WO2014104713A1 - Microsphère magnétique ayant une finition à l'or - Google Patents

Microsphère magnétique ayant une finition à l'or Download PDF

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Publication number
WO2014104713A1
WO2014104713A1 PCT/KR2013/012107 KR2013012107W WO2014104713A1 WO 2014104713 A1 WO2014104713 A1 WO 2014104713A1 KR 2013012107 W KR2013012107 W KR 2013012107W WO 2014104713 A1 WO2014104713 A1 WO 2014104713A1
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WO
WIPO (PCT)
Prior art keywords
gold
magnetic
film
finish
microspheres
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PCT/KR2013/012107
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English (en)
Korean (ko)
Inventor
박세진
Original Assignee
주식회사 노마디엔
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Priority claimed from KR1020130162067A external-priority patent/KR20140082576A/ko
Publication of WO2014104713A1 publication Critical patent/WO2014104713A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G01N33/5434—Magnetic particles using magnetic particle immunoreagent carriers which constitute new materials per se
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/32—Micromanipulators structurally combined with microscopes
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes

Definitions

  • 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.
  • biochemicals such as nucleic acids and proteins
  • biological materials such as cells and viruses.
  • spherical beads that is, magnetic elements in the form of spheres are used.
  • nanoparticles having a core / shell structure having a magnetic core and a gold shell surrounding the same have been reported.
  • the core / shell structured nanoparticles have an average diameter of 5 to 150 nm.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the size of the magnetic beads has a size of a micrometer ( ⁇ m) unit that can be observed and operated by an optical microscope.
  • ⁇ m micrometer
  • 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.
  • 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.
  • 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.
  • 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.
  • magnetic beads are often used in multiplex analysis to mix, transport and separate in a fluid.
  • 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.
  • 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.
  • 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.
  • microbe-adjusted magnetic beads with gold finishes rarely have monodisperse particle sizes.
  • 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.
  • the spherical support is a spherical diameter of 1 ⁇ 100 ⁇ m.
  • the spherical support is composed of a polymer
  • the spherical support is made of poly (methyl methacrylate) (PMMA) or a copolymer thereof
  • 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.
  • the magnetic material film is iron oxide.
  • the magnetic material film is nickel, the magnetic material film is made of a film thickness of 20 ⁇ 2000 nm.
  • 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.
  • the gold-finish microspheres have a specific gravity greater than 1 g / cm 3 and less than 3 g / cm 3 .
  • 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.
  • 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.
  • FIG. 1 is a schematic view of a gold finish magnetic microsphere of the present invention.
  • FIGS. 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.
  • FIG. 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.
  • 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.
  • Gold finish microsphere of the present invention is formed through the following process.
  • polymeric microspheres can be made according to well-known methods and commercially available as industrial materials.
  • polymer beads having a diameter of 1 to 00 ⁇ m and a relative standard deviation of 10% or less were purchased and used.
  • 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.
  • 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.
  • the surface of poly (methylmethacrylate (PMMA)) is modified with an amine functional group.
  • PMMA methylmethacrylate
  • a monodisperse PMMA having a diameter of 15 ⁇ m was added thereto, followed by agitation at 80 ° C. for 6 hours, and then washed several times with distilled water.
  • 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.
  • a high magnetic microsphere was obtained by performing nickel plating in the following plating bath.
  • reports of high magnetic nickel plating conditions already exist.
  • 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.
  • the solution is mixed with monodisperse polymer microspheres to adsorb the iron oxide nanoparticles on the surface of the polymer microspheres.
  • 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 ⁇ m thus obtained is shown in FIG. 2B.
  • 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.
  • 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.
  • the spherical support is a spherical diameter of 1 ⁇ 100 ⁇ m.
  • the spherical support is composed of a polymer
  • the spherical support is made of poly (methyl methacrylate) (PMMA) or a copolymer thereof
  • 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.
  • the magnetic material film is iron oxide.
  • the magnetic material film is nickel, the magnetic material film is made of a film thickness of 20 ⁇ 2000 nm.
  • 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.
  • the gold-finish microspheres have a specific gravity greater than 1 g / cm 3 and less than 3 g / cm 3 .
  • the magnetic material film is nickel.
  • the magnetic material film has a film thickness of 20 to 2000 nm.
  • a magnetic material film of a predetermined thickness or more should be formed.
  • a thickness of 20 nm or more is required.
  • the specific gravity of the gold-finish magnetic microspheres is excessively increased, so the maximum diameter of the 100 ⁇ m 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.
  • 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.
  • 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.
  • 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.
  • the thiol molecular film may be composed of a self-assembled monolayer (self-assembled monolayer).
  • 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.
  • 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.
  • FIG. 6a is an optical micrograph of a gold-finish magnetic microsphere according to the present invention
  • FIG. 6b is a fluorescence micrograph photographed simultaneously with an optical micrograph.
  • gold-finish magnetic microspheres are formed in black dots.
  • 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.
  • the gold-finish microsphere formed by the present invention 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.

Abstract

L'invention concerne une microsphère magnétique ayant une finition à l'or et comprenant : a) un support sphérique servant de cœur ; b) une couche magnétique recouvrant la surface du cœur ; et c) une couche d'or recouvrant la couche magnétique. Le support sphérique, la couche magnétique et la couche d'or forment une structure en couche définitive.
PCT/KR2013/012107 2012-12-24 2013-12-24 Microsphère magnétique ayant une finition à l'or WO2014104713A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR20120152369 2012-12-24
KR10-2012-0152369 2012-12-24
KR1020130162067A KR20140082576A (ko) 2012-12-24 2013-12-24 금 마감 자성 마이크로스피어
KR10-2013-0162067 2013-12-24

Publications (1)

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WO2014104713A1 true WO2014104713A1 (fr) 2014-07-03

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5648124A (en) * 1993-07-09 1997-07-15 Seradyn, Inc. Process for preparing magnetically responsive microparticles
KR20060050750A (ko) * 2004-11-11 2006-05-19 삼성전자주식회사 다층구조의 나노결정 및 그의 제조방법
US20060269751A1 (en) * 2005-05-20 2006-11-30 Winstead J Magnetically-responsive microparticles with improved response times
KR20090000859A (ko) * 2007-06-28 2009-01-08 연세대학교 산학협력단 진단 및 치료용 자성 메탈 나노 복합체
JP2009509132A (ja) * 2005-09-16 2009-03-05 コンセホ・スペリオール・デ・インベスティガシオネス・シエンティフィカス ナノ粒子バイオセンサとその製造方法および使用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5648124A (en) * 1993-07-09 1997-07-15 Seradyn, Inc. Process for preparing magnetically responsive microparticles
KR20060050750A (ko) * 2004-11-11 2006-05-19 삼성전자주식회사 다층구조의 나노결정 및 그의 제조방법
US20060269751A1 (en) * 2005-05-20 2006-11-30 Winstead J Magnetically-responsive microparticles with improved response times
JP2009509132A (ja) * 2005-09-16 2009-03-05 コンセホ・スペリオール・デ・インベスティガシオネス・シエンティフィカス ナノ粒子バイオセンサとその製造方法および使用
KR20090000859A (ko) * 2007-06-28 2009-01-08 연세대학교 산학협력단 진단 및 치료용 자성 메탈 나노 복합체

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