JP6893644B2 - Sugar chain-immobilized magnetic metal nanoparticles, virus capture, concentration, purification and detection - Google Patents

Description

The present invention relates to sugar chain-immobilized magnetic metal nanoparticles having a uniform particle size, capture of viruses by sugar chain-immobilized magnetic metal nanoparticles, concentration and purification method of captured viruses, and real-time quantitative reverse transcription polymerase chain. It is a technique relating to a method for detecting a highly sensitive virus by a reaction (RT-PCR) or a real-time quantitative polymerase chain reaction (PCR).

A sugar chain is a third life chain after nucleic acids and proteins, and is an important molecule for living organisms like nucleic acids and proteins. In particular, viruses are known to recognize sugar chains on the cell surface and infect them. For example, it is known that influenza virus recognizes a sialic acid structure having a specific binding mode on the surface layer of a target cell as a host, inserts RNA having its own genetic information into the host cell, and proliferates. ..

On the other hand, in recent years, there are concerns about the emergence of highly pathogenic viruses and mutant viruses that cause a global pandemic, and in the production of livestock and livestock products such as bird flu and pig breeding / respiratory disorder syndrome. Affected viral diseases have also been reported. Therefore, development of virus capture technology for virus research and early diagnosis technology for viral diseases for treatment is 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, detection of antigens, separation / purification, and the like. In addition, analytical reagents such as drug delivery systems (DDS) using nanoparticles on which drugs or genes are immobilized, markers for analysis using labeled nanoparticles, and tracers are also widely used.

Due to its magnetic nature, magnetic nanoparticles can be used for cell purification, biomarkers, etc. because the state of the particles can be controlled by an appropriate external magnetic field. Further, in the medical field, magnetic particles are highly practical elements having excellent features such as being able to image living organisms, cells, etc. when used as a sensitizer such as magnetic resonance imaging. .. In addition, silver is a highly versatile metal that can be bonded to sulfur atoms like metals such as gold and can be complexed with biomolecules and organic compounds.

For example, Patent Document 1 describes a method in which an antigen protein on the surface of a virus particle that specifically binds to a sugar chain is separated by a sugar chain-immobilized gold nanoparticles, and the antigen protein is used as an immune antigen to obtain an antibody. Are listed. Further, Patent Document 2 describes a method for concentrating a virus using sugar chain-immobilized magnetic metal nanoparticles and a predetermined second magnetic substance.

Japanese Unexamined Patent Publication No. 2013-159567 Japanese Unexamined Patent Publication No. 2011-45358

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 detection, isolation, purification, etc. of sugar chain-binding proteins. ..

The 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 reddish purple color. Therefore, the binding activity with sugar chain-binding proteins such as lectins can be visually measured by using color change, agglutination reaction, etc. as indicators. It can be analyzed with.

However, isolation and purification of the components bound to the sugar chain-immobilized gold nanoparticles require a large-scale device such as an ultracentrifugator.

Sugar chain-immobilized magnetic gold nanoparticles (for example, Patent Document 2) do not require a large-scale device when bound to a target, and isolate and purify binding components such as proteins or viruses only by magnetic separation. be able to. This technology is applied not only for research, but also in bedside medical care, farms, livestock farms, etc. as a simple test / diagnostic tool for bacteria and viruses.

It has been found that sugar chain-immobilized magnetic gold nanoparticles can 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 a problem that the particle size and dispersion stability after production change depending on the type of sugar chain to be immobilized.

So far, manufacturing methods have already been established for "sugar chain-immobilized gold nanoparticles" and "sugar chain-immobilized magnetic gold nanoparticles" whose constituent atoms are gold, and their use in research institutes, medical institutions, etc. has been expanded. There is. Among them, the sugar chain-immobilized magnetic gold nanoparticles have high versatility, but have problems such as the particle size and dispersion stability changing depending on the type of sugar chain to be immobilized, the manufacturing technique, and the like. .. That is, there is a problem in controlling the particle size.

Therefore, in the present specification, various sugar chains can be immobilized, the dispersion stability is high regardless of the type of sugar chain, and the magnetism of the sugar chain-immobilized magnetic metal nanoparticles is utilized. A novel sugar chain-immobilized magnetic metal nanoparticles capable of concentrating a virus and a method for preparing the same, a method for capturing, concentrating, and purifying binding components using the novel sugar chain-immobilized magnetic metal nanoparticles, and a method for detecting a virus will be described.

That is, in one embodiment of the present invention, the surface of magnetic nanoparticles is coated with gold and / or silver capable of immobilizing sugar chains via thiols similar to gold, and a metal having magnetism to maintain magnetism is used. This is a method for preparing inherent sugar chain-immobilized magnetic metal nanoparticles having gold and / or silver, a method for preparing sugar chain-immobilized magnetic metal nanoparticles, and a technique for capturing and separating a virus in a sample.

The present invention includes the following aspects.

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

<2> The sugar chain-immobilized magnetic metal nanoparticles according to <1>, wherein the sugar chain captures a virus by binding to a protein on the surface of the virus particle.

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

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

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

<6> The RNA of the virus concentrated by the virus concentration method according to any one of <3> to <5> can be amplified by real-time quantitative RT-PCR, or <3> to <5. > The virus detection method, which comprises amplifying the virus DNA concentrated by the virus concentration method according to any one of> by real-time quantitative PCR.

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

Further, since the RNA in the virus can be purified and extracted easily in a short time by simply capturing and crushing the virus particles using the sugar chain-immobilized magnetic metal nanoparticles. 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, which 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 sugar chain to be immobilized, and can quickly detect and identify viruses. It has the effect of being able to be performed easily and with high sensitivity.

A sugar containing α2-3 bound sialyllactose (2,3SL), dextran sulfate 2500 (DS25), β1-3 bound N-acetylgalactosamine-galactose, or α1-6 bound mannose-glucose and a linker compound. It is a figure which shows the structure of a chain ligand complex. It is a figure which shows the transmission electron microscope image and the average particle diameter of 2,3-sialyll lactose-immobilized magnetic silver nanoparticles. It is a figure which shows the dynamic light scattering measurement value, and the average particle diameter of 2,3-sialyll lactose-immobilized magnetic silver nanoparticles. It is a figure which shows the ultraviolet-visible absorption spectrum of 2,3-sialyll lactose-immobilized magnetic silver nanoparticles. It is a figure which shows the transmission electron micrograph of the virus particle captured by 2,3-sialyll lactose-immobilized magnetic silver nanoparticles. It is a figure which shows the amplification curve of real-time quantitative RT-PCR of RNA possessed by the virus captured by 2,3-sialyll lactose-immobilized magnetic silver nanoparticles. It is a figure which shows the transmission electron micrograph and the average particle diameter of DS25 immobilized magnetic gold silver nanoparticles. It is a figure which shows the dynamic light scattering measurement value of the DS25 immobilized magnetic gold and silver nanoparticles, and the average particle diameter. It is a figure which shows the ultraviolet-visible absorption spectrum of the DS25 immobilized magnetic gold and silver nanoparticles.

Embodiments of the present invention will be described below, but the present invention is not limited thereto. Hereinafter, by "sugar chain-immobilized magnetic metal nanoparticles", "method for concentrating virus by 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 an embodiment of the present invention are a linker compound or sugar chain having an amino group at one end of a hydrocarbon chain or a hydrocarbon induction chain as a main chain and a sulfur atom at the other end. , And the amino group is bonded to the reducing end of the sugar chain, the sulfur atom is bonded to the magnetic nanoparticles, and the magnetic nanoparticles contain gold and / or silver. Further, the magnetic nanoparticles are characterized in that their surfaces are coated with gold and / or silver.

As used herein, the term "sugar chain-immobilized magnetic metal nanoparticles" is intended to be a combination of a sugar chain ligand complex described in detail below and magnetic nanoparticles. A "sugar chain ligand complex" is a sugar capable of specifically interacting with a linker compound capable of binding to magnetic nanoparticles containing gold and / or silver and a surface protein of a virus to be captured. It is a compound composed of chains. Therefore, the sugar chain ligand complex is required not to form a non-specific interaction based on hydrophobicity with a substance such as a protein.

The sugar chain-immobilized magnetic metal nanoparticles according to an 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. Shown. The surface of the sugar chain-immobilized magnetic metal nanoparticles is modified with a sugar chain ligand complex, and the average particle size is preferably 1 to 200 nm, more preferably 1 nm to 50 nm.

Further, the sugar chain-immobilized magnetic metal nanoparticles according to the embodiment of the present invention are formed on the sugar chain ligand complex, that is, an amino group existing at one end of a linker compound having a hydrocarbon chain or a hydrocarbon induction chain. Sugar chain-immobilized magnetic metal nanoparticles that are bonded to a sugar chain having a reducing end and are bonded to silver, gold, or other metal in a hydrocarbon structure containing a sulfur atom present at the other end of the linker compound. Is.

The sugar chain-immobilized magnetic metal nanoparticles according to the embodiment of the present invention can capture a virus by binding the sugar chain to a protein on the surface of the 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 induction chain as a main chain, and a sulfur atom at the other end.

The above-mentioned "hydrocarbon-induced chain" means that a part of the carbon-carbon bond (CC bond), which is the main chain structure of the hydrocarbon chain, is a carbon-nitrogen bond (CN bond) or a carbon-oxygen bond (CC bond). It refers to a bond that may be replaced with a CO bond), an amide bond (CO-NH bond), or the like.

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

Since the linker compound has an amino group at the end of the hydrocarbon chain or the hydrocarbon induction 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, etc.), an aromatic amino group, or an unmodified amino group. Good.

Above all, the amino group is preferably an aromatic amino group. Under the optimum conditions of the reductive amination reaction, pH 3-4, it is necessary that the amino group is not protonated. Therefore, it is preferable that the unshared electron pair is an aromatic amino group that can exist on the nitrogen atom even under the condition of pH 3 to 4 due to the conjugate with the aromatic.

A sugar chain having a reducing end is introduced into the amino group of the linker compound in the sugar chain ligand complex which is a complex of the linker compound and the sugar chain. That is, the sugar chain ligand complex shows a structure in which a sugar chain having a reducing end is bound to the linker compound 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 the 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. By continuing to reduce the Schiff base formed by this reaction, it is possible to easily introduce a sugar chain into the amino group.

The above-mentioned "sugar chain having a reducing end" is a saccharide capable of forming a ketone or aldehyde having a reducing property at the molecular end. 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 the reducing end is a reducing sugar chain. The sugar chain having a reducing end may be a commercially available product or a natural product, and a synthetic product or a product prepared by decomposing a commercially available or natural polysaccharide chain may be used. it can.

Examples of sugar chains having a reducing end include glucose, galactose, mannose, maltose, isomaltose, lactose, panose, cellobiose, melibiose, mannooligosaccharide, chito-oligo, etc., laminari-oligosaccharide, glucosamine, N-acetylglucosamine, glucuronic acid, heparin, etc. Examples thereof include heparan sulfate, chondroitin sulfate, dermatane sulfate, N-acetylgalactosamine, and fucose.

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

For example, 2 and 3SL are sugar chains required for avian influenza infection, and DS25 is highly negatively charged by a large number of sulfate groups and exhibits strong binding to the surface of virus particles such as influenza and herpes. .. In addition, 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 sugar chain-immobilized magnetic metal nanoparticles.

Dextran sulfate is a polyanionic dextran derivative consisting mainly of a glucose skeleton bound by an α1-6 bond and is synthesized from various high-purity dextran fractions. Of this dextran sulfate, DS25 retains a large number of sulfate groups necessary for binding to a virus by adjusting the molecular weight to about 2500, and controls the particle size of the product nanoparticles to about 10 nm. It is possible. The "product nanoparticles" are sugar chain-immobilized magnetic metal nanoparticles in which the ligand complex is bound to the magnetic nanoparticles. As the DS25, one prepared by decomposing a commercially available or natural polysaccharide chain can be used.

In addition, the linker compound is a metal with a gold-sulfur (Au-S) bond, a silver-sulfur (Ag-S) bond, or another metal by means of a sulfur atom provided at the other end of the hydrocarbon chain or the hydrocarbon induction chain. -Sulfur (MS) bonds 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 can form metal-sulfur bonds (eg Au-S bonds, Ag-S bonds) with surface metals (gold and / or silver) in magnetic nanoparticles to strengthen the bond with the metal. it can. As a result, in the above linker compound, a sugar chain is bonded to the amino group and the sulfur atom is bonded to the magnetic nanoparticles, so that the sugar chain molecules can be aggregated and arranged on the magnetic nanoparticles.

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 sulfur of the SH group. Therefore, the sugar chain can be easily immobilized on the surface of the magnetic nanoparticles. Furthermore, the high hydration of the sugar chain thus bound on the magnetic nanoparticles makes it possible to stabilize the magnetic nanoparticles in a colloidal state.

As the linker compound, conventionally known linker compounds, for example, the linker compounds developed by the present inventors, the linker compounds described in WO2005 / 077965, and US Pat. No. 7,320,867B2 can be used. 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 independently integers of 0 or more and 6 or less, and X has an amino group or an aromatic amino group at the terminal and a carbon-nitrogen bond in the main chain. It is a structure containing one chain, two chains or three chains of a hydrocarbon induction chain which may be present, Y is a hydrocarbon structure containing a sulfur atom or a sulfur atom, and Z is a carbon-carbon bond or carbon. -Structure with a straight chain or a branch with 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 a heterocycle containing an element other than carbon. The 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 independently integers of 0 or more and 6 or less.)

(In the chemical formula (6), n ³ is an integer of 1 or more and 100 or less.)

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

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

The linker compound may be 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 structures represented may be more preferably used:

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

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 above-mentioned linker compound can be obtained by condensing the carboxyl group and the aromatic amino group of the thioctic acid by the condensation reaction of the thioctic acid and the aromatic amino group to form an amide bond. 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 whose aromatic amino group terminal is protected by a protecting group, and the protective group at the aromatic amino group terminal is deprotected.

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

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

The above thioctic acid

It has a structure represented by. Further, the amine compound is not particularly limited as long as it has an aromatic amino group 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) does not react by the condensation reaction. Such a protecting group is not particularly limited, but is, for example, a t-butoxycarbonyl group (-COOC (CH ₃ ) ₃ groups; hereinafter also referred to as a Boc group), a benzyl group, and an allylcarbamate group (-). COOCH ₂ CH = CH ₂ , Alloc group) and the like can be mentioned.

A sugar chain ligand complex can be produced using the linker compound as described above. For example, by introducing a sugar chain into a 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 X of the chemical formula (1)) and a sugar chain. A sugar chain ligand complex can be obtained.

FIG. 1 shows α2-3 bound sialyllactose (2,3SL), dextran sulfate 2500 (DS25), β1-3 bound N-acetylgalactosamine-galactose, or α1-6 bound mannose-glucose and a linker compound. It is a figure which shows the structure of the sugar chain ligand complex containing. 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 thus obtained is composed of sulfur (S) contained in the linker compound (for example, contained in the above Y of the chemical formula (1)) and gold and / or silver contained in the magnetic nanoparticles. It can be firmly and easily immobilized on magnetic nanoparticles through a metal-sulfur bond formed between the two. Therefore, the sugar chain ligand complex can be easily immobilized on the surface of the magnetic nanoparticles.

[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 for cell purification, biomarkers, and the like. Further, magnetic nanoparticles are highly practical elements having excellent features such as being able to image living organisms or cells when used as a sensitizer for nuclear magnetic resonance imaging in the medical field. Is.

The magnetic nanoparticles are metal nanoparticles having magnetism, and are intended to be dispersed in an aqueous solution to form a colloidal solution. Therefore, 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.

When the average particle size 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. Further, since the capture efficiency is dramatically increased when nanoparticles smaller in size than the target virus particles are used, the average particle size of the magnetic nanoparticles is more preferably 1 nm to 50 nm.

As used herein, the term "particle size" is intended to be the diameter of the maximum inscribed circle with respect to the two-dimensional shape of a particle when the particle is observed with a transmission electron microscope. For example, if the two-dimensional shape of a particle is substantially circular, the diameter of the circle is intended, and if it is substantially elliptical, the minor axis of the rectangle is intended, and it is substantially square. In the case, the length of the side of the square is intended, and in the case of a substantially rectangular shape, the length of the short side of the rectangle is intended.

The "average particle size" means the average value of the particle size of a plurality of particles. In the present specification, whether or not the average particle size has a value in a predetermined range is determined by observing 20 particles with a transmission electron microscope and measuring the particle size of each particle. It was confirmed by obtaining the average value of the above particle sizes or by the dynamic light scattering method. Furthermore, it was confirmed by the dynamic light scattering method that the particle size of the sugar chain-immobilized magnetic metal nanoparticles in which the sugar chains were immobilized on the magnetic nanoparticles was increased by the amount of the sugar chains.

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

Above all, since the magnetism is strong, the first magnetic material is preferably iron oxide and cobalt. In this specification, iron oxide refers to an oxide of trivalent iron. Magnetite is a divalent and trivalent iron oxide (Fe ₂ ⁺ Fe ^{3 +} 2 O ₄ ).

The magnetic nanoparticles contain gold and / or silver. Silver is a highly versatile metal that can be bonded to a sulfur atom and can be complexed with a biomolecule, an organic compound, or the like, like a metal such as gold.

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

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

Further, 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 , ZrO _{2 and the} like.

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 from the center, the first magnetic material, and then gold and / or silver.

For example, Japanese Patent Application Laid-Open No. 2016-153519 describes a metal composite having a metal core such as gold, a first shell which is a magnetic material for coating the metal core, and a second shell such as gold for coating the first shell. The particles are described, and it is described that the metal complex 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 the 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 containing the above-mentioned metal complex particles. Further, no relation between the above-mentioned metal complex particles and the above-mentioned problem of the present invention has been suggested. That is, by using the above magnetic nanoparticles, the particle size, dispersion stability, etc. change depending on the type of sugar chain to be immobilized, the manufacturing technique, etc., that is, the conventional sugar chain-immobilized gold nanoparticles and sugar. It is a unique finding found by the present inventor that the problems of chain-immobilized magnetic gold nanoparticles can be solved.

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

[4. Preparation method of sugar chain-immobilized magnetic metal nanoparticles]
The sugar chain-immobilized magnetic metal nanoparticles according to an embodiment of the present invention are obtained by mixing a solution containing the magnetic nanoparticles with a linker compound (sugar chain ligand complex) bonded to a sugar chain by a reducing agent treatment. be able to. The sugar chain-immobilized magnetic metal nanoparticles are formed by binding each S atom of the SS bond of the sugar chain ligand complex to gold, silver, or another metal on the magnetic nanoparticles by a metal-sulfur bond. It is formed.

The sugar chain-immobilized magnetic metal nanoparticles show 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, more preferably in the range of 1 nm to 50 nm.

When the average particle size is 1 nm or more, it is possible to manufacture at low cost, which is practical. Further, when the average particle size is 200 nm or less, the change in the dispersion stability of the sugar chain-immobilized magnetic metal nanoparticles with time is easily suppressed, which is preferable.

Further, when the average particle size is 1 nm to 50 nm, nanoparticles having a size smaller than the target virus particles are used, which is preferable because the virus capture efficiency is dramatically increased.

As a method for binding 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.

Specific examples of the method for preparing sugar chain-immobilized magnetic metal nanoparticles include a solution containing a sugar chain ligand complex in a solution of silver-containing magnetic nanoparticles (hereinafter, also referred to as magnetic silver nanoparticles). Can be mentioned as a method of adding. Examples of the sugar chain ligand complex include a sugar chain ligand complex in which 2,3-sialyllactose and a linker compound are bound by a reducing agent treatment. Thereby, the SS bond of the sugar chain ligand complex can be converted into a metal-sulfur bond on the magnetic nanoparticles to obtain sugar chain-immobilized magnetic metal nanoparticles.

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, as in the case of using potassium instead of sodium, salts having a cation component different from that of 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, and examples thereof include ultrapure water, methanol, ethanol, propanol, and a mixed solvent thereof. Can be done.

In addition, the sugar chain-immobilized magnetic metal nanoparticles obtained by the above mixing are subjected to washing by ultrafiltration, centrifugation, etc., and by removing components such as low-molecular-weight salts, stable sugar chain immobilization in a solution state is performed. Magnetic metal nanoparticles can be obtained.

The mixing ratio of gold, silver, the above-mentioned sugar chain ligand complex, and the reducing agent used for preparing the sugar chain-immobilized magnetic metal nanoparticles is not particularly limited, but the sugar chain-immobilized magnetic metal nanoparticles are used. The final concentration of gold and / or silver in the contained solution is preferably 0.1 to 1 mM, respectively.

The concentration of the sugar chain ligand complex is preferably 0.1 to 10 mM at the final concentration in the solution containing the sugar chain-immobilized magnetic metal nanoparticles. The concentration of the reducing agent used is preferably 1 to 10 mM at 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-mentioned 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 have a feature of being dispersed in an aqueous solution. That is, this production method is also a method for preparing a colloidal solution of sugar chain-immobilized magnetic metal nanoparticles.

[5. Virus capture and concentration method using sugar chain-immobilized magnetic metal nanoparticles]
The method for capturing and concentrating a virus with sugar chain-immobilized magnetic metal nanoparticles according to an embodiment of the present invention allows a solution containing sugar chain-immobilized magnetic metal nanoparticles to interact with a solution containing virus particles. After that, the bonded body and the non-bonded body are separated by magnetism.

That is, the method for concentrating a virus according to an embodiment of the present invention includes a step of applying a magnetic force to an admixture obtained by mixing sugar chain-immobilized magnetic metal nanoparticles and a sample containing a virus. A high-concentration virus solution can be obtained by subjecting the sugar chain-immobilized magnetic metal nanoparticles according to the embodiment of the present invention in which a virus is captured to magnetic separation and removing the supernatant.

The sample is not particularly limited as long as it can be brought into contact with the sugar chain-immobilized magnetic metal nanoparticles. For example, saliva, nasal mucosa, plant, animal body fluid and the like can be mentioned.

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

The above-mentioned "solution containing sugar chain-immobilized magnetic metal nanoparticles" is intended to be a colloidal solution in which sugar chain-immobilized magnetic metal nanoparticles according to an embodiment of the present invention are dispersed in a liquid. The solution may contain other salts or the like as long as it contains sugar chain-immobilized magnetic metal nanoparticles. As the liquid, for example, ultrapure water or a buffer solution can be used.

Further, the above-mentioned "solution containing virus particles" (sample containing virus) is intended to be a colloidal solution in which a virus is dispersed in a liquid in the present invention. Further, the virus is intended to be a virus that exhibits binding property to the sugar chain ligand complex of the sugar chain-immobilized magnetic metal nanoparticles according to the embodiment of the present invention. The particle size of the virus particles is preferably 10-200 nm.

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

In the mixture obtained by mixing the sugar chain-immobilized magnetic metal nanoparticles and the sample containing the virus, the sugar chain-immobilized magnetic metal nanoparticles and the virus interact with each other to obtain the sugar chain-immobilized magnetic metal. There are sugar chains of nanoparticles, conjugates that are components to which viruses are bound, and non-conjugates that are components that are not bound to sugar chain-immobilized magnetic metal nanoparticles. By applying a magnetic force to the admixture, the conjugate can be separated from the non-conjugate. As a result, the virus can be concentrated. The components separated and concentrated in the above step are designated as nanoparticles-virus conjugates.

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

The second magnetic material is preferably, but not limited to, 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, more preferably in the range of 1 nm to 50 nm. The average particle size of the sugar chain-immobilized magnetic metal nanoparticles in which the sugar chain is fixed to 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 size of the second magnetic material is not particularly limited as long as the upper limit of the average particle size is 100 μm and is larger than the average particle size of the sugar chain-immobilized magnetic metal nanoparticles. However, it is preferably 100 nm or more and 100 μm or less, more preferably 100 nm or more and 50,000 nm or less, and further preferably 1000 nm or more and 10000 nm or less.

By using the second magnetic material, a concentration effect comparable to that when centrifugation is used can be obtained more efficiently than when only the first magnetic material is used. Then, by setting the average particle size of the sugar chain-immobilized magnetic metal nanoparticles in the above range and adjusting the average particle size of the second magnetic material as in the above range, the average particle size of both is optimized. Guess. As a result, by applying a magnetic force to the admixture containing the sugar chain-immobilized magnetic nanoparticles, the second magnetic substance, and the virus-containing sample, a small amount of virus in the sample is centrifuged. It can be concentrated more efficiently, easily and safely without the need for.

The weight ratio of the sugar chain-immobilized magnetic metal nanoparticles to the second magnetic material is preferably ^{1: 1 × 10 3} to 1: 1 × 10 ^11. The concentration efficiency can be further improved by adjusting the ratio of the amount of the sugar chain-immobilized magnetic metal nanoparticles having the average particle size as described above to the amount of the second magnetic material used in such a range. ..

Regarding the above-mentioned "applying magnetic force", the method of applying the magnetic force is not particularly limited. For example, a magnetic force can be applied to the admixture 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 admixture. The strength of the magnetic force is preferably 100 to 500 millistella in order to obtain the same concentration efficiency as when centrifugation is used.

When a second magnetic material is used, at the end of the step of applying a magnetic force to the admixture, the coloration exhibited by the dispersion of the sugar chain-immobilized magnetic metal nanoparticles in the liquid is sugar chain-immobilized. Judgment may be made based on the phenomenon that the magnetic metal nanoparticles are thinned by being adsorbed on the second magnetic material.

When the second magnetic material is not used, the end time of the step of applying the magnetic force to the admixture is based on the phenomenon that the sugar chain-immobilized magnetic metal nanoparticles to which the virus is bound are accumulated by the magnetic force and the coloring becomes lighter. As a result, it may be judged visually.

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

The virus to be concentrated is not particularly limited. For example, influenza virus, simple herpesvirus, sexually transmitted disease herpesvirus, AIDS virus, hepatitis B virus, hepatitis C virus, vesicular virus (VZV), cytomegalovirus (CMV), adult T-cell leukemia virus (HTLV-1), Lentivirus, Koiherpesvirus, adenovirus, norovirus, rotavirus and the like can be mentioned.

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

Further, in the method for concentrating a virus according to an embodiment of the present invention, the surface of the second magnetic material may be coated with silica. As an embodiment of the coating, for example, an embodiment in which a silica film is formed on the surface of a second magnetic material containing iron oxide as a component can be mentioned. According to this configuration, it is possible to prevent the nucleic acid from being adsorbed on the second magnetic substance, so that the recovery rate of the nucleic acid derived from the virus can be significantly improved.

As a method of coating the surface of the second magnetic material 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.} Examples include methods of treating with excess amounts of tetraethyl orthosilicate and ammonia in ethanol. Further, as a method for confirming that the surface of the second magnetic material is coated with silica, observation by composition analysis (EPMA) and scanning electron microscope (SEM) can be mentioned.

[6. High-sensitivity virus detection method by real-time quantitative RT-PCR using nanoparticles-virus conjugate or real-time quantitative PCR]
The virus detection method according to the embodiment of the present invention is to amplify the RNA of the virus concentrated by the above-mentioned virus concentration method by real-time quantitative RT-PCR, or to concentrate by the above-mentioned virus concentration method. It is characterized in that the DNA of the virus is amplified by real-time quantitative PCR.

In the high-sensitivity virus detection method by real-time PCR using the nanoparticles-virus conjugate, the nanoparticles-virus conjugate separated and concentrated by the above step is crushed and RNA or DNA contained in the virus is real-time quantitative RT-PCR. Amplify by.

The sugar chain-immobilized magnetic metal nanoparticles that have captured the virus are particularly bound to proteins on the surface layer of the virus particles. Therefore, it is assumed that a large number of virus-derived molecules such as nucleic acids and proteins are present in the solution after crushing the virus particles. The RNA of the virus bound to the sugar chain-immobilized magnetic metal nanoparticles is amplified by real-time quantitative RT-PCR, or the viral DNA bound to the sugar chain-immobilized magnetic metal nanoparticles is amplified by real-time quantitative PCR. By amplifying, it is possible to determine the presence or absence of a specific virus, the number of copies of the virus, and the like.

In real-time quantitative RT-PCR and real-time quantitative PCR, the Ct (Threshold Cycle) value when the sugar chain-immobilized magnetic metal nanoparticles are not used is obtained, and the Ct value is used by the method according to the embodiment of the present invention. The degree of concentration of the virus was evaluated by obtaining the difference obtained by subtracting the Ct value when concentrated.

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

(1) Magneticity in which a sulfur atom present at the other end of the linker compound is bonded to a sugar chain having a reducing end at an amino group existing at one end of the linker compound having a hydrocarbon-induced chain and has silver as a surface structure. Sugar chain-immobilized magnetic silver nanoparticles characterized by being bonded to particles.

(2) The sugar chain-immobilized magnetic silver nanoparticles according to (1), which captures a virus by binding a sugar chain to a protein on the surface of the virus particle.

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

The present invention will be described in more detail 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 gold-silver nanoparticles", and "influenza with 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 breeding / respiratory disorder syndrome in pig saliva by sugar chain-immobilized magnetic gold and silver nanoparticles", "Concentration by magnetic separation of sugar chain-immobilized magnetic silver nanoparticles-" The explanation will be divided into 6 steps: disruption of virus conjugate and detection of virus by real-time quantitative RT-PCR.

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

The obtained solution is stirred while being heated by a mantle heater under an argon atmosphere, and after the liquid temperature reaches 170 ° C., a mixed solution of oleylamine and toluene (3.0 ml, 2: 1) is added, and an additional 250 is added. Heated to ° C. The mixture contains cobalt (II) acetylacetoneate (70.6 mg, 0.2 mmol, aldrich) and iron (III) acetylacetoneate (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 were washed by performing centrifugation (3,700 g, 5 min) twice after adding excess acetone in order to remove residual organic components, and then concentrated under reduced pressure to make them organic. The solvent was removed. Then, 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 above hexane solution consisted of a sugar chain ligand complex on which 2,3-sialyllactose was immobilized and an aqueous solution of sodium borohydride (final concentrations of 2.5 mM and 1.0 ml, respectively) at 10 mM. In addition to the aqueous solution prepared to pH 12 with an aqueous sodium hydroxide solution, ultrasonic waves were irradiated.

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 ultrafiltered (3 k, 13,000 g, 15 min, 3 times). , Unreacted reagents, salts, etc. were removed to synthesize 2,3-sialyllactose-immobilized magnetic silver nanoparticles (2,3SL-MSNP).

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

FIG. 2 shows a transmission electron microscope image of 2,3-sialyllactose-immobilized magnetic silver nanoparticles (2,3SL-MSNP) and an average particle size (measured by observing 20 particles with a transmission electron microscope). It is a figure which shows 9.24 nm).

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

The dispersity of the average particle size of 2,3SL-MSNP measured by the dynamic light scattering method is less than 30%. Although the data is not shown, the degree of dispersion is lower than that of 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, it is said that 2,3SL-MSNP has a uniform particle size as that of the conventional sugar chain-immobilized gold nanoparticles and sugar chain-immobilized magnetic gold nanoparticles, and has excellent particle size and dispersion stability after production. I can say.

FIG. 4 is a diagram showing an ultraviolet-visible absorption spectrum of 2,3-sialyllactose-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]
Among the sugar chain-immobilized magnetic silver nanoparticles, virus particles were captured using 2,3SL-MSNP prepared as described above. A few SL-MSNP solutions (10 μl) were added to an influenza virus diluted solution (500 μl) obtained by diluting the culture supernatant of type A avian influenza (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 a second magnetic substance, mixed well, and allowed to stand on a magnet stand (TAKARA Magnetic Stand (6tubes)) for 1 minute for magnetic separation. After magnetic separation, the supernatant of the mixed solution was removed, and the precipitate magnetically separated by a few SL-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 2,3-sialyllactose-immobilized magnetic silver nanoparticles. The right figure of FIG. 5 is an enlarged view of a portion surrounded by a frame in the left figure of FIG.

[Crushing of sugar chain-immobilized magnetic silver nanoparticles-virus conjugate and detection of virus by real-time quantitative RT-PCR]
To the precipitate obtained by magnetic separation, 20 μl of a 0.1% SDS solution was added to crush the virus particles to obtain a precipitate fraction. In addition, as a control, 10 μl of each was collected from the virus solution that did not use the sugar chain-immobilized magnetic silver nanoparticles and the supernatant fraction after magnetic separation, and 10 μl of the 0.1% SDS solution was added to the control image. Minutes were prepared.

For the precipitate and control fractions, 2 μl of each of the obtained solutions was added to 23 μl of PCR reagent 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 are 12.5 μl of 2 × One Step SYBR RT-PCR Buffer 4, 1 μl of PrimeScript 1 step Enzyme Mix 2, 1 μl of PCR Forward Primer (10 μM), 1 μl of PCR Forward Primer (10 μM), and 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 apparatus, Thermal Cycler Dice Real Time System II (manufactured by Takara Bio Inc.) was used.

Primers include Type A / MPgene (217-236) Forward GGACTGCAGCGACGCTT (20bp, SEQ ID NO: 1) and TypeA / MP gene (382-405) ReverseCATYCTGCAT (382-405) Reverse CATYCTGTGTGT, which are primers for detecting influenza Type A / M gene. ) Was used to amplify the M protein region 188 bp of the RNA of influenza A virus.

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

FIG. 6 is a diagram showing an amplification curve of real-time quantitative RT-PCR of RNA contained in a virus captured by 2,3-sialyllactose-immobilized magnetic silver nanoparticles. Table 1 shows the Ct values of the precipitated 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 from the Ct value (31.8) of the sample in which the sugar chain-immobilized magnetic silver nanoparticles were not used. It was also confirmed that 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 by 2,3SL-MSNP had a virus concentration of 7.21 times and 22.31 times that of the sample solution and the supernatant fraction without 2,3SL-MSNP, respectively. It was shown that it was ready.

[Example 2]
[Preparation of sugar chain-immobilized magnetic gold and silver nanoparticles]
Silver nitrate (17.0 mg, 0.1 mmol, Wako Pure Chemical Industries, Ltd.) and 1,2-hexadecanediol (260 mg, 1.0 mmol, Tokyo Chemical Industry), oleic acid (2.3 ml, 8.0 mmol, aldrich), oleylamine It was dissolved in (3.2 ml, 10.0 mmol, Tokyo Chemical Industry) and tetraethylene glycol (10 ml, Tokyo Chemical Industry).

The obtained solution is stirred while being heated by a mantle heater under an argon atmosphere, and after the liquid temperature reaches 170 ° C., a mixed solution of oleylamine and toluene (3.0 ml, 2: 1) is added, and an additional 250 is added. Heated to ° C. The mixed solution contains cobalt (II) acetylacetoneate (70.6 mg, 0.2 mmol, aldrich) and iron (III) acetylacetoneate (53.4 mg, 0.2 mmol, aldrich).

Next, by adding a mixed solution (2.0 ml, 1: 1) of oleylamine and toluene in which sodium tetrachloroauric (III) dihydrate (39.7 mg, 0.1 mmol, nacalai tesque) was dissolved. , Hydrophobic magnetic gold and silver nanoparticles containing gold atoms were synthesized.

The obtained hydrophobic magnetic gold-silver nanoparticles were washed by performing centrifugation (3,700 g, 5 min) twice after adding excess acetone in order to remove the remaining organic components, and then concentrated under reduced pressure to make them organic. The solvent was removed. Then, the obtained hydrophobic magnetic gold and silver nanoparticles were dispersed in a hexane solution (200 μl) so as to have a concentration of 20.4 mg / ml.

The hydrophobic magnetic gold-silver nanoparticles containing gold atoms dispersed in the above hexane solution consisted of 10 mM of a sugar chain ligand complex on which DS25 was immobilized and an aqueous solution of sodium borohydride (final concentrations of 2.5 mM and 1.0 ml, respectively). In addition to the aqueous solution prepared to pH 11 with the aqueous sodium hydroxide solution of the above, ultrasonic waves were irradiated.

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 ultrafiltered (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 is a reducing agent containing 1.2 equivalents of the linker compound represented by the chemical formula (14) with respect to sodium dextran sulfate (DS25 / Na) in a mixed solvent of water, methanol and acetic acid. It was prepared by adding 10 equivalents of sodium cyanoborohydride and carrying out a reductive amination reaction at 40 ° C. in the dark for 2 days.

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

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

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

[Capture of influenza virus by sugar chain-immobilized magnetic gold and silver nanoparticles and concentration by magnetic separation]
Among the sugar chain-immobilized magnetic metal nanoparticles, virus particles were captured using DS25-MSGNP prepared as described above. A DS25-MSGNP solution (10 μl) was mixed with an influenza virus diluted solution (500 μl) obtained by diluting the culture supernatant of type A avian influenza (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 (6tubes)) for 23 hours for magnetic separation. After magnetic separation, the supernatant of the mixed solution was removed, and the precipitate magnetically separated by DS25-MSGNP and a magnet was collected.

[Crushing of sugar chain-immobilized magnetic gold and silver nanoparticles-virus conjugate and detection of virus by real-time quantitative RT-PCR]
To the precipitate obtained by magnetic separation, 10 μl of a 0.1% SDS solution was added to crush the virus particles to obtain a precipitate fraction. In addition, as a control, 10 μl each of a virus solution that did not use sugar chain-immobilized magnetic gold and silver nanoparticles and a supernatant fraction after magnetic separation were collected, and 10 μl of a 0.1% SDS solution was added as a control. Fractions were prepared.

For the precipitate and control fractions, 2 μl of each of the obtained solutions was added to 23 μl of PCR reagent 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 are 12.5 μl of 2 × One Step SYBR RT-PCR Buffer4, 1 μl of PrimeScript 1step Enzyme Mix2, 1 μl of PCR Forward Primer (10 μM), PCR Reverse Primer (10 μM) of 1 μl of PCR River Primer (10 μM), and PCR River Primer (10 μM). Was used as a mixture of 5.5 μl and 6.25% Tween 20 in 2 μl.

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

Primers include Type A / MPgene (217-236) Forward GGACTGCAGCGACGCTT (20bp, SEQ ID NO: 1) and TypeA / MP gene (382-405) ReverseCATYCTGCAT (382-405) Reverse CATYCTGTGTGT, which are primers for detecting influenza Type A / M gene. ) Was used to amplify the M protein region 188 bp of the RNA of influenza A virus.

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

Table 2 shows the Ct value and Tm value of the precipitate fraction obtained by capturing and magnetically separating the virus from the RNA amplification curve obtained from RT-PCR. As a result, the Ct value (26.17) of the precipitation fraction using the sugar chain-immobilized magnetic gold-silver nanoparticles (DS25-MSGNP) was the sample in which the sugar chain-immobilized magnetic gold-silver nanoparticles (DS25-MSGNP) were not used. It was confirmed that it was 3.88 cycles earlier than the Ct value (30.05) and 4.52 cycles earlier than the supernatant fraction. The plasmid is a positive control for the target gene.

From this, it was found that the virus solution magnetically separated by DS25-MSGNP could be prepared to have a virus concentration of 14.72 times and 22.94 times, respectively, as compared with the sample solution and the supernatant fraction without DS25-MSGNP. Shown.

[Example 3]
[Capture of pig breeding / respiratory disorder syndrome virus (PRRSV) in oral fluid by sugar chain-immobilized magnetic gold and silver nanoparticles and concentration by magnetic separation]
PRRSV particles were captured using the DS25-MSGNP prepared in Example 2. The clinical isolate PRRSV was cultured using alveolar macrophage cells isolated from piglets, and the culture was diluted 300-fold, 1000-fold, and 10000-fold with PBS (containing 1% PS).

Oral fluid (saliva) collected by the rope method from a group of pigs (about 80 pigs) about 60 days old is diluted twice with PBS (containing 1% PS), and further diluted with PBS (containing 1% PS) to PBS (1) containing 10% DTT (dithiothereitol). Diluted 2-fold with% PS).

300 μL of each virus diluted solution and 300 μL of oral solution diluted solution were mixed and centrifuged (10000 G, 30 seconds) to obtain 500 μL of a supernatant without solid matter. To this, a DS25-MSGNP solution (10 μl) was mixed, and about 10 mg of a second magnetic substance was further added, and then the mixture was mixed by pipetting several times to obtain a mixed solution. As the second magnetic material, triiron tetroxide coated with silica was used. The particle size of the second magnetic material was 10 to 40 μm.
Next, the mixed solution was allowed to stand on a magnet stand (TAKARA Magnetic Stand (6tubes)) for 5 seconds to perform magnetic separation. After magnetic separation, the supernatant of the mixed solution was removed, and the precipitate magnetically separated by DS25-MSGNP and a magnet was collected.

[Crushing of sugar chain-immobilized magnetic gold and silver nanoparticles-virus conjugate and detection of virus by real-time quantitative RT-PCR]
To the precipitate obtained by 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 the PCR reagent 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 are 12.5 μl of 2 × One Step SYBR RT-PCR Buffer4, 1 μl of PrimeScript 1 step Enzyme Mix2, 1 μl of PCR Forward Primer (10 μM), PCR Reverse Primer (10 μM) of 1 μl of PCR River Was used as a mixture of 5.5 μl and 2.25% Tween 20 in 2 μl.

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

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

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

Table 3 shows the Ct value and Tm value of the precipitate fraction obtained by capturing the virus and magnetically separating it from the amplification curve of the cDNA obtained from RT-PCR. Since the difference in the amount of virus concentrated and purified from PRRSV diluted 300 times, diluted 1000 times, and 10000 times and the oral fluid appears as a difference in the number of PCR cycles, the solution diluted 300 times and the solution diluted 1000 times The difference in the Ct value of was 2.3, and the difference in the amount of virus was 4.9 times. The difference in Ct value between the 1000-fold diluted solution and the 10000-fold diluted solution was 3.22, and the viral load was 9.3 times different.

Simply, the difference in the amount of virus between the 300-fold diluted solution and the 1000-fold diluted solution is 3.3 times, and the difference in the virus amount between the 1000-fold diluted solution and the 10000-fold diluted solution is 10-fold. 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.

Unexpected outbreaks of viral diseases can occur in medical institutions, farms, and livestock farms, but in order to prevent enormous damage, it is required to detect trace amounts of viruses and prevent epidemics. On the other hand, not all institutions have an ultracentrifuge, which is a large device for concentrating trace viruses, and it is difficult to detect dilute viruses from body fluids with conventional detection kits and PCR methods. is there.

The present invention utilizes the binding property between sugar chains and viruses, and by using a second magnetic substance in combination, virus particles can be captured from a dilute virus solution and efficiently concentrated. Therefore, the present invention is highly resistant to viral infections of animals and plants. It can be used for sensitivity tests and can prevent economic damage in the livestock, bio, and pharmaceutical industries.

Claims (5)

It has a linker compound, a sugar chain, and magnetic nanoparticles having an amino group at one end and a sulfur atom at the other end of the main chain hydrocarbon chain or hydrocarbon induction chain.
The amino group is bonded to the reducing end of the sugar chain, and the sulfur atom is bonded to the magnetic nanoparticles.
The magnetic nanoparticles contain gold and / or silver, as well as the first magnetic material, and further.
The surface of the magnetic nanoparticles is coated with gold and / or silver .
It has a core-shell-shell structure consisting of gold and / or silver from the center, the first magnetic material, and then gold and / or silver.
The sugar chain is a sugar chain-immobilized magnetic metal nanoparticle, which is characterized by being able to specifically interact with a surface protein of a virus to be captured. A method for concentrating a virus, which comprises a step of applying a magnetic force to an admixture obtained by mixing the sugar chain-immobilized magnetic metal nanoparticles according to claim 1 with a sample containing a virus. The method for concentrating a virus according to claim 2 , wherein the admixture contains a second magnetic substance having a larger average particle size than the sugar chain-immobilized magnetic metal nanoparticles. The method for concentrating a virus according to claim 3 , wherein the surface of the second magnetic material is coated with silica. Amplify the RNA of the virus concentrated by the method for concentrating the virus according to any one of claims 2 to 4 by real-time quantitative RT-PCR, or according to any one of claims 2 to 4. A virus detection method, which comprises amplifying virus DNA concentrated by the above-described virus concentration method by real-time quantitative PCR.

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