WO2019117518A1 - Nanoparticule magnétique et procédé d'amplification de signal dans une analyse par débit latéral à l'aide de celle-ci - Google Patents

Nanoparticule magnétique et procédé d'amplification de signal dans une analyse par débit latéral à l'aide de celle-ci Download PDF

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WO2019117518A1
WO2019117518A1 PCT/KR2018/015083 KR2018015083W WO2019117518A1 WO 2019117518 A1 WO2019117518 A1 WO 2019117518A1 KR 2018015083 W KR2018015083 W KR 2018015083W WO 2019117518 A1 WO2019117518 A1 WO 2019117518A1
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platinum
analyte
magnetic
antibody
nanoparticles
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Korean (ko)
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이진우
도준상
권순호
김민수
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주식회사 포스코
포항공과대학교 산학협력단
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Publication of WO2019117518A1 publication Critical patent/WO2019117518A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G01N33/5434—Magnetic particles using magnetic particle immunoreagent carriers which constitute new materials per se
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/553—Metal or metal coated
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01F—MAGNETS; INDUCTANCES; TRANSFORMERS; SELECTION OF MATERIALS FOR THEIR MAGNETIC PROPERTIES
    • H01F1/00—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01F—MAGNETS; INDUCTANCES; TRANSFORMERS; SELECTION OF MATERIALS FOR THEIR MAGNETIC PROPERTIES
    • H01F1/00—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties
    • H01F1/0036—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties showing low dimensional magnetism, i.e. spin rearrangements due to a restriction of dimensions, e.g. showing giant magnetoresistivity
    • H01F1/0045—Zero dimensional, e.g. nanoparticles, soft nanoparticles for medical/biological use

Definitions

  • the present invention relates to a magnetic nanoparticle for detecting an analyte with high sensitivity and a signal amplification method in an analysis of a lateral flow method using the same. More particularly, the present invention relates to a technique for effectively amplifying a signal in a sandwich assay by an antigen amplification process using magnetic nanoparticles and magnetism excellent in catalytic activity.
  • Immunochromatography is a method that can qualitatively and quantitatively inspect analytes in a short period of time using the property that biological substances or chemical substances adhere to each other in a specific manner.
  • sandwich immunoassay is an object It is well known that a first antibody capable of binding specifically to a first epitope of an analyte is immobilized on a solid support and a second antibody specific for a second epitope of the analyte is used.
  • an analytical strip or an analytical apparatus in which the analytical strip is assembled inside the housing is generally used.
  • the analyte is bound to the antibody including the label and the immobilized antibody while the fluid flows by the capillary phenomenon.
  • a lateral flow device generally has an antibody immobilized on a membrane, which is a porous membrane through which a liquid sample can flow as a capillary phenomenon.
  • a sample pad and a bonding pad are provided on the upstream side of the membrane. Pads are connected.
  • the sample pad absorbs the liquid sample containing the analyte and ensures uniform flow, and the binding pad is dried with a label attached with an antibody capable of selectively binding to the analyte.
  • the immobilized antibody selectively binding to the analyte and the substance capable of binding to the antibody immobilized on the label are immobilized on the membrane at different positions to form a detection unit and a control unit, respectively.
  • An antibody immobilized on a membrane capable of selectively binding with the analyte and an antibody immobilized on the label are configured to be sandwich-bound to the analyte.
  • the absorbent pad is made of a material capable of absorbing a liquid sample.
  • FIG. 1 schematically shows the structure of a conventional LFIA chip.
  • the immunoassay method is the most widely used bio-diagnosis system in the point-of-care market due to the simplicity of the diagnostic method, the diversity of the detection substance, and the economical merit. It mainly detects the analyte based on the immune detection method Since it can detect the presence of an antigen in a short time, it is developed and used as a home medical diagnostic kit as well as a hospital or an institution.
  • one aspect of the present invention is to provide magnetic nanoparticles having excellent catalytic activity.
  • another aspect of the present invention is to provide a method for effectively amplifying a signal in a sandwich analysis method by an antigen amplification process using magnetic nanoparticles and magnetism.
  • a magnetic nanoparticle comprising: a core comprising magnetic nanoparticles; And a magnetic nanoparticle-platinum core shell composite disposed on the surface of the core and consisting of a shell comprising platinum (Pt) nanoparticles, wherein platinum is included in an amount of 7 wt% to 30 wt% do.
  • a magnetic nanoparticle comprising: (a) preparing magnetic nanoparticles; (b) dispersing the magnetic nanoparticles in a dispersion solvent to prepare a solution in which the magnetic nanoparticles are dispersed; And (c) adding a reducing agent that simultaneously performs the function of a stabilizing agent to the solution prepared in step (b), wherein the molar ratio of Pt to the magnetic nanoparticles is 1 to 10 To be less than moles To produce a magnetic nanoparticle-platinum core shell composite. ≪ Desc / Clms Page number 5 >
  • a magnetic nanoparticle-platinum core shell composite comprising a first antibody or a first antibody capable of specifically binding to a first epitope or first binding site of an analyte, 1 specific binding substance; Combining the analyte with the magnetic nanoparticle-platinum core shell complex to which the first antibody or the first specific binding substance is immobilized; Concentrating the magnetic nanoparticle-platinum core shell complex to which the analyte is bound by magnetic separation; And a step of applying a magnetic nanoparticle-platinum core shell complex with a concentrated analyte to a lateral flow immunochromatography. The method of signal amplification in a lateral flow analysis is provided.
  • an antibody is immobilized on a magnetic nanoparticle-platinum core shell complex, the antigen is concentrated using the magnetism of nanoparticles, and signal amplification is carried out with platinum having enzyme-mimicking activity.
  • the gold nanoparticle-based immunoassay (LFIA, an immunodiagnostic system with a higher sensitivity than the lateral flow immunoassay method can be obtained.
  • the nanoparticle-based immunoassay of the core-shell structure is a field diagnostic biosensor that is simple enough to be used in general households, and has a rapid and high sensitivity, and its application fields are heart disease diagnosis, hormone test, Various infectious diseases and so on, it is expected that it can be put to practical use through technology transfer to a specialized company of diagnostic apparatuses.
  • FIG. 1 schematically shows a structure of a conventional immunoassay (LFIA) immunochromatography apparatus.
  • LFIA immunoassay
  • LFIA Lateral flow immunoassay
  • FIG. 3 schematically illustrates an enzyme mimetic nanoparticle-based LFIA method according to the present invention.
  • FIG. 4 shows TEM images of (a) Fe 3 O 4 NPs, (b) MPt / CS-7 NPs, (c) MPt / CS-15 NPs and (d) MPt / CS-30 NPs.
  • FIG. 5 shows the XRD pattern of (a) Fe3O4 NPs, (b) MPt / CS-7 NPs, (c) MPt / CS-15 NPs and (d) MPt / CS-30 NPs.
  • FIG. 6 shows the amount of hCG antibody fixation of Fe 3 O 4 NPs, MPt / CS-7 NPs, MPt / CS-15 NPs and MPt / CS-30 NPs.
  • Figure 7 shows a magnetic separation optical optical image of MPt / CS-7 NPs over time.
  • Figure 8 shows the relative absorbance in the supernatant of magnetic separated Fe 3 O 4 NPs, MPt / CS-7 NPs, MPt / CS-15 NPs and MPt / absorbance.
  • 10 is a graph showing the results of the detection limit test by the imaging program analysis.
  • the signal amplification method of the present invention relates to a technique for effectively amplifying a signal by an antigen amplification process using magnetic nanoparticles and magnetism excellent in catalytic activity in a lateral flow.
  • the magnetic nanoparticle-platinum core shell composite of the present invention comprises a core comprising magnetic nanoparticles; And a shell located on the surface of the core and containing platinum (Pt) nanoparticles, wherein platinum is contained in an amount of 7 wt% to 30 wt%.
  • the magnetic nanoparticle-platinum core shell composite of the present invention comprises a core comprising magnetic nanoparticles and a shell located on the surface of the core and comprising platinum (Pt) nanoparticles .
  • the magnetic nanoparticles are selected from the group consisting of iron oxide selected from Fe 2 O 3 and Fe 3 O 4 ; And ferrite selected from CoFe 2 O 4 and MnFe 2 O 4 .
  • the magnetic nanoparticles may be any one of iron oxide and ferrite, wherein the ferrite is one in which one of Fe and Fe is substituted with another metal element.
  • the ferrite may be CoFe 2 O 4 , MnFe 2 O 4 , or the like.
  • the iron oxide may be Fe 2 O 3 , Fe 3 O 4 , or the like.
  • the magnetic nanoparticles of the present invention may have an average diameter of 0.1 to 5 nm, preferably 0.5 to 4 nm, and more preferably 0.5 to 3 nm.
  • the diameter of the core may be 8 to 12 nm, preferably 9 to 11 nm, and more preferably 9.5 to 10.5 nm.
  • the magnetic nanoparticle-platinum core shell composite may further include a functional group on its surface, and the functional group may be derived from a carboxylic acid of sodium citrate, which is a surface stabilizer.
  • the functional groups can provide covalent binding or ionic electrical attraction as a means of immobilizing the detection antibody.
  • the functional group may be a carboxyl group.
  • the method for producing the magnetic nanoparticle-platinum core shell composite of the present invention comprises the steps of: (a) preparing magnetic nanoparticles; (b) dispersing the magnetic nanoparticles in a dispersion solvent to prepare a solution in which the magnetic nanoparticles are dispersed; And (c) adding a reducing agent that simultaneously performs the function of a stabilizing agent to the solution prepared in step (b), wherein the molar ratio of Pt to the magnetic nanoparticles is 1 to 10 Mol, so as to prepare a magnetic nanoparticle-platinum core shell composite.
  • the magnetic nanoparticle-platinum core shell composite of the present invention firstly forms a core containing magnetic nanoparticles, coating a surface of the core with platinum (Pt) nanoparticles to form a platinum shell .
  • magnetic nanoparticles are prepared (step a).
  • step a-1 A solution containing a salt of a metal having magnetic properties is prepared (step a-1). Next, a basic solution is added to the solution to precipitate the magnetic nanoparticles under basic conditions to prepare magnetic nanoparticles (step a-2).
  • the salt of the magnetic metal may be FeCl 3 or / and FeCl 2 , and preferably FeCl 3 and FeCl 2 may be mixed in an appropriate ratio.
  • the precipitation can be carried out at 50 to 100 ⁇ ⁇ , preferably at 60 to 95 ⁇ ⁇ , more preferably at 70 to 90 ⁇ ⁇ .
  • the precipitation can be carried out for 2 to 6 hours, preferably 3 to 5 hours, more preferably 3 to 30 minutes to 4 hours and 30 minutes.
  • the time for performing the precipitation is not limited thereto, and may be varied depending on the temperature of the precipitation.
  • the basic solution may be an aqueous solution such as ammonia, sodium carbonate, potassium carbonate, sodium hydroxide, or potassium hydroxide, and preferably an aqueous ammonia solution.
  • the basic condition may be a pH of 8 to 12, preferably 9 to 11, more preferably 9.5 to 10.5.
  • the magnetic nanoparticles are dispersed in a dispersion solvent to prepare a solution in which the magnetic nanoparticles are dispersed (step b).
  • the dispersion solvent used may be possible, such as hydroxylamine hydrochloride (NH 2 OH ⁇ HCl), tetramethylammonium hydroxide (TMAOH), preferably a mixture of hydroxylamine hydrochloride and tetramethylammonium hydroxide Can be used.
  • hydroxylamine hydrochloride NH 2 OH ⁇ HCl
  • TMAOH tetramethylammonium hydroxide
  • a mixture of hydroxylamine hydrochloride and tetramethylammonium hydroxide can be used.
  • a platinum (Pt) salt and a reducing agent are added to the solution in which the magnetic nanoparticles are dispersed to prepare a magnetic nanoparticle-platinum core shell composite (step c).
  • the platinum salt may be H 2 PtCl 6 .6H 2 O, or K 2 PtCl 4 Or the like may be possible, and preferably H 2 PtCl 6 .6H 2 O.
  • the reducing agent can simultaneously perform the function of a stabilizer, and sodium citrate, ascorbic acid, and the like can be used.
  • the reducing agent is preferably sodium citrate and ascorbic acid.
  • the addition can be carried out slowly for 1 to 3 hours, preferably 1 hour 30 minutes to 2 hours 30 minutes, more preferably 1 hour 45 minutes to 2 hours 15 minutes.
  • the reaction time may be 2 hours to 6 hours, preferably 3 hours to 5 hours, more preferably 3 hours to 5 hours, more preferably 3 hours to 5 hours, so that the platinum nanoparticles can be coated on the surface of the magnetic nanoparticles after the addition.
  • the time may be from 30 minutes to 4 hours and 30 minutes.
  • the reducing agent that simultaneously performs the function of the platinum (Pt) salt and the stabilizer in the solution prepared in (b) above is such that the molar ratio of Pt and the magnetic nanoparticles exceeds 1 mole of the magnetic nano- To less than 10 moles, so that platinum can be contained in an amount of 7 to 30% by weight in a shell containing platinum (Pt) nanoparticles.
  • the platinum content is less than 7% by weight, the catalytic activity of the magnetic nanoparticle-platinum core shell composite is low, which is problematic. .
  • a method of effectively amplifying a signal in a sandwich assay by an antigen amplification process using magnetic nanoparticles and magnetism excellent in catalytic activity is provided.
  • the signal amplification method in the lateral flow analysis of the present invention is characterized in that the magnetic nanoparticle-platinum core shell complex of the present invention is specifically bound to the first epitope or first binding site of the analyte Immobilizing the first antibody or the first specific binding material capable of binding to the first antibody; Combining the analyte with the magnetic nanoparticle-platinum core shell complex to which the first antibody or the first specific binding substance is immobilized; Concentrating the magnetic nanoparticle-platinum core shell complex to which the analyte is bound by magnetic separation; And a second antibody or a second specific binding substance that specifically binds the second epitope or second binding site of the analyte to the magnetic nanoparticle-platinum core shell complex to which the concentrated analyte is bound, to a lateral flow immunochromatography comprising a detection site; And attaching the chromogenic substrate to the detection unit after the magnetic nanoparticle-platinum core shell complex to which the concentrated analyte is bound is
  • Lateral flow chromatography uses a magnetic nanoparticle-platinum core shell complex with a first antibody or a first specific binding material immobilized thereon, wherein the labeled complex comprises enzyme mimetic inorganic nanoparticles ,
  • the protein enzyme inorganic nanoparticle is immobilized in the reaction unit after binding with the target substance, and then reacts with the chromogenic substrate to catalyze the oxidation reaction, thereby amplifying the detection signal.
  • the enzyme-mimicking inorganic nanoparticles bind to the target substance contained in the liquid sample and move to the reaction part together with the liquid sample, so that the second epitope or the second binding site of the enzyme is specific
  • the second antibody or the second specific binding substance that binds to the target is immobilized on the detection site by binding to the immobilized detection site with the analyte and thus the detection signal is displayed.
  • the nanoparticles oxidize the chromogenic substrate to amplify the detection signal.
  • Enzyme-mimicking Inorganic nanoparticles have an overall reaction surface that is unlike protein enzymes, where enzymatic-substrate reactions, which are used in signal amplification in conventional immunoassays, can mostly react with substrates, The coloring property is remarkably improved.
  • protein enzymes are highly affected by environmental characteristics (temperature, pH, etc.) due to their protein characteristics, but inorganic nanoparticles can be maintained in a relatively stable state for a long period of time.
  • the magnetic nanoparticle-platinum core-shell composite having the analyte bound thereto is subjected to magnetic separation and concentration, and then the magnetic nanoparticle-platinum core-shell complex, to which the concentrated analyte is bound, Lateral flow) immunochromatography.
  • the signal amplification method in the lateral flow analysis of the present invention includes a step of magnetically separating and concentrating the magnetic nanoparticle-platinum core shell complex to which the analyte is bound, thereby significantly improving the color development .
  • the step of applying to the lateral flow immunochromatography can be carried out by applying the magnetic nanoparticle-platinum core shell complex with the concentrated analyte combined with the liquid sample.
  • the liquid sample that can be used here is not particularly limited, For example, sodium acetate buffer, phosphate buffer and the like can be used, but any component which does not affect the results of the assay, color reaction and overall immunoassay can be used.
  • the 'enzyme-mimicking inorganic nanoparticle' refers to a substance that catalyzes chemical reactions of various substrates similar to protein enzymes.
  • the term " chromogenic substrate " means a substance in which color change of a substance before reaction and a substance after reaction are caused by the catalytic action of the enzyme-mimicking nanoparticle.
  • &quot color development change " or " amplification " is understood to mean at least one of color development, color development wavelength change, and color development intensity change.
  • 'analyte (target substance)' may be any one selected from the group consisting of an antigen protein, a ligand, DNA, an environmental hormone, an environmental pollutant, and a virus. However, Is not limited, and preferably the analyte is selected from the group consisting of DNA, environmental hormones, antigenic proteins.
  • the binding site is at least one selected from the group consisting of a protein ligand, a DNA sequence and an RNA sequence.
  • the specific binding substance is a protein capable of specifically binding to the binding site, a viral phage, Nucleic acid molecule aptamer, and hapten (DNP).
  • first antibody or first specific binding substance means an antibody capable of binding to a target substance, a Fab or a recombinant substance of an antibody, which is a fragment of an antibody. Binding can be both chemical and physical.
  • second antibody or second specific binding substance means a target substance, or a substance capable of binding with a reactive substance bound to the target substance. Bonding is possible both for chemical bonding and physical bonding, but for physical bonding, bonding can be made without special chemical reaction.
  • the reactive substance may be an antibody, a Fab fragment of an antibody, a recombinant scFv, or the like, and may be a fragment of a receptor or a receptor.
  • &quot enzyme-substrate reaction " is understood to include not only the reaction of a substrate catalyzed by an enzyme, but also the reaction of a chromogenic substrate catalyzed by enzyme-mimicking nanoparticles.
  • the chromogenic substrate is color-coded by precipitating and insolubilizing the enzyme-mimicking inorganic nano-particles at the position where the inorganic nanoparticles exist after the oxidation, serving as an oxidation catalyst, and amplifying the detection signal to such a degree that the detection signal can be visually recognized .
  • the coloring substrate comprises a coloring substrate selected from the group consisting of AEC (3-amino-9-ethylcarbazole), DAB (3,3'-Diaminobenzidine) and TMB (3,3 ' Tetramethylbenzidine).
  • AEC 3-amino-9-ethylcarbazole
  • functional groups capable of fixing the detection antibody may be present on the surface of the magnetic nanoparticle-platinum core shell complex.
  • the functional group present on the surface of the magnetic nanoparticle-platinum core shell complex is preferably a carboxylic acid group capable of providing a covalent bond or an ionic electrical attraction as a fixing means for the detection antibody.
  • an amine group (-NH 2 , amine group) abundantly present in an IgG antibody can be used.
  • the magnetic nanoparticle-platinum core shell composite according to the present invention is preferably monodispersed nanoparticles included in the liquid phase so as to be able to move to the junction of the lateral flow chromatography through a capillary tube having a diameter of several micrometers.
  • the term " monodisperse " means that the size and structure of the nanoparticles are uniform, indicating substantially uniformity.
  • the synthesis of the above-described monodisperse inorganic nanoparticles can be carried out using a synthetic method in which an organic solvent is improved in monodispersibility by using a surfactant.
  • the detection antibody was immobilized on the surface of inorganic nanoparticles and dispersed in physiological saline containing sucrose and bovine serum albumin.
  • the antibody was absorbed evenly into a joint made of glass fibers and dried. Since glass fibers have a low affinity for proteins, dried antibody-inorganic nanoparticles can easily escape to the test pads after being hydrated when they meet the analyte fluid in glass fiber.
  • the body fluid containing the analyte is flowed into the lateral flow immunochromatographic chip and the enzyme-substrate reaction is carried out using the precipitable insoluble substrate, whereby the detection sensitivity can be successfully improved by amplifying the color signal.
  • the lateral flow immunochromatography may comprise, as shown in Figure 2, a sample pad to which a liquid sample containing an analyte is applied; A membrane comprising a detection site and a control section immobilized with a second antibody or a second specific binding substance which specifically binds to a second epitope or a second binding site of the analyte in contact with the sample pad; And an absorption pad disposed downstream of the membrane and capable of absorbing the liquid sample by capillary phenomenon.
  • the lateral flow chromatography refers to a kit in which a liquid sample containing a target substance reacts with a fixed detection substance while moving through a porous medium and displays a color signal upon detection so as to be visually distinguishable.
  • the lateral flow immunochromic chip may include a sample pad including a sample portion into which a sample containing a target substance is injected, a detection pad having a detection substance capable of binding with the target substance to which the inorganic nanoparticles are bound, And an absorption pad including a membrane (test pad) including a control part for error checking and an absorption part capable of absorbing the liquid sample by capillary phenomenon.
  • Each of the sample portion, the detection portion, the control portion, and the absorption portion of the assay kit of the lateral flow chromatography is connected to each other through microtubules, or connected to each other by a membrane.
  • the membrane may be a porous material of natural or synthetic material, Nitrocellulose, but is not limited thereto.
  • the signal amplification method of the present invention can be applied to a lateral flow method, and the lateral flow method includes a vertical flow method and a flow though method.
  • the samples are vertically stacked on the porous membrane arranged in parallel.
  • the lateral flow system is not limited to the antibody-antigen reaction, and the 'binding site (ligand)' referred to in the present specification refers to a protein ligand, nucleic acid (DNA or RNA) Specific binding substance "includes a protein, a virus phage, a nucleic acid molecule aptamer, hapten (DNP), etc. which can selectively and specifically bind to the binding site, and the like. Biomolecules, and is not particularly limited to those described above.
  • the analyte that can be detected by the method of the present invention is not particularly limited as long as it is capable of binding to the first and second antibodies and forming a sandwich-type immunoconjugate by an immunological reaction, that is, an antigen-antibody reaction, It can be applied to environmental pollutants, viruses, etc., including proteins or DNA, environmental hormones and the like.
  • the antigen and the antibody are not particularly limited as long as they are substances capable of specifically binding to an analyte by an antigen-antibody reaction.
  • the analyte is an antibody
  • a substance specifically binding to the analyte can be used as an antigen have.
  • the antigens and the antibodies may adopt known antigens and antibodies depending on the analyte.
  • the reaction of a 'specific binding substance' selectively recognizing an analyte as a 'binding site (ligand)' is considered to be included in an antigen-antibody reaction in a broad sense.
  • Fig. 2 is a schematic diagram showing an exemplary structure of each configuration in a lateral flow analyzer according to the present invention, wherein (10) is a sample pad, (12) is a membrane, (13) 14 denotes a control unit, and 15 denotes an absorbing pad, and the bonding pad 11 can be omitted in comparison with the conventional lateral flow analyzing apparatus of FIG.
  • the sample pad is a portion where the liquid sample containing the analyte is first absorbed.
  • the sample pad may be formed by using one end of the membrane as it is or by a separate member.
  • the sample pad is absorbed by the capillary phenomenon Thereby moving the analyte to the bonding pad.
  • the membrane is not particularly limited as long as it is a material capable of securing fluidity of a liquid sample, and is preferably formed of a porous membrane. More specifically, the present invention relates to an antibody or a specific binding substance, and a method for immobilizing an enzyme protein and minimizing nonspecific reaction, such as nitrocellulose, cellulose, polyvinylidene fluoride (PVDF), poly (ethylene terephthalate) Hydrophobic porous membranes, such as fibers, nylons, and the like, which can be adjusted to a certain micropore size, can be used.
  • PVDF polyvinylidene fluoride
  • Hydrophobic porous membranes such as fibers, nylons, and the like, which can be adjusted to a certain micropore size, can be used.
  • the membrane may include a detection site on which a second antibody or a second specific binding substance that specifically binds to a second site of the analyte to which the conjugate is bound is immobilized, and a control site, And a control site for controlling the operation of the apparatus.
  • the detector is the part that shows the results for reading the test results.
  • the control unit is configured to identify errors of the gold nanoparticle conjugate and capture antibody or immobilized second specific binding material and to confirm whether or not the substances with mobility are properly reacted to the detection unit / control unit .
  • the absorbing pad is not particularly limited as long as it can sufficiently absorb the residue remaining after the reaction by the capillary phenomenon, for example, cellulose, cotton. Hydrophilic porous polymer and the like can be used.
  • the existence of inorganic Fe 3 O 4 NPs was confirmed by TEM (JEOL EM-2010 microscope) image as shown in FIG. 4, and the presence of Fe 3 O 4 and Pt was confirmed through XRD (Rigaku D / Max 2500) Respectively.
  • the peroxidase mimic activity of MPt / CS was measured by using an UV-Visible meter using 3,3 ', 5,5'-tetramethylbenzidine (TMB).
  • the activity for TMB was determined by cuvetteing 100 ⁇ L H2O2 (1 M), 100 ⁇ L TMB (31.25 ⁇ M to 1200 ⁇ M), and 30 ⁇ L of 770 ⁇ L sodium acetate buffer and measuring the absorbance difference at 652 nm for 1 min Were measured.
  • the LFI chip (BioCard, Ilyang Bio), which has been commercialized in the past, has been modified and used.
  • the bonding pad was washed and used to use a commercialized pregnancy diagnostic test.
  • the bonding pad can be omitted in the chip analysis chip to which the present invention can be applied.
  • ⁇ -hCG monoclonal antibody 5 mg / ml
  • MPt / CS NPs 7.0 mg / ml
  • 0.1 wt% of tween20 and 3 wt% of BSA diluted in PBS were added to block the portion of the antibody not immobilized with BSA. If necessary, a subsequent reaction can be carried out after attaching to the rotator at 4 ° C for one day.
  • the cells were then washed three times with 0.1 wt% tween20 and 3 wt% BSA diluted in PBS (14,000 rpm, 30 min, 4 ° C) and stored at a concentration of 7.0 mg / ml.
  • the amount of immobilized hCG antibody per particle was analyzed using a commercial kit, Bradford assay, and the result is shown in FIG.
  • FIG. 8 shows the results of confirming changes in absorbance in the supernatant with time.
  • the supernatant was then discarded and the antigen-antibody-MPt / CS NPs complex was suspended in 50 ⁇ l of PBS containing 10 wt% sucrose, 0.1 wt% tween 20, and 3 wt% BSA.
  • LFIA was performed using MPt / CS-15 NPs. As can be seen from FIG. 9, it was visually confirmed up to 0.1 ng / ml. And the detection limit of the present invention.
  • LFIA was carried out using MPt / CS-7 NPs, and as a result of the imaging program, the detection limit of 40 pg / ml was confirmed.
  • LFIA was performed using MPt / CS-30 NPs, and as a result of the analysis with the imaging program, the detection limit of 40 pg / ml was confirmed.
  • Control section 15 Absorption pad

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Abstract

La présente invention concerne une nanoparticule magnétique et un procédé d'amplification d'un signal dans une analyse par débit latéral à l'aide de celle-ci et, plus spécifiquement, un composite à noyau de nanoparticules magnétiques/enveloppe de nanoparticules de platine, un procédé de production de celui-ci, et un procédé d'amplification d'un signal dans une analyse par débit latéral à l'aide de celui-ci, le composite à noyau de nanoparticules magnétiques/enveloppe de nanoparticules de platine étant constitué d'un noyau comprenant des nanoparticules magnétiques et d'une enveloppe positionnée sur la surface du noyau et comprenant des nanoparticules de platine (Pt), le platine étant inclus dans une quantité de 7 % à 30 % en poids.
PCT/KR2018/015083 2017-12-13 2018-11-30 Nanoparticule magnétique et procédé d'amplification de signal dans une analyse par débit latéral à l'aide de celle-ci WO2019117518A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021065144A1 (fr) * 2019-09-30 2021-04-08 富士フイルム株式会社 Immunochromatographie
US20220351289A1 (en) * 2019-09-30 2022-11-03 Centiglobe Ab A real-time trust distributed multi asset converter
WO2023001839A2 (fr) 2021-07-19 2023-01-26 Tissue Click Limited Kit de détection et procédés de détection d'agents infectieux

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102513309B1 (ko) * 2021-02-17 2023-03-24 성균관대학교 산학협력단 생물학적 표적의 분리를 위한 나노 입자 복합체 및 이의 제조 방법

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005520125A (ja) * 2001-05-25 2005-07-07 ノースウエスタン ユニバーシティ 非合金のコアシェル型ナノ粒子
US20080226917A1 (en) * 2007-02-20 2008-09-18 Research Foundation Of State University Of New York Core-shell nanoparticles with multiple cores and a method for fabricating them
KR20150092443A (ko) * 2014-02-04 2015-08-13 포항공과대학교 산학협력단 효소 모방 무기 나노입자를 이용한 고감도 측면유동 면역 발색칩 및 이를 이용한 검출 방법
KR20170034504A (ko) * 2015-09-21 2017-03-29 포항공과대학교 산학협력단 자성 나노입자-백금 코어쉘 복합체 및 그의 제조방법

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005520125A (ja) * 2001-05-25 2005-07-07 ノースウエスタン ユニバーシティ 非合金のコアシェル型ナノ粒子
US20080226917A1 (en) * 2007-02-20 2008-09-18 Research Foundation Of State University Of New York Core-shell nanoparticles with multiple cores and a method for fabricating them
KR20150092443A (ko) * 2014-02-04 2015-08-13 포항공과대학교 산학협력단 효소 모방 무기 나노입자를 이용한 고감도 측면유동 면역 발색칩 및 이를 이용한 검출 방법
KR20170034504A (ko) * 2015-09-21 2017-03-29 포항공과대학교 산학협력단 자성 나노입자-백금 코어쉘 복합체 및 그의 제조방법

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
"Development of a Simple and Fast Immunoassay Biosensor Using a Pt-Fe3O4-graphene Oxide as a Peroxidase-mimetic", POSTECH ACADEMY-INDUSTRY FOUNDATION, November 2015 (2015-11-01), pages 1 - 64 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021065144A1 (fr) * 2019-09-30 2021-04-08 富士フイルム株式会社 Immunochromatographie
JPWO2021065144A1 (fr) * 2019-09-30 2021-04-08
US20220351289A1 (en) * 2019-09-30 2022-11-03 Centiglobe Ab A real-time trust distributed multi asset converter
JP7274595B2 (ja) 2019-09-30 2023-05-16 富士フイルム株式会社 イムノクロマトグラフィー
WO2023001839A2 (fr) 2021-07-19 2023-01-26 Tissue Click Limited Kit de détection et procédés de détection d'agents infectieux

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