit II (Takara Bio, product code RR086A) was used.

  The reagents per reaction were 1 × 12.5 μl of 2 × One Step SYBR RT-PCR Buffer 4, 1 μl of PrimeScript 1step Enzyme Mix 2, 1 μl of PCR Forward Primer (10 μM), and 1 μl of PCR Reverse Primer (10 μM). A mixture of 5.5 μl of water and 2 μl of 6.25% Tween 20 was used.

  As a real-time quantitative RT-PCR device, Thermal Cycler Dice Real Time System II (manufactured by Takara Bio) was used.

  As primers, Type A / MPgene (217-236) Forward GGACTGCAGCGTAGGACGCTT (20 bp, SEQ ID NO: 1) which is a primer for detecting the influenza Type A / M gene and Type A / MP gene (382-405) Reverse CATYCTGTGATGATCATGTGATCATGTGATC ) Was used to amplify the 188 bp M protein region of the influenza A virus RNA.

  The conditions of RT-PCR were as follows: reverse transcription reaction was set at 45 ° C. for 5 minutes, initial thermal denaturation treatment was set at 95 ° C. for 10 seconds, and PCR cycle was set at 95 ° C. for 5 seconds and 60 ° C. for 30 seconds, and 45 cycles were performed. .

  FIG. 6 is a diagram showing an amplification curve of real-time quantitative RT-PCR of RNA of a virus captured by 2,3-sialyl lactose-immobilized magnetic silver nanoparticles. Table 1 shows the Ct values and the like of the precipitate fraction and the control fraction. From FIG. 6 and Table 1, the Ct value (28.95) of the precipitate fraction obtained by capturing the virus and magnetically separating it is based on the Ct value (31.8) of the sample not using the sugar chain-immobilized magnetic silver nanoparticles. Was also confirmed to be 2.85 cycles earlier and 4.48 cycles earlier than the Ct value (33.43) of the supernatant fraction.

  From this, the virus solution magnetically separated with 2,3SL-MSNP has a virus concentration 7.21 times and 22.31 times that of the sample solution and the supernatant fraction without using 2,3SL-MSNP, respectively. It was shown that it could be prepared.

[Example 2]
[Preparation of magnetic gold-silver nanoparticles immobilized on sugar chains]
Silver nitrate (17.0 mg, 0.1 mmol, Wako Pure Chemical Industries) and 1,2-hexadecanediol (260 mg, 1.0 mmol, Tokyo Chemical Industry) were converted into oleic acid (2.3 ml, 8.0 mmol, Aldrich), oleylamine (3.2 ml, 10.0 mmol, Tokyo Chemical Industry) and tetraethylene glycol (10 ml, Tokyo Chemical Industry).

  The obtained solution was stirred while being heated with a mantle heater under an argon atmosphere, and after the temperature of the solution reached 170 ° C., a mixed solution of oleylamine and toluene (3.0 ml, 2: 1) was added, and the solution was further heated for 250 minutes. Heated to ° C. The mixed solution contains cobalt (II) acetylacetonate (70.6 mg, 0.2 mmol, Aldrich) and iron (III) acetylacetonate (53.4 mg, 0.2 mmol, Aldrich).

  Next, a mixed solution (2.0 ml, 1: 1) of oleylamine and toluene in which sodium tetrachloroaurate (III) dihydrate (39.7 mg, 0.1 mmol, Nacalai Tesque) was dissolved was added. And hydrophobic magnetic gold-silver nanoparticles containing gold atoms were synthesized.

  The obtained hydrophobic magnetic gold / silver nanoparticles are washed by adding excess acetone and then centrifuging (3,700 g, 5 min) twice to remove the remaining organic components, and concentrating under reduced pressure. The solvent was removed. Thereafter, the obtained hydrophobic magnetic gold-silver nanoparticles were dispersed in a hexane solution (200 μl) to a concentration of 20.4 mg / ml.

  The hydrophobic magnetic gold-silver nanoparticles containing gold atoms dispersed in the hexane solution were prepared by mixing a sugar chain ligand complex having DS25 immobilized thereon and an aqueous sodium borohydride solution (final concentration 2.5 mM, 1.0 ml, respectively) at 10 mM. To an aqueous solution adjusted to pH 11 with an aqueous solution of sodium hydroxide, and irradiated with ultrasonic waves.

  After adding 2.0 ml of ultrapure water while irradiating ultrasonic waves for 30 minutes, the reaction solution is centrifuged (3,700 g, 5 min), and the aqueous layer is subjected to ultrafiltration (3 k, 13,000 g, 15 min, 3 times) ) To remove unreacted reagents, salts, etc., to synthesize DS25-immobilized magnetic gold-silver nanoparticles (DS25-MSGNP).

  The sugar chain ligand complex was prepared by adding 1.2 equivalents of a linker compound represented by the chemical formula (14) to dextran sulfate sodium salt (DS25 · Na) in a mixed solvent of water, methanol, and acetic acid, Was added by adding 10 equivalents of sodium cyanoborohydride, and subjected to reductive amination reaction at 40 ° C. in a dark place for 2 days.

  FIG. 7 is a diagram showing a transmission electron microscope image of DS25-immobilized magnetic gold-silver nanoparticles and an average particle diameter (13.11 nm) measured by observing 20 particles with a transmission electron microscope. The center diagram in FIG. 7 is an enlarged view of the left diagram in FIG.

  FIG. 8 is a diagram showing the particle size distribution of DS25-immobilized magnetic gold-silver nanoparticles and the average particle size (27.56 ± 7.65 nm) measured by the dynamic light scattering method.

  FIG. 9 shows an ultraviolet-visible absorption spectrum of the DS25-immobilized magnetic gold-silver nanoparticles. The vertical axis represents the absorbance, and the horizontal axis represents the wavelength.

[Capture of influenza virus by magnetic gold-silver nanoparticles immobilized on sugar chains and concentration by magnetic separation]
Virus particles were captured using DS25-MSGNP prepared as described above among the sugar chain-immobilized magnetic metal nanoparticles. A DS25-MSGNP solution (10 μl) was mixed with an influenza virus dilution (500 μl) obtained by diluting a culture supernatant of avian influenza A (DUCK A / spot-billed duck / kagoshima / KU57 / 2015 (H11N9)) with PBS. .

  The mixed solution was allowed to stand on a magnet stand (TAKARA Magnetic Stand (6 tubes)) for 23 hours to perform magnetic separation. After the magnetic separation, the supernatant of the mixed solution was removed, and the precipitate subjected to magnetic separation using DS25-MSGNP and a magnet was collected.

[Disruption of Sugar Chain-Immobilized Magnetic Gold / Silver Nanoparticle-Virus Conjugate and Virus Detection by Real-Time Quantitative RT-PCR]
To the precipitate obtained by the magnetic separation, 10 μl of a 0.1% SDS solution was added to crush the virus particles to obtain a precipitate fraction. As a control, 10 μl of each of the virus solution in which the sugar chain-immobilized magnetic gold-silver nanoparticles were not used and the supernatant fraction after magnetic separation were collected, and 10 μl of a 0.1% SDS solution was added. Fractions were prepared.

  For the precipitated fraction and the control fraction, 2 μl of each of the obtained solutions was added to 23 μl of a reagent for PCR, and subjected to real-time quantitative RT-PCR. As a reagent, One Step SYBR PrimeScript RT-PCR Kit II (Takara Bio, product code RR086A) was used.

  The reagents per reaction were 1 × 2 Step SYBR RT-PCR Buffer 4, 12.5 μl, PrimeScript 1 Step Enzyme Mix2, 1 μl, PCR Forward Primer (10 μM), 1 μl of PCR Reverse Primer, and 1 μm of PCR Reverse Primer. 5.5 μl and 6.25% Tween 20 mixed in 2 μl were used.

  As a real-time quantitative RT-PCR device, Thermal Cycler Dice Real Time System II (manufactured by Takara Bio) was used.

  As primers, Type A / MPgene (217-236) Forward GGACTGCAGCGTAGGACGCTT (20 bp, SEQ ID NO: 1) which is a primer for detecting the influenza Type A / M gene and Type A / MP gene (382-405) Reverse CATYCTGTGATGATCATGTGATCATGTGATC ) Was used to amplify the 188 bp M protein region of the influenza A virus RNA.

  The conditions of RT-PCR were as follows: reverse transcription reaction was set at 45 ° C. for 5 minutes, initial thermal denaturation treatment was set at 95 ° C. for 10 seconds, and PCR cycle was set at 95 ° C. for 5 seconds and 60 ° C. for 30 seconds, and 45 cycles were performed. .

  Table 2 shows the Ct value and Tm value of the precipitate fraction magnetically separated by capturing the virus from the RNA amplification curve obtained by RT-PCR. As a result, the Ct value (26.17) of the precipitated fraction using the sugar chain-immobilized magnetic gold-silver nanoparticles (DS25-MSGNP) was the sample without the sugar chain-immobilized magnetic gold-silver nanoparticles (DS25-MSGNP). 3.88 cycles earlier than the Ct value of (30.05) and 4.52 cycles earlier than the supernatant fraction. The plasmid is a positive control for the target gene.

  This indicates that the virus solution magnetically separated with DS25-MSGNP could be adjusted to 14.72 times and 22.94 times the virus concentration of the sample solution and the supernatant fraction without DS25-MSGNP, respectively. Indicated.

[Example 3]
[Capture of Porcine Reproductive and Respiratory Disorder Syndrome Virus (PRRSV) in Oral Fluid by Sugar Chain-Immobilized Magnetic Gold / Silver Nanoparticles and Concentration by Magnetic Separation]
PRRSV particles were captured using DS25-MSGNP prepared in Example 2. PRRSV, a clinical isolate, was cultured using alveolar macrophage cells isolated from piglets, and the supernatant was diluted 300-fold, 1000-fold, and 10000-fold with PBS (containing 1% PS).

  Oral fluid (saliva) collected by a rope method from a group of about 60 days old pigs (about 80) was diluted twice with PBS (containing 1% PS), and further diluted with PBS (1%) containing 10% DTT (dithiothereitol). % PS).

300 μL of each virus diluent and oral fluid diluent were mixed and centrifuged (10,000 G, 30 seconds) to obtain 500 μL of a supernatant from which solids were omitted. To this, a DS25-MSGNP solution (10 μl) was mixed, and about 10 mg of the second magnetic substance was further added, followed by mixing by pipetting several times to obtain a mixed solution. As the second magnetic material, triiron tetroxide coated with silica was used. The particle diameter of the second magnetic body was 10 to 40 μm,
Next, the mixed solution was allowed to stand for 5 seconds on a magnet stand (TAKARA Magnetic Stand (6 tubes)) to perform magnetic separation. After the magnetic separation, the supernatant of the mixed solution was removed, and the precipitate subjected to magnetic separation using DS25-MSGNP and a magnet was collected.

[Disruption of Sugar Chain-Immobilized Magnetic Gold / Silver Nanoparticle-Virus Conjugate and Virus Detection by Real-Time Quantitative RT-PCR]
To the precipitate obtained by the magnetic separation, 10 μl of a 0.1% SDS solution was added to crush the virus particles to obtain a precipitate fraction. Each 2 μl of the obtained solution was added to 23 μl of a reagent for PCR, and subjected to real-time quantitative RT-PCR. As a reagent, One Step SYBR PrimeScript RT-PCR Kit II (Takara Bio, product code RR086A) was used.

  Reagents per reaction were 12.5 μl of 2 × One Step SYBR RT-PCR Buffer 4, 1 μl of PrimeScript 1step Enzyme Mix2, 1 μl of PCR Forward Primer (10 μM), and 1 μl of PCR Reverse Primer with 1 μm of PCR Reverse Primer. Was mixed with 5.5 μl and 6.25% Tween 20 in 2 μl.

  As a real-time quantitative RT-PCR device, Thermal Cycler Dice Real Time System II (manufactured by Takara Bio) was used.

  As a primer, Forward ATTCTGGCCCCTGCCCACCA (20 bp, SEQ ID NO: 3) and Reverse TGCCACCCAACACGAGGGC (18 bp, SEQ ID NO: 4) were used to amplify the 151 bp M protein coding region of PRSSV RNA.

  The conditions of RT-PCR were as follows: reverse transcription reaction was set at 45 ° C. for 5 minutes, initial thermal denaturation treatment was set at 95 ° C. for 10 seconds, and PCR cycle was set at 95 ° C. for 5 seconds and 60 ° C. for 30 seconds, and 45 cycles were performed. .

  Table 3 shows the Ct value and Tm value of the precipitate fraction magnetically separated by capturing the virus from the amplification curve of the cDNA obtained by RT-PCR. Since the difference in the amount of virus concentrated and purified from PRRSV and oral fluid diluted 300-fold, 1000-fold, and 10,000-fold appears as a difference in the number of PCR cycles, the difference between the 300-fold diluted solution and the 1000-fold diluted solution The difference between the Ct values was 2.3 and the virus load was 4.9-fold. In addition, the difference in Ct value between the 1000-fold diluted solution and the 10,000-fold diluted solution was 3.22, and the virus load was 9.3-fold.

  Simply, the difference in the amount of virus between the 300 times diluted solution and the 1000 times diluted solution is 3.3 times, and the difference in the virus amount between the 1000 times diluted solution and the 10,000 times diluted solution is 10 times. is there. Therefore, it was suggested that virus concentration by magnetic separation using DS25-MSGNP was efficiently performed even from a solution containing oral fluid.

  At medical institutions, farms, and livestock farms, unexpected viral diseases can occur. However, in order to prevent enormous damage, it is required to detect a trace amount of virus and prevent epidemics. On the other hand, not all institutions have ultracentrifuges, which are large devices for concentrating trace viruses, and it is difficult to detect dilute viruses from body fluids using conventional detection kits and PCR methods. is there.

  The present invention utilizes the binding property between a sugar chain and a virus and captures virus particles from a dilute virus solution by using a second magnetic substance in combination, thereby efficiently concentrating the virus particles. It can be used for sensitivity tests and can prevent economic damage in the livestock, bio and pharmaceutical industries.

Claims (6)

With an amino group at one end of a hydrocarbon chain or a hydrocarbon derived chain that is a main chain, a linker compound having a sulfur atom at the other end, a sugar chain, and a magnetic nanoparticle,
The amino group is bonded to the reducing end of the sugar chain, the sulfur atom is bonded to the magnetic nanoparticle,
The magnetic nanoparticles contain gold and / or silver, and
A sugar chain-immobilized magnetic metal nanoparticle, wherein the surface of the magnetic nanoparticle is coated with gold and / or silver.   The sugar chain-immobilized magnetic metal nanoparticles according to claim 1, wherein the virus captures the virus by binding the sugar chain to a protein on the surface of the virus particle.   A method for concentrating a virus, comprising a step of applying a magnetic force to a mixture obtained by mixing the sugar chain-immobilized magnetic metal nanoparticles according to claim 1 and a specimen containing a virus.   The method according to claim 3, wherein the admixture contains a second magnetic substance having an average particle diameter larger than the sugar chain-immobilized magnetic metal nanoparticles.   The method according to claim 4, wherein the surface of the second magnetic material is coated with silica.   The method according to any one of claims 3 to 5, wherein the viral RNA concentrated by the virus concentration method according to any one of claims 3 to 5 is amplified by real-time quantitative RT-PCR. A virus detection method, comprising amplifying, by real-time quantitative PCR, virus DNA concentrated by the virus concentration method described above.