WO2014104713A1

WO2014104713A1 - Gold-finished magnetic microsphere

Info

Publication number: WO2014104713A1
Application number: PCT/KR2013/012107
Authority: WO
Inventors: 박세진
Original assignee: 주식회사 노마디엔
Priority date: 2012-12-24
Filing date: 2013-12-24
Publication date: 2014-07-03

Abstract

The present invention relates to a gold-finished magnetic microsphere comprising: a) a spherical support that serves as a core; b) a magnetic layer formed at the surface of the core; and c) a gold layer formed at the magnetic layer. The spherical support, the magnetic layer, and the gold layer form a definite layer structure.

Description

Gold finish magnetic microspheres

The present invention relates to a gold-finish magnetic microsphere, and more particularly, the microsphere has a size that can be observed and manipulated by a general optical microscope, has a very uniform particle distribution, excellent chemical stability, In addition to providing a variety of material recognition capabilities, its excellent spectroscopic analytical properties and low specific gravity make it a gold finish that can be applied to a wide variety of in vitro diagnostics, including microsphere-based multiplexing. It relates to magnetic microspheres.

Magnetic particles are widely used for the separation, concentration and detection of various substances ranging from atomic and molecular chemicals to biochemicals such as nucleic acids and proteins, and to biological materials such as cells and viruses. In particular, spherical beads, that is, magnetic elements in the form of spheres are used.

According to Mirkin et al. (US 7,238,472 B2), nanoparticles having a core / shell structure having a magnetic core and a gold shell surrounding the same have been reported. Here, the core / shell structured nanoparticles have an average diameter of 5 to 150 nm. Here, a method of detecting oligonucleotides by binding oligonucleotides to core / shell nanoparticles and using the same has been proposed. The magnetic core is limited to metal oxide, iron (Fe), nickel (Ni), cobalt (Co), iron-platinum (FePt), and iron-gold (FeAu).

Albrecht et al. (US 2011/0256621 A1) also report molecular analysis using similar core / shell nanoparticles. Wherein the core material is selected from iron, cobalt, zinc, nickel and the shell is selected from gold, silver, palladium, rhodium, molybdenum, rudenium. The diameter of the core / shell nanoparticles is limited to 1-100 nm.

On the other hand, Sucholeiki et al. (US Pat. No. 5,834,121) report that magnetic polymers and polymer composites have a structure in which a plurality of magnetic nanoparticles encapsulated in a polymer are trapped in a matrix of a much larger sized second polymer bead. Modahl et al. (US 2012/0141798 A1) also report the formation of magnetic nanoparticles inside the polymer beads and then polymer coating the polymer bead surface again, reporting the finished polymer particles and the polymer composite beads.

As reported, magnetic beads presently have a core / shell structure in which one magnetic core is surrounded by one metal shell, or a complex structure in which a plurality of magnetic nanoparticles are contained in one polymer bead. These prior arts have several problems.

The most commonly used method for measuring the amount of a substance to be detected in the process of detecting a substance with magnetic beads is to measure the fluorescence intensity generated by the substance to be separated and concentrated on the magnetic beads. For this purpose, the magnetic beads are identified with an optical microscope, and the incident light of an appropriate wavelength is irradiated to the magnetic beads, and the resulting light is measured, and the intensity is divided by the area of the magnetic beads. In order to perform this process, it is preferable that the size of the magnetic beads has a size of a micrometer (μm) unit that can be observed and operated by an optical microscope. Thus, magnetic beads in the form of monodisperse microspheres are desired.

As described above, the magnetic beads of the core / shell structure are small in diameter and are difficult to observe and manipulate with an optical microscope. Even if the magnetic beads of this structure are manufactured in a micrometer size, there is inconvenience in use because they have a very heavy weight because the materials constituting the core / shell are all metals or metal oxides. In comparison, the magnetic microbead of the present technology has a size of a micrometer scale, but the low-density polymer occupies most of the volume, thereby reducing the weight.

In order for an analytical process for measuring the fluorescence intensity generated by the substance to be separated and concentrated on the magnetic beads to be performed with high sensitivity and precision, it is required that the fluorescence does not occur in the magnetic beads in which the substance to be detected is not present. That is, background fluorescence generated in the magnetic beads itself should be minimized.

As described above, the magnetic beads of the composite structure have their surfaces coated with a polymer. Most of these organic polymer materials often generate their own fluorescence. In addition, when the organic polymer material is in contact with the sample, the organic polymer material easily binds to a material other than the analyte, which is called non-specific binding. Non-specific binding may also cause fluorescence of substances present on the surface of the magnetic beads, causing background fluorescence to increase, thereby reducing the accuracy of analysis. In comparison, the gold finish magnetic microbeads of this technology have a gold finish on the surface. Gold is a material that does not have its own fluorescence, and is very inactive as an inert metal and can minimize nonspecific adsorption, thereby minimizing background fluorescence.

Compared with the polymer outer membrane, the gold outer membrane has another advantage. In general, it is often necessary to chemically modify the magnetic bead surface to increase selectivity for the analyte.

Gold has a very strong bond with thiol molecules dissolved in water or alcohol. Since a wide variety of thiol molecules have already been synthesized and sold, these thiol molecules can be obtained and dissolved in water or alcohol, infused with a gold-finish magnetic microsphere, and then collected and washed with magnets. The surface can be modified with On the other hand, no binding of thiol molecules occurs on the polymer surface.

As can be seen in an example such as the magnetic-activated cell sorting (MACS) invented by Miltenyi Biotec, magnetic beads are often used in multiplex analysis to mix, transport and separate in a fluid. For these applications, the magnetic beads should have a specific gravity greater than the water specific gravity of 1 g / cm ³ but should not be too large. Magnetic particles composed of only magnetic materials that are metals or metal oxides inevitably have a large specific gravity. In comparison, the magnetic microbeads of the present technology have an advantage of reducing weight by forming a spherical core therein with a polymer having a low density.

Despite these distinct advantages, rarely reported micrometer-scale magnetic beads with gold have been reported, and moreover, gold-finish magnetic beads, most of which consist of spherical structures of low-density material, can reduce the overall density. rare. In addition, microbe-adjusted magnetic beads with gold finishes rarely have monodisperse particle sizes.

An object of the present invention has been proposed to improve the conventional properties as described above, the microsphere has a size that can be observed and manipulated by a general optical microscope, has a very uniform particle distribution, and recognizes a variety of materials on the surface In addition to being functional, it has excellent chemical stability, excellent spectroscopic and analytical properties, and low specific gravity, which can be applied to a wide variety of in vitro diagnosis including microsphere-based multiplexing. To offer a gold finish microsphere.

The present invention has the following configuration to achieve the above object.

According to the invention, a) a spherical support as a core; b) a magnetic material film formed on the surface of the core; And c) a gold film formed on the magnetic material film, wherein the spherical support, the magnetic material film, and the gold film form a clear layered structure.

And the spherical support is a spherical diameter of 1 ~ 100㎛.

In addition, the spherical support is composed of a polymer, the spherical support is made of poly (methyl methacrylate) (PMMA) or a copolymer thereof, or the spherical support is polystyrene or a copolymer thereof. It is composed of any one selected.

The magnetic material film is made of any one selected from a paramagnetic metal or a metal oxide.

In addition, the magnetic material film is iron oxide.

And the magnetic material film is nickel, the magnetic material film is made of a film thickness of 20 ~ 2000 nm.

Further, the gold film has a film thickness of 2 to 100 nm.

The magnetic material film is preferably formed in a ratio of 0.02 to 0.2 relative to the diameter of the spherical support.

It is also preferred that the gold-finish microspheres have a specific gravity greater than 1 g / cm ^{3 and} less than 3 g / cm ³ .

According to the present invention, the proposed microsphere has a size that can be observed and manipulated by a general optical microscope, has a very uniform particle distribution, can impart various material recognition functions to its surface, and also has chemical stability. Its excellent, high spectroscopic analytical properties and low specific gravity make it effective for a wide range of in vitro diagnostics, including microsphere-based multiplexing.

In addition, according to the present invention, the gold finish magnetic microspheres and the material recognition gold finish magnetic microspheres have a strong magnetic property, and have the effect of being separated and collected again by an external magnetic force after being dispersed in a solution.

1 is a schematic view of a gold finish magnetic microsphere of the present invention.

2A and 2B are photographs showing electron microscope images of the magnetic microspheres according to the present invention.

3a to 3c are photographs showing the electron microscope image of the gold-finish magnetic microspheres according to the present invention.

4 is a graph showing the magnetic properties of the gold finish magnetic microspheres according to the present invention.

5A is a photograph showing an electron microscope image and an elemental analysis image of a cross section of a gold finish magnetic microsphere according to the present invention.

5b is a photograph showing only a magnetic material portion of the elemental analysis image of the gold finish magnetic microsphere cross section according to the present invention.

5c is a photograph showing only a gold film portion of the elemental analysis image of the gold finish magnetic microsphere cross section according to the present invention.

6A is an optical micrograph of a gold finished magnetic microsphere in accordance with the present invention.

6b is a light micrograph of a gold-finish magnetic microsphere in accordance with the present invention.

The objects, features, and advantages of the present invention described above will become more apparent through the following embodiments in conjunction with the accompanying drawings.

The following specific structures or functional descriptions are only illustrated for the purpose of describing embodiments according to the inventive concept, and the embodiments according to the inventive concept may be embodied in various forms and may be described in detail herein. It should not be construed as limited to the examples.

Embodiments in accordance with the concepts of the present invention can be variously modified and have a variety of forms, specific embodiments will be illustrated in the drawings and described in detail herein. However, this is not intended to limit the embodiments in accordance with the concept of the present invention to a particular disclosed form, it should be understood to include all modifications, equivalents, and substitutes included in the spirit and scope of the present invention.

Terms such as first and / or second may be used to describe various components, but the components are not limited to the terms. The terms are only for the purpose of distinguishing one component from other components, for example, without departing from the scope of the rights according to the inventive concept, the first component may be called a second component, and For example, the second component may also be referred to as a first component.

When a component is referred to as being "connected" or "connected" to another component, it may be directly connected or connected to that other component, but it may be understood that other components may be present in the middle. Should be. On the other hand, when a component is said to be "directly connected" or "directly connected" to another component, it should be understood that no other component exists in the middle. Other expressions for describing the relationship between components, such as "between" and "immediately between" or "adjacent to" and "directly adjacent to", should be interpreted as well.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Singular expressions include plural expressions unless the context clearly indicates otherwise. The terms "comprise" or "having" herein are intended to indicate that there is a feature, number, step, action, component, part, or combination thereof that is practiced, and that one or more other features or numbers, It is to be understood that it does not exclude in advance the possibility of the presence or addition of steps, actions, components, parts or combinations thereof.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art. Terms such as those defined in the commonly used dictionaries should be construed as having meanings consistent with the meanings in the context of the related art, and are not construed in ideal or excessively formal meanings unless expressly defined herein. Do not.

Gold finish microsphere of the present invention is formed through the following process.

The preparation of polymeric microspheres can be made according to well-known methods and commercially available as industrial materials. In this example, polymer beads having a diameter of 1 to 00 µm and a relative standard deviation of 10% or less were purchased and used.

(a) Modification of the surface of the monodisperse spherical support as a core: A functional group capable of acting as a ligand is generated on the surface of the spherical support. Ligands bind strongly to metal ions and play a critical role in coating a stable magnetic film. As a method of generating a ligand on the surface of the spherical support, a method of adsorbing a polymer such as PEI or PEG containing a ligand may be used, but in this case, since the adsorption force is not strong enough, it is difficult to expect a stable membrane. Instead, a ligand-containing molecule is bound to the support surface with strong covalent bonds. An exemplary method is to bind amine functional groups to the surface of PMMA.

In the present embodiment, the surface of poly (methylmethacrylate (PMMA)) is modified with an amine functional group. Into a solution of boric acid (boric acid) and ethylene diamine at an appropriate concentration, a monodisperse PMMA having a diameter of 15 μm was added thereto, followed by agitation at 80 ° C. for 6 hours, and then washed several times with distilled water.

(b) coating a magnetic material film on the surface of the modified simple acid spherical support: in the present embodiment, tin ions (Sn ²⁺ ) are bound to the ligands, and palladium ions (Pd ²⁺ ) are bound to the It was reduced to the palladium-coated surface to produce a magnetic nickel film by using an electroless plating reaction occurring on the surface of the palladium.

The specific process in this embodiment is as follows.

The monodisperse PMMA modified with the amine obtained in 1 (a) was added to a tin chloride (SnCl ₂ ) solution, stirred for 1 hour to bind tin ions, filtered, and the excess tin chloride was washed with distilled water. This was dispersed in a solution in which palladium chloride (PdCl ₂ ) was dissolved in a 10% hydrochloric acid solution and reacted at 60 ° C. for 30 minutes to prepare a palladium membrane.

In carrying out the nickel plating on the spherical support on which the palladium film is formed, special attention is paid to the conditions under which the high magnetic nickel film is formed. In this embodiment, a high magnetic microsphere was obtained by performing nickel plating in the following plating bath. Of course, reports of high magnetic nickel plating conditions already exist. However, there have been no reports on the magnetic support of gold-finished magnetic microspheres by forming a layered structure of a core support, a nickel magnetic film, and a gold film thereon using these plating conditions. 5 is measurement data showing the magnetic properties of the magnetic nickel microspheres produced according to the present embodiment. The highly magnetic nickel film electroless plating method according to the present embodiment is as follows.

After dissolving polyvinylpiperidone and dodecyltrimethylammonium bromide in 1000 ml of water to prepare a dispersion solution, the palladium-coated PMMA microspheres are added thereto, the concentration of the solution is corrected to pH 10, heated to 60 ° C, and stirred. Nickel sulfate (II), glycine, and sodium hypophosphate are added thereto to conduct electroless plating. After the plating is completed, the filtration and washing with distilled water several times to collect the microspheres with a magnetic nickel film is collected and dried in a vacuum oven. The scanning electron microscope (SEM) image of the magnetic microspheres thus obtained is shown in FIG. 2A.

11.75 g FeCl ₃ and 4.3 g FeCl ₂ are dissolved in distilled water, heated to 85 ° C., and nitrogen is blown to remove air. NH ₄ OH was added thereto and the mixture was stirred at rpm 600 while maintaining 85 ° C. When black particles are formed, 1.5 ml oleic acid is added several times, followed by stirring and heating for 10 minutes to stop. When the temperature is lowered to room temperature, it is washed with distilled water and dried to collect iron oxide nanoparticles. After preparing a solution in which the iron oxide nanoparticles are dispersed in cyclohexane, the solution is mixed with monodisperse polymer microspheres to adsorb the iron oxide nanoparticles on the surface of the polymer microspheres. When this process is repeated and a sufficient amount of iron oxide film is formed on the surface of the polymer microsphere, it is washed several times with clean cyclohexane, sequentially washed with ethanol and distilled water, and then dried in a vacuum oven. The scanning electron microscope (SEM) image of the magnetic microspheres having a diameter of 1.3 μm thus obtained is shown in FIG. 2B.

In 50 ml of distilled water, 5.6 g of EDTA-Na and 0.3 g of KH ₂ PO ₄ were added and stirred. Add 1 m Na ₃ Au (SO ₃ ) ₂ solution and 2.5% catechol solution, add 0.1 g of magnetic microspheres, react for 10 minutes, form a gold film, collect and wash several times with distilled water. To dry. A scanning electron microscope (SEM) image of the magnetic microspheres having a diameter of 15 μm thus obtained is shown in FIG. 3A.

After stirring 0.1 g of magnetic microspheres in 1 L of distilled water, 10% HAuCl ₄ was added thereto, 400 mM NH ₂ OH was added thereto, and the mixture was stirred for 20 minutes (rpm 200). After the reaction, the mixture was washed with distilled water and dried in a vacuum oven. The scanning electron microscope (SEM) images of the magnetic microspheres having the diameters of 5 μm and 15 μm are as shown in FIGS. 3B and 3C, respectively.

After the gold finish magnetic microspheres were prepared, their magnetic properties were measured using a SQUID magnetometer. 4 is measurement data showing the magnetic properties of the gold-finish magnetic microsphere produced by the present embodiment.

The x-axis of the graph represents the applied magnetic field (Oe) and the y-axis represents the magnetization (unit: emu / g), and the measured data shows that the gold finish magnetic microsphere is saturated magnetization. It shows paramagnetism above 15 emu / g.

After mixing a certain amount of gold-finish magnetic microspheres with a predetermined volume of distilled water, the volume change was measured, and the specific gravity was measured by dividing the weight of the gold-finish magnetic microspheres by the volume change amount. The specific gravity of the gold finish magnetic microsphere can be adjusted according to the thickness of the magnetic film and the gold film, and was maintained in the range of 1 to 3 g / cm ³ .

Gold finish microsphere of the present invention produced through the manufacturing process as described above is made of the following configuration.

According to the invention as shown in Figure 1, a) a spherical support as a core; b) a magnetic material film formed on the surface of the core; And c) a gold film formed on the magnetic material film, wherein the spherical support, the magnetic material film, and the gold film form a clear layered structure.

And the spherical support is a spherical diameter of 1 ~ 100㎛.

In addition, the magnetic material film is iron oxide.

Further, the gold film has a film thickness of 2 to 100 nm.

According to another preferred embodiment of the present invention, the magnetic material film is nickel.

The magnetic material film has a film thickness of 20 to 2000 nm.

Hereinafter, the present invention will be described in more detail.

As the thickness of the magnetic material film increases, the magnetic strength of the magnetic microspheres increases. In order for the magnetic properties of the magnetic microspheres to be sufficient, a magnetic material film of a predetermined thickness or more should be formed. For spherical supports having a diameter of 1 μm, a thickness of 20 nm or more is required. As the diameter of the spherical support increases, a thicker magnetic film is needed. However, if the thickness of the magnetic material film is unconditionally increased, the specific gravity of the gold-finish magnetic microspheres is excessively increased, so the maximum diameter of the 100 μm spherical support is set to 2000 nm.

The gold film has a film thickness of 2 to 100 nm. To eliminate background fluorescence and prevent nonspecific binding, the gold film should completely cover the outside of the magnetic microspheres so that the inner spherical support core and the magnetic material film are not exposed to the outside. To do this, the gold film must be at least 2 nm thick. The thicker the gold film, the more it can act as an outer film. However, if the thickness is thicker than necessary, there is a problem in that the weight of the magnetic gold magnetic finish is increased and the manufacturing cost is increased. A gold film thickness of up to 100 nm is sufficient.

In addition, the thickness of the magnetic material film is formed in a ratio of 0.02 ~ 0.2 relative to the diameter of the spherical support. In order to maintain sufficient magnetism in proportion to the weight of the spherical support, a ratio of at least 0.02 is maintained, but not more than 0.2 so that the total specific gravity of the magnetic microspheres is not too large. Maintaining this ratio allows the gold finish magnetic microspheres to be sufficiently magnetic and at the same time low in weight.

The gold finish magnetic microspheres also have specific gravity values greater than 1 g / cm ^{3 and} less than 3 g / cm ³ . Since the specific gravity is much smaller than that of metals or metal oxides, the process of mixing, moving, screening and collecting mixed fluids can be effectively performed in multiplex analysis such as MACS.

In addition, the material-recognized gold-finish magnetic microspheres with a thiol functional group attached to the outermost gold membrane of the gold-finish magnetic microspheres according to the present invention are dispersible in solution according to the type of the molecule having a thiol functional group, Affinity to certain substances is controlled. The material recognizing gold-finish magnetic microsphere according to the present invention comprises a) a spherical support as a core, b) a magnetic film formed on the surface of the core, c) a gold film formed on the magnetic material film, and d) a surface of the gold film. It comprises a thiol molecular film adsorbed on, the spherical support, the magnetic material film and the gold film forms a clear layered structure, the thiol molecular film may be composed of a self-assembled monolayer (self-assembled monolayer).

Preferably, the gold finish magnetic microspheres and the material recognition gold finish magnetic microspheres use a monodisperse spherical support as a core.

The gold-finish magnetic microspheres and the material recognition gold-finish magnetic microspheres possess strong magnetic properties, and can be separated by being dispersed in a solution and collected again by an external magnetic force.

5A is a photograph showing an electron microscope image and an elemental analysis image of a cross section of a gold finish magnetic microsphere according to the present invention, where a layered image of a gold film, a magnetic material film, and a spherical support can be seen. The dark area of the lower right is the polymer area, which is a spherical support. A band-shaped region having a thickness of about 500 nm formed from the lower left ear to the upper right ear corresponds to the magnetic material film. Above the magnetic material film, a gold film is formed to a thickness of about 30 nm.

Figure 5b is a photograph showing only the magnetic material portion of the elemental analysis image of the gold finish magnetic microsphere cross-section according to the present invention, as shown in Figure 5a the magnetic material film is formed from the lower left to the upper right ear of about 500 nm thick Observed in the form of a band.

Figure 5c is a photograph showing only the gold film portion of the elemental analysis image of the gold finish magnetic microsphere cross-section according to the present invention, as shown in Figure 5a, the gold film is observed in the form of about 30 nm thick.

6a is an optical micrograph of a gold-finish magnetic microsphere according to the present invention, and FIG. 6b is a fluorescence micrograph photographed simultaneously with an optical micrograph. As shown in FIG. 6a, gold-finish magnetic microspheres are formed in black dots. At the same time, the background fluorescence from the gold-finish magnetic microspheres was measured as shown in FIG. 6b. As a result, no background fluorescence was generated and no image was shown in FIG. 6b.

As such, when using the gold-finish microsphere formed by the present invention, it is possible to increase the specific gravity through the relatively high-density spherical support, and to realize the precise size and spherical shape. Since the reactivity can be excellent, it is possible to prevent false positives through fluorescence with the detection target.

As mentioned above, although this invention was demonstrated through the preferable embodiment, this is only to help understanding of the technical content of this invention, and is not intended to limit the technical scope of this invention to this.

That is, those skilled in the art without departing from the technical gist of the present invention can be variously modified or modified, as well as such changes or modifications, the technical scope of the present invention in the interpretation of the claims. It is hard to say that I am within.

Claims

a) spherical support as core;

b) a magnetic material film formed on the surface of the core; And

c) a gold film formed on the magnetic material film;

The spherical support, the magnetic material film and the gold film are gold-finish magnetic microspheres, characterized in that to form a clear layered structure.
The method of claim 1,

The spherical support is a gold finish magnetic microspheres, characterized in that the diameter of 1 ~ 100㎛ sphere.
The method of claim 2,

The spherical support is a gold finish magnetic microspheres, characterized in that consisting of a polymer.
The method of claim 3,

The spherical support is a gold-finish magnetic microsphere, characterized in that consisting of any one selected from poly (methyl methacrylate, PMMA) or copolymers thereof.
The method of claim 3,

The spherical support is a gold finish magnetic microspheres, characterized in that composed of any one selected from polystyrene or copolymers thereof.
The method of claim 1,

The magnetic material film is a gold-finish magnetic microspheres, characterized in that composed of any one of a paramagnetic (paramagnetic) metal or a metal oxide.
The method of claim 6,

The magnetic material film is a gold finish magnetic microspheres, characterized in that the iron oxide.
The method of claim 6,

The magnetic material film is gold finish magnetic microspheres, characterized in that the nickel.
The method according to claim 1 or 6, 7 or 8,

The magnetic material film is a gold finish magnetic microspheres, characterized in that the film thickness of 20 ~ 2000 ㎚.
The method of claim 1,

The gold film is a gold finish magnetic microspheres, characterized in that consisting of a film thickness of 2 ~ 100nm.
The method of claim 9,

The magnetic material film is a gold finish magnetic microspheres, characterized in that the film is formed in a ratio of 0.02 to 0.2 relative to the diameter of the spherical support.
The method according to any one of claims 1 to 8 or 10 to 11,

The gold finish magnetic microspheres are gold finish magnetic microspheres, characterized in that the specific gravity is greater than 1 g / cm 3 and less than 3 g / cm 3 .