WO2010151085A2 - Zinc-containing magnetic nanoparticle-based magnetic separation systems and magnetic sensors - Google Patents

Zinc-containing magnetic nanoparticle-based magnetic separation systems and magnetic sensors Download PDF

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Publication number
WO2010151085A2
WO2010151085A2 PCT/KR2010/004158 KR2010004158W WO2010151085A2 WO 2010151085 A2 WO2010151085 A2 WO 2010151085A2 KR 2010004158 W KR2010004158 W KR 2010004158W WO 2010151085 A2 WO2010151085 A2 WO 2010151085A2
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magnetic
zinc
nanoparticles
nanoparticle
separation system
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PCT/KR2010/004158
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French (fr)
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WO2010151085A3 (en
Inventor
Jin Woo Cheon
Jae Hyun Lee
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Industry-Academic Cooperation Foundation, Yonsei University
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Publication of WO2010151085A2 publication Critical patent/WO2010151085A2/en
Publication of WO2010151085A3 publication Critical patent/WO2010151085A3/en

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C1/00—Magnetic separation
    • B03C1/005—Pretreatment specially adapted for magnetic separation
    • B03C1/01—Pretreatment specially adapted for magnetic separation by addition of magnetic adjuvants
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/72—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating magnetic variables
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C2201/00—Details of magnetic or electrostatic separation
    • B03C2201/18—Magnetic separation whereby the particles are suspended in a liquid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C2201/00—Details of magnetic or electrostatic separation
    • B03C2201/26—Details of magnetic or electrostatic separation for use in medical applications

Definitions

  • the present invention relates to zinc-containing magnetic nanopartide-based magnetic separation systems and magnetic sensors.
  • Magnetic nanoparticles have been found to be applied to very numerous application fields.
  • MRI magnetic resonance imaging
  • material separation systems using magnetism include: (a) material separation systems using magnetism; (c) drug delivery systems using magnetism; (d) material sensors using magnetism; and (e) thermotherapies using a high-frequency magnetic field (HFMF).
  • HFMF high-frequency magnetic field
  • iron oxide nanoparticles have some advantages such as (a) feasible synthesis and (b) relatively low cost in its synthesis according to the development of various synthesis methods.
  • a few iron oxide nanoparticles ⁇ e.g., MRI contrast agent) are commercially accessible as iron oxide has been known to exhibit very low toxicity in a human body.
  • a nanoparticle having high saturation magnetism (AZ 5 ) is as follows:
  • MnFe 2 O 4 nanopartides are synthesized by substituting Mn 2+ for Fe 2+ in conventional iron oxide nanopartides.
  • the nanopartides with a size of 12 nm have saturation magnetism of 125 emu/g.
  • FeCo nanopartides (Seo eta/., Nature Materials, 5: 971 (2006))
  • FeCo nanopartides are produced by alloy of iron and cobalt, and their saturation magnetisms are 215 emu/g.
  • nanopartides developed in aforementioned references as MRI contrast agent their contrast effects were improved several ten times compared with iron oxide-based contrast agents, supposing that the nanopartides may be utilized in early diagnosis of diseases such as cancer more excellent than present techniques.
  • Zn ⁇ 4 Fe 2-6 O 4 nanoparticle using a zinc-containing iron oxide have lower toxicity than FeCo nanoparticle, it exhibits the most excellent application probability in human body and bio-experiments.
  • magnetic moment of nanopartides is crucial for enhancement of contrast effect.
  • nanoparticles having high magnetic moment contribute to enhancement of contrast effect in several other application fields including magnetic separation, magnetoresistance sensor, magnetic relaxation sensor, heat release by high- frequency magnetic field, and so forth.
  • the nanoparticle cluster may be utilized.
  • the present inventors have made intensive studies to develop a magnetic separation system having improved separation efficiency for isolating a target material.
  • a magnetic nanoparticle used in the magnetic separation system zinc-containing magnetic nanoparticle having high saturation magnetism or cluster thereof may be applied to the magnetic separation system for providing a magnetic separation system to achieve the aforementioned purpose.
  • a magnetic sensor for detecting or quantitating an analyte in more improved sensitive and accurate manner.
  • a magnetic nanopartide used in the magnetic sensor zinc-containing magnetic nanopartide having specific stoichiometry and high saturation magnetism or cluster thereof may be applied to the magnetic sensor for providing a magnetic sensor to achieve the aforementioned purpose.
  • a zinc-containing magnetic nanopartide-based magnetic separation system or magnetic sensor comprising a zinc-containing magnetic nanopartide represented by the following formula 1 or 2 or a cluster thereof:
  • Zn f M a-f O b (0 ⁇ f ⁇ 8, 0 ⁇ a ⁇ 16, 0 ⁇ b ⁇ 8, 0 ⁇ f/(a-f) ⁇ 10, M represents a magnetic metal atom or an alloy thereof)
  • Zn g M c-g M' d O e (0 ⁇ g ⁇ 8, 0 ⁇ c ⁇ 16, 0 ⁇ d ⁇ 16, 0 ⁇ e ⁇ 8, 0 ⁇ g/ ⁇ (c-g)+d ⁇ 10
  • M represents a magnetic metal atom or an alloy thereof
  • M' represents one or more elements selected from the group consisting of Group 1 elements, Group 2 elements, Group 12 elements, Group 13 elements, Group 14 elements, Group 15 elements, transition metal elements, Lanthanide elements and Actinide elements)
  • the magnetic separation system and magnetic sensor of the present invention exhibit utilize a zinc-containing magnetic nanoparticle having specific stoichiometry and high saturation magnetism or cluster thereof, and both working principles are similar.
  • Zinc-containing magnetic nanoparticles used in the magnetic separation system of the present invention are represented by the following formula 1 or 2:
  • M represents a magnetic metal atom or an alloy thereof
  • M represents a magnetic metal atom or an alloy thereof;
  • M' represents one or more elements selected from the group consisting of Group 1 elements, Group 2 elements, Group 12 elements, Group 13 elements, Group 14 elements, Group 15 elements, transition metal elements, Lanthanide elements and Actinide elements)
  • High saturation magnetism of zinc-containing nanoparticles described in the formula 1 or 2 leads to enhanced separation efficiency on the magnetic separation system to which magnetic field is applied.
  • M represents preferably transition metal elements
  • Lanthanide metal elements and Actinide metal elements more preferably, transition metal elements selected from the group consisting of Ba, Mn, Co, Ni and Fe, Lanthanide metal elements selected from the group consisting of Gd, Er, Ho, Dy, Tb,
  • M' preferably represents one or more elements selected from the group consisting of Li, Na, K, Rb, Cs, Be, Mg, Ca, Sr, Ba, Ra, Ge, Ga, Bi, In, Si, Ge, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Y, Zr, Nb, Mo, Tc, Ru, Rh, Pd, Ag, Cd, Hf, Ta, W, Re, Os, Ir, Pt, Au, Hg, Lanthanide elements and Actinide elements.
  • the zinc-containing magnetic nanopartide used in the magnetic separation system of this invention is represented by the following formula 3:
  • M" represents a magnetic metal atom or an alloy thereof
  • M" represents preferably transition metal elements, Lanthanide metal elements and Actinide metal elements; more preferably, transition metal elements selected from the group consisting of Ba, Mn, Co, Ni and Fe, Lanthanide metal elements selected from the group consisting of Gd, Er, Ho, Dy, Tb, Sm and Nd; and most preferably, Mn, Co, Ni, Fe, Gd, Er, Ho, Dy, Tb, Sm or Nd, or an alloy thereof.
  • the zinc-containing magnetic nanopartide used in the magnetic separation system of this invention is represented by the following formula 4 or 5:
  • a stoichiometric content ratio of zinc and other metals is as follows: 0.001 ⁇ 'zinc/ (entire metal component - zinc)' ⁇ 10, more preferably 0.01 ⁇ 'zinc/ (entire metal component - zinc)' ⁇ 1, and most preferably 0.03 ⁇ 'zinc/ (entire metal component - zinc)' ⁇ 0.5.
  • zinc is contained as the above, high saturation magnetism can be obtained, resulting in significant improvement of separation efficiency in the present magnetic separation system.
  • the zinc-containing magnetic nanopartides contained in the cluster of the present invention are aggregated in a number of preferably 2-10,000, more preferably 2-1,000, and most preferably 2-100.
  • Each zinc-containing magnetic nanoparticles in the cluster is linked to each other by an intermolecular interaction, or encapsulated by an organic or inorganic carrier.
  • a binding agent with a binding affinity to a target material may be linked to the surface of a zinc-containing magnetic nanopartide or cluster thereof used in the present invention, or not.
  • the material of interest may be directly linked to a metal element contained in the zinc-containing magnetic nanopartide of the present invention.
  • nickel may be directly linked to a histidine residue of a protein or peptide, whereby protein is able to be isolated.
  • a gas- selective metal contained in the zinc-containing magnetic nanopartide of the present invention contributes to gas separation without a further binding agent.
  • the gas- selective metal includes Pt, Pd, Au, Ag, Nb, Ir, Rh, Ru and an alloy thereof, but is not limited to.
  • a hydrogen-selective metal includes Pt, Pd, Au, Ag and an alloy thereof.
  • a binding agent with a binding affinity to a target material is linked to the surface of a zinc-containing magnetic nanopartide or cluster thereof.
  • the binding agent is directly or indirectly, covalently or non-covalently linked to the surface of zinc-containing magnetic nanopartide of the present invention.
  • the binding agent may be directly bound to the surface of zinc- containing magnetic nanopartide by a covalent or non-covalent linkage such as an ionic bond, an electrostatic bond, a hydrophobic interaction, a hydrogen bond, a covalent bond, a hydrophilic interaction or a Van der Waal force.
  • the binding agent may be indirectly bound to the surface of zinc-containing magnetic nanopartide through an intervening agent.
  • target material refers to a material in a sample to be separated (or separated and detected, or separated, detected or quantified).
  • the target material includes, but is not limited to, a nucleic acid molecule (DNA or RNA), a protein, a peptide, an antigen, a sugar, a lipid, a bacterium, a virus, a cell, an organic compound, an inorganic compound, a metal and an inorganic ion.
  • the sample includes a biological sample, a chemical sample and an environmental sample, but is not limited to.
  • the biological sample includes blood, plasma, serum, virus, bacterium, tissue, cell, lymph, bone marrow, saliva, milk, urine, feces, eyeball fluid, seminal fluid, brain extract, spinal fluid, synovial fluid, thymus fluid, ascites, amniotic fluid, cell tissue fluid and cell culture, but is not limited to.
  • binding agent means a substance having a specific affinity to a target material to be separated. Any material having a binding affinity to a target material to be separated may be utilized as the binding agent.
  • a non- limiting example of a binding agent includes a nucleic acid molecule (DNA or RNA), a protein, an antibody, an antigen, an aptamer (RNA, DNA and peptide aptamer), a receptor, a hormone, a streptavidin, an avidin, a biotin, a lectin, a ligand, an agonist, an antagonist, an enzyme, a coenzyme, an inorganic ion, a cofactor, a sugar, a lipid, a substrate for an enzyme, a hapten, a neutravidin, a protein A, a protein G, a selectin, calcium sulfate, a gas binding agent (example: Pt, Pd, Au, Ag, Nb, Ir, Rh and Ru).
  • the binding agent is bound to the surface of zinc-containing magnetic nanopartide or cluster thereof through an intervening agent.
  • the intervening agent includes various linkers and nanopartide-coating materials known to those ordinarily skilled in the art.
  • the surface of the zinc-containing magnetic nanopartides or cluster thereof is coated with a water-soluble multifunctional ligand for nanopartide solubilization, whereby the binding agent is linked to nanopartide.
  • water-soluble multi-functional ligand refers to a ligand that may be bound to zinc-containing nanoparticles or cluster thereof to solublize in water and stabilize the nanoparticles, and may allow the nanoparticles to be bound by binding agent having a specific affinity to a target material.
  • the water-soluble multi-functional ligand can include (a) an adhesive region (Li), and further can include (b) a binding region (L M ), (c) a cross-linking region (Lm ), or (d) a binding & cross-linking region (L M -Lm) which includes both the binding region (L n ) and the cross-linking region (L,,,).
  • an adhesive region Li
  • L M an adhesive region
  • L M a binding region
  • Lm cross-linking region
  • Lm binding & cross-linking region
  • the term "adhesive region (U)" refers to a portion of a multi-functional ligand including a functional group capable of binding to the nanoparticles, and preferably to an end portion of the functional group. Accordingly, it is preferable that the adhesive region including the functional group should have high affinity with the surface of the nanoparticles.
  • the nanopartide can be attached to the adhesive region by an ionic bond, a covalent bond, a hydrogen bond, a hydrophobic interaction or a metal-ligand coordination bond.
  • the adhesive region of the multifunctional ligand may be varied depending on the substances constituting the nanoparticles.
  • binding region (Ln) means a portion of the multi-functional ligand containing a functional group capable of binding to binding agents having a specific affinity to a target material, and preferably the other end portion located at the opposite side from the adhesive region.
  • the functional group of the reactive region may be varied depending on the type of binding agents and their chemical formulae (Table 2).
  • cross-linking region (Lm) refers to a portion of the multi-functional ligand including a functional group capable of cross-linking to an adjacent multifunctional ligand, and preferably a side chain attached to a central portion.
  • cross-linking means that the multi-functional ligand is bound to another adjacent multi-functional ligand by intermolecular interaction.
  • the intermolecular interaction includes, but is not limited to, a hydrophobic interaction, a hydrogen bond, a covalent bond ⁇ e.g., a disulfide bond), a Van der Waals force and an ionic bond. Therefore, the cross-linkable functional group may be variously selected according to the kind of the intermolecular interaction.
  • the compound which originally contains the above-described functional group may be used as a water-soluble multi-functional ligand, but a compound modified or prepared so as to have the above-described functional group by a chemical reaction known in the art may be also used as a water-soluble multifunctional ligand.
  • the preferable multi-functional ligand of the present invention includes a monomer, a polymer, a carbohydrate, a protein, a peptide, a nucleic acid, a lipid or an amphiphilic ligand.
  • one example of the preferable multi-functional ligand is a monomer which contains the functional group described above, and preferably dimercaptosuccinic acid, since it originally contains the adhesive region, the cross-linking region and the binding region. That is, -COOH on one side of dimercaptosuccinic acid is bound to the magnetic nanoparticle, and -COOH and -SH on the other end portion functions to bind to a binding agent.
  • -SH of dimercaptosuccinic acid acts as the cross-linking region by disulfide bond with another -SH.
  • dimercaptosuccinic acid in addition to the dimercaptosuccinic acid, other compounds having -COOH as the functional group of the adhesive region and - COOH, -NH 2 or -SH as the functional group of the binding region may be utilized as the preferable multi-functional ligand, but not limited to.
  • Still another example of the preferable water-soluble multi-functional ligand according to the present invention includes, but not limited to, one or more polymers selected from the group consisting of polyphosphagen, polylactide, polylactide-co- glycolide, polycaprolactone, polyanhydride, polymaleic acid, a derivative of polymaleic acid, polyalkylcyanoacrylate, polyhydroxybutylate, polycarbonate, polyorthoester, polyethylene glycol, poly-L-lysine, polyglycolide, polymethyl methacrylate and polyvinylpyrrolidone.
  • polymers selected from the group consisting of polyphosphagen, polylactide, polylactide-co- glycolide, polycaprolactone, polyanhydride, polymaleic acid, a derivative of polymaleic acid, polyalkylcyanoacrylate, polyhydroxybutylate, polycarbonate, polyorthoester, polyethylene glycol, poly-L-lysine, polyglycolide, polymethyl
  • preferable other examples of the multi-functional ligand are a peptide.
  • Peptide is oligomer/polymer consisting of several amino acids. Since the amino acids have -COOH and -NH 2 functional groups in both ends thereof, peptides naturally have the adhesive region and the binding region.
  • the peptide that contains one or more amino acids having one or more of -SH, -COOH, -NH 2 and -OH as the side chain may be utilized as the preferable water-soluble multifunctional ligand.
  • still another example of the preferable multi-functional ligand is a protein.
  • Protein is a polymer composed of more amino acids than peptides, that is, composed of several hundreds to several hundred thousands of amino acids. Proteins contains -COOH and -NH 2 functional group at both ends, and also contains a lot of functional groups such as -COOH, -NH 2 , -SH, -OH, -CONH 2 , and so on. Proteins may be used as the water-soluble multi-functional ligand because they naturally contain the adhesive region, the cross-linking region and the binding region according to its structure as the above-described peptide.
  • the representative examples of proteins which are preferable as the water-soluble multi-functional ligand include a structural protein, a storage protein, a transport protein, a hormone protein, a receptor protein, a contraction protein, a defense protein and an enzyme protein, and more specifically, albumin, antibody, antigen, avidin, cytochrome, casein, myosin, glycinin, carotene, collagen, globular protein, light protein, streptavidin, protein A, protein G, protein S, lectin, selectin, angiopoietin, anti-cancer protein, antibiotic protein, hormone antagonist protein, interleukin, interferon, growth factor protein, tumor necrosis factor protein, endotoxin protein, lymphotoxin protein, tissue plasminogen activator, urokinase, streptokinase, protease inhibitor, alkyl phosphocholine, surfactant, cardiovascular pharmaceutical protein, neuro pharmaceuticals protein and gastrointestinal pharmaceuticals, but not limited to.
  • nucleic acid is oligomer consisting of many nucleotides. Since the nucleic acids have -PO 4 " and -OH functional groups in their both ends, they naturally have the adhesive region and the binding region (L 1 - L n ), or the adhesive region and the cross-linking region (L r L m ). Therefore, the nucleic acids may be useful as the water-soluble multi-functional ligand in this invention. In some cases, the nucleic acid is preferably modified to have the functional group such as -SH, -NH 2 , -COOH or -OH at 3'- or 5'-terminal ends.
  • the preferable multi-functional ligand is an amphiphilic ligand including both a hydrophobic and a hydrophilic region.
  • hydrophobic ligands having long carbon chains coat the surface.
  • the hydrophobic region of the amphiphilic ligand and the hydrophobic ligand on the nanoparticles are bound to each other through intermolecular interaction to stabilize the nanoparticles.
  • the outermost part of the nanoparticles shows the hydrophilic functional group, and consequently water-soluble nanoparticles can be prepared.
  • the intermolecular interaction includes a hydrophobic interaction, a hydrogen bond, a Van der Waals force, and so forth.
  • the portion which binds to the nanoparticles by the hydrophobic interaction is an adhesive region (Li), and further the binding region (Ln) and the cross-linking region (Lm) can be introduced therewith by an organo-chemical method.
  • an adhesive region Li
  • the binding region (Ln) and the cross-linking region (Lm) can be introduced therewith by an organo-chemical method.
  • amphiphilic polymer ligands with multiple hydrophobic and hydrophilic regions can be used.
  • Cross-linking between the amphiphilic ligands can be also performed by a linker for enhancement of stability in an aqueous solution.
  • hydrophobic region of the amphiphilic ligand can be a linear or branched structure composed of chains containing two or more carbon atoms, more preferably an alkyl functional group such as ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, tetradecyl, hexadecyl, icosyl, tetracosyl, dodecyl, cyclopentyl or cyclohexyl; a functional group having an unsaturated carbon chain containing a carbon-carbon double bond, such as ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, octenyl, decenyl or oleyl; or a functional group having an unsaturated carbon chain containing a carbon-carbon triple bond, such as propyl
  • examples of the hydrophilic region include a functional group being neutral at a specific pH, but being positively or negatively charged at a higher or lower pH such as -SH, -COOH, -NH 2 , -OH, -PO 3 H, -PO 4 H 2 , -SO 3 H, -OSO 3 H-NR 3 + X ' and so on.
  • preferable examples thereof include a polymer and a block copolymer, wherein monomers used therefor include ethylglycol, acrylic acid, alkylacrylic acid, ataconic acid, maleic acid, fumaric acid, acrylamidomethylpropane sulfonic acid, vinylsulfonic acid, vinylphophoric acid, vinyl lactic acid, styrenesulfonic acid, allylammonium, acrylonitrile, N-vinylpyrrolidone and N-vinylformamide, but not limited to.
  • monomers used therefor include ethylglycol, acrylic acid, alkylacrylic acid, ataconic acid, maleic acid, fumaric acid, acrylamidomethylpropane sulfonic acid, vinylsulfonic acid, vinylphophoric acid, vinyl lactic acid, styrenesulfonic acid, allylammonium, acrylonitrile, N-vinylpyrrolidone and N-vinyl
  • the preferable water-soluble multi-functional ligand in the nanoparticle of the present invention is a carbohydrate. More preferably, the carbohydrate includes, but not limited to, glucose, mannose, fucose, N-acetyl glucomine, N-acetyl galactosamine, N-acetylneuraminic acid, fructose, xylose, sorbitol, sucrose, maltose, glycoaldehyde, dihydroxyacetone, erythrose, erythrulose, arabinose, xylulose, lactose, trehalose, mellibose, cellobiose, rafflnose, melezitose, maltoriose, starchyose, estrodose, xylan, araban, hexosan, fructan, galactan, mannan, agaropectin, alginic acid, carrageenan, hemicelluloses, hyprome
  • the surface of the zinc-containing magnetic nanoparticles used in the present invention is coated with a water-soluble multi-functional organic ligand, and linked to the binding agent having a specific affinity to a target material through the binding region (Lu).
  • the zinc-containing nanoparticles may be synthesized according to various methods.
  • the zinc-containing nanoparticles of the present invention may be produced using a nanoparticle synthesis method in gas phase or in liquid phase ⁇ e.g., aqueous solution, organic solution or multiple solution system, etc.) known to those ordinarily skilled in the art.
  • the water-soluble zinc-containing nanoparticle coated with a water-soluble multi-functional organic ligand may be synthesized according to a chemical reaction in an aqueous solution.
  • This method is to synthesize the zinc- containing water-soluble metal oxide nanoparticles by adding zinc ion precursor materials to the reaction solution containing the water-soluble multi-functional ligand. It may be performed according to a synthesis method ⁇ e.g., a coprecipitation method, a sol-gel method, a micelle method, etc.) of a conventional water-soluble nanoparticle known to those skilled in the art.
  • a metal nitrate-based compound, a metal sulfate-based compound, a metal acetylacetonate-based compound, a metal fluoroacetoacetate-based compound, a metal halide-based compound, a metal perchlorate-based compound, a metal alkyloxide-based compound, a metal sulfamate-based compound, a metal stearate-based compound, a metal alkoxide- based compound or an organometallic compound may be used, but not limited to.
  • a benzene-based solvent a hydrocarbon solvent, an ether-based solvent, a polymer solvent, an ionic liquid solvent, an alcohol-based solvent, a sulfoxide-based solvent or water
  • benzene, toluene, halobenzene, octane, nonane, decane, benzyl ether, phenyl ether, hydrocarbon ether, a polymer solvent, diethylene glycol (DEG), water or an ionic liquid solvent may be used, but not limited to.
  • the zinc-containing metal oxide nanopartides according to the above- described method have a uniform size distribution ( ⁇ ⁇ 10%) and a high crystallinity.
  • zinc-content in the nanoparticle may be precisely controlled. In other words, by changing the ratio of zinc to other metal precursor material, the zinc-content in the nanoparticle can be controlled between 0.001 ⁇ 'zinc/(entire metal material - zinc)' ⁇ 10 in a stiochiometric ratio.
  • the hydrodynamic diameter of the final nanopartides prepared by the present invention is in a size range of preferably 1-1000 nm, more preferably 1-800 nm, much more preferably 2-100 nm and most preferably 10-50 nm.
  • the hydrodynamic diameter of the final nanoparticle clusters prepared by the present invention is in a size range of preferably 1-1000 nm, more preferably 1-800 nm, much more preferably 2-100 nm and most preferably 10-50 nm.
  • the nanoparticles of this invention have a saturation magnetization (M s ) value in a range of 100-200 emu/g and more preferably 120-200 emu/g.
  • the zinc-containing magnetic nanoparticle-based magnetic separation system further includes a magnetic field- generating means.
  • the magnetic field-generating means include a conventional magnet such as a permanent magnet and electromagnet known to those ordinarily skilled in the art.
  • the permanent magnet and electromagnet may be modified depending on a separation system, and have a magnetic field strength of preferably 10-10,000 mT, more preferably 100-5,000 mT, and most preferably 200-1,000 mT.
  • a method for separating a target material in a sample comprising the steps of:
  • M represents a magnetic metal atom or an alloy thereof
  • M represents a magnetic metal atom or an alloy thereof
  • M' represents one or more elements selected from the group consisting of Group 1 elements, Group 2 elements, Group 12 elements, Group 13 elements, Group 14 elements, Group 15 elements, transition metal elements, Lanthanide elements and Actinide elements) (2);
  • the zinc-containing magnetic nanopartide-target material complex is separated from other components in the sample by induction of magnetic field.
  • the complex may be removed from other components in the sample by various methods (pipetting, draining, skimming, pouring, etc.) to obtain a sample concentration solution containing only the zinc-containing magnetic nanopartide-target material complex.
  • various methods pipetting, draining, skimming, pouring, etc.
  • zinc-containing magnetic nanopartides may be added to a reactor containing a sample and mixed, they may be separated in a portion of the reactor by induction of magnetic field.
  • only the zinc-containing magnetic nanopartides may be separated by pipetting the sample under magnetic field induction.
  • the zinc-containing magnetic nanopartides are washed and resuspended in a suitable solvent, enabling to produce a sample concentration solution containing only the zinc-containing magnetic nanopartide-target material complex.
  • the separation method of the present invention may be combined with a detection or quantification process.
  • the method of the present invention further includes a step (c) which detects a formation of the zinc-containing magnetic nanopartide-target material complex in the sample.
  • a method to detect a formation of the zinc-containing magnetic nanopartide- target material complex in the sample may be carried out according to the conventional methods known to those ordinarily skilled in the art. For instance, the detection described above may be accomplished by measuring an absorbance change for a sample containing the zinc-containing magnetic nanopartide-target material complex. In addition, the zinc-containing magnetic nanoparticle complex conjugated with a signal generating label may be detected by measuring the fluorescent signal.
  • the signal generating label includes, but is not limited to, chemical labels
  • biotin ⁇ e.g., biotin
  • enzyme labels ⁇ e.g., alkaline phosphatase, peroxidase, ⁇ -galactosidase and ⁇ -glucosidase
  • radioisotopes ⁇ e.g., I 125 and C 14
  • fluorescent labels e.g., luminescent labels, chemiluminescent labels, FRET (fluorescence resonance energy transfer) labels and heavy metals ⁇ e.g., gold).
  • the non-limiting example of the above-described fluorescent label includes fluorescein, rhodamine, lucifer yellow, B-phytoerythrin, 9-acrydine isothiocyanate, lucifer yellow VS, 4-acetamido-4'-isothio-cyanatostilbene-2,2'-disulfonate, 7- diethylamino-3-(4'-isothiocyatophenyl)-4-methylcoumarin, succinimidyl- pyrenebutyrate, 4-acetoamido-4'-isothio-cyanatostilbene-2,2'-disulfonate derivatives, LCTM-Red 640, LCTM-Red 705, Cy5, Cy5.5, resamine, isothiocyanate, erythrin isothiocyanate, diethyltriamine pentaacetate, l-dimethylaminonaphthyl-5-sulfonate, l
  • a method to detect the formation of zinc-containing magnetic nanoparticle-target material complex conjugated with the label may be carried out according to the methods such as fluorometer, spectrophotometer, colorimetric detection or radioactivity detection.
  • the present invention may be used in separation, concentration and detection of a specific target material in a sample.
  • Using zinc-containing magnetic nanoparticles synthesized depending on a type of binding agent to be bound to a target material an economic system may be designed to separate and/or detect various target materials in a high efficient manner. According to the present invention, it is also unnecessary to perform further procedures such as complicated pre-enrichment, purification or processing needed in common detection.
  • the nanopartides of the present invention may be rapidly distributed into a biological sample (generally, an aqueous sample), i.e., numerous opportunities to contact a target material in an aqueous sample by Brown's diffusion, and furthermore may be harvested/separated under magnetic field induction.
  • a biological sample generally, an aqueous sample
  • numerous opportunities to contact a target material in an aqueous sample by Brown's diffusion and furthermore may be harvested/separated under magnetic field induction.
  • the present invention may be utilized in various application fields including: (a) diagnosis - disease-related substances (for example, proteins, antigens, nucleic acid molecules, viruses, bacteria, carbohydrates and lipids) may be separated/detected by applying the zinc-containing magnetic nanopartides to a biological sample; (b) separation - a final product may be separated by applying the nanopartides to a synthesis of a chemical compound; (c) water or seawater desalination using zinc-containing magnetic nanopartides modified with a chelating /complexing agent to selectively remove a specific salt, ion or metal.
  • diagnosis - disease-related substances for example, proteins, antigens, nucleic acid molecules, viruses, bacteria, carbohydrates and lipids
  • separation - a final product may be separated by applying the nanopartides to a synthesis of a chemical compound
  • water or seawater desalination using zinc-containing magnetic nanopartides modified with a chelating /complexing agent to selectively remove a specific salt, ion or metal
  • the present magnetic separation system using the same has much more improved separation efficiencies.
  • an economic system may be designed to separate and/or detect various target materials in a high efficient manner. According to the present invention, it is also unnecessary to perform further procedures such as complicated pre-enrichment, purification or processing needed in common detection.
  • a zinc-containing magnetic nanopartide-based magnetic sensor comprising: (a) a zinc-containing magnetic nanopartide or a cluster thereof represented by the following formula 1 or 2:
  • Zn f M a-f O b (0 ⁇ f ⁇ 8, 0 ⁇ a ⁇ 16, 0 ⁇ b ⁇ 8, 0 ⁇ f/(a-f) ⁇ 10, M represents a magnetic metal atom or an alloy thereof)
  • Zn g M c-g M' d O e (0 ⁇ g ⁇ 8, 0 ⁇ c ⁇ 16, 0 ⁇ d ⁇ 16, 0 ⁇ e ⁇ 8, 0 ⁇ g/ ⁇ (c-g)+d ⁇ 10
  • M represents a magnetic metal atom or an alloy thereof
  • M' represents one or more elements selected from the group consisting of Group 1 elements, Group 2 elements, Group 12 elements, Group 13 elements, Group 14 elements, Group 15 elements, transition metal elements, Lanthanide elements and Actinide elements)
  • the present magnetic sensor comprises the zinc-containing magnetic nanoparticles described in the magnetic separation system of this invention, the common descriptions between them, are omitted in order to avoid undue redundancy leading to the complexity of this specification.
  • the zinc-containing magnetic nanopartide or the cluster thereof used in the magnetic sensor of this invention plays a role in a signal generator.
  • the zinc-containing magnetic nanopartide used in the magnetic sensor of this invention is represented by the following formula 1 or 2:
  • M represents a magnetic metal atom or an alloy thereof
  • M represents a magnetic metal atom or an alloy thereof;
  • M' represents one or more elements selected from the group consisting of Group 1 elements, Group 2 elements, Group 12 elements, Group 13 elements, Group 14 elements, Group 15 elements, transition metal elements, Lanthanide elements and Actinide elements) (2) Since the zinc-containing magnetic nanoparticles of the formula 1 or 2 have high saturation magnetism, the present magnetic sensor using the same may exhibit much more enhanced sensitivity under magnetic field induction.
  • M represents preferably transition metal elements, Lanthanide metal elements and Actinide metal elements; more preferably, transition metal elements selected from the group consisting of Ba, Mn, Co, Ni and Fe, Lanthanide metal elements selected from the group consisting of Gd, Er, Ho, Dy, Tb, Sm and Nd; and most preferably, Mn, Co, Ni, Fe, Gd, Er, Ho, Dy, Tb, Sm or Nd, or an alloy thereof.
  • M' preferably represents one or more elements selected from the group consisting of Li, Na, K, Rb, Cs, Be, Mg, Ca, Sr, Ba, Ra, Ge, Ga, Bi, In, Si, Ge, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Y, Zr, Nb, Mo, Tc, Ru, Rh, Pd, Ag, Cd, Hf, Ta, W, Re, Os, Ir, Pt, Au, Hg, Lanthanide elements and Actinide elements.
  • the zinc-containing magnetic nanoparticle used in the magnetic sensor of this invention is represented by the following formula 3:
  • M'V k FeiO j (0 ⁇ k ⁇ 8, 0 ⁇ h ⁇ 16, 0 ⁇ i ⁇ 8, 0 ⁇ j ⁇ 8, 0 ⁇ k/ ⁇ (h-k)+i ⁇ 10, M" represents a magnetic metal atom or an alloy thereof)
  • M represents preferably transition metal elements, Lanthanide metal elements and Actinide metal elements; more preferably, transition metal elements selected from the group consisting of Ba, Mn, Co, Ni and Fe, Lanthanide metal elements selected from the group consisting of Gd, Er, Ho, Dy, Tb, Sm and Nd; and most preferably, Mn, Co, Ni, Fe, Gd, Er, Ho, Dy, Tb, Sm or Nd, or an alloy thereof. More preferably, the zinc-containing magnetic nanoparticle used in the magnetic sensor of this invention is represented by the following formula 4 or 5:
  • a stoichiometric content ratio of zinc and other metals is as follows: 0.001 ⁇ 'zinc/ (entire metal component - zinc)' ⁇ 10, more preferably 0.01 ⁇ 'zinc/ (entire metal component - zinc)' ⁇ 1, and most preferably 0.03 ⁇ 'zinc/ (entire metal component - zinc)' ⁇ 0.5.
  • zinc is contained as the above, high saturation magnetism can be obtained, resulting in remarkable improvement of sensor sensitivity in the present magnetic sensor.
  • the zinc-containing magnetic nanopartides contained in the cluster of the present invention are clustered in the number of preferably 2-10,000, more preferably 2-1,000, and most preferably 2-100.
  • Each zinc-containing magnetic nanopartides in the duster is linked each other by their intermolecular interactions, or encapsulated by organic or inorganic carrier.
  • the surface of the zinc-containing magnetic nanopartides or cluster thereof is coated with a water-soluble multi- functional ligand.
  • water-soluble multi-functional ligand refers to a ligand that may be bound to zinc-containing nanopartides or cluster thereof to solublize in water and stabilize the nanopartides, and may allow the nanopartides to be bound by binding agent having a specific affinity to an analyte.
  • the water-soluble multi-functional ligand can include (a) an adhesive region
  • the compound which originally contains the above-described functional group may be used as a water-soluble multi-functional ligand, but a compound modified or prepared so as to have the above-described functional group by a chemical reaction known in the art may be also used as a water-soluble multifunctional ligand.
  • the preferable multi-functional ligand of the present invention includes a monomer, a polymer, a carbohydrate, a protein, a peptide, a nucleic acid, a lipid or an amphiphilic ligand.
  • the hydrodynamic diameter of the final nanopartides prepared by the present invention is in a size range of preferably 1-1000 nm, more preferably 1-800 nm, much more preferably 2-100 nm and most preferably 10-50 nm.
  • the hydrodynamic diameter of the final nanopartide clusters prepared by the present invention is in a size range of preferably 1-1000 nm, more preferably 1-800 nm, much more preferably 2-100 nm and most preferably 10-50 nm.
  • the nanopartides of this invention have a saturation magnetization ( ⁇ / s ) value in a range of 100-300 emu/g and more preferably 120-200 emu/g.
  • the zinc-containing magnetic nanopartide used in the present invention is linked to a binding agent having a binding affinity to an analyte. More preferably, the zinc-containing magnetic nanopartide used in the present invention is coated with a water-soluble multifunctional organic ligand, and linked to the binding agent having a specific affinity to an analyte through the binding region (Ln).
  • analyte refers to a substance or chemical constituent in a sample ⁇ e.g., a liquid or gas sample, preferably a liquid sample, and most preferably an aqueous sample) to be separated (detected or quantified).
  • the analyte includes a nucleic acid molecule (DNA or RIMA), a protein, a peptide, an antigen, a bacterium, a virus, a sugar, a lipid, an organic compound ⁇ e.g., chiral compounds, therapeutic compounds), an inorganic compound, a metal and an inorganic ion, but is not limited to.
  • binding agent means a substance having a specific affinity to an analyte to be separated. Any material having a binding affinity to a target material to be separated may be utilized as the binding agent.
  • a non-limiting example of a binding agent includes a nucleic acid molecule (DNA or RNA), an antibody, an aptamer, a receptor, a streptavidin, an avidin, a biotin, a lectin, a ligand, an enzyme, a coenzyme, an inorganic ion, a cofactor, a sugar, a lipid, a substrate for an enzyme, a hapten, a neutravidin, a protein A, a protein G, a selectin, an inorganic compound, a metal, a semiconductor and an organic compound.
  • the magnetic sensor of this invention may be prepared as a variety of methods.
  • the zinc-containing magnetic nanopartide-based magnetic sensor includes all types of sensors containing magnetic nanoparticles such as a magneto-resistance sensor, a magnetic relaxation sensor, a magnetic micro- cantilever sensor, a magneto-electronic sensor and magnetophoresis sensor.
  • the magnetic sensor of the present invention may further include a signal processing means.
  • the signal processing means may include at least one amplifier.
  • the signal processing means may further include a linearizing circuit, and thus have potential to normalize a non-linear R-H property of a sensor device.
  • a magnetic sensor device, a signal processing means and a magnetic field-generating means may be composed as a single integrated circuit.
  • the magnetic field-generating means may include a conductor and a current source to generate current through the conductor.
  • the sensor of the present invention further includes a means measuring current via a magnetic sensor device.
  • the present invention using a magnetic sensor as a magneto-resistance sensor includes the steps of: (a) generating magnetic field in the vicinity of a magnetic sensor device; (b) inducing regular voltage across the magnetic sensor device; and (c) measuring total signal currents of the magnetic sensor device.
  • the zinc-containing magnetic nanoparticles are preferably used as a label of an analyte.
  • the sample containing an analyte is reacted with the zinc- containing magnetic nanopartides coated with a binding agent having a specific affinity to the analyte for labeling, and then the signal from the magnetic nanopartides is measured to determine the presence or amount of the analyte in the sample.
  • an antibody immobilized on a substrate is contacted with a sample containing an analyte, and washed.
  • the substrate is reacted with a detecting antibody linked to the surface of zinc-containing magnetic nanoparticle, and then washed. After stopping reaction, it may be concluded that the analyte is involved in the sample with the proviso that the signal from the zinc-containing magnetic nanopartides is detected (by the magnetic sensor device).
  • the changes in the Tl relaxation time are measured to determine contacting between zinc-containing magnetic nanopartides and a reactant, i.e., the presence or amount of a reactant).
  • a reactant i.e., the presence or amount of a reactant.
  • the reactants are linked to the binding agents on the surface of nanopartides, leading to form nanoparticle aggregates.
  • the magnetic moment of nanoparticle is changed, and T2 relaxation time of water is decreased.
  • the changes in the T2 relaxation time may be measured using a MR (magnetic resonance) imaging machine. It may be concluded that the analyte is involved in the sample with the proviso that the changes in the T2 relaxation time are detected.
  • the amount of reactants may be easily determined.
  • the changes in the Tl relaxation time may be measured using T2-weighted spin echo sequence and 1.5 T superconduction magnet under the condition of fixed echo time (TE) and repetition time (TR).
  • MR imaging instruments may be utilized as a magnetic sensor device and magnetic field-generating means in a magnetic relaxation sensor.
  • MR imaging method and devices are disclosed in D. M. Kean and M. A. Smith,
  • a magnetic micro-cantilever sensor is composed of a micro-sized cantilever and magnetic nanopartides conjugated with a substance ⁇ e.g., antigen) capable of binding to a reactant of interest.
  • the reactant in a sample is attached to the cantilever and nanopartides according to a sandwich method.
  • the cantilever is bent by the magnetic nanopartides under magnetic field induction. The bending force is measured as current by a piezoelectric device, enabling to determine exact amount of reactant.
  • a magnetophoresis sensor is a sensor employing the motion of dispersed magnetic nanopartides relative to a fluid under the influence of a magnetic field.
  • the movement of magnetic particles can be used to detect or isolate specific components in the fluid, using specific binding and/or capture.
  • the magnetophoresis sensor using the magnetic nanopartide or cluster thereof of the present invention is described in Example 8.
  • the sensors using magnetic nanopartides include, but is not limited to, a Maxwell bridge, a frequency dependent magnetometer, a magnetic remanence measurement using a superconducting quantum interference device, a Hall effect measurement, a micro fluidic system, and so forth.
  • the magnetic field-generating means used in the present invention may include a permanent magnet and electromagnet and may be varied by modulation depending on the morphology and size of nanoparticle.
  • Zn f M a - f O b (0 ⁇ f ⁇ 8, 0 ⁇ a ⁇ 16, 0 ⁇ b ⁇ 8, 0 ⁇ f/(a-f) ⁇ 10, M represents a magnetic metal atom or an alloy thereof)
  • Zn g M c-g M' d O e (0 ⁇ g ⁇ 8, 0 ⁇ c ⁇ 16, 0 ⁇ d ⁇ 16, 0 ⁇ e ⁇ 8, 0 ⁇ g/ ⁇ (c-g)+d ⁇ 10
  • M represents a magnetic metal atom or an alloy thereof
  • M' represents one or more elements selected from the group consisting of Group 1 elements, Group 2 elements, Group 12 elements, Group 13 elements, Group 14 elements, Group 15 elements, transition metal elements, Lanthanide elements and Actinide elements) (2); and
  • the present method comprises the magnetic sensor of this invention described above, the common descriptions between them are omitted in order to avoid undue redundancy leading to the complexity of this specification.
  • the zinc-containing magnetic nanoparticle-based magnetic sensor of the present invention may be utilized in various application fields including molecular diagnosis, analysis for a biological sample and chemical sample, and so on. Since the magnetic sensor of the present invention utilizes zinc-containing magnetic nanopartide having very high saturation magnetism, it may exhibit much more enhanced sensitivity under magnetic field induction and detect an infinitesimal amount of analytes ⁇ e.g., proteins or nucleic acid molecules in blood).
  • the present magnetic sensor using the same may detect analytes in a much more sensitive manner.
  • the nanopartides having improved sensitivity ⁇ e.g., sensitivity in fM level) prepared by the present invention may permit to detect a trace amount of analytes such as anti-cancer marker proteins or nucleic acid molecules, and prion proteins in blood.
  • the present sensor may be constituted by all types of sensors including a magneto-resistance sensor, a magnetic relaxation sensor, a magnetic micro-cantilever sensor, a magnetophoresis sensor, a magneto-electronic sensor, and so forth.
  • Fig. Ia shows a TEM (transmission electron microscope) image of synthesized Zno. 4 Fe 2 . 6 O 4 nanopartides.
  • Fig. Ib shows a high resolution TEM image of synthesized Zn 0-4 Fe 2-6 O 4 nanopartides.
  • the internal image of Fig. Ib is a FFT (fast fourier transformation) image of the high resolution TEM image.
  • Fig. Ic is a XRD (X-ray diffraction) pattern of synthesized Zn 0-4 Fe 2-6 O 4 nanopartides.
  • Fig. 2 represents TEM images of (A) ZnxMn 1-x Fe 2 0 4 and (B) Zn x Fe 3-x 0 4 .
  • the zinc content ratio (x) may be modulated from 0 to 0.8.
  • Fig. 3 is a graph representing a saturation magnetism of zinc-containing magnetic nanopartides and several nanopartides containing no zinc.
  • Fig. 4 represents a TEM image of synthesized zinc-containing nanopartide cluster.
  • the cluster is prepared by encapsulating zinc-containing nanopartides in a micelle consisting of polystyrene-polyacryl acid copolymers.
  • Fig. 5 is a graph measuring a magnetic field strength depending on the distance from the surface of NdFeB magnet used as external magnetic field- generating means.
  • Fig. 6 schematically represents the principle of a magnetic separation system using zinc-containing magnetic nanoparticles.
  • a solution containing a target material is mixed with magnetic nanoparticles conjugated with a binding agent capable of selectively binding to the target material, resulting in binding of the target material and magnetic nanoparticles. Afterwards, the target material may be separated by magnet attraction.
  • Fig. 7 shows a graph comparing separation efficiency of a fluorescent material (rhodamine) using zinc-containing nanoparticles (Zno. 4 Fe 2 .eO 4 ) or nanoparticles containing no zinc (Fe 3 O 4 ).
  • Fig. 7A represents a graph measuring the fluorescent material (rhodamine) absorbance at 580 nm depending on magnetic separation time
  • Fig. 7B represents a graph measuring separation efficiency of the fluorescent material (rhodamine) with the passage of time.
  • Fig. 8 is a graph comparing magnetic separation efficiency between zinc- containing nanoparticles (Zn 0-4 Fe 2-6 O 4 ) and nanoparticles containing no zinc (Fe 3 O 4 ) according to changes of external magnetic field strength.
  • Fig. 9 shows a graph comparing antibody separation using nanoparticles containing no zinc and zinc-containing nanoparticles.
  • Fig. 10 represents results comparing: (A) cell amounts separated with the passage of time in zinc-containing nanoparticles and nanoparticles containing no zinc; and (B) cell separation efficiency of zinc-containing nanoparticles and nanoparticles containing no zinc.
  • Fig. lla shows a TEM image of synthesized Zn 0-4 Fe 2-6 O 4 nanoparticles.
  • Fig. lib shows a high resolution TEM image of synthesized Zn 0-4 Fe 2-6 O 4 nanoparticles.
  • the internal image of Fig. Ib is a FFT (fast fourier transformation) image of the high resolution TEM image.
  • Fig. lie is a XRD (X-ray diffraction) pattern of synthesized Zn 0-4 Fe 2-6 O 4 nanoparticles.
  • Fig. 12 represents TEM images of (A) ZnxMni -x Fe 2 0 4 and (B) Zn x Fe 3-x 0 4 .
  • the zinc content ratio (x) may be modulated from 0 to 0.8.
  • Fig. 13 is a graph analyzing a saturation magnetism of zinc-containing magnetic nanoparticles and several nanoparticles containing no zinc.
  • Fig. 14 represents a TEM image of synthesized zinc-containing nanopartide cluster.
  • the cluster is prepared by encapsulating zinc-containing nanoparticles in a micelle consisting of polystyrene-polyacryl acid copolymers.
  • Fig. 15 schematically represents the principle of a magnetic relaxation sensor using zinc-containing magnetic nanoparticles.
  • the magnetic nanoparticles exhibit high spin-spin relaxation time (T2) due to rare interaction each other at a distance enough to be dispersed, but the formation of magnetic nanopartide aggregates mediated by samples of interest allows magnetic particles having very low TZ relaxation time.
  • T2 relaxation time may be measured using MRI or magnetic relaxation system.
  • Fig. 16 shows DNA detection using in zinc-containing nanoparticles
  • Fig. 16A represents a colorimetric MRI image merging ⁇ T2 with image measured by MRI in each nanoparticles.
  • Fig. 16B is a graph expressing Fig. 16A numerically (i.e., ⁇ T2 value to
  • Fig. 17 shows protein (avidin) detection using in zinc-containing nanoparticles
  • Fig. 17A represents a colorimetric MRI image merging ⁇ T2 with image measured by MRI in each nanoparticles.
  • Fig. 17B is a graph expressing Fig. 17A numerically (i.e., ⁇ T2 value to protein amount analyzed).
  • Fig. 18 schematically represents a magnetophoresis sensor using zinc- containing magnetic nanoparticles.
  • Fig. 19 represents a graph analyzing a cell migration rate (panel A) and attractive force toward a magnetic tip (panel B).
  • Fig. 20 shows microscopic images observing practical operation of the magnetophoresis sensor. It could be appreciated that the cells bound with zinc- containing magnetic nanoparticles are migrated toward a magnetic tip more rapidly than those bound with magnetic nanoparticles containing no zinc.
  • DMSA Dimercaptosuccinic Acid
  • ZnCI 2 Aldrich, USA
  • FeCI 2 Aldrich, USA
  • MnCI 2 Aldrich, USA
  • Fe(acac) 3 Aldrich, USA
  • the synthesized zinc-containing ferrite nanoparticles were precipitated by excess ethanol and then the precipitated nanoparticles were again dispersed in toluene, obtaining a colloid solution.
  • nanoparticles synthesized according to the method aforementioned have globular structure with a homogeneous size of 15 nm (size distribution s ⁇ 10%). As demonstrated in a high resolution electron microscope (Fig.
  • the nanoparticles exhibit higher crystallinity as a spinel structure.
  • the amount of zinc was determined using Inductively Coupled Plasma- Atomic Emission Spectroscopy (ICP-AES, OPTIMA-3000, Perkin Elmer) and Energy Dispersive X-ray (EDAX, Gatan).
  • nanoparticles with different sizes ⁇ e.g., 9 or 12 nm
  • equal amount of precursors were mixed with trioctylamine solvent containing various ratios of oleic acid and oleylamine depending on the sizes of nanoparticles, and the same process described above was carried out.
  • the synthesized nanoparticles (20 mg/ml in toluene) were mixed with DMSO solution containing excess DMSA, and incubated for 2 hrs. Subsequently, the nanoparticles were centrifuged and re-dispersed in water. As results, the synthesized nanoparticles have globular structure with a homogeneous size, and their zinc contents were analyzed using ICP-MS and EDAX.
  • ZnCI 2 , CoCI 2 or NiCI 2 , and Fe(acac) 3 as precursors of nanoparticles were added to trioctylamine solvent containing 20 mmol oleic acid and 20 mmol oleylamine as capping molecules.
  • M Co or Ni
  • the synthesized nanoparticles (20 mg/ml in toluene) were mixed with DMSO solution containing excess DMSA, and incubated for 2 hrs. Subsequently, the nanoparticles were centrifuged and re-dispersed in water. As results, the nanoparticles have globular structure with a homogeneous size, and zinc contents were analyzed using ICP-MS and EDAX.
  • the synthesized nanopartides (50 mg/ml in 1 ml toluene) were precipitated by excess ethanol and then the precipitated nanopartides were again dispersed in 5 ml TMAOH solution, obtaining a soluble solution.
  • the synthesized nanopartides have the composition of Zn 0-4 M 26 O 4 , Zn 0 . 2 Co 0 . 8 ⁇ or Zn 0-2 Ni 0-8 O with the core size of 6, 7 or 10 nm, respectively.
  • Zinc contents were analyzed using ICP-MS and EDAX.
  • each saturation magnetization of zinc-containing nanopartides, conventionally accessible metal oxide nanopartides, CLJO (cross-linked iron oxide) and FeridexTM (Taejoon Co Ltd.), and several ferrite nanopartides containing no zinc was measured at room temperature.
  • CLJO was synthesized according to the method described in Weissleder et a/., Journal of Magnetic Resonance in Medicine 29: 599 (1993).
  • Several ferrite nanopartides were produced according to the method described in Korean Pat. Nos. 10-0604975, 10-0652251 and 10-0713745, PCT/KR2004/002509, Korean Pat. No.
  • saturation magnetization of each zinc-containing nanoparticles, Zn 0-4 Fe 2 ⁇ O 4 and Zn 0-4 Mn 0-6 Fe 2 O 4 was 161 and 175 emu/g higher than that of conventionally accessible metal oxide nanoparticles ⁇ e.g., CLIO, 64 emu/g;
  • Feridex 82 emu/g. Furthermore, these values are enhanced to 47 and 61 emu/g compared with nanoparticles containing no zinc, Fe 3 O 4 and MnFe 2 O 4 , respectively.
  • zinc-containing nanoparticles having enhanced saturation magnetization may highly increase conventional sensor sensitivity in the senses that saturation magnetization is proportional to the square of sensor sensitivity.
  • Exemplified nanoparticle cluster using zinc-containing nanoparticles includes a magnetic nanoparticle cluster which is encapsulated in micelles prepared by polystyrene-polyacryl acid copolymers.
  • Basic preparation method of polymer and cluster used was carried out according to the method described in Taton et a/. Nano Leters 5: 1987 (2005).
  • Zinc-containing nanoparticles coated with oleic acid and oleylamine were prepared according to the method described in Korean Pat. Nos. 10- 0604975, 10-0652251 and 10-0713745, PCT/KR2004/002509, Korean Pat. No. 10- 0604975, PCT/KR2004/003088, PCT/KR2007/001001, and Korean Pat. Appln. No.
  • the polystyrene-polyacryl acid copolymers were dissolved in dimethylformamide (DMF; Aldrich) at a concentration of 0.1 mg/ml, and further added with tetrahydrofuran (THF; Aldrich) to be at a final concentration of 50%.
  • DMF dimethylformamide
  • THF tetrahydrofuran
  • the solution was vigorously stirred and the nanoparticles were gradually added to be at a concentration of 0.1 mg/ml, followed by adding 40 ml water at a rate of 5 ml/hr.
  • micelles are stabilized in a soluble solution due to exposure of carboxylic acid on their surface, which serves as a functional group to attach a binding agent such as antibodies.
  • the micelles were cross-linked to enhance their stability and dispersion in solution.
  • 5 mM EDC 1- ethyl-3-(3-dimethylaminopropyl) carbodiimide; Sigma
  • 2 mM sulfo-NHS N- hydroxysulfosuccinimide; Sigma
  • 5 mM 2,2'-(ethylenedioxy)bis(ethylamine) Aldrich
  • the synthesized micelles have a total size of about 120 nm, and contain numbers of 15 zinc-containing nanoparticles. As described above, it is expected that the synthesized nanoparticle clusters exhibit enhanced saturation magnetization, leading to significantly improve separation efficiencies of magnetic separation systems.
  • EXAMPLE 7 Magnetic Field Measurement of NdFeB Magnets Used as a Means for Generating External Magnetic Field
  • NdFeB magnets were used in the magnetic separation system of the present invention.
  • the magnetic separation system used in the present invention has a working principle shown in Fig. 6. Through mixing a solution containing materials of interest with magnetic nanoparticles including binding agents capable of selectively binding to materials of interest, they may be linked to magnetic nanoparticles, enabling to be separated by intermolecular interaction of magnets.
  • Each zinc-containing nanoparticles and nanoparticles containing no zinc were synthesized according to the methods described in Example 1 and Korean Pat. Nos. 10-0604975, 10-0652251 and 10- 0713745, PCT/KR2004/002509, Korean Pat. No.
  • each nanoparticles were solubilized in water by surface modifications with TMAOH according to the method described in Example 4. Afterwards, these nanoparticles were coated with bovine serum albumin (BSA) according to the methods described in Korean Pat. Nos. 10-0604975, 10-0652251 and 10-0713745.
  • BSA bovine serum albumin
  • fluorescent dye rhodamine; Pierce
  • Fig. 7 represents a graph measuring rhodamine absorbance at 580 nm by monitoring with the passage of time.
  • rhodamine molecules in solution are decreased with the passage of time, resulting in gradual reduction of their absorbance.
  • almost all rhodamine molecules were separated at about 4 min in zinc- containing nanoparticles, and their absorbance was measured in the level of approximate 0.02.
  • the absorbance of rhodamine was unchangeable until 6 min in nanoparticles containing no zinc, and was reduced at 10 min to the level of about 0.12, reaching at the level of almost 0.02 after 50 min.
  • Fig. 7B is a graph representing separation rate (%) of Fig. 7A.
  • Zinc-containing magnetic nanoparticles and magnetic nanoparticles containing no zinc with a concentration 0.2 mg/ml was added to MACS ® column under external magnetic fields, respectively.
  • the magnetic nanoparticles were captured in MACS ® column by external magnetic fields, and the removal of external magnetic fields causes their release from the column.
  • nanoparticle magnetism is varied depending on a type of nanoparticle, the amount of nanoparticles captured in the column is different.
  • the captured nanoparticles were quantified using an absorption spectrophotometer.
  • Fig. 8 is a graph comparing magnetic separation efficiency between zinc- containing nanoparticles (Zn 0-4 Fe 2-6 O 4 ) and nanoparticles containing no zinc (Fe 3 O 4 ) according to changes of external magnetic field strength. As shown in Fig. 8, the amount of both separated magnetic nanoparticles was similar under very strong external magnetic fields (300 mT). However, the separated amount of zinc- containing nanoparticles or nanoparticles containing no zinc was significantly different depending on reduction of external magnetic field. That is, the amount of zinc-containing nanoparticles captured in the column was increased in 17% and 22% under external magnetic fields of 150 and 100 mT higher than that of nanoparticles containing no zinc, respectively.
  • EXAMPLE 10 Comparison and Quantification of Magnetic Separation Efficiency in Zinc-Containing Nanoparticles and Nanoparticles Containing no Zinc for Anti-Mouse IgG-FITC in the Presence of External Magnetic Field
  • MACS ® column (Miltenyi Biotech, Germany) was used as a magnetic separation system.
  • Nanopartides containing no zinc and zinc-containing nanopartides are Fe 3 O 4 and Zn 0-4 Fe 2-6 O 4 , respectively. Both nanopartides were particles with a diameter of 15 nm.
  • the surface of each nanopartides was coated with dimercaptosuccinic acid (DMSA) and then linked to protein A (Sigma).
  • DMSA dimercaptosuccinic acid
  • the synthesis of nanopartides was carried out according to the method described in Example 1. To bind protein A on the surface of nanopartides, it was reacted with sulfo-SMCC (Sulfosuccinimidyl-4-(N- maleimidomethyl)cydohexane-l-carboxylate; Pierce) for 30 min, and then mixed with nanopartides.
  • sulfo-SMCC Sulfosuccinimidyl-4-(N- maleimidomethyl)cydohexane-l-carboxylate; Pierce
  • Target protein of the present invention is goat anti-mouse IgG-FlTC (Sigma). Under the condition of external magnetic field, each zinc-containing nanopartides (0.08 mg/ml) and nanopartides containing no zinc (0.08 mg/ml) were added to MACS ® column, and subsequently to antibody protein (1 ml of 0.01 M anti-mouse IgG-FITC) labeled with fluorescent dye, resulting in binding of antibody protein on the surface of nanopartides by specific interactions between antibody protein and protein A coated on the surface of nanopartides.
  • Magnetic nanopartides are captured in MACS ® column due to external magnetic field, and thus the removal of external magnetic field allows magnetic nanopartides to be released from the column.
  • the captured nanopartides may be quantified using an absorption spectrophotometer.
  • the amount of separated nanoparticles may be inversely determined by measuring fluorescent signal intensity as antibody protein with fluorescent dye is bound to the surface of captured nanoparticles. Consequently, separation efficiency to antibody protein may be analyzed by measuring magnetic values of two nanoparticles.
  • Fig. 9 is a graph comparing magnetic separation efficiency between zinc- containing nanoparticles (Zno.
  • the higher fluorescence intensity refers to the large amount of nanoparticles.
  • FITC has physical potential to emit the strongest fluorescence intensity at 521 nm by absorbing the light with a wavelength of 495 nm.
  • each zinc-containing nanoparticles and nanoparticles containing no zinc was coated with dimercaptosuccinic acid (DMSA) and bound to antibody proteins.
  • DMSA dimercaptosuccinic acid
  • the antibody proteins utilized Cetuximab (Merck) conventionally known as Erbitux, which is capable of binding to an EGFR receptor on the surface of U87MG cells.
  • antibody proteins were activated using sulfo-SMCC (Pierce) and then mixed with nanoparticles coated with DMSA according to the method described in Example 10.
  • sulfo-SMCC Pieris-maleimidomab
  • DMSA DMSA
  • Each nanoparticles linked to Cetuximab was incubated with cells in PBS buffer at room temperature for 1 hr, followed by adding external magnetic field. Afterwards, Cetuximab antibodies on the nanoparticles were bound to cells. Cells were attracted toward magnetic field under external magnetic field by the nanoparticles bound to the surface of cells. Thus, samples in reaction solution were harvested depending on time in certain region apart from magnets, and cell number was observed.
  • Fig. 10 is graphs comparing cell separation using zinc-containing nanoparticles (Zno. 4 Fe 2 .eO 4 ) and nanoparticles containing no zinc (Fe 3 ⁇ 4 ) as described above. Since magnetism of zinc-containing nanoparticles is higher than that of nanoparticles containing no zinc, cells were attracted in stronger and faster manner. Therefore, it could be appreciated that cell number separated from solution to a region adjacent to magnet were much more rapidly increased with the passage of time (Fig. 10A).
  • Fig. 1OB represents cell separation efficiency which is determined by counting cell number separated at 5 min after addition of external magnetic field. The cell separation efficiency is calculated by a ratio of cell number after to cell number before cell separation. It was demonstrated that the efficiency of cell separation in zinc-containing nanoparticles may be about 2.9-fold higher than that in nanoparticles containing no zinc, and the amount of separated cells may be quantified using a hematocytometer.
  • ZnCI 2 Aldrich, USA
  • FeCI 2 Aldrich, USA
  • MnCI 2 Aldrich, USA
  • Fe(acac) 3 Aldrich, USA
  • the synthesized zinc-containing ferrite nanoparticles were precipitated by excess ethanol and then the precipitated nanoparticles were again dispersed in toluene, obtaining a colloid solution.
  • nanoparticles synthesized according to the method aforementioned have globular structure with a homogeneous size of 15 nm (size distribution s ⁇ 10%). As demonstrated in a high resolution electron microscope (Fig.
  • the nanoparticles exhibit higher cr ⁇ stallinity as a spinel structure.
  • the amount of zinc was determined using Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES, OPT ⁇ MA-3000, Perkin Elmer) and Energy Dispersive X-ray (EDAX, Gatan).
  • nanopartides with different sizes e.g., 9 or 12 nm
  • equal amount of precursors were mixed with trioctylamine solvent containing various ratios of oleic acid and oleylamine depending on the sizes of nanopartides, and the same process described above was carried out.
  • the synthesized nanopartides (20 mg/ml in toluene) were mixed with DMSO solution containing excess DMSA, and incubated for 2 hrs. Subsequently, the nanopartides were centrifuged and re-dispersed in water. As results, the synthesized nanopartides have globular structure with a homogeneous size, and their zinc contents were analyzed using ICP-MS and EDAX.
  • ZnCI 2 , CoCI 2 or NiCI 2 , and Fe(acac) 3 as precursors of nanopartides were added to trioctylamine solvent containing 20 mmol oleic acid and 20 mmol oleylamine as capping molecules.
  • M Co or Ni
  • a ratio of ZnCI 2 , CoCI 2 or NiCI 2 as precursors of nanoparticles was modulated depending on the composition of Zn.
  • the synthesized nanoparticles (20 mg/ml in toluene) were mixed with DMSO solution containing excess DMSA, and incubated for 2 hrs. Subsequently, the nanoparticles were centrifuged and re-dispersed in water. As results, the nanoparticles have globular structure with a homogeneous size, and zinc contents were analyzed using ICP-MS and EDAX.
  • the synthesized nanoparticles (50 mg/ml in 1 ml toluene) were precipitated by excess ethanol and then the precipitated nanoparticles were again dispersed in 5 ml TMAOH solution, obtaining a soluble solution.
  • the synthesized nanoparticles have the composition of Zn 0-4 M 2-6 O 4 , Zn 0-2 Co 0-8 O or Zn 0 2 Ni 0-8 O with the core size of 6, 7 or 10 nm, respectively.
  • Zinc contents were analyzed using ICP-MS and EDAX.
  • each saturation magnetization of zinc-containing nanoparticles, conventionally accessible metal oxide nanoparticles, CLJO and FeridexTM, and several ferrite nanoparticles containing no zinc was measured at room temperature.
  • CLJO was synthesized according to the method described in Weissleder et a/., Journal of Magnetic Resonance in Medicine 29: 599 (1993).
  • Several ferrite nanoparticles were produced according to the method described in Korean Pat. Nos. 10-0604975, 10- 0652251 and 10-0713745, PCT/KR2004/002509, Korean Pat. No.
  • saturation magnetization of each zinc-containing nanoparticles was 161 and 175 emu/g higher than that of conventionally accessible metal oxide nanoparticles ⁇ e.g., CLJO, 64 emu/g; Feridex, 82 emu/g). Furthermore, these values are enhanced to 47 and 61 emu/g compared with nanoparticles containing no zinc, Fe 3 O 4 and MnFe 2 O 4 , respectively.
  • zinc-containing nanoparticles having enhanced saturation magnetization may highly increase conventional sensor sensitivity in the senses that saturation magnetization is proportionate to the square of sensor sensitivity.
  • Exemplified nanoparticle cluster using zinc-containing nanoparitldes includes a magnetic nanoparticle cluster which is encapsulated in micelles prepared by polystyrene-polyacryl acid copolymers.
  • Basic preparation method of polymer and cluster used was carried out according to the method described in Taton et a/. Nano Leters 5: 1987 (2005).
  • Zinc-containing nanopartides coated with oleic acid and oleylamine were prepared according to the method described in Korean Pat. Nos. 10- 0604975, 10-0652251 and 10-0713745, PCT/KR2004/002509, Korean Pat. No. 10- 0604975, PCT/KR2004/003088, PCT/KR2007/001001, and Korean Pat. Appln. No.
  • the polystyrene-polyacryl acid copolymers were dissolved in dimethylformamide (DMF; Aldrich) at a concentration of 0.1 mg/ml, and further added with tetrahydrofuran (THF; Aldrich) to be at a final concentration of 50%.
  • DMF dimethylformamide
  • THF tetrahydrofuran
  • the solution was vigorously stirred and the nanopartides were gradually added to be at a concentration of 0.1 mg/ml, followed by adding 40 ml water at a rate of 5 ml/hr.
  • micelles are stabilized in a soluble solution due to exposure of carboxylic acid on their surface, which serves as a functional group to attach a binding agent such as antibodies.
  • the micelles were cross-linked to enhance their stability and dispersion in solution.
  • 5 mM EDC 1- ethyl-3-(3-dimethylaminopropyl) carbodiimide; Sigma
  • 2 mM sulfo-NHS N- hydroxysulfosuccinimide; Sigma
  • 5 mM 2,2'-(ethylenedioxy)bis(ethylamine) Aldrich
  • the synthesized micelles have a total size of about 120 nm, and contain 15 numbers of zinc-containing nanopartides. As described above, it is expected that the synthesized nanopartide clusters exhibit enhanced saturation magnetization, leading to significantly improve separation efficiencies of magnetic sensor systems.
  • the magnetic relaxation sensor system used in the present invention has a working principle shown in Fig. 15.
  • the magnetic nanoparticles have high spin-spin relaxation time (T2) at a distance enough to be dispersed, whereas the aggregation of magnetic nanoparticles through samples of interest allows magnetic particles having very low T2 relaxation time.
  • T2 relaxation time The presence and quantification of samples of interest may be determined by measurement of T2 relaxation time using MRI or magnetic relaxation system.
  • Each zinc-containing nanoparticles and nanoparticles containing no zinc was synthesized according to the methods described in Example 1 and Korean Pat. Nos. 10-0604975, 10-0652251 and 10-0713745, PCT/KR2004/002509, Korean Pat. No. 10-0604975,
  • each nanoparticles were solubilized in water by surface modifications with dimercaptosuccinic acid according to the method described in
  • DNAl or DNA2 having a nucleotide sequence complementary to DNA of interest is bound to the surface of nanoparticles to produce nanopartide 1 or nanopartide 2, respectively.
  • the nucleotide sequence of DNA to be analyzed, and DNAl and DNA2 complementary to DNA are as follows:
  • DNA of interest 5'-TAC GAG TTG AGA ATC CTG AAT GCG-3'
  • DNAl HS-(CH 2 ) 6 -5'-CGC ATT CAG GAT-3'
  • DNA2 HS-(CH 2 ) 6 -3'-ATG CTC AAC TCT-5'
  • T2 time changes were measured using MRI (See, Rg. 16).
  • 3T system (Acheiva; Philips Medical Systems. Best, the Netherlands) equipped with sense-flex-M coil was used for MRI analysis.
  • Fig. 16A represents a colorimetric plot merging ⁇ T2 with image measured by MRI.
  • T2 time changes depending on addition of DNA to be analyzed, red and blue color indicate low and high ⁇ T2, respectively.
  • ⁇ T2 was changed to yellow color in zinc-containing nanopartides (Zn 0-4 Fe 2 ⁇ O 4 ) by addition of 10 fmol DNA.
  • ⁇ T2 was gradually enhanced depending on increase of DNA amounts (from 100 to 2,000 fmol), leading to color changes from yellow color to green or blue color.
  • the color was unchanged in nanopartides containing no zinc (Fe 3 O 4 ) by addition of DNA (10-100 fmol), and slightly changed to yellow color by treatment of 1000 fmol DNA.
  • FIG. 16B is a graph expressing Fig. 16A numerically, suggesting that the use of zinc- containing nanopartides in a magnetic relaxation sensor contributes to striking enhancement of sensor sensitivity.
  • ⁇ T2 of about 10 is measured in zinc- containing nanopartides by addition of 10 fmol DNA, whereas in nanopartides containing no zinc by treatment of 1000 fmol DNA. Consequently, it could be appreciated that the sensor sensitivity of zinc-containing nanopartides is enhanced 100-fold higher than that of nanopartides containing no zinc.
  • Fig. 17A represents a colorimetric plot merging ⁇ T2 with image measured by
  • EXAMPLE 8 Magnetophoresis Sensor Using Zinc-Containing Magnetic Nanopartides; Comparison of Magnetic Potential Strength between Zinc- Containing Magnetic Nanopartides and Magnetic Nanopartides Containing No Zinc by Measuring Migration Rate of Cells Bound with Magnetic Nanopartides in the Presence of External Magnetic Field
  • Zinc-containing nanopartides and nanopartides containing no zinc used in the sensor of the present invention are Zn 0 ⁇ Fe 26 O 4 and Fe 3 O 4 , respectively.
  • DMSA dimercaptosuccinic acid
  • sulfo-SMCC Sulfosuccinimidyl-4-(N-maleimidomethyl)cydohexane-l- carboxylate; Pierce
  • the antibody proteins enable to bind to a EGFR receptor on the surface of U87MG cells.
  • Cell migration toward a magnetic tip was recorded as a video using a computer linked with a microscope. Further, a positioning coordinate of a specific cell was observed in a frame with regular interval to measure migration distance depending on the time.
  • Fig. 19A is a graph representing mean of cell migration rate obtained from 20 specific cells using zinc-containing magnetic nanoparticles and magnetic nanoparticles containing no zinc.
  • the cell migration rate of cells bound with zinc- containing magnetic nanoparticles was attracted toward a magnetic tip about 4.14- fold more rapid than that that of cells bound with magnetic nanoparticles containing no zinc.
  • Fig. 19B is a graph calculating the strength by stokes' law to determine how powerful cells bound with magnetic nanoparticles are practically affected by a magnetic tip.
  • the magnetic attraction of cells bound with zinc-containing magnetic nanoparticles was about 4.12-fold stronger than that that of cells bound with magnetic nanoparticles containing no zinc.
  • Fig. 19A is a graph representing mean of cell migration rate obtained from 20 specific cells using zinc-containing magnetic nanoparticles and magnetic nanoparticles containing no zinc.
  • the cell migration rate of cells bound with zinc- containing magnetic nanoparticles was attracted toward a magnetic tip about 4.14- fold more rapid than that that

Abstract

The present invention relates to a zinc-containing magnetic nanoparticle-based magnetic separation system or magnetic sensor comprising a zinc-containing magnetic nanoparticle (preferably, a zinc-containing magnetic nanoparticle of which a binding agent having a binding affinity to a target material or analyte is bound on the surface) or a cluster thereof. Since the zinc-containing magnetic nanoparticles used in this invention have very high saturation magnetism, the present magnetic separation system using the same has much more improved separation efficiencies. Due to higher saturation magnetism of the zinc-containing magnetic nanoparticle used in this invention, the present magnetic sensor using the same may also detect analytes in a much more sensitive manner. The nanoparticles having improved sensitivity (e.g., sensitivity in fM level) prepared by the present invention may permit to detect a trace amount of analytes such as anti-cancer marker proteins or nucleic acid molecules, and prion proteins in blood.

Description

ZINC-CONTAINING MAGNETIC NANOPARTICLE-BASED MAGNETIC SEPARATION SYSTEMS AND MAGNETIC SENSORS
BACKGROUND OF THE INVENTION FIELD OF THE INVENTION
The present invention relates to zinc-containing magnetic nanopartide-based magnetic separation systems and magnetic sensors.
DESCRIPTION OF THE RELATED ART Magnetic nanoparticles have been found to be applied to very numerous application fields. First of all, several application areas using magnetism have been recently developed in biomedicine field, leading to focus a spotlight on magnetic nanoparticles. These applications include: (a) magnetic resonance imaging (MRI) contrast agents; (b) material separation systems using magnetism; (c) drug delivery systems using magnetism; (d) material sensors using magnetism; and (e) thermotherapies using a high-frequency magnetic field (HFMF).
Until recent year, a variety of magnetic nanoparticles have been developed and utilized in various application fields. In particular, most of available magnetic nanoparticles are iron oxide nanoparticles. The iron oxide nanoparticle has some advantages such as (a) feasible synthesis and (b) relatively low cost in its synthesis according to the development of various synthesis methods. In addition, a few iron oxide nanoparticles {e.g., MRI contrast agent) are commercially accessible as iron oxide has been known to exhibit very low toxicity in a human body. Irrespectively of aforementioned merits, there is a severe drawback that saturation magnetism of iron oxide nanoparticle is low in itself (AZ5 = 110 emu/g). Low saturation magnetism leads to decrease performance and sensitivity of nanoparticles in diverse applications described above.
Several formulae are listed in the following table 1 to express a relative correlation with performances of various application techniques.
Figure imgf000003_0001
In table 1, the performance of all application techniques is directly proportional to magnetic moment (μ), or proportional to the square of magnetic moment. Magnetic moment (μ) refers to a magnetic moment of a particle under constant magnetic field, and saturation magnetism (AZ5) of a magnetic nanoparticle suggests that magnetic moment (μ) is saturated in higher magnetic field. Therefore, as a nanoparticle having high saturation magnetism enables to significantly enhance the performance of application technique, its development has been urgently demanded.
Of magnetic nanopartides developed in the art, a nanoparticle having high saturation magnetism (AZ5) is as follows:
(a) MnFe2O4 nanopartides (Lee eta/., Nature Medicine, 13: 95 (2007))
MnFe2O4 nanopartides are synthesized by substituting Mn2+ for Fe2+ in conventional iron oxide nanopartides. The nanopartides with a size of 12 nm have saturation magnetism of 125 emu/g. (b) FeCo nanopartides (Seo eta/., Nature Materials, 5: 971 (2006))
FeCo nanopartides are produced by alloy of iron and cobalt, and their saturation magnetisms are 215 emu/g.
(c) Zn0.4Fe2.6θ4 nanopartides (Jang et a/., Angewandte Chemie International Edition, 48: 1234 (2009)) Fe2+ of iron oxide nanopartides is substituted by partial doping of Zn2+, producing Zn0.4Fe2.6O4 nanopartides with saturation magnetism of 161 emu/g.
Using nanopartides developed in aforementioned references as MRI contrast agent, their contrast effects were improved several ten times compared with iron oxide-based contrast agents, supposing that the nanopartides may be utilized in early diagnosis of diseases such as cancer more excellent than present techniques. In particular, as Zn^4Fe2-6O4 nanoparticle using a zinc-containing iron oxide have lower toxicity than FeCo nanoparticle, it exhibits the most excellent application probability in human body and bio-experiments. As shown in table 1, magnetic moment of nanopartides is crucial for enhancement of contrast effect. Likewise, nanoparticles having high magnetic moment contribute to enhancement of contrast effect in several other application fields including magnetic separation, magnetoresistance sensor, magnetic relaxation sensor, heat release by high- frequency magnetic field, and so forth.
As an alternative method enhancing magnetic moment of nanoparticle, and improving its performance in view of application, the nanoparticle cluster may be utilized. The term "cluster" used herein refers to a bundle in which nanoparticles of from a few to several hundred numbers are aggregated. It has been known that total magnetic moment of cluster is increased much higher than that of a single nanoparticle. In the senses that magnetic moment is proportional to volume of nanoparticle, a correlation between magnetic moment (μ) and total magnetic moment (M) is represented by the following equation: M = μ • V. Therefore, utilization of a nanoparticle cluster instead of a single nanoparticle may allow enhancement of performance in described-above applications.
Throughout this application, various patents and publications are referenced and citations are provided in parentheses. The disclosure of these patents and publications in their entities are hereby incorporated by references into this application in order to more fully describe this invention and the state of the art to which this invention pertains.
DETAILED DESCRIPTION OF THIS INVETNION
The present inventors have made intensive studies to develop a magnetic separation system having improved separation efficiency for isolating a target material. As results, we have discovered that as a magnetic nanoparticle used in the magnetic separation system, zinc-containing magnetic nanoparticle having high saturation magnetism or cluster thereof may be applied to the magnetic separation system for providing a magnetic separation system to achieve the aforementioned purpose. Meanwhile, we have made intensive studies to develop a magnetic sensor for detecting or quantitating an analyte in more improved sensitive and accurate manner. As results, we have discovered that as a magnetic nanopartide used in the magnetic sensor, zinc-containing magnetic nanopartide having specific stoichiometry and high saturation magnetism or cluster thereof may be applied to the magnetic sensor for providing a magnetic sensor to achieve the aforementioned purpose.
Accordingly, it is an object of this invention to provide a zinc-containing magnetic nanopartide-based magnetic separation system and magnetic sensor. It is another object of this invention to provide a method for isolating a target material in a sample.
It is still another object of this invention to provide a method for detecting an analyte in a sample.
Other objects and advantages of the present invention will become apparent from the following detailed description together with the appended claims and drawings.
In one aspect of this invention, there is provided a zinc-containing magnetic nanopartide-based magnetic separation system or magnetic sensor, comprising a zinc-containing magnetic nanopartide represented by the following formula 1 or 2 or a cluster thereof:
ZnfMa-fOb (0<f<8, 0<a<16, 0<b<8, 0<f/(a-f)<10, M represents a magnetic metal atom or an alloy thereof) (1) ZngMc-gM'dOe (0<g<8, 0<c<16, 0<d<16, 0<e<8, 0<g/{(c-g)+d}<10, M represents a magnetic metal atom or an alloy thereof; M' represents one or more elements selected from the group consisting of Group 1 elements, Group 2 elements, Group 12 elements, Group 13 elements, Group 14 elements, Group 15 elements, transition metal elements, Lanthanide elements and Actinide elements) (2)
The magnetic separation system and magnetic sensor of the present invention exhibit utilize a zinc-containing magnetic nanoparticle having specific stoichiometry and high saturation magnetism or cluster thereof, and both working principles are similar.
Z. Magnetic Separation System and Isolation Method of Target Material
Zinc-containing magnetic nanoparticles used in the magnetic separation system of the present invention are represented by the following formula 1 or 2:
ZnfMa-f0b (0<f<8, 0<a<16, 0<b<8, 0<f/(a-f)<10, M represents a magnetic metal atom or an alloy thereof) (1)
ZngMc-gM'dOe (0<g<8, 0<c<16, 0<d<16, 0<e<8, 0<g/{(c-g)+d}<10, M represents a magnetic metal atom or an alloy thereof; M' represents one or more elements selected from the group consisting of Group 1 elements, Group 2 elements, Group 12 elements, Group 13 elements, Group 14 elements, Group 15 elements, transition metal elements, Lanthanide elements and Actinide elements)
(2)
High saturation magnetism of zinc-containing nanoparticles described in the formula 1 or 2 leads to enhanced separation efficiency on the magnetic separation system to which magnetic field is applied.
In formulae 1 and 2, M represents preferably transition metal elements,
Lanthanide metal elements and Actinide metal elements; more preferably, transition metal elements selected from the group consisting of Ba, Mn, Co, Ni and Fe, Lanthanide metal elements selected from the group consisting of Gd, Er, Ho, Dy, Tb,
Sm and Nd; and most preferably, Mn, Co, Ni, Fe, Gd, Er, Ho, Dy, Tb, Sm or Nd, or an alloy thereof. In formula 2, M' preferably represents one or more elements selected from the group consisting of Li, Na, K, Rb, Cs, Be, Mg, Ca, Sr, Ba, Ra, Ge, Ga, Bi, In, Si, Ge, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Y, Zr, Nb, Mo, Tc, Ru, Rh, Pd, Ag, Cd, Hf, Ta, W, Re, Os, Ir, Pt, Au, Hg, Lanthanide elements and Actinide elements. According to a preferable embodiment, the zinc-containing magnetic nanopartide used in the magnetic separation system of this invention is represented by the following formula 3:
ZnkM"h-kFe,Oj (0<k<8, 0<h<16, 0<i<8, 0<j<8, 0<k/{(h-k)+i}<10, M" represents a magnetic metal atom or an alloy thereof) (3) In formula 3, M" represents preferably transition metal elements, Lanthanide metal elements and Actinide metal elements; more preferably, transition metal elements selected from the group consisting of Ba, Mn, Co, Ni and Fe, Lanthanide metal elements selected from the group consisting of Gd, Er, Ho, Dy, Tb, Sm and Nd; and most preferably, Mn, Co, Ni, Fe, Gd, Er, Ho, Dy, Tb, Sm or Nd, or an alloy thereof.
More preferably, the zinc-containing magnetic nanopartide used in the magnetic separation system of this invention is represented by the following formula 4 or 5:
ZnqFei-qO,,, (0<q<8, 0<l<8, 0<m<8, 0<q/(l-q)<10) (4) ZnrMnn-rFeoOp (0<r<8, 0<n<8, 0<o<8, 0<p<8, 0<r/{(n-r)+o}<10) (5)
In the zinc-containing magnetic nanopartide of the present invention, a stoichiometric content ratio of zinc and other metals is as follows: 0.001 < 'zinc/ (entire metal component - zinc)' < 10, more preferably 0.01 < 'zinc/ (entire metal component - zinc)' < 1, and most preferably 0.03 < 'zinc/ (entire metal component - zinc)' < 0.5. When zinc is contained as the above, high saturation magnetism can be obtained, resulting in significant improvement of separation efficiency in the present magnetic separation system.
The zinc-containing magnetic nanopartides contained in the cluster of the present invention are aggregated in a number of preferably 2-10,000, more preferably 2-1,000, and most preferably 2-100. Each zinc-containing magnetic nanoparticles in the cluster is linked to each other by an intermolecular interaction, or encapsulated by an organic or inorganic carrier. A binding agent with a binding affinity to a target material may be linked to the surface of a zinc-containing magnetic nanopartide or cluster thereof used in the present invention, or not. The material of interest may be directly linked to a metal element contained in the zinc-containing magnetic nanopartide of the present invention. For instance, where nickel is involved in the zinc-containing magnetic nanopartide of the present invention, it may be directly linked to a histidine residue of a protein or peptide, whereby protein is able to be isolated. In addition, a gas- selective metal contained in the zinc-containing magnetic nanopartide of the present invention contributes to gas separation without a further binding agent. The gas- selective metal includes Pt, Pd, Au, Ag, Nb, Ir, Rh, Ru and an alloy thereof, but is not limited to. A hydrogen-selective metal includes Pt, Pd, Au, Ag and an alloy thereof.
According to a preferable embodiment, a binding agent with a binding affinity to a target material is linked to the surface of a zinc-containing magnetic nanopartide or cluster thereof. The binding agent is directly or indirectly, covalently or non-covalently linked to the surface of zinc-containing magnetic nanopartide of the present invention.
For example, the binding agent may be directly bound to the surface of zinc- containing magnetic nanopartide by a covalent or non-covalent linkage such as an ionic bond, an electrostatic bond, a hydrophobic interaction, a hydrogen bond, a covalent bond, a hydrophilic interaction or a Van der Waal force. In addition, the binding agent may be indirectly bound to the surface of zinc-containing magnetic nanopartide through an intervening agent.
The term "target material" used herein refers to a material in a sample to be separated (or separated and detected, or separated, detected or quantified). The target material includes, but is not limited to, a nucleic acid molecule (DNA or RNA), a protein, a peptide, an antigen, a sugar, a lipid, a bacterium, a virus, a cell, an organic compound, an inorganic compound, a metal and an inorganic ion. The sample includes a biological sample, a chemical sample and an environmental sample, but is not limited to. The biological sample includes blood, plasma, serum, virus, bacterium, tissue, cell, lymph, bone marrow, saliva, milk, urine, feces, eyeball fluid, seminal fluid, brain extract, spinal fluid, synovial fluid, thymus fluid, ascites, amniotic fluid, cell tissue fluid and cell culture, but is not limited to.
The term "binding agent" used herein means a substance having a specific affinity to a target material to be separated. Any material having a binding affinity to a target material to be separated may be utilized as the binding agent. A non- limiting example of a binding agent includes a nucleic acid molecule (DNA or RNA), a protein, an antibody, an antigen, an aptamer (RNA, DNA and peptide aptamer), a receptor, a hormone, a streptavidin, an avidin, a biotin, a lectin, a ligand, an agonist, an antagonist, an enzyme, a coenzyme, an inorganic ion, a cofactor, a sugar, a lipid, a substrate for an enzyme, a hapten, a neutravidin, a protein A, a protein G, a selectin, calcium sulfate, a gas binding agent (example: Pt, Pd, Au, Ag, Nb, Ir, Rh and Ru).
According to a preferable embodiment, the binding agent is bound to the surface of zinc-containing magnetic nanopartide or cluster thereof through an intervening agent.
The intervening agent includes various linkers and nanopartide-coating materials known to those ordinarily skilled in the art.
According to a preferable embodiment, the surface of the zinc-containing magnetic nanopartides or cluster thereof is coated with a water-soluble multifunctional ligand for nanopartide solubilization, whereby the binding agent is linked to nanopartide.
The term "water-soluble multi-functional ligand" refers to a ligand that may be bound to zinc-containing nanoparticles or cluster thereof to solublize in water and stabilize the nanoparticles, and may allow the nanoparticles to be bound by binding agent having a specific affinity to a target material.
The water-soluble multi-functional ligand can include (a) an adhesive region (Li), and further can include (b) a binding region (LM), (c) a cross-linking region (Lm ), or (d) a binding & cross-linking region (L M -Lm) which includes both the binding region (Ln) and the cross-linking region (L,,,). Hereinafter, the water-soluble multifunctional ligand will be described in detail.
The term "adhesive region (U)" refers to a portion of a multi-functional ligand including a functional group capable of binding to the nanoparticles, and preferably to an end portion of the functional group. Accordingly, it is preferable that the adhesive region including the functional group should have high affinity with the surface of the nanoparticles. The nanopartide can be attached to the adhesive region by an ionic bond, a covalent bond, a hydrogen bond, a hydrophobic interaction or a metal-ligand coordination bond. The adhesive region of the multifunctional ligand may be varied depending on the substances constituting the nanoparticles. For example, the adhesive region (U) using ionic bond, covalent bond, hydrogen bond or metal-ligand coordination bond may include -COOH, -NH2, -SH, - CONH2, -PO3H, -OPO3H2, -SO3H, -OSO3H,, -N3, -NR3OH (R= CnH2n+I, 0<n<16), -OH, - SS-, -NO2, -CHO, -COX (X = F, Cl, Br or I), -COOCO-, -CONH- or -CN, and the adhesive region (Li) using hydrophobic interaction may include a hydrocarbon chain having two or more carbon atoms, but is not limited to.
The term "binding region (Ln)" means a portion of the multi-functional ligand containing a functional group capable of binding to binding agents having a specific affinity to a target material, and preferably the other end portion located at the opposite side from the adhesive region. The functional group of the reactive region may be varied depending on the type of binding agents and their chemical formulae (Table 2). In this invention, the binding region includes, but is not limited to, -SH, - COOH, -CHO, -NH2, -OH, -PO3H, -OPO3H2, -SO3H, -OSO3H, -NR3 +X" (R= CnHm, 0<n<16, 0<m<34, X = OH, Cl or Br), NR4 +X' (R= CnH01, 0<n<16, 0<m<34, X = OH, Cl or Br), -N3, -SCOCH3, -SCN, -NCS, -NCO, -CN, -F, -Cl, -Br, -I, an epoxy group, a sulfonate group, a nitrate group, a phosphonate group, an aldehyde group, a hydrazone group, -C=C- and -C≡C-.
The term "cross-linking region (Lm)" refers to a portion of the multi-functional ligand including a functional group capable of cross-linking to an adjacent multifunctional ligand, and preferably a side chain attached to a central portion. The term "cross-linking" means that the multi-functional ligand is bound to another adjacent multi-functional ligand by intermolecular interaction. The intermolecular interaction includes, but is not limited to, a hydrophobic interaction, a hydrogen bond, a covalent bond {e.g., a disulfide bond), a Van der Waals force and an ionic bond. Therefore, the cross-linkable functional group may be variously selected according to the kind of the intermolecular interaction. For example, the cross-linking region may include -SH, -COOH,-^CHOr^NH2, -OH, -PO3H, -OPO3H2, -SO3H, -OSO3H, Si-OH, Si(MeO)3, -NR3 +X" (R= CnH171, 0<n<16, 0<m<34, X = OH, Cl or Br), NR4 +X" (R= CnHm, 0<n<16, 0<m<34, X = OH, Cl or Br), -N3, -SCOCH3, -SCN, -NCS, -NCO, -CN, -F, -Cl, -Br, -I, an epoxy group, -ONO2, -PO(OH)2, -C=NNH2, -C=C- and -C≡C- as the functional ligand, but is not limited to. TABLE 2. Exemplary functional groups of binding region in multifunctional ligand
Figure imgf000012_0001
Figure imgf000013_0001
(I: functional group of binding region in multi-functional ligand, II: binding agent, III: exemplary bonds by reaction of I and II)
In this invention, the compound which originally contains the above-described functional group may be used as a water-soluble multi-functional ligand, but a compound modified or prepared so as to have the above-described functional group by a chemical reaction known in the art may be also used as a water-soluble multifunctional ligand.
The preferable multi-functional ligand of the present invention includes a monomer, a polymer, a carbohydrate, a protein, a peptide, a nucleic acid, a lipid or an amphiphilic ligand.
For the water-soluble nanoparticle of the present invention, one example of the preferable multi-functional ligand is a monomer which contains the functional group described above, and preferably dimercaptosuccinic acid, since it originally contains the adhesive region, the cross-linking region and the binding region. That is, -COOH on one side of dimercaptosuccinic acid is bound to the magnetic nanoparticle, and -COOH and -SH on the other end portion functions to bind to a binding agent. In addition, -SH of dimercaptosuccinic acid acts as the cross-linking region by disulfide bond with another -SH. In addition to the dimercaptosuccinic acid, other compounds having -COOH as the functional group of the adhesive region and - COOH, -NH2 or -SH as the functional group of the binding region may be utilized as the preferable multi-functional ligand, but not limited to. Still another example of the preferable water-soluble multi-functional ligand according to the present invention includes, but not limited to, one or more polymers selected from the group consisting of polyphosphagen, polylactide, polylactide-co- glycolide, polycaprolactone, polyanhydride, polymaleic acid, a derivative of polymaleic acid, polyalkylcyanoacrylate, polyhydroxybutylate, polycarbonate, polyorthoester, polyethylene glycol, poly-L-lysine, polyglycolide, polymethyl methacrylate and polyvinylpyrrolidone.
In the zinc-containing water-soluble metal oxide nanomaterial according to the present invention, preferable other examples of the multi-functional ligand are a peptide. Peptide is oligomer/polymer consisting of several amino acids. Since the amino acids have -COOH and -NH2 functional groups in both ends thereof, peptides naturally have the adhesive region and the binding region. In addition, the peptide that contains one or more amino acids having one or more of -SH, -COOH, -NH2 and -OH as the side chain may be utilized as the preferable water-soluble multifunctional ligand. In the water-soluble nanopartides according to the present invention, still another example of the preferable multi-functional ligand is a protein. Protein is a polymer composed of more amino acids than peptides, that is, composed of several hundreds to several hundred thousands of amino acids. Proteins contains -COOH and -NH2 functional group at both ends, and also contains a lot of functional groups such as -COOH, -NH2, -SH, -OH, -CONH2, and so on. Proteins may be used as the water-soluble multi-functional ligand because they naturally contain the adhesive region, the cross-linking region and the binding region according to its structure as the above-described peptide. The representative examples of proteins which are preferable as the water-soluble multi-functional ligand include a structural protein, a storage protein, a transport protein, a hormone protein, a receptor protein, a contraction protein, a defense protein and an enzyme protein, and more specifically, albumin, antibody, antigen, avidin, cytochrome, casein, myosin, glycinin, carotene, collagen, globular protein, light protein, streptavidin, protein A, protein G, protein S, lectin, selectin, angiopoietin, anti-cancer protein, antibiotic protein, hormone antagonist protein, interleukin, interferon, growth factor protein, tumor necrosis factor protein, endotoxin protein, lymphotoxin protein, tissue plasminogen activator, urokinase, streptokinase, protease inhibitor, alkyl phosphocholine, surfactant, cardiovascular pharmaceutical protein, neuro pharmaceuticals protein and gastrointestinal pharmaceuticals, but not limited to.
Still another example of the preferable water-soluble multi-functional ligand in the present invention is a nucleic acid. The nucleic acid is oligomer consisting of many nucleotides. Since the nucleic acids have -PO4 " and -OH functional groups in their both ends, they naturally have the adhesive region and the binding region (L1- Ln), or the adhesive region and the cross-linking region (LrLm). Therefore, the nucleic acids may be useful as the water-soluble multi-functional ligand in this invention. In some cases, the nucleic acid is preferably modified to have the functional group such as -SH, -NH2, -COOH or -OH at 3'- or 5'-terminal ends. For the water-soluble nanoparticles according to the present invention, still another example of the preferable multi-functional ligand is an amphiphilic ligand including both a hydrophobic and a hydrophilic region. In the nanoparticles synthesized in an organic solvent, hydrophobic ligands having long carbon chains coat the surface. When amphophilic ligands are added to the nanopartide solution, the hydrophobic region of the amphiphilic ligand and the hydrophobic ligand on the nanoparticles are bound to each other through intermolecular interaction to stabilize the nanoparticles. Further, the outermost part of the nanoparticles shows the hydrophilic functional group, and consequently water-soluble nanoparticles can be prepared. The intermolecular interaction includes a hydrophobic interaction, a hydrogen bond, a Van der Waals force, and so forth. The portion which binds to the nanoparticles by the hydrophobic interaction is an adhesive region (Li), and further the binding region (Ln) and the cross-linking region (Lm) can be introduced therewith by an organo-chemical method. In addition, in order to increase the stability in an aqueous solution, amphiphilic polymer ligands with multiple hydrophobic and hydrophilic regions can be used. Cross-linking between the amphiphilic ligands can be also performed by a linker for enhancement of stability in an aqueous solution. As the phase transfer ligands, hydrophobic region of the amphiphilic ligand can be a linear or branched structure composed of chains containing two or more carbon atoms, more preferably an alkyl functional group such as ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, tetradecyl, hexadecyl, icosyl, tetracosyl, dodecyl, cyclopentyl or cyclohexyl; a functional group having an unsaturated carbon chain containing a carbon-carbon double bond, such as ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, octenyl, decenyl or oleyl; or a functional group having an unsaturated carbon chain containing a carbon-carbon triple bond, such as propynyl, isopropynyl, butynyl, isobutynyl, octynyl or decynyl. In addition, examples of the hydrophilic region include a functional group being neutral at a specific pH, but being positively or negatively charged at a higher or lower pH such as -SH, -COOH, -NH2, -OH, -PO3H, -PO4H2, -SO3H, -OSO3H-NR3 +X' and so on. Furthermore, preferable examples thereof include a polymer and a block copolymer, wherein monomers used therefor include ethylglycol, acrylic acid, alkylacrylic acid, ataconic acid, maleic acid, fumaric acid, acrylamidomethylpropane sulfonic acid, vinylsulfonic acid, vinylphophoric acid, vinyl lactic acid, styrenesulfonic acid, allylammonium, acrylonitrile, N-vinylpyrrolidone and N-vinylformamide, but not limited to.
Another example of the preferable water-soluble multi-functional ligand in the nanoparticle of the present invention is a carbohydrate. More preferably, the carbohydrate includes, but not limited to, glucose, mannose, fucose, N-acetyl glucomine, N-acetyl galactosamine, N-acetylneuraminic acid, fructose, xylose, sorbitol, sucrose, maltose, glycoaldehyde, dihydroxyacetone, erythrose, erythrulose, arabinose, xylulose, lactose, trehalose, mellibose, cellobiose, rafflnose, melezitose, maltoriose, starchyose, estrodose, xylan, araban, hexosan, fructan, galactan, mannan, agaropectin, alginic acid, carrageenan, hemicelluloses, hypromellose, amylose, deoxyacetone, glyceraldehyde, chitin, agarose, dextran, ribose, ribulose, galactose, carboxy methylcellulose, glycogen dextran, carbodextran, polysaccharide, cyclodextrin, carboxymethyl dextran, pullulan, cellulose, starch and glycogen. According to a preferable embodiment, the surface of the zinc-containing magnetic nanoparticles used in the present invention is coated with a water-soluble multi-functional organic ligand, and linked to the binding agent having a specific affinity to a target material through the binding region (Lu).
The zinc-containing nanoparticles may be synthesized according to various methods. For instance, the zinc-containing nanoparticles of the present invention may be produced using a nanoparticle synthesis method in gas phase or in liquid phase {e.g., aqueous solution, organic solution or multiple solution system, etc.) known to those ordinarily skilled in the art.
For example, the water-soluble zinc-containing nanoparticle coated with a water-soluble multi-functional organic ligand may be synthesized according to a chemical reaction in an aqueous solution. This method is to synthesize the zinc- containing water-soluble metal oxide nanoparticles by adding zinc ion precursor materials to the reaction solution containing the water-soluble multi-functional ligand. It may be performed according to a synthesis method {e.g., a coprecipitation method, a sol-gel method, a micelle method, etc.) of a conventional water-soluble nanoparticle known to those skilled in the art.
As the nanoparticle precursors, a metal nitrate-based compound, a metal sulfate-based compound, a metal acetylacetonate-based compound, a metal fluoroacetoacetate-based compound, a metal halide-based compound, a metal perchlorate-based compound, a metal alkyloxide-based compound, a metal sulfamate-based compound, a metal stearate-based compound, a metal alkoxide- based compound or an organometallic compound may be used, but not limited to. As the organic solvents, a benzene-based solvent, a hydrocarbon solvent, an ether-based solvent, a polymer solvent, an ionic liquid solvent, an alcohol-based solvent, a sulfoxide-based solvent or water may be used, and preferably benzene, toluene, halobenzene, octane, nonane, decane, benzyl ether, phenyl ether, hydrocarbon ether, a polymer solvent, diethylene glycol (DEG), water or an ionic liquid solvent may be used, but not limited to.
The zinc-containing metal oxide nanopartides according to the above- described method have a uniform size distribution (δ < 10%) and a high crystallinity. In addition, with the method, zinc-content in the nanoparticle may be precisely controlled. In other words, by changing the ratio of zinc to other metal precursor material, the zinc-content in the nanoparticle can be controlled between 0.001 < 'zinc/(entire metal material - zinc)' < 10 in a stiochiometric ratio.
The hydrodynamic diameter of the final nanopartides prepared by the present invention is in a size range of preferably 1-1000 nm, more preferably 1-800 nm, much more preferably 2-100 nm and most preferably 10-50 nm. The hydrodynamic diameter of the final nanoparticle clusters prepared by the present invention is in a size range of preferably 1-1000 nm, more preferably 1-800 nm, much more preferably 2-100 nm and most preferably 10-50 nm. According to a preferable embodiment, the nanoparticles of this invention have a saturation magnetization (Ms) value in a range of 100-200 emu/g and more preferably 120-200 emu/g.
According to a preferable embodiment, the zinc-containing magnetic nanoparticle-based magnetic separation system further includes a magnetic field- generating means. The magnetic field-generating means include a conventional magnet such as a permanent magnet and electromagnet known to those ordinarily skilled in the art. The permanent magnet and electromagnet may be modified depending on a separation system, and have a magnetic field strength of preferably 10-10,000 mT, more preferably 100-5,000 mT, and most preferably 200-1,000 mT.
In another aspect of this invention, there is provided a method for separating a target material in a sample, comprising the steps of:
(a) forming a zinc-containing magnetic nanopartide-target material complex by contacting the target material in the sample with the magnetic nanopartide or cluster thereof represented by the following formula 1 or 2, wherein a binding agent having a binding affinity to the target material is bound on the surface of the magnetic nanopartide or cluster thereof;
ZnfMa-fOb (0<f<8, 0<a<16, 0<b<8, 0<f/(a-f)<10, M represents a magnetic metal atom or an alloy thereof) (1)
ZngMc-gM'dOe (0<g<8, 0<c<16, 0<d<16, 0<e<8, 0<g/{(c-g)+d}<10, M represents a magnetic metal atom or an alloy thereof; M' represents one or more elements selected from the group consisting of Group 1 elements, Group 2 elements, Group 12 elements, Group 13 elements, Group 14 elements, Group 15 elements, transition metal elements, Lanthanide elements and Actinide elements) (2); and
(b) separating the zinc-containing magnetic nanopartide-target material complex from other components in the sample by magnetic field induction. Since the present method comprises the magnetic separation system of this invention described above, the common descriptions between them are omitted in order to avoid undue redundancy leading to the complexity of this specification.
The zinc-containing magnetic nanopartide-target material complex is separated from other components in the sample by induction of magnetic field. The complex may be removed from other components in the sample by various methods (pipetting, draining, skimming, pouring, etc.) to obtain a sample concentration solution containing only the zinc-containing magnetic nanopartide-target material complex. For example, after zinc-containing magnetic nanopartides are added to a reactor containing a sample and mixed, they may be separated in a portion of the reactor by induction of magnetic field. Subsequently, only the zinc-containing magnetic nanopartides may be separated by pipetting the sample under magnetic field induction. And then, the zinc-containing magnetic nanopartides are washed and resuspended in a suitable solvent, enabling to produce a sample concentration solution containing only the zinc-containing magnetic nanopartide-target material complex.
According to a preferable embodiment, the separation method of the present invention may be combined with a detection or quantification process. In this case, the method of the present invention further includes a step (c) which detects a formation of the zinc-containing magnetic nanopartide-target material complex in the sample.
A method to detect a formation of the zinc-containing magnetic nanopartide- target material complex in the sample may be carried out according to the conventional methods known to those ordinarily skilled in the art. For instance, the detection described above may be accomplished by measuring an absorbance change for a sample containing the zinc-containing magnetic nanopartide-target material complex. In addition, the zinc-containing magnetic nanoparticle complex conjugated with a signal generating label may be detected by measuring the fluorescent signal.
The signal generating label includes, but is not limited to, chemical labels
{e.g., biotin), enzyme labels {e.g., alkaline phosphatase, peroxidase, β-galactosidase and β-glucosidase), radioisotopes {e.g., I125 and C14), fluorescent labels, luminescent labels, chemiluminescent labels, FRET (fluorescence resonance energy transfer) labels and heavy metals {e.g., gold).
The non-limiting example of the above-described fluorescent label includes fluorescein, rhodamine, lucifer yellow, B-phytoerythrin, 9-acrydine isothiocyanate, lucifer yellow VS, 4-acetamido-4'-isothio-cyanatostilbene-2,2'-disulfonate, 7- diethylamino-3-(4'-isothiocyatophenyl)-4-methylcoumarin, succinimidyl- pyrenebutyrate, 4-acetoamido-4'-isothio-cyanatostilbene-2,2'-disulfonate derivatives, LC™-Red 640, LC™-Red 705, Cy5, Cy5.5, resamine, isothiocyanate, erythrin isothiocyanate, diethyltriamine pentaacetate, l-dimethylaminonaphthyl-5-sulfonate, l-anilino-8-naphthalene, 2-p-toluidinyl-6-naphthalene, 3-phenyl-7- isocyanatocoumarin, 9-isothiocyanatoacridine, acridine orange, N-(p-(2- benzoxazolyl)phenyl)meleimide, benzoxadiazol, stilbene and pyrene and a derivative thereof.
A method to detect the formation of zinc-containing magnetic nanoparticle- target material complex conjugated with the label may be carried out according to the methods such as fluorometer, spectrophotometer, colorimetric detection or radioactivity detection.
The present invention may be used in separation, concentration and detection of a specific target material in a sample. Using zinc-containing magnetic nanoparticles synthesized depending on a type of binding agent to be bound to a target material, an economic system may be designed to separate and/or detect various target materials in a high efficient manner. According to the present invention, it is also unnecessary to perform further procedures such as complicated pre-enrichment, purification or processing needed in common detection.
Since the nanopartides of the present invention have small size and very high saturation magnetism, they may be rapidly distributed into a biological sample (generally, an aqueous sample), i.e., numerous opportunities to contact a target material in an aqueous sample by Brown's diffusion, and furthermore may be harvested/separated under magnetic field induction.
In this connection, the present invention may be utilized in various application fields including: (a) diagnosis - disease-related substances (for example, proteins, antigens, nucleic acid molecules, viruses, bacteria, carbohydrates and lipids) may be separated/detected by applying the zinc-containing magnetic nanopartides to a biological sample; (b) separation - a final product may be separated by applying the nanopartides to a synthesis of a chemical compound; (c) water or seawater desalination using zinc-containing magnetic nanopartides modified with a chelating /complexing agent to selectively remove a specific salt, ion or metal.
Since the zinc-containing magnetic nanopartides used in this invention have very high saturation magnetism, the present magnetic separation system using the same has much more improved separation efficiencies. Using zinc-containing magnetic nanopartides synthesized depending on a type of binding agent to be bound to a target material, an economic system may be designed to separate and/or detect various target materials in a high efficient manner. According to the present invention, it is also unnecessary to perform further procedures such as complicated pre-enrichment, purification or processing needed in common detection.
II. Magnetic Sensors and Methods for Detecting an Analyte
In another aspect of this invention, there is provided a zinc-containing magnetic nanopartide-based magnetic sensor, comprising: (a) a zinc-containing magnetic nanopartide or a cluster thereof represented by the following formula 1 or 2:
ZnfMa-fOb (0<f<8, 0<a<16, 0<b<8, 0<f/(a-f)<10, M represents a magnetic metal atom or an alloy thereof) (1) ZngMc-gM'dOe (0<g<8, 0<c<16, 0<d<16, 0<e<8, 0<g/{(c-g)+d}<10, M represents a magnetic metal atom or an alloy thereof; M' represents one or more elements selected from the group consisting of Group 1 elements, Group 2 elements, Group 12 elements, Group 13 elements, Group 14 elements, Group 15 elements, transition metal elements, Lanthanide elements and Actinide elements) (2)
(b) at least one magnetic sensor devices; and
(c) at least one magnetic field-generating means.
Since the present magnetic sensor comprises the zinc-containing magnetic nanoparticles described in the magnetic separation system of this invention, the common descriptions between them, are omitted in order to avoid undue redundancy leading to the complexity of this specification.
The zinc-containing magnetic nanopartide or the cluster thereof used in the magnetic sensor of this invention plays a role in a signal generator.
The zinc-containing magnetic nanopartide used in the magnetic sensor of this invention is represented by the following formula 1 or 2:
ZnfMa-fOb (0<f<8, 0<a<16, 0<b<8, 0<f/(a-f)<10, M represents a magnetic metal atom or an alloy thereof) (1)
ZngMc-gM'dOe (0<g<8, 0<c<16, 0<d<16, 0<e<8, 0<g/{(c-g)+d}<10, M represents a magnetic metal atom or an alloy thereof; M' represents one or more elements selected from the group consisting of Group 1 elements, Group 2 elements, Group 12 elements, Group 13 elements, Group 14 elements, Group 15 elements, transition metal elements, Lanthanide elements and Actinide elements) (2) Since the zinc-containing magnetic nanoparticles of the formula 1 or 2 have high saturation magnetism, the present magnetic sensor using the same may exhibit much more enhanced sensitivity under magnetic field induction.
In formulae 1 and 2, M represents preferably transition metal elements, Lanthanide metal elements and Actinide metal elements; more preferably, transition metal elements selected from the group consisting of Ba, Mn, Co, Ni and Fe, Lanthanide metal elements selected from the group consisting of Gd, Er, Ho, Dy, Tb, Sm and Nd; and most preferably, Mn, Co, Ni, Fe, Gd, Er, Ho, Dy, Tb, Sm or Nd, or an alloy thereof. In formula 2, M' preferably represents one or more elements selected from the group consisting of Li, Na, K, Rb, Cs, Be, Mg, Ca, Sr, Ba, Ra, Ge, Ga, Bi, In, Si, Ge, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Y, Zr, Nb, Mo, Tc, Ru, Rh, Pd, Ag, Cd, Hf, Ta, W, Re, Os, Ir, Pt, Au, Hg, Lanthanide elements and Actinide elements.
According to a preferable embodiment, the zinc-containing magnetic nanoparticle used in the magnetic sensor of this invention is represented by the following formula 3:
ZnkM'VkFeiOj (0<k<8, 0<h<16, 0<i<8, 0<j<8, 0<k/{(h-k)+i}<10, M" represents a magnetic metal atom or an alloy thereof) (3)
In formula 3, M" represents preferably transition metal elements, Lanthanide metal elements and Actinide metal elements; more preferably, transition metal elements selected from the group consisting of Ba, Mn, Co, Ni and Fe, Lanthanide metal elements selected from the group consisting of Gd, Er, Ho, Dy, Tb, Sm and Nd; and most preferably, Mn, Co, Ni, Fe, Gd, Er, Ho, Dy, Tb, Sm or Nd, or an alloy thereof. More preferably, the zinc-containing magnetic nanoparticle used in the magnetic sensor of this invention is represented by the following formula 4 or 5:
ZnqFei-qOm (0<q<8, 0<l<8, 0<m<8, 0<q/(l-q)<10) (4)
ZnrMnn-rFe00p (0<r<8, 0<n<8, 0<o<8, 0<p<8, 0<r/{(n-r)+o}<10) (5) In the zinc-containing magnetic nanoparticle of the present invention, a stoichiometric content ratio of zinc and other metals is as follows: 0.001 < 'zinc/ (entire metal component - zinc)' < 10, more preferably 0.01 < 'zinc/ (entire metal component - zinc)' < 1, and most preferably 0.03 < 'zinc/ (entire metal component - zinc)' < 0.5. When zinc is contained as the above, high saturation magnetism can be obtained, resulting in remarkable improvement of sensor sensitivity in the present magnetic sensor.
The zinc-containing magnetic nanopartides contained in the cluster of the present invention are clustered in the number of preferably 2-10,000, more preferably 2-1,000, and most preferably 2-100. Each zinc-containing magnetic nanopartides in the duster is linked each other by their intermolecular interactions, or encapsulated by organic or inorganic carrier.
According to a preferable embodiment, the surface of the zinc-containing magnetic nanopartides or cluster thereof is coated with a water-soluble multi- functional ligand.
The term "water-soluble multi-functional ligand" refers to a ligand that may be bound to zinc-containing nanopartides or cluster thereof to solublize in water and stabilize the nanopartides, and may allow the nanopartides to be bound by binding agent having a specific affinity to an analyte. The water-soluble multi-functional ligand can include (a) an adhesive region
(L1), and further can include (b) a binding region (Ln ), (c) a cross-linking region (Lm ), or (d) a binding & cross-linking region (Ln -Lm) which includes both the binding region (LM) and the cross-linking region (Lm).
In this invention, the compound which originally contains the above-described functional group may be used as a water-soluble multi-functional ligand, but a compound modified or prepared so as to have the above-described functional group by a chemical reaction known in the art may be also used as a water-soluble multifunctional ligand. The preferable multi-functional ligand of the present invention includes a monomer, a polymer, a carbohydrate, a protein, a peptide, a nucleic acid, a lipid or an amphiphilic ligand.
The hydrodynamic diameter of the final nanopartides prepared by the present invention is in a size range of preferably 1-1000 nm, more preferably 1-800 nm, much more preferably 2-100 nm and most preferably 10-50 nm.
The hydrodynamic diameter of the final nanopartide clusters prepared by the present invention is in a size range of preferably 1-1000 nm, more preferably 1-800 nm, much more preferably 2-100 nm and most preferably 10-50 nm. According to a preferable embodiment, the nanopartides of this invention have a saturation magnetization (Λ/s) value in a range of 100-300 emu/g and more preferably 120-200 emu/g.
According to a preferable embodiment, the zinc-containing magnetic nanopartide used in the present invention is linked to a binding agent having a binding affinity to an analyte. More preferably, the zinc-containing magnetic nanopartide used in the present invention is coated with a water-soluble multifunctional organic ligand, and linked to the binding agent having a specific affinity to an analyte through the binding region (Ln).
The term "analyte" used herein refers to a substance or chemical constituent in a sample {e.g., a liquid or gas sample, preferably a liquid sample, and most preferably an aqueous sample) to be separated (detected or quantified). The analyte includes a nucleic acid molecule (DNA or RIMA), a protein, a peptide, an antigen, a bacterium, a virus, a sugar, a lipid, an organic compound {e.g., chiral compounds, therapeutic compounds), an inorganic compound, a metal and an inorganic ion, but is not limited to.
The term "binding agent" used herein means a substance having a specific affinity to an analyte to be separated. Any material having a binding affinity to a target material to be separated may be utilized as the binding agent. A non-limiting example of a binding agent includes a nucleic acid molecule (DNA or RNA), an antibody, an aptamer, a receptor, a streptavidin, an avidin, a biotin, a lectin, a ligand, an enzyme, a coenzyme, an inorganic ion, a cofactor, a sugar, a lipid, a substrate for an enzyme, a hapten, a neutravidin, a protein A, a protein G, a selectin, an inorganic compound, a metal, a semiconductor and an organic compound.
The magnetic sensor of this invention may be prepared as a variety of methods. Preferably, the zinc-containing magnetic nanopartide-based magnetic sensor includes all types of sensors containing magnetic nanoparticles such as a magneto-resistance sensor, a magnetic relaxation sensor, a magnetic micro- cantilever sensor, a magneto-electronic sensor and magnetophoresis sensor.
As a magneto-resistance sensor, the magnetic sensor of the present invention may further include a signal processing means. The signal processing means may include at least one amplifier. The signal processing means may further include a linearizing circuit, and thus have potential to normalize a non-linear R-H property of a sensor device. According to an illustrative example, a magnetic sensor device, a signal processing means and a magnetic field-generating means may be composed as a single integrated circuit. The magnetic field-generating means may include a conductor and a current source to generate current through the conductor. According to a preferable embodiment, the sensor of the present invention further includes a means measuring current via a magnetic sensor device.
The present invention using a magnetic sensor as a magneto-resistance sensor includes the steps of: (a) generating magnetic field in the vicinity of a magnetic sensor device; (b) inducing regular voltage across the magnetic sensor device; and (c) measuring total signal currents of the magnetic sensor device.
In a magnetic sensor of this invention prepared as a magneto-resistance sensor, the zinc-containing magnetic nanoparticles are preferably used as a label of an analyte. For example, the sample containing an analyte is reacted with the zinc- containing magnetic nanopartides coated with a binding agent having a specific affinity to the analyte for labeling, and then the signal from the magnetic nanopartides is measured to determine the presence or amount of the analyte in the sample. For instance, in the biosensor using the method of the present invention, an antibody immobilized on a substrate is contacted with a sample containing an analyte, and washed. The substrate is reacted with a detecting antibody linked to the surface of zinc-containing magnetic nanoparticle, and then washed. After stopping reaction, it may be concluded that the analyte is involved in the sample with the proviso that the signal from the zinc-containing magnetic nanopartides is detected (by the magnetic sensor device).
General descriptions of a magneto-resistance sensor applicable in the present invention are disclosed in US Patent No. 6,057,167, Baselt et a/., A biosensor based on magnetoresistance technology, Journal of Biosens. Bioelectron., 13 (7-8): 731 (1998)), WO 2007/014322, WO 2007/046051 and US Patent Publn. No. 20080054896 in detail, which are incorporated herein by reference.
In a magnetic relaxation sensor, the changes in the Tl relaxation time are measured to determine contacting between zinc-containing magnetic nanopartides and a reactant, i.e., the presence or amount of a reactant). Fundamental principle is as follows: the reactants are linked to the binding agents on the surface of nanopartides, leading to form nanoparticle aggregates. As a result, the magnetic moment of nanoparticle is changed, and T2 relaxation time of water is decreased. In general, the changes in the T2 relaxation time may be measured using a MR (magnetic resonance) imaging machine. It may be concluded that the analyte is involved in the sample with the proviso that the changes in the T2 relaxation time are detected. Based on a standard curve plotted using a basal amount of reactants, the amount of reactants may be easily determined. For example, the changes in the Tl relaxation time may be measured using T2-weighted spin echo sequence and 1.5 T superconduction magnet under the condition of fixed echo time (TE) and repetition time (TR).
Conventional MR imaging instruments may be utilized as a magnetic sensor device and magnetic field-generating means in a magnetic relaxation sensor. MR imaging method and devices are disclosed in D. M. Kean and M. A. Smith,
Magnetic Resonance Imaging: Principles and Applications (William and Wilkins, Baltimore 1986), U.S. Pat. Nos. 6,151,377, 6,144,202, 6,128,522, 6,127,825, 6,121,775, 6,119,032, 6,115,446, 6,111,410 and 602,891, which are incorporated herein by reference. A magnetic micro-cantilever sensor is composed of a micro-sized cantilever and magnetic nanopartides conjugated with a substance {e.g., antigen) capable of binding to a reactant of interest. The reactant in a sample is attached to the cantilever and nanopartides according to a sandwich method. Subsequently, the cantilever is bent by the magnetic nanopartides under magnetic field induction. The bending force is measured as current by a piezoelectric device, enabling to determine exact amount of reactant.
The basic concept and device of a magnetic micro-cantilever sensor are disclosed in Baselt et a/., Journal of Vacuum Science and Technology B 14: 789 (1996) and Weizmann et a/., Journal of American Chemical Society 126: 1073 (2004).
A magnetophoresis sensor is a sensor employing the motion of dispersed magnetic nanopartides relative to a fluid under the influence of a magnetic field. The movement of magnetic particles can be used to detect or isolate specific components in the fluid, using specific binding and/or capture. The magnetophoresis sensor using the magnetic nanopartide or cluster thereof of the present invention is described in Example 8.
Besides, the sensors using magnetic nanopartides include, but is not limited to, a Maxwell bridge, a frequency dependent magnetometer, a magnetic remanence measurement using a superconducting quantum interference device, a Hall effect measurement, a micro fluidic system, and so forth.
The basic concept and device of the sensors described above are disclosed in C. R. Tamanaha eta/., Biosensors and Bioelectronics, 24, 1 (2008). The magnetic field-generating means used in the present invention may include a permanent magnet and electromagnet and may be varied by modulation depending on the morphology and size of nanoparticle.
In still another aspect of this invention, there is provided a method for detecting an analyte in a sample, comprising the steps of:
(a) contacting the analyte in the sample with the magnetic nanoparticle or cluster thereof represented by the following formula 1 or 2;
ZnfMa-fOb (0<f<8, 0<a<16, 0<b<8, 0<f/(a-f)<10, M represents a magnetic metal atom or an alloy thereof) (1) ZngMc-gM'dOe (0<g<8, 0<c<16, 0<d<16, 0<e<8, 0<g/{(c-g)+d}<10, M represents a magnetic metal atom or an alloy thereof; M' represents one or more elements selected from the group consisting of Group 1 elements, Group 2 elements, Group 12 elements, Group 13 elements, Group 14 elements, Group 15 elements, transition metal elements, Lanthanide elements and Actinide elements) (2); and
(b) measuring a magnetic signal from a reaction product of the step (a). Since the present method comprises the magnetic sensor of this invention described above, the common descriptions between them are omitted in order to avoid undue redundancy leading to the complexity of this specification. The zinc-containing magnetic nanoparticle-based magnetic sensor of the present invention may be utilized in various application fields including molecular diagnosis, analysis for a biological sample and chemical sample, and so on. Since the magnetic sensor of the present invention utilizes zinc-containing magnetic nanopartide having very high saturation magnetism, it may exhibit much more enhanced sensitivity under magnetic field induction and detect an infinitesimal amount of analytes {e.g., proteins or nucleic acid molecules in blood). Due to higher saturation magnetism of the zinc-containing magnetic nanopartides used in this invention, the present magnetic sensor using the same may detect analytes in a much more sensitive manner. The nanopartides having improved sensitivity {e.g., sensitivity in fM level) prepared by the present invention may permit to detect a trace amount of analytes such as anti-cancer marker proteins or nucleic acid molecules, and prion proteins in blood. The present sensor may be constituted by all types of sensors including a magneto-resistance sensor, a magnetic relaxation sensor, a magnetic micro-cantilever sensor, a magnetophoresis sensor, a magneto-electronic sensor, and so forth.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. Ia shows a TEM (transmission electron microscope) image of synthesized Zno.4Fe2.6O4 nanopartides.
Fig. Ib shows a high resolution TEM image of synthesized Zn0-4Fe2-6O4 nanopartides. The internal image of Fig. Ib is a FFT (fast fourier transformation) image of the high resolution TEM image.
Fig. Ic is a XRD (X-ray diffraction) pattern of synthesized Zn0-4Fe2-6O4 nanopartides.
Fig. 2 represents TEM images of (A) ZnxMn1-xFe204 and (B) ZnxFe3-x04. The zinc content ratio (x) may be modulated from 0 to 0.8. Fig. 3 is a graph representing a saturation magnetism of zinc-containing magnetic nanopartides and several nanopartides containing no zinc.
Fig. 4 represents a TEM image of synthesized zinc-containing nanopartide cluster. The cluster is prepared by encapsulating zinc-containing nanopartides in a micelle consisting of polystyrene-polyacryl acid copolymers.
Fig. 5 is a graph measuring a magnetic field strength depending on the distance from the surface of NdFeB magnet used as external magnetic field- generating means. Fig. 6 schematically represents the principle of a magnetic separation system using zinc-containing magnetic nanoparticles. A solution containing a target material is mixed with magnetic nanoparticles conjugated with a binding agent capable of selectively binding to the target material, resulting in binding of the target material and magnetic nanoparticles. Afterwards, the target material may be separated by magnet attraction.
Fig. 7 shows a graph comparing separation efficiency of a fluorescent material (rhodamine) using zinc-containing nanoparticles (Zno.4Fe2.eO4) or nanoparticles containing no zinc (Fe3O4). Fig. 7A represents a graph measuring the fluorescent material (rhodamine) absorbance at 580 nm depending on magnetic separation time, and Fig. 7B represents a graph measuring separation efficiency of the fluorescent material (rhodamine) with the passage of time.
Fig. 8 is a graph comparing magnetic separation efficiency between zinc- containing nanoparticles (Zn0-4Fe2-6O4) and nanoparticles containing no zinc (Fe3O4) according to changes of external magnetic field strength. Fig. 9 shows a graph comparing antibody separation using nanoparticles containing no zinc and zinc-containing nanoparticles.
Fig. 10 represents results comparing: (A) cell amounts separated with the passage of time in zinc-containing nanoparticles and nanoparticles containing no zinc; and (B) cell separation efficiency of zinc-containing nanoparticles and nanoparticles containing no zinc.
Fig. lla shows a TEM image of synthesized Zn0-4Fe2-6O4 nanoparticles. Fig. lib shows a high resolution TEM image of synthesized Zn0-4Fe2-6O4 nanoparticles. The internal image of Fig. Ib is a FFT (fast fourier transformation) image of the high resolution TEM image.
Fig. lie is a XRD (X-ray diffraction) pattern of synthesized Zn0-4Fe2-6O4 nanoparticles.
Fig. 12 represents TEM images of (A) ZnxMni-xFe204 and (B) ZnxFe3-x04. The zinc content ratio (x) may be modulated from 0 to 0.8.
Fig. 13 is a graph analyzing a saturation magnetism of zinc-containing magnetic nanoparticles and several nanoparticles containing no zinc.
Fig. 14 represents a TEM image of synthesized zinc-containing nanopartide cluster. The cluster is prepared by encapsulating zinc-containing nanoparticles in a micelle consisting of polystyrene-polyacryl acid copolymers.
Fig. 15 schematically represents the principle of a magnetic relaxation sensor using zinc-containing magnetic nanoparticles. The magnetic nanoparticles exhibit high spin-spin relaxation time (T2) due to rare interaction each other at a distance enough to be dispersed, but the formation of magnetic nanopartide aggregates mediated by samples of interest allows magnetic particles having very low TZ relaxation time. T2 relaxation time may be measured using MRI or magnetic relaxation system.
Fig. 16 shows DNA detection using in zinc-containing nanoparticles
Zn0.4Fe2.6θ4) and nanoparticles containing no zinc (Fe3O4). Fig. 16A represents a colorimetric MRI image merging ΔT2 with image measured by MRI in each nanoparticles. Fig. 16B is a graph expressing Fig. 16A numerically (i.e., ΔT2 value to
DNA amount analyzed).
Fig. 17 shows protein (avidin) detection using in zinc-containing nanoparticles
Zn0.4Fe2.6θ4) and nanoparticles containing no zinc (Fe3O4). Fig. 17A represents a colorimetric MRI image merging ΔT2 with image measured by MRI in each nanoparticles. Fig. 17B is a graph expressing Fig. 17A numerically (i.e., ΔT2 value to protein amount analyzed).
Fig. 18 schematically represents a magnetophoresis sensor using zinc- containing magnetic nanoparticles.
Fig. 19 represents a graph analyzing a cell migration rate (panel A) and attractive force toward a magnetic tip (panel B).
Fig. 20 shows microscopic images observing practical operation of the magnetophoresis sensor. It could be appreciated that the cells bound with zinc- containing magnetic nanoparticles are migrated toward a magnetic tip more rapidly than those bound with magnetic nanoparticles containing no zinc.
The present invention will now be described in further detail by examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the present invention as set forth in the appended claims is not limited to or by the examples.
EXAMPLES
I. Zinc-Containing Magnetic Nanoparticle-Based Magnetic Systems
EXAMPLE 1: Synthesis of Zinc-Containing Ferrite Nanoparticles with the Composition of ZnxMi.xFe2O4 (M = Fe or Mn, x = 0.1, 0.2, 0.3, 0.4, 0.8) and the Core Size of 15 nm Coated with Dimercaptosuccinic Acid (DMSA) for Magnetic Separation Systems
As an example of zinc-containing metal oxide nanoparticles described in the present invention, zinc-containing ferrite nanoparitldes with the composition of ZnxMi-XFe2O4 (M = Fe or Mn; x = 0.1, 0.2, 0.3, 0.4, 0.8) and the core size of 15 nm were produced according to the method described in Korean Pat. Nos. 10-0604975, 10-0652251 and 10-0713745, PCT/KR2004/002509, Korean Pat. No. 10-0604975, PCT/KR2004/003088, PCT/KR2007/001001, and Korean Pat. Appln. No. 2006- 0018921. To prepare 15 nm-sized nanoparticles, ZnCI2 (Aldrich, USA), FeCI2 (Aldrich, USA) or MnCI2 (Aldrich, USA), and Fe(acac)3 (Aldrich, USA) as precursors of nanoparticles were added to trioctylamine solvent (Aldrich, USA) containing 20 mmol oleic acid (Aldrich, USA) and 20 mmol oleylamine (Aldrich, USA) as capping molecules. The mixture was incubated at 2000C under argon gas atmosphere and further reacted at 3000C, synthesizing 15 nm-sized ZnxM1-XFe2O4 (M = Fe or Mn; x = 0.1, 0.2, 0.3, 0.4, 0.8) nanoparticles. In addition, a ratio of ZnCI2 or MCI2 as precursors of nanoparticles was modulated depending on the composition of Zn (x = 0.1-0.8) and reacted as described above. The synthesized zinc-containing ferrite nanoparticles were precipitated by excess ethanol and then the precipitated nanoparticles were again dispersed in toluene, obtaining a colloid solution. The synthesized nanoparticles (20 mg/ml in toluene) were mixed with DMSO solution containing excess dimercaptosuccinic acid (DMSA; Aldrich, USA), and incubated for 2 hrs. Subsequently, the nanoparticles were centrifuged and re-dispersed in water. As shown in Fig. Ia and Fig. 2, ZnxM1-xFe204 (M = Fe or Mn; x = 0.1, 0.2, 0.3, 0.4, 0.8) nanoparticles synthesized according to the method aforementioned have globular structure with a homogeneous size of 15 nm (size distribution s < 10%). As demonstrated in a high resolution electron microscope (Fig. Ib) and X-ray diffraction pattern (Fig. Ic) analysis, the nanoparticles exhibit higher crystallinity as a spinel structure. The amount of zinc was determined using Inductively Coupled Plasma- Atomic Emission Spectroscopy (ICP-AES, OPTIMA-3000, Perkin Elmer) and Energy Dispersive X-ray (EDAX, Gatan).
EXAMPLE 2: Synthesis of Zinc-Containing Ferrite Nanoparticles with the Composition of Zn014M016Fe2O4 (M = Fe or Mn) and the Core Size of 6, 9 or 12 nm Coated with Dimercaptosuccinic Acid (DMSA) for Magnetic Separation Systems
ZnCI2, FeCI2 or MnCI2, and Fe(acac)3 as precursors of nanoparticles were added to trioctylamine solvent containing 10 mmol oleic acid and 30 mmol oleylamine as capping molecules. The mixture was incubated at 18O0C for 3 hrs and further reacted at 2500C, synthesizing 6 nm-sized Zn014MCeFe2O4 (M = Fe or Mn) nanoparticles. To prepare nanoparticles with different sizes {e.g., 9 or 12 nm), equal amount of precursors were mixed with trioctylamine solvent containing various ratios of oleic acid and oleylamine depending on the sizes of nanoparticles, and the same process described above was carried out. The synthesized nanoparticles (20 mg/ml in toluene) were mixed with DMSO solution containing excess DMSA, and incubated for 2 hrs. Subsequently, the nanoparticles were centrifuged and re-dispersed in water. As results, the synthesized nanoparticles have globular structure with a homogeneous size, and their zinc contents were analyzed using ICP-MS and EDAX.
EXAMPLE 3: Synthesis of Zinc-Containing Ferrite Nanoparticles with the Composition of ZnxMi-xFe2O4 (M = Co or Ni, x = 0.3, 0.4) and the Core Size of 12 nm Coated with Dimercaptosuccinic Acid (DMSA) for Magnetic Separation Systems
As an example of zinc-containing metal oxide nanoparticles described in the present invention, zinc-containing ferrite nanoparticles with the composition of ZnxM1-xFe204 (M = Fe or Mn; x = 0.1, 0.2, 0.3, 0.4, 0.8) and the core size of 12 nm were produced according to the following method. ZnCI2, CoCI2 or NiCI2, and Fe(acac)3 as precursors of nanoparticles were added to trioctylamine solvent containing 20 mmol oleic acid and 20 mmol oleylamine as capping molecules. The mixture was incubated at 2000C for 3 hrs and further reacted at 3000C, synthesizing 12 nm-sized Zn0JM0JFe2O4 (M = Co or Ni) nanoparticles. In addition, a ratio of ZnCI2, CoCI2 or NiCI2 as precursors of nanoparticles was modulated depending on composition of Zn (x = 0.3-0.4). The synthesized nanoparticles (20 mg/ml in toluene) were mixed with DMSO solution containing excess DMSA, and incubated for 2 hrs. Subsequently, the nanoparticles were centrifuged and re-dispersed in water. As results, the nanoparticles have globular structure with a homogeneous size, and zinc contents were analyzed using ICP-MS and EDAX.
EXAMPLE 4: Synthesis of Zinc-Containing Oxide Nanoparitlcles with the Composition of ZnxMyOz (M = Mn, Co, Ni) Coated with Tetramethylammoniumhydroxide (TMAOH) for Magnetic Separation Systems
As an example of zinc-containing metal oxide nanopartides described in the present invention, zinc-containing manganese oxide nanoparitlcles with the composition of ZnxMyOz (M = Mn, Co, Ni) used in the present invention were produced according to the following method. ZnCI2 and MCI2 (M = Mn, Co, Ni) as precursors of nanopartides were added to trioctylamine solvent containing 0.5 mmol oleic acid and 6.5 mmol oleylamine as capping molecules. The mixture was decomposed at 2700C for 1 hrs, synthesizing ZnxMxO2 (M = Mn, Co, Ni) nanopartides. The synthesized nanopartides (50 mg/ml in 1 ml toluene) were precipitated by excess ethanol and then the precipitated nanopartides were again dispersed in 5 ml TMAOH solution, obtaining a soluble solution. The synthesized nanopartides have the composition of Zn0-4M26O4, Zn0.2Co0.8θ or Zn0-2Ni0-8O with the core size of 6, 7 or 10 nm, respectively. Zinc contents were analyzed using ICP-MS and EDAX.
EXAMPLE 5: Comparison of Saturation Magnetization (Ms) between Zinc- Containing Nanopartides and Nanopartides Containing no Zinc
To determine to what extent zinc increases saturation magnetization (/1Z5) of nanopartides, each saturation magnetization of zinc-containing nanopartides, conventionally accessible metal oxide nanopartides, CLJO (cross-linked iron oxide) and Feridex™ (Taejoon Co Ltd.), and several ferrite nanopartides containing no zinc was measured at room temperature. CLJO was synthesized according to the method described in Weissleder et a/., Journal of Magnetic Resonance in Medicine 29: 599 (1993). Several ferrite nanopartides were produced according to the method described in Korean Pat. Nos. 10-0604975, 10-0652251 and 10-0713745, PCT/KR2004/002509, Korean Pat. No. 10-0604975, PCT/KR2004/003088, PCT/KR2007/001001, and Korean Pat. Appln. No. 2006-0018921, and zinc-containing metal oxide nanoparticles were synthesized according to the method described in Example 1. These saturation magnetizations were measured at room temperature using a MPMS Superconducting Quantum Interference Device (SQUID) Magnetometer (MPMS-XL, Quantum Design). As shown in Rg. 3, zinc-containing nanoparticles have more excellent saturation magnetization than nanoparticles containing no zinc. Interestingly, saturation magnetization of each zinc-containing nanoparticles, Zn0-4Fe2^O4 and Zn0-4Mn0-6Fe2O4, was 161 and 175 emu/g higher than that of conventionally accessible metal oxide nanoparticles {e.g., CLIO, 64 emu/g;
Feridex, 82 emu/g). Furthermore, these values are enhanced to 47 and 61 emu/g compared with nanoparticles containing no zinc, Fe3O4 and MnFe2O4, respectively.
Accordingly, it could be expected that zinc-containing nanoparticles having enhanced saturation magnetization may highly increase conventional sensor sensitivity in the senses that saturation magnetization is proportional to the square of sensor sensitivity.
EXAMPLE 6: Preparation of Nanoparticle Cluster Using Zinc-Containing Nanoparticles
Exemplified nanoparticle cluster using zinc-containing nanoparticles includes a magnetic nanoparticle cluster which is encapsulated in micelles prepared by polystyrene-polyacryl acid copolymers. Basic preparation method of polymer and cluster used was carried out according to the method described in Taton et a/. Nano Leters 5: 1987 (2005). Zinc-containing nanoparticles coated with oleic acid and oleylamine were prepared according to the method described in Korean Pat. Nos. 10- 0604975, 10-0652251 and 10-0713745, PCT/KR2004/002509, Korean Pat. No. 10- 0604975, PCT/KR2004/003088, PCT/KR2007/001001, and Korean Pat. Appln. No. 2006-0018921. Subsequently, the polystyrene-polyacryl acid copolymers were dissolved in dimethylformamide (DMF; Aldrich) at a concentration of 0.1 mg/ml, and further added with tetrahydrofuran (THF; Aldrich) to be at a final concentration of 50%. The solution was vigorously stirred and the nanoparticles were gradually added to be at a concentration of 0.1 mg/ml, followed by adding 40 ml water at a rate of 5 ml/hr. The polymer micelles, in which the synthesized zinc-containing nanoparticles were encapsulated, were separated and concentrated using a centrifugation. These micelles are stabilized in a soluble solution due to exposure of carboxylic acid on their surface, which serves as a functional group to attach a binding agent such as antibodies. In addition, the micelles were cross-linked to enhance their stability and dispersion in solution. For the purpose, 5 mM EDC (1- ethyl-3-(3-dimethylaminopropyl) carbodiimide; Sigma), 2 mM sulfo-NHS (N- hydroxysulfosuccinimide; Sigma) and 5 mM 2,2'-(ethylenedioxy)bis(ethylamine) (Aldrich) were used as cross-linkers. As shown in Fig. 4, the synthesized micelles have a total size of about 120 nm, and contain numbers of 15 zinc-containing nanoparticles. As described above, it is expected that the synthesized nanoparticle clusters exhibit enhanced saturation magnetization, leading to significantly improve separation efficiencies of magnetic separation systems.
EXAMPLE 7: Magnetic Field Measurement of NdFeB Magnets Used as a Means for Generating External Magnetic Field
Of conventional ferromagnetic magnets well known to generate external magnetic field, NdFeB magnets were used in the magnetic separation system of the present invention. In general, the magnetic field of magnets is inversely proportional to the distance from the surface. Therefore, the magnetic field suitable for magnetic separation was examined by measuring magnetic field strength depending on the distance. The results were represented in Fig. 5. Magnetic field was measured using a Kanetec TM-601 tesla meter. As a result, the magnetic field strength was inversely proportional to the distance according to a linear expression with the slope (=dB/dx) of 4.7 T/m.
EXAMPLE 8: Comparison of Magnetic Separation Efficiency Using Zinc- Containing Nanoparticles or Nanoparticles Containing no Zinc
It was demonstrated in the present invention that zinc-containing nanoparticles may much more remarkably improve the efficiency of magnetic separation system than nanoparticles containing no zinc: The magnetic separation system used in the present invention has a working principle shown in Fig. 6. Through mixing a solution containing materials of interest with magnetic nanoparticles including binding agents capable of selectively binding to materials of interest, they may be linked to magnetic nanoparticles, enabling to be separated by intermolecular interaction of magnets. Each zinc-containing nanoparticles and nanoparticles containing no zinc were synthesized according to the methods described in Example 1 and Korean Pat. Nos. 10-0604975, 10-0652251 and 10- 0713745, PCT/KR2004/002509, Korean Pat. No. 10-0604975, PCT/KR2004/003088, PCT/KR2007/001001, and Korean Pat. Appln. No. 2006-0018921. In addition, each nanoparticles were solubilized in water by surface modifications with TMAOH according to the method described in Example 4. Afterwards, these nanoparticles were coated with bovine serum albumin (BSA) according to the methods described in Korean Pat. Nos. 10-0604975, 10-0652251 and 10-0713745. As an illustrative material of interest, fluorescent dye (rhodamine; Pierce) was utilized. In other words, 0.1 mg/ml of each zinc-containing nanoparticles (Zno.4Fe2.eO4) and nanoparticles containing no zinc (Fe3O4) coated with BSA was added to an aqueous solution containing 100 nM rhodamine. One end of rhodamine is linked to N- hydrosuccinimide, contributing to selective binding to a primary amine group of amino acids {e.g., lysine) on the surface of nanoparticles coated with BSA. In this connection, each separation rate and efficiency of zinc-containing nanoparticles or nanoparticles containing no zinc linked to rhodamine was measured by magnet attraction (Fig. 7). Fig. 7A represents a graph measuring rhodamine absorbance at 580 nm by monitoring with the passage of time. As beginning of magnetic separation using magnets, rhodamine molecules in solution are decreased with the passage of time, resulting in gradual reduction of their absorbance. As shown in a graph, almost all rhodamine molecules were separated at about 4 min in zinc- containing nanoparticles, and their absorbance was measured in the level of approximate 0.02. On the contrary, the absorbance of rhodamine was unchangeable until 6 min in nanoparticles containing no zinc, and was reduced at 10 min to the level of about 0.12, reaching at the level of almost 0.02 after 50 min. Fig. 7B is a graph representing separation rate (%) of Fig. 7A. Similarly, the separation rate of not less than 90% was observed only at 4 min in zinc-containing nanoparticles, whereas the separation rate was even 20% at 10 min and reached to not less than 90% after 50 min in nanoparticles containing no zinc. Accordingly, it is suggested that zinc-containing nanoparticles enable to decrease magnetic separation time in an excellent manner.
EXAMPLE 9: Comparison of Magnetic Separation Efficiency in Zinc- Containing Nanoparticles and Nanoparticles Containing no Zinc According to Changes of External Magnetic Field Strength
By changing external magnetic field strength, magnetic separation efficiency of zinc-containing nanoparticles was compared with that of nanoparticles containing no zinc. The modulation of magnetic field strength was carried out by changing the distance between magnetic separation system and magnets based on the results observed in [Example 7. MACS® column (Miltenyi Biotech, Germany) was used as a magnetic separation system, and each zinc-containing nanoparticles and nanoparticles containing no zinc was synthesized according to the methods described in Example 1 and Korean Pat. Nos. 10-0604975, 10-0652251 and 10- 0713745, PCT/KR2004/002509, Korean Pat. No. 10-0604975, PCT/KR2004/003088, PCT/KR2007/001001, and Korean Pat. Appln. No. 2006-0018921.
Zinc-containing magnetic nanoparticles and magnetic nanoparticles containing no zinc with a concentration 0.2 mg/ml was added to MACS® column under external magnetic fields, respectively. The magnetic nanoparticles were captured in MACS® column by external magnetic fields, and the removal of external magnetic fields causes their release from the column. As nanoparticle magnetism is varied depending on a type of nanoparticle, the amount of nanoparticles captured in the column is different. The captured nanoparticles were quantified using an absorption spectrophotometer.
Fig. 8 is a graph comparing magnetic separation efficiency between zinc- containing nanoparticles (Zn0-4Fe2-6O4) and nanoparticles containing no zinc (Fe3O4) according to changes of external magnetic field strength. As shown in Fig. 8, the amount of both separated magnetic nanoparticles was similar under very strong external magnetic fields (300 mT). However, the separated amount of zinc- containing nanoparticles or nanoparticles containing no zinc was significantly different depending on reduction of external magnetic field. That is, the amount of zinc-containing nanoparticles captured in the column was increased in 17% and 22% under external magnetic fields of 150 and 100 mT higher than that of nanoparticles containing no zinc, respectively. Meanwhile, where external magnetic field is reduced below 100 mT, the difference of the separated amount between both nanoparticles is gradually decreased. In addition, both nanoparticles are not separated in the absence of external magnetic field. Therefore, the present results suggest that zinc- containing nanoparticles exhibit more excellent separation efficiency than conventional nanoparticles in spite of using relatively low external magnetic field.
EXAMPLE 10: Comparison and Quantification of Magnetic Separation Efficiency in Zinc-Containing Nanoparticles and Nanoparticles Containing no Zinc for Anti-Mouse IgG-FITC in the Presence of External Magnetic Field
Using difference of magnetic strength between zinc-containing nanopartides and nanopartides containing no zinc in the presence of external magnetic field, antibody proteins were isolated and their separation efficiencies were compared.
MACS® column (Miltenyi Biotech, Germany) was used as a magnetic separation system.
Nanopartides containing no zinc and zinc-containing nanopartides are Fe3O4 and Zn0-4Fe2-6O4, respectively. Both nanopartides were particles with a diameter of 15 nm. The surface of each nanopartides was coated with dimercaptosuccinic acid (DMSA) and then linked to protein A (Sigma). The synthesis of nanopartides was carried out according to the method described in Example 1. To bind protein A on the surface of nanopartides, it was reacted with sulfo-SMCC (Sulfosuccinimidyl-4-(N- maleimidomethyl)cydohexane-l-carboxylate; Pierce) for 30 min, and then mixed with nanopartides. Electrophoresis was performed to determine whether protein A was stably bound to the surface of nanopartides. Target protein of the present invention is goat anti-mouse IgG-FlTC (Sigma). Under the condition of external magnetic field, each zinc-containing nanopartides (0.08 mg/ml) and nanopartides containing no zinc (0.08 mg/ml) were added to MACS® column, and subsequently to antibody protein (1 ml of 0.01 M anti-mouse IgG-FITC) labeled with fluorescent dye, resulting in binding of antibody protein on the surface of nanopartides by specific interactions between antibody protein and protein A coated on the surface of nanopartides. Magnetic nanopartides are captured in MACS® column due to external magnetic field, and thus the removal of external magnetic field allows magnetic nanopartides to be released from the column. As nanopartide magnetism is varied depending on a type of nanopartide, the amount of nanopartides captured in the column is changed. The captured nanopartides may be quantified using an absorption spectrophotometer. In this context, the amount of separated nanoparticles may be inversely determined by measuring fluorescent signal intensity as antibody protein with fluorescent dye is bound to the surface of captured nanoparticles. Consequently, separation efficiency to antibody protein may be analyzed by measuring magnetic values of two nanoparticles. Fig. 9 is a graph comparing magnetic separation efficiency between zinc- containing nanoparticles (Zno.4Fe2.eO4) and nanoparticles containing no zinc (Fe3O4). As shown in Fig. 9, very high fluorescent signal intensity was observed in zinc- conlaining nanoparlides after MACS® column separation, suggesting that zinc- containing nanoparticles are highly bound to antibodies linked to fluorescent materials. In this connection, zinc-containing nanoparticles with strong magnetic value are massively captured in MACS® column, as magnetism of zinc-containing nanoparticles has higher than that of nanoparticles containing no zinc. In other words, the amount of magnetic nanoparticles may be determined as the fluorescent signal intensity because antibody protein conjugated with FITC as a fluorescent dye is linked to the surface of magnetic nanoparticles. The higher fluorescence intensity refers to the large amount of nanoparticles. FITC has physical potential to emit the strongest fluorescence intensity at 521 nm by absorbing the light with a wavelength of 495 nm. By calculating the difference of separation efficiency to antibody proteins of each magnetic nanoparticle under the standard of fluorescence intensity at 521 nm, it was demonstrated that the amount of antibody proteins is separated in zinc- containing nanoparticles about 4.71-fold higher than in nanoparticles containing no zinc (After MACS® column separation, fluorescence signal intensity value of each zinc-containing nanoparticles and nanoparticles containing no zinc is measured as 10.00 au and 2.13 au at 521 nm). Given the amount of fluorescent material linked to antibody (about 50 molecules/antibody), about 3 μg antibodies were separated from zinc-containing nanoparticles. Therefore, the present results suggest that more excellent separation efficiency and quantification may be obtained in zinc-containing nanoparticles than in conventional nanoparticles. EXAMPLE 11: Comparison of Magnetic Separation Efficiency in Zinc- Containing Nanoparticles and Nanoparticles Containing no Zinc for Cells (U87MG, Human Glioblastoma Cell Line) in the Presence of External Magnetic Field
Using difference of magnetic strength between zinc-containing nanoparticles and nanoparticles containing no zinc in the presence of external magnetic field, cells were isolated and their separation efficiencies were compared. -External magnetic field was generated using NdFeB permanent magnets. To bind nanoparticles to U87MG cell line (human glioblastoma cell line; ATCC), each zinc-containing nanoparticles and nanoparticles containing no zinc was coated with dimercaptosuccinic acid (DMSA) and bound to antibody proteins. The antibody proteins utilized Cetuximab (Merck) conventionally known as Erbitux, which is capable of binding to an EGFR receptor on the surface of U87MG cells. For binding of nanoparticles and antibodies, antibody proteins were activated using sulfo-SMCC (Pierce) and then mixed with nanoparticles coated with DMSA according to the method described in Example 10. Each nanoparticles linked to Cetuximab was incubated with cells in PBS buffer at room temperature for 1 hr, followed by adding external magnetic field. Afterwards, Cetuximab antibodies on the nanoparticles were bound to cells. Cells were attracted toward magnetic field under external magnetic field by the nanoparticles bound to the surface of cells. Thus, samples in reaction solution were harvested depending on time in certain region apart from magnets, and cell number was observed. As results, cells were uniformly dispersed in total reaction solution before addition of external magnetic field, whereas cell numbers observed were decreased depending on time as cells in reaction solution were attracted toward magnets under the conditions adding external magnetic field. Practical experiments was carried out as follows: NdFeB magnets of 5 cm in width, 5 cm in length, and 1 cm in height were placed at a position adjacent to vials containing zinc-containing nanoparticles or nanopartides containing no zinc in reaction solution. And then, the solution was harvested from a portion of the vial at the longest distance from the magnets in a time-dependent manner. The time interval harvesting reaction solution was determined as 0, 0.5, 1, 2.5, 5, 10, 15 and 60 min. Thirty μl of reaction solution per indicated time was collected, and stained with 0.4% tryphan blue, followed by counting cell number with a hematocytometer.
Fig. 10 is graphs comparing cell separation using zinc-containing nanoparticles (Zno.4Fe2.eO4) and nanoparticles containing no zinc (Fe3θ4) as described above. Since magnetism of zinc-containing nanoparticles is higher than that of nanoparticles containing no zinc, cells were attracted in stronger and faster manner. Therefore, it could be appreciated that cell number separated from solution to a region adjacent to magnet were much more rapidly increased with the passage of time (Fig. 10A).
Fig. 1OB represents cell separation efficiency which is determined by counting cell number separated at 5 min after addition of external magnetic field. The cell separation efficiency is calculated by a ratio of cell number after to cell number before cell separation. It was demonstrated that the efficiency of cell separation in zinc-containing nanoparticles may be about 2.9-fold higher than that in nanoparticles containing no zinc, and the amount of separated cells may be quantified using a hematocytometer.
II. Zinc-Containing Magnetic Nanoparticle-Based Magnetic Sensors
EXAMPLE 1: Synthesis of Zinc-Containing Ferrite Nanoparticles with the Composition of ZnxM1-XFe2O4 (M = Fe or Mn, x = 0.1, 0.2, 0.3, 0.4, 0.8) and the Core Size of 15 nm Coated with Dimercaptosuccinic Acid (DMSA) Used in the Magnetic Sensor Systems
As an example of zinc-containing metal oxide nanoparticles described in the present invention, zinc-containing ferrite nanoparitldes with the composition of ZnxM1-xFe204 (M = Fe or Mn; x = 0.1, 0.2, 0.3, 0.4, 0.8) and the core size of 15 nm were produced according to the method described in Korean Pat. Nos. 10-0604975, 10-0652251 and 10-0713745, PCT/KR2004/002509, Korean Pat. No. 10-0604975, PCT/KR2004/003088, PCT/KR2007/001001, and Korean Pat. Appln. No. 2006- 0018921. To prepare 15 nm-sized nanoparticles, ZnCI2 (Aldrich, USA), FeCI2 (Aldrich, USA) or MnCI2 (Aldrich, USA), and Fe(acac)3 (Aldrich, USA) as precursors of nanoparticles were added to trioctylamine solvent (Aldrich, USA) containing 20 mmol oleic acid (Aldrich, USA) and 20 mmol oleylamine (Aldrich, USA) as capping molecules. The mixture was incubated at 2000C under argon gas atmosphere and further reacted at 3000C, synthesizing 15 nm-sized ZnxMi-xFe204 (M = Fe or Mn; x = 0.1, 0.2, 0.3, 0.4, 0.8) nanoparticles. In addition, a ratio of ZnCI2 or MCI2 as precursors of nanoparticles was modulated depending on the composition of Zn (x = 0.1-0.8) and reacted as described above. The synthesized zinc-containing ferrite nanoparticles were precipitated by excess ethanol and then the precipitated nanoparticles were again dispersed in toluene, obtaining a colloid solution. The synthesized nanoparticles (20 mg/ml in toluene) were mixed with DMSO solution containing excess dimercaptosuccinic acid (DMSA; Aldrich, USA), and incubated for 2 hrs. Subsequently, the nanoparticles were centrifuged and re-dispersed in water. As shown in Fig. 11a and Fig. 12, ZnxM1-xFe204 (M = Fe or Mn; x = 0.1, 0.2, 0.3, 0.4, 0.8) nanoparticles synthesized according to the method aforementioned have globular structure with a homogeneous size of 15 nm (size distribution s < 10%). As demonstrated in a high resolution electron microscope (Fig. lib) and X-ray diffraction pattern (Fig. lie) analysis, the nanoparticles exhibit higher crγstallinity as a spinel structure. The amount of zinc was determined using Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES, OPTΪMA-3000, Perkin Elmer) and Energy Dispersive X-ray (EDAX, Gatan).
EXAMPLE 2: Synthesis of Zinc-Containing Ferrite Nanoparticles with the Composition of Zn04M06Fe2O4 (M = Fe or Mn) and the Core Size of 6, 9 or 12 nm Coated with Dimercaptosuccinic Acid (DMSA) Used in the Magnetic Sensor Systems
ZnCI2, FeCI2 or MnCI2, and Fe(acac)3 as precursors of nanopartides were added to trioctylamine solvent containing 10 mmol oleic acid and 30 mmol oleylamine as capping molecules. The mixture was incubated at 1800C for 3 hrs and further reacted at 2500C, synthesizing 6 nm-sized Zn0.4M0.6Fe2O4 (M = Fe or Mn) nanopartides. To prepare nanopartides with different sizes (e.g., 9 or 12 nm), equal amount of precursors were mixed with trioctylamine solvent containing various ratios of oleic acid and oleylamine depending on the sizes of nanopartides, and the same process described above was carried out. The synthesized nanopartides (20 mg/ml in toluene) were mixed with DMSO solution containing excess DMSA, and incubated for 2 hrs. Subsequently, the nanopartides were centrifuged and re-dispersed in water. As results, the synthesized nanopartides have globular structure with a homogeneous size, and their zinc contents were analyzed using ICP-MS and EDAX.
EXAMPLE 3: Synthesis of Zinc-Containing Ferrite Nanopartides with the Composition of ZnxM1-xFe204 (M = Co or Ni, x = 0.3, 0.4) and the Core Size of 12 nm Coated with Dimercaptosuccinic Acid (DMSA) Used in the Magnetic Sensor Systems
As an example of zinc-containing metal oxide nanopartides described in the present invention, zinc-containing ferrite nanoparitldes with the composition of ZnxMi-xFe204 (M = Fe or Mn; x = 0.1, 0.2, 0.3, 0.4, 0.8) and the core size of 12 nm were produced according to the following method. ZnCI2, CoCI2 or NiCI2, and Fe(acac)3 as precursors of nanopartides were added to trioctylamine solvent containing 20 mmol oleic acid and 20 mmol oleylamine as capping molecules. The mixture was incubated at 2000C for 2 hrs and further reacted at 3000C, synthesizing 12 nm-sized Zn0^Ma7Fe2O4 (M = Co or Ni) nanopartides. In addition, a ratio of ZnCI2, CoCI2 or NiCI2 as precursors of nanoparticles was modulated depending on the composition of Zn. The synthesized nanoparticles (20 mg/ml in toluene) were mixed with DMSO solution containing excess DMSA, and incubated for 2 hrs. Subsequently, the nanoparticles were centrifuged and re-dispersed in water. As results, the nanoparticles have globular structure with a homogeneous size, and zinc contents were analyzed using ICP-MS and EDAX.
EXAMPLE 4: Synthesis of Zinc-Containing Oxide Nanoparitlcles with the Composition of ZnxMyO2 (M = Mn, Co, Ni) Coated with Tetramethylammoniumhydroxide (TMAOH) Used in the Magnetic Sensor Systems
As an example of zinc-containing metal oxide nanoparticles described in the present invention, zinc-containing manganese oxide nanoparitlcles with the composition of ZnxMyOz (M = Mn, Co, Ni) used in the present invention were produced according to the following method. ZnCI2 and MCI2 (M = Mn, Co, Ni) as precursors of nanoparticles were added to trioctyiamine solvent containing 0.5 mmol oleic acid and 6.5 mmol oleylamine as capping molecules. The mixture was decomposed at 2700C for 1 hrs, synthesizing ZnxMyOz (M = Mn, Co, Ni) nanoparticles. The synthesized nanoparticles (50 mg/ml in 1 ml toluene) were precipitated by excess ethanol and then the precipitated nanoparticles were again dispersed in 5 ml TMAOH solution, obtaining a soluble solution. The synthesized nanoparticles have the composition of Zn0-4M2-6O4, Zn0-2Co0-8O or Zn0 2Ni0-8O with the core size of 6, 7 or 10 nm, respectively. Zinc contents were analyzed using ICP-MS and EDAX.
EXAMPLE 5: Comparison of Saturation Magnetization between Zinc- Containing Nanoparticles and Nanoparticles Containing no Zinc
To determine to what extent zinc increases saturation magnetization of nanoparticles, each saturation magnetization of zinc-containing nanoparticles, conventionally accessible metal oxide nanoparticles, CLJO and Feridex™, and several ferrite nanoparticles containing no zinc was measured at room temperature. CLJO was synthesized according to the method described in Weissleder et a/., Journal of Magnetic Resonance in Medicine 29: 599 (1993). Several ferrite nanoparticles were produced according to the method described in Korean Pat. Nos. 10-0604975, 10- 0652251 and 10-0713745, PCT/KR2004/002509, Korean Pat. No. 10-0604975, PCT/KR2004/003088, PCT/KR2007/001001, and Korean Pat. Appln. No. 2006- 0018921, and zinc-containing metal oxide nanoparticles were synthesized according to the method described in Example 1. These saturation magnetizations were measured at room temperature using a MPMS Superconducting Quantum Interference Device (SQUID) Magnetometer (MPMS-XL, Quantum Design). As shown in Fig. 13, zinc-containing nanoparticles have more excellent saturation magnetization than nanoparticles containing no zinc. Interestingly, saturation magnetization of each zinc-containing nanoparticles, Zn0-4Fe^eO4 and Zn0-4MnCeFe2O4, was 161 and 175 emu/g higher than that of conventionally accessible metal oxide nanoparticles {e.g., CLJO, 64 emu/g; Feridex, 82 emu/g). Furthermore, these values are enhanced to 47 and 61 emu/g compared with nanoparticles containing no zinc, Fe3O4 and MnFe2O4, respectively.
Accordingly, it could be expected that zinc-containing nanoparticles having enhanced saturation magnetization may highly increase conventional sensor sensitivity in the senses that saturation magnetization is proportionate to the square of sensor sensitivity.
EXAMPLE 6: Preparation of Nanopartϊcle Cluster Using Zinc-Containing Nanoparticles
Exemplified nanoparticle cluster using zinc-containing nanoparitldes includes a magnetic nanoparticle cluster which is encapsulated in micelles prepared by polystyrene-polyacryl acid copolymers. Basic preparation method of polymer and cluster used was carried out according to the method described in Taton et a/. Nano Leters 5: 1987 (2005). Zinc-containing nanopartides coated with oleic acid and oleylamine were prepared according to the method described in Korean Pat. Nos. 10- 0604975, 10-0652251 and 10-0713745, PCT/KR2004/002509, Korean Pat. No. 10- 0604975, PCT/KR2004/003088, PCT/KR2007/001001, and Korean Pat. Appln. No. 2006-0018921. Subsequently, the polystyrene-polyacryl acid copolymers were dissolved in dimethylformamide (DMF; Aldrich) at a concentration of 0.1 mg/ml, and further added with tetrahydrofuran (THF; Aldrich) to be at a final concentration of 50%. The solution was vigorously stirred and the nanopartides were gradually added to be at a concentration of 0.1 mg/ml, followed by adding 40 ml water at a rate of 5 ml/hr. The polymer micelles, in which the synthesized zinc-containing nanopartides were encapsulated, were separated and concentrated using a centrifugation. These micelles are stabilized in a soluble solution due to exposure of carboxylic acid on their surface, which serves as a functional group to attach a binding agent such as antibodies. In addition, the micelles were cross-linked to enhance their stability and dispersion in solution. For the purpose, 5 mM EDC (1- ethyl-3-(3-dimethylaminopropyl) carbodiimide; Sigma), 2 mM sulfo-NHS (N- hydroxysulfosuccinimide; Sigma) and 5 mM 2,2'-(ethylenedioxy)bis(ethylamine) (Aldrich) were used as cross-linkers. As shown in Fig. 14, the synthesized micelles have a total size of about 120 nm, and contain 15 numbers of zinc-containing nanopartides. As described above, it is expected that the synthesized nanopartide clusters exhibit enhanced saturation magnetization, leading to significantly improve separation efficiencies of magnetic sensor systems.
EXAMPLE 7: Comparison of Magnetic Relaxation Sensor Potential Using Zinc-Containing Nanopartides or Nanopartides Containing no Zinc
It was demonstrated in the present invention that zinc-containing nanopartides may much more remarkably improve potential of magnetic relaxation sensor potential than nanoparticles containing no zinc: The magnetic relaxation sensor system used in the present invention has a working principle shown in Fig. 15.
The magnetic nanoparticles have high spin-spin relaxation time (T2) at a distance enough to be dispersed, whereas the aggregation of magnetic nanoparticles through samples of interest allows magnetic particles having very low T2 relaxation time. The presence and quantification of samples of interest may be determined by measurement of T2 relaxation time using MRI or magnetic relaxation system. Each zinc-containing nanoparticles and nanoparticles containing no zinc was synthesized according to the methods described in Example 1 and Korean Pat. Nos. 10-0604975, 10-0652251 and 10-0713745, PCT/KR2004/002509, Korean Pat. No. 10-0604975,
PCT/KR2004/003088, PCT/KR2007/001001, and Korean Pat. Appln. No. 2006-
0018921. In addition, each nanoparticles were solubilized in water by surface modifications with dimercaptosuccinic acid according to the method described in
Example 1.
DNA detection
To bind DNA of interest to zinc-containing nanoparticles and nanoparticles containing no zinc surface-modified with dimercaptosuccinic acid, DNAl or DNA2 having a nucleotide sequence complementary to DNA of interest is bound to the surface of nanoparticles to produce nanopartide 1 or nanopartide 2, respectively. The nucleotide sequence of DNA to be analyzed, and DNAl and DNA2 complementary to DNA are as follows:
DNA of interest; 5'-TAC GAG TTG AGA ATC CTG AAT GCG-3' DNAl: HS-(CH2)6-5'-CGC ATT CAG GAT-3' DNA2: HS-(CH2)6-3'-ATG CTC AAC TCT-5'
The linkage of nanopartide with DNAl and DNA2 was carried out using sulfo- SMCC (Sulfosuccinimidyl-4-(N-maleimidomethyl)cydohexane-l-carboxylate, Pierce) (G. Hermanson. Bioconjugate Technique, Academic Press, 1996). Prior to reaction with nanopartides, DNAl and DNA2 were reacted with DTT (dithiotreitol; Aldrich) to cut off disulfide bonds forming dimer between DNAs. Isolation and purification of nanopartides bound to DNA were performed using Biospin 6 column (Biorad). The resulting nanopartide 1 and nanopartide 2 containing zinc or no zinc was mixed at an equal amount. For comparative analysis of magnetic relaxation sensor sensitivity, DNA of interest was added at a concentration of 0, 10, 100, 1000 and 2000 fmol and incubated for 30 min. T2 time changes (ΔT2) were measured using MRI (See, Rg. 16). 3T system (Acheiva; Philips Medical Systems. Best, the Netherlands) equipped with sense-flex-M coil was used for MRI analysis. The magnetic resonance imaging results were obtained by T2 FSE (fast spin echo sequence). The practical parameters are as follows: resolution 256 x 256 μm, slice thickness = 1 mm, TE (echo time) = 100 ms, TR (repetition time) = 4000 ms, FOV= 10 x 10 cm2, number of excitation = 2.
Fig. 16A represents a colorimetric plot merging ΔT2 with image measured by MRI. As T2 time changes depending on addition of DNA to be analyzed, red and blue color indicate low and high ΔT2, respectively. It was shown that the color representing ΔT2 is changed to yellow color in zinc-containing nanopartides (Zn0-4Fe2^O4) by addition of 10 fmol DNA. In addition, ΔT2 was gradually enhanced depending on increase of DNA amounts (from 100 to 2,000 fmol), leading to color changes from yellow color to green or blue color. On the contrary, the color was unchanged in nanopartides containing no zinc (Fe3O4) by addition of DNA (10-100 fmol), and slightly changed to yellow color by treatment of 1000 fmol DNA. Fig. 16B is a graph expressing Fig. 16A numerically, suggesting that the use of zinc- containing nanopartides in a magnetic relaxation sensor contributes to striking enhancement of sensor sensitivity. In detail, ΔT2 of about 10 is measured in zinc- containing nanopartides by addition of 10 fmol DNA, whereas in nanopartides containing no zinc by treatment of 1000 fmol DNA. Consequently, it could be appreciated that the sensor sensitivity of zinc-containing nanopartides is enhanced 100-fold higher than that of nanopartides containing no zinc.
Protein detection
Using zinc-containing nanopartides and nanopartides containing no zinc surface-modified with dimercaptosuccinic acid, avidin as a target protein was detected. Biotin having high affinity to avidin was linked to the surface of nanopartides. One end of the biotin is conjugated with a maleimide group capable of binding to a thiol group on the surface of nanopartides through covalent bond (G. Hermanson. Bioconjugate Technique, Academic Press, 1996). Isolation and purification of nanopartide-biotin complexes were performed using MACS® column (Miltenyi Biotech). The resulting nanopartide 1 and nanopartide 2 containing zinc or no zinc was mixed with avidin (Sigma) at various concentrations (0, 10, 100, 1000, 2000 fmol). After incubation for 30 min, ΔT2 of each samples was measured using MRI (See, Fig. 17). Fig. 17A represents a colorimetric plot merging ΔT2 with image measured by
MRI. As T2 time changes depending on addition of avidin to be analyzed, red and blue color indicate low and high ΔT2, respectively. It was shown that the color representing ΔT2 is changed to yellow color in zinc-containing nanopartides (Zno.4Fe2.6O4) by addition of 10 fmol avidin. In addition, ΔT2 was gradually enhanced depending on increase of avidin amounts (from 100 to 2,000 fmol), leading to color changes from yellow color to green or blue color. On the contrary, T2 relaxation time was unchanged in nanopartides containing no zinc (Fe3O4) by addition of avidin (10- 100 fmol), and slightly changed to yellow color by treatment of 1000 fmol DNA. Fig. 17B is a graph expressing Fig. 17A numerically, suggesting that the use of zinc- containing nanopartides in a magnetic relaxation sensor contributes to striking enhancement of sensor sensitivity. In detail, ΔT2 of about 10 is measured in zinc- containing nanopartides by addition of 10 fmol avidin, whereas in nanopartides containing no zinc by treatment of 1000 fmol avidin. Accordingly, it could be appreciated that the sensor sensitivity of zinc-containing nanopartides is enhanced 100-fold higher than that of nanopartides containing no zinc.
EXAMPLE 8: Magnetophoresis Sensor Using Zinc-Containing Magnetic Nanopartides; Comparison of Magnetic Potential Strength between Zinc- Containing Magnetic Nanopartides and Magnetic Nanopartides Containing No Zinc by Measuring Migration Rate of Cells Bound with Magnetic Nanopartides in the Presence of External Magnetic Field
As U87MG cells (ATCC) bound with magnetic nanopartides were moved toward a magnet at a position adjacent to a magnetic tip, cell migration in the presence of external magnetic field was observed under a microscope. Magnetic tip was composed of irons magnetized by a NdFeB permanent magnet. As shown in Fig. 18, migration rate of cells bound with zinc-containing magnetic nanopartide was higher than that of cells bound with magnetic nanopartides containing no zinc due to high magnetic potential.
Zinc-containing nanopartides and nanopartides containing no zinc used in the sensor of the present invention are Zn0^Fe26O4 and Fe3O4, respectively. To bind nanopartides to U87MG cell line, the surface of each zinc-containing nanopartides and nanopartides containing no zinc was coated with dimercaptosuccinic acid (DMSA), and then U87MG cells were reacted with Cetuximab (Merck) antibodies treated with sulfo-SMCC (Sulfosuccinimidyl-4-(N-maleimidomethyl)cydohexane-l- carboxylate; Pierce), inducing binding of nanopatides on U87MG cells. The antibody proteins enable to bind to a EGFR receptor on the surface of U87MG cells. Cell migration toward a magnetic tip was recorded as a video using a computer linked with a microscope. Further, a positioning coordinate of a specific cell was observed in a frame with regular interval to measure migration distance depending on the time.
Fig. 19A is a graph representing mean of cell migration rate obtained from 20 specific cells using zinc-containing magnetic nanoparticles and magnetic nanoparticles containing no zinc. The cell migration rate of cells bound with zinc- containing magnetic nanoparticles was attracted toward a magnetic tip about 4.14- fold more rapid than that that of cells bound with magnetic nanoparticles containing no zinc. Fig. 19B is a graph calculating the strength by stokes' law to determine how powerful cells bound with magnetic nanoparticles are practically affected by a magnetic tip. The magnetic attraction of cells bound with zinc-containing magnetic nanoparticles was about 4.12-fold stronger than that that of cells bound with magnetic nanoparticles containing no zinc. Fig. 20 represents practical images observed under a microscope and each three image was obtained at an interval of 3 sec. It could be appreciated that the cells bound with zinc-containing magnetic nanoparticles (ZnMEIO) are migrated toward a magnetic tip during the same period longer than those bound with magnetic nanoparticles containing no zinc.
Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents.

Claims

What is claimed is:
1. A zinc-containing magnetic nanopartide-based magnetic separation system or magnetic sensor, comprising a zinc-containing magnetic nanopartide represented by the following formula 1 or 2 or a cluster thereof: ZnfMa-fOb (0<f<8, 0<a<16, 0<b<8, 0<f/(a-f)<10, M represents a magnetic metal atom or an alloy thereof) (1)
ZngMc-gM'dOe (0<g<8, 0<c<16, 0<d<16, 0<e<8, 0<g/{(c-g)+d}<10, M represents a magnetic metal atom or an alloy thereof; M' represents one or more elements selected from the group consisting of Group 1 elements, Group 2 elements, Group 12 elements, Group 13 elements, Group 14 elements, Group 15 elements, transition metal elements, Lanthanide elements and Actinide elements) (2)
2. The magnetic separation system or magnetic sensor according to claim 1, wherein the zinc-containing magnetic nanoparticle-based magnetic separation system further comprises a magnetic field-generating means.
3. The magnetic separation system or sensor according to claim 1, wherein the zinc-containing magnetic nanoparticle-based magnetic sensor further comprises a magnetic sensor device and a magnetic field-generating means.
4. The magnetic separation system or magnetic sensor according to claim 1, wherein the M' of the formula 2 represents one or more elements selected from the group consisting of Li, Na, K, Rb, Cs, Be, Mg, Ca, Sr, Ba, Ra, Ge, Ga, Bi, In, Si, Ge, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Y, Zr, Nb, Mo, Tc, Ru, Rh, Pd, Ag, Cd, Hf, Ta, W, Re, Os, Ir, Pt, Au, Hg, Lanthanide elements and Actinide elements.
5. The magnetic separation system or sensor according to claim 1, wherein the zinc-containing magnetic nanoparticle is represented by the following formula 3:
ZnkM"h-κFei0j (0<k<8, 0<h<16, 0<i<8, 0<j<8, 0<k/{(h-k)+i}<10, M" represents a magnetic metal atom or an alloy thereof) (3)
6. The magnetic separation system or magnetic sensor according to claim 5, wherein the zinc-containing magnetic nanoparticle is represented by the following formula 4 or 5:
ZnqFeι-qOm (0<q<8, 0<l<8, 0<m<8, 0<q/(l-q)<10) (4) Zn1-Mn1VrFe0Op (0<r<8, 0<n<8, 0<o<8, 0<p<8, 0<r/{(n-r)+o}<10) (5)
7. The magnetic separation system or magnetic sensor according to claim 1, wherein a stoichiometric ratio of zinc and other metal in the zinc-containing magnetic nanoparticles is in a range of 0.001 < zinc/(entire metal material - zinc) < 10.
8. The magnetic separation system or magnetic sensor according to claim 1, wherein the zinc-containing magnetic nanoparticle cluster is a single nanoparticle complex aggregated with zinc-containing magnetic nanoparticles in a number range of 2-1,000.
9. The magnetic separation system or magnetic sensor according to claim 1, wherein the zinc-containing magnetic nanoparticles in the cluster are linked to each other by an intermolecular interaction or encapsulated by an organic or inorganic carrier.
10. The magnetic separation system or magnetic sensor according to claim 1, wherein a binding agent having a binding affinity to a target material or analyte is bound on the surface of the zinc-containing magnetic nanoparticle.
11. The magnetic separation system or magnetic sensor according to claim 10, wherein the surface of the zinc-containing magnetic nanoparticle or cluster thereof is coated with a water-soluble multi-functional organic ligand for nanoparticle solubilization.
12. The magnetic separation system or magnetic sensor according to claim 11, wherein the water-soluble multi-functional organic ligand comprises an attachment region (L1) to be linked to the surface of an inorganic nanoparticle.
13. The magnetic separation system or magnetic sensor according to claim 12, wherein the attachment region is bound to the surface of the nanoparticle through one or more bonds selected from the group consisting of an ionic bond, a covalent bond, a hydrogen bond and a coordination bond.
14. The magnetic separation system or magnetic sensor according to claim 11, wherein the water-soluble multi-functional organic ligand comprises an binding region (LM ) for bonding of a binding agent having a binding affinity to a target material or analyte, or a cross-linking region (Lm ) for cross-linking between water- soluble multi-functional organic ligands, or both the binding region (Ln) and the cross-linking region (Lm ).
15. The magnetic separation system or magnetic sensor according to claim 12, wherein the attachment region (LO comprises a functional group selected from the group consisting of -CHO, -COOH, -NH2, -SH, -CONH2, -PO3H, -OPO4H, -SO3H, - OSO3H, -N3, -NR3OH (R = CnH2n+1, 0<n<16), -OH, -SS-, -NO2, -COX (X = F, Cl, Br or I), -COOCO-, -CONH-, -CN and hydrocarbon having two or more carbon atoms.
16. The magnetic separation system or magnetic sensor according to claim 14, wherein the binding region (LM ) comprises a functional group selected from the group consisting of -CHO, -SH, -COOH, -NH2, -OH, -PO3H, -PO4H2, -SO3H, -OSO3H, - NR3 +X" (R = CnHm, 0<n<16, 0<m<34, X = OH, Cl or Br), NR4 +X" (R = CnHm, 0<n<16, 0<m<34, X = OH, Cl, Br), -N3, -SCOCH3, -SCN, -NCS, -NCO, -CN, -F, -Cl, -I, -Br, an epoxy group, -ONO2, -PO(OH)2, -C=NNH2, -HC=CH- and -C≡C-.
17. The magnetic separation system or magnetic sensor according to claim 14, wherein the cross-linking region (Lm ) comprises a functional group selected from the group consisting of -SH, -CHO, -COOH, -NH2, -OH, -PO3H, -PO4H2, -SO3H, -OSO3H, - NR3 +X" (R = CnHm, 0<n<16, 0<m<34, X = OH, Cl or Br), NR4 +X" (R = CnHm, 0<n<16, 0<m<34, X = OH, Cl, Br), -N3, -SCOCH3, -SCN, -NCS, -NCO, -CN, -F, -Cl, -I, -Br, an epoxy group, -ONO2, -PO(OH)2, -C=NNH2, -HC=CH- and -C≡C-.
18. The magnetic separation system or magnetic sensor according to claim 11, wherein the water-soluble multi-functional organic ligand comprises a chemical monomer, a polymer, an amphiphilic ligand, a carbohydrate, a peptide, a protein, a nucleic acid or a lipid.
19. The magnetic separation system or magnetic sensor according to claim 11, wherein the zinc-containing magnetic nanoparticles or cluster thereof is coated with the water-soluble multi-functional organic ligand and linked to the binding agent having the binding affinity for the target material or analyte through the binding region (LM ).
20. The magnetic separation system or magnetic sensor according to claim 1, wherein the zinc-containing magnetic nanoparticle-based magnetic sensor comprises a magneto-resistance sensor, a magnetic relaxation sensor, a magnetic micro-cantilever sensor, a magnetophoresis sensor or a magneto-electronic sensor.
21. A method for separating a target material in a sample, comprising the steps of: (a) forming a zinc-containing magnetic nanoparticle-target material complex by contacting the target material in the sample with the magnetic nanoparticle or cluster thereof of the magnetic separation system according to any one of claims 1-19, wherein a binding agent having a binding affinity to the target material is bound on the surface of the magnetic nanoparticle or cluster thereof; and
(b) separating the zinc-containing magnetic nanoparticle-target material complex from other components in the sample by magnetic field induction.
21. A method for detecting an analyte in a sample, comprising the steps of: (a) contacting the sample with the magnetic nanoparticle or duster thereof of the magnetic sensor according to any one of claims 1-20, wherein a binding agent having a binding affinity to the analyte is bound on the surface of the magnetic nanoparticle or cluster thereof; and (b) measuring a magnetic signal from a reaction product of the step (a) to analyze the analyte in the sample.
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