WO2007115444A1 - Composition de séparation ou d'analyse à nanostructure active et procédé de séparation ou d'analyse - Google Patents

Composition de séparation ou d'analyse à nanostructure active et procédé de séparation ou d'analyse Download PDF

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Publication number
WO2007115444A1
WO2007115444A1 PCT/CN2006/001374 CN2006001374W WO2007115444A1 WO 2007115444 A1 WO2007115444 A1 WO 2007115444A1 CN 2006001374 W CN2006001374 W CN 2006001374W WO 2007115444 A1 WO2007115444 A1 WO 2007115444A1
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nanostructure
activated
group
functionalized
composition
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PCT/CN2006/001374
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English (en)
Chinese (zh)
Inventor
Fanglin Zou
Chunsheng Chen
Jianxia Wang
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Chengdu Kuachang Medical Industrial Limited
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Priority to PCT/CN2006/002659 priority Critical patent/WO2007085156A1/fr
Publication of WO2007115444A1 publication Critical patent/WO2007115444A1/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
    • G01N33/54366—Apparatus specially adapted for solid-phase testing

Definitions

  • the present invention relates to an isolated or analytical composition comprising activated nanostructures, and a method of separation or analysis associated with the compositions of the present invention.
  • the present invention is a continuation of International Patent Application PCT/CN2004/000437.
  • the primary object of the present invention is to increase the efficiency of the functionalized nanostructures including sensitivity or/and functional stability.
  • a composition for separation or analysis comprising an activated nanostructure comprising at least a nanostructure and an activated structure covalently bonded to the nanostructure,
  • the activating structure comprises at least an activating group useful for binding to a functional agent, the activating group comprising an amino group-containing polyfunctional group based on a polypeptide synthesis reagent, or/and a derivative group thereof.
  • a composition for separation or analysis comprising a functionalized nanostructure comprising at least an activated nanostructure and a functional reagent immobilized on the activated nanostructure,
  • the activated nanostructure comprises at least a nanostructure and an activated structure covalently bonded to the nanostructure, the activated structure comprising at least an activating group useful for binding a functional reagent, the activating group comprising An amino group-containing polyfunctional group, or/and a derivative group thereof, based on a polypeptide synthesis reagent.
  • a composition for separation or analysis comprising an activated nanostructure carrier comprising at least a conventional support and an activated nanostructure, the activated nanostructure comprising at least a nanostructure and an activated structure covalently bonded to the nanostructure; the activation structure comprising at least an activating group useful for binding a functional reagent, the activating group comprising an amino group-containing based on a polypeptide synthesis reagent A polyfunctional group, or/and a derivative group thereof.
  • a composition for separation or analysis comprising a functionalized nanostructure carrier comprising at least a conventional support and a functionalized nanostructure, said functionalized nanostructure
  • the structure comprises at least an activated nanostructure and a functional agent immobilized on the activated nanostructure, wherein the activated nanostructure comprises at least a nanostructure and an activated structure covalently bonded to the nanostructure, the activated structure comprising at least There is an activating group for binding to a functional agent, the activating group comprising an amino group-containing polyfunctional group based on a polypeptide synthesis reagent, or/and a derivative group thereof.
  • a method of separating or analyzing composition comprising activated nanostructures comprising the steps of providing and determining the separation or analysis composition of the first to fourth aspects of the invention.
  • isolated or analytical composition refers to a composition for separation or analysis, such as a device or kit, or a component thereof; "analysis” refers to qualitative in vitro or in vivo. Or / and quantitative analysis; “separation” means that one or more components are separated from other components of the sample; “device” refers to articles with specific functions, such as instruments containing functional reagents, such as: analysis chips, enzymes Markers, affinity electrophoresis strips, affinity chromatography columns, planar chromatography reagent strips, and more.
  • nanostructure refers to a structure in which at least one dimension is nanometer-sized in a three-dimensional structure, such as a particle size, a tube diameter, a wire diameter, or the like, which is a nanometer-sized structure, and its preferred size is 3 nm- 300 nm ;
  • Nanoparticles refers to particles having a particle size of nanometer size, preferably particles having a particle diameter of 3 nm to 300 nm;
  • Nano bead refers to a nanostructured composite in which more than one nanoparticle is covalently linked with an organic group;
  • Nano-convex refers to any nanostructure on the surface of a non-nanomaterial, such as: immobilized nanoparticles, immobilized nanotubes, immobilized nanofibers, and nanostructures formed by self-assembly of nanoparticles on a carrier, and the like.
  • Coupled nanostructure refers to a composition that contains nanostructures and A structurally fixed coupling group.
  • the “activated structure” as used in the present invention means a composition which contains at least an activating group and may further contain a coupling group;
  • activating group means a group for providing a binding to a functional agent, for example A group providing an amino group, a carboxyl group or the like such as an amino acid, or a complex group such as an amino acid derivative, a synthetic peptidyl group, a synthetic peptide derivative group;
  • activated nanostructure means a composition which contains a nanostructure and thereon Fixed activated structure;
  • activated nanoparticle refers to a composition that contains nanoparticles and an activated structure immobilized thereon;
  • activated nanoprotrusion refers to a composition that contains nano-convex and The activated structure immobilized thereon;
  • activated nanobead means a composition comprising nanobeads and an activated structure immobilized thereon.
  • the "functional reagent” as used in the present invention refers to a reagent which is reactive with the nanostructure, for example, an agent reactive with a target.
  • the functional agent captures the target by interaction including affinity, ion exchange, lipophilic action, etc., and it includes a ligand (equivalent to Ligand in English), an ion exchanger, and the like.
  • Ion exchangers include: diethylaminoethyl (DEAE), diethyl mono(2-hydroxypropyl)aminoethyl (QAE), carboxymethyl (CM), sulfonic acid propyl (SP), oxime ethyl Pyridyl (MEP), one NH 2 , one RCOOH, a siloxane group, a thiol group, an alkyl group.
  • Ligands include: antigens, antibodies, ligands, ligand-enhancing phylogenetic techniques, screening of adaptor molecules, polypeptides, polysaccharides, co-enzymes, cofactors, antibiotics, steroids, viruses, cells, and the like.
  • the term "functionalized nanostructure” as used in the present invention refers to a composition comprising: a nanostructure and a functional reagent immobilized thereon; "functionalized nanoparticle” means a composition which contains nanoparticles and a functional reagent immobilized thereon; “functionalized nanoprotrusion” means a composition comprising: a nanoprotrusion and a functional reagent immobilized thereon; “functionalized nanobead” means a composition comprising: Nanobeads and functional agents immobilized thereon.
  • the "activated nanostructure carrier” as used in the present invention means a composition which contains at least an activated nanostructure such as an activated nanoprotrusion and a conventional carrier;
  • “functionalized nanostructure carrier” means a composition which contains at least Functionalized nanostructures such as functionalized nanoprotrusions and conventional carriers;
  • sheet base refers to conventional carriers having a fixed function with a macroscopic plane on one side, such as analytical chip base, ELISA plate base, electrophoretic film, planar layer Analysis of carriers and the like.
  • the “analytical chip” described in the present invention is a detecting device in the designation and/or quantitative analysis, and the micro function in the reactor
  • the result of the reaction of the reagent with the target molecule in the sample can be identified in an addressable manner;
  • a “nanostructured chip” is a chip containing at least one nanostructured active region (eg, a sample in a functional reagent microarray).
  • One chip can have multiple reactors, and one reactor can have multiple samples containing active reagents (functional reagent points), only If one of the samples is the nanostructure active region of the present invention, the chip is the nanostructure active carrier or nanostructure chip of the present invention.
  • chromatography described in the present invention is equivalent to the English “Chromatogmphy”, including affinity chromatography, reversed phase chromatography, hydrophobic chromatography, ion exchange chromatography, etc., which is divided into planar chromatography (for example, a rapid detection reagent strip) And quick test kits) and column chromatography.
  • polypeptide in the present invention is equivalent to "polypeptide” in English, and includes natural or synthetic proteins, protein fragments, synthetic peptides, and the like, and the usual targets in immunoassays and ligands commonly used in detection, such as antigens and antibodies. And so on belong to the polypeptide;
  • “molecularly labeled substance” refers to a substance used to form or participate in the formation of a detection signal and has a molecular morphology at the time of labeling, such as rhodamine, CY3, CY5, etc. in commonly used labels for chip detection.
  • the analytical or separation compositions of the first to fourth aspects of the invention each comprise an activated nanostructure comprising at least an activating group useful for binding a functional agent, the activating group Amino-containing polyfunctional groups based on a polypeptide synthesis reagent, or/and derivative groups thereof are included.
  • the activating group comprises an aminoguanidine group or/and an aminoguanidine derivative group; the activating group comprises an amino acid a group or/and an amino acid derivative group; the activating group includes a synthetic peptide group or/and a synthetic peptide derivative group.
  • Amino acids commonly used as passivating agents can provide reactive groups, particularly activating groups.
  • the amino acid group comprises an arginine group, an asparaginyl group, a glutamyl group, a glycine group, a lysine group, a glutamine group; the number of amino acids in the synthetic peptide group is greater than Or equal to 2 (for example, 2-5), wherein the amino acid species are the same (for example, a single amino acid peptide group formed by arginine, asparagine, glutamine, etc.) or different (for example, arginine and asparagine) Amino acid peptidyl group formed by ammonia, asparagine and glycine, glutamylamine and lysine, etc.).
  • the activating group is typically provided by an activator.
  • the activator used including a base activator that provides at least a partial activating group that binds to the coupling group, and an activating group when the activating group is composed of not only a group provided by the base activator (eg, a derivative group) The other part of the second activator.
  • the above reagent synthesis peptide synthesis such as a peptide coupling reagent and an amino acid group-containing peptide synthesis reagent, can be used as both a base activator and a second activator.
  • the peptide synthesis reagent is preferably a polyfunctional reagent, more preferably a polyfunctional reagent containing an amino group (- ⁇ 1 or/and a carboxyl group (-COOH). Further, it is also preferred to contain a peptide synthesis protecting group (for example, Fmoc). Peptide linkers and peptide synthesis reagents containing amino acid groups, such as Fmoc-aminoguanidine and Fmoc-amino acids.
  • the activation structure further contains a coupling group linking the nanostructure and an activating group, the coupling group Includes silane groups.
  • the silane coupling agent used comprises:
  • the nanostructure in the activated nanostructure, comprises a nanostructure comprising an inorganic or/and an organic material (e.g., plastic, polysaccharide, latex, resin).
  • the inorganic material includes a non-magnetic inorganic material and a magnetic inorganic material.
  • the non-magnetic inorganic material includes metallic materials (e.g., gold, vanadium, lead) and non-metallic inorganic materials.
  • the non-metallic inorganic material includes an inorganic oxide.
  • the inorganic oxide used includes silicon oxide, aluminum oxide, and titanium oxide.
  • the nanostructures include: nanoparticles, nanobeads, nanoprotrusions.
  • the activated nanostructures include the nanostructures and the activated structures, which are: activated nanoparticles, activated nanobeads, activated nanoprotrusions, respectively.
  • the analytical or separation composition of the first aspect of the invention comprises: activating nanoparticles, activating nanobeads, and activating nano-protrusions.
  • the functionalized nanostructure comprises the nanostructure, the activated structure and a functional reagent, respectively: functionalized nanoparticles, functionalized nanobeads, functionalized Nano convex body.
  • the analytical or separation composition of the second aspect of the invention comprises: functionalized nanoparticles, functionalized nanobeads, functionalized nanoprotrusions.
  • the analysis or separation composition of the second aspect of the invention further comprises a composition comprising a combination of the above functionalized nanostructures, such as a complex of more than one of said plurality of said functionalized nanoparticles, more than one of said plurality of said functionalizations A system composed of nanoparticles and so on.
  • the functional reagent includes any substance that can be immobilized on the activating group without losing its function, for example Nucleic acids or/and polypeptides.
  • the polypeptide used includes: an antigen, an antibody, and other ligands.
  • the antigens used include: EBV-VCA-P18 antigen, hepatitis C virus antigen (HCVAg), HIV antigen (HIVAg), syphilis antigen; antibodies used include anti-hepatitis B virus surface antibody (HBs Ab), monoclonal or polyclonal secondary antibodies Other ligands used include protein incorporation.
  • the conventional carrier comprises at least one of two or more sets of materials or derivatives thereof, having a size of at least two dimensions greater than 100 nm Carriers: glass, silicon wafers, silica gel, ceramics, metal oxides, metals, polymeric materials and their composites.
  • Such conventional carriers also include conventional carrier derivatives.
  • the derivative includes a derivative that incorporates a surface group or/and an organic coating.
  • the conventional carrier comprises one of the following carriers: a granular conventional carrier (for example, a chromatographic gel, in particular, a microparticle chromatography gel), a planar conventional carrier (for example, a biochip, a microplate) Ordinary substrate) and membranous conventional carrier (eg, planar chromatography strip).
  • a granular conventional carrier for example, a chromatographic gel, in particular, a microparticle chromatography gel
  • a planar conventional carrier for example, a biochip, a microplate
  • Ordinary substrate for example, a biochip, a microplate
  • membranous conventional carrier eg, planar chromatography strip
  • the activated nanostructure carrier comprises an activated nanoprojection carrier.
  • the activated nano-convex support comprises the activated nano-protrusion and the conventional carrier, for example: activated nanostructure carrier, activated nanoparticle/sheet-based composite, activated nanoparticle/microparticle composite, activated nanoparticle/micron Particle/sheet based composites and the like.
  • the analysis or separation composition of the third aspect of the present invention comprises any one of the following groups: an analysis chip nanostructure substrate, a nanostructure enzyme label, a microplate, a planar chromatography nanostructure sheet, and a nanostructure activation. Chromatographic stationary phase.
  • the activated nanostructures are distributed on part or all of the substrate. Moreover, on at least a portion of the substrate, the nanoprotrusions have a distribution density greater than 1 nanoprotrusion / ⁇ 2 , preferably greater than 5 nanoprotrusions / ⁇ 2 .
  • some of the methods of the present invention can also be used to generate nanostructure carriers, such as nanostructure carriers for use in devices such as computers, cell phones, microchip cards, and the like.
  • the functionalized nanostructure carrier comprises a functionalized nano-convex support.
  • the functionalized nanoprotrusion carrier comprises the functionalized nanoprotrusion and the conventional carrier, for example: a functionalized nanostructure carrier, a functionalized nanoparticle/sheet based composite, a functionalized nanoparticle/microparticle composite, Functionalized nanoparticles/microparticles/sheet based composites and the like.
  • the analytical or separation composition of the fourth aspect of the invention further comprises a composition comprising a plurality of the above functionalized nanostructures and a conventional carrier, for example: one or more of a plurality of functionalized nanoparticles/microparticle composites, more than one or more A system of functionalized nanoparticles and one or more microparticles, a composite of more than one functionalized nanoparticle and a substrate, more than one multifunctional nanoparticle and affinity A system consisting of a base, a composite of more than one of a plurality of functional reagents and a nanostructured base, and the like.
  • a composition comprising a plurality of the above functionalized nanostructures and a conventional carrier, for example: one or more of a plurality of functionalized nanoparticles/microparticle composites, more than one or more A system of functionalized nanoparticles and one or more microparticles, a composite of more than one functionalized nanoparticle and a substrate, more than one multifunctional nanoparticle and affinity A system consist
  • one or more nanostructures in the functionalized nanostructure carrier may have one or more ligands, or/and one or more There is a heavy or multiple nanostructure between the base and the conventional support, or/and a heavy or multiple ligand between the at least one heavy nanostructure and the other heavy nanostructure.
  • the nanostructured active carrier having multiple ligands between the one or more nanoparticles and the carrier prepared by the method respectively, the carrier is coated with a ligand 1 to form a ligand 1 coated carrier, and the nanoparticles are coated with one weight and the other Ligand 2 (pairing reaction between ligands 1 and 2) forms a ligand 2/nanoparticle complex, and the ligand 2/nanoparticle complex is coated or spotted onto the ligand 1 coated carrier.
  • the number of base layers is greater than 2, and so on.
  • nanostructured active carriers having multiple ligands between one nanoparticle and another nanoparticle, for example, ligand 3 - nanoparticle - ligand 3 - ligand 2 - nanoparticle - ligand 2 - ligand 1 A carrier.
  • a nanostructured active carrier having multiple nanoparticles between one or more ligands and a carrier can be prepared by first combining one or more of the nanoparticles with a plurality of such ligands to form a plurality of activities.
  • Nanoparticles eg, ligand 2 - nanoparticle-ligand 2, ligand 3 - nanoparticle-ligand 2, ligand 1 - nanoparticle-ligand 1, etc.
  • a ligand such as a ligand 2 - a nanoparticle - a ligand 2 - a ligand 1 - a nanoparticle - a ligand 1 - a carrier, a ligand 3 - a nanoparticle - a ligand 2 - a ligand 1 - a nanoparticle
  • the analytical or separation composition of the second or fourth aspect of the invention comprises a separation system comprising the functionalized nanostructure or/and a functionalized nanostructure carrier.
  • the functionalized nanostructures are: functionalized nanoparticles (eg, functionalized nanoparticles in a functionalized nanomagnetic separation system), functionalized nanobeads (eg, functions in a functionalized nanomagnetic separation system) Functionalized nano-beads, functionalized nano-protrusions (eg, functionalized nano-protrusions on affinity chromatography nano-stationary phases in nanoaffinity chromatography systems).
  • the analytical or isolated composition of the second or fourth aspect of the invention comprises a labeling system comprising said functionalized nanostructures or/and functionalized nanostructure carriers.
  • the functionalized nanostructures are: functionalized nanoparticles (eg, functionalized nanoparticles in nanomarkers), functionalized nanobeads (eg, functionalized nanobeads in nanomarkers).
  • the analytical or separation composition of the second or fourth aspect of the invention comprises a reaction system comprising a functionalized nanostructure or/and a functionalized nanostructure carrier.
  • the functionalized nanostructure is a functionalized nanoprotrusion.
  • the analytical or separation composition of the fourth aspect of the invention comprises a device comprising the reaction system, such as a sensor, an analytical chip, an ELISA plate, a rapid test strip, and the like.
  • the device comprises: a nanostructure analysis chip, a nanostructure ELISA plate, and a nanostructure planar chromatography reagent strip.
  • the activated nanostructures are only distributed on some or all of the functional reagent points.
  • the nano-protrusion has a distribution density greater than 1 Nanoprotrusions / ⁇ 2 , preferably greater than 5 nanoprotrusions / ⁇ 2 .
  • the analytical or isolated composition of the second or fourth aspect of the invention comprises a kit comprising one, two or three of the following: said nanoreactor system, said nanolabeling system, said nanoseparation system.
  • the kit comprises one of the following groups: a nanostructure analysis chip kit, a nanostructure ELISA kit, and a nanostructure planar chromatography reagent strip kit.
  • the assay or separation composition of the first to fourth aspects of the invention wherein the target of the separation or/and analysis comprises a polypeptide or/and a drug that interacts with the polypeptide, or/and a nucleic acid or/and a drug that interacts with the nucleic acid .
  • the analysis or separation method according to the fifth aspect of the present invention comprises the steps of providing and applying the analysis or separation composition of the first to fourth aspects of the invention.
  • the providing or separating the composition of the composition comprises providing the nanostructure, and covalently immobilizing the activated structure to the nanostructure to form the activated nanoparticle structure.
  • one of the methods of forming or/and introducing an activating group in the activated structure is a method of synthesizing a peptide.
  • the synthetic peptide method comprises one or more of the following steps: providing a protecting group-containing reactant and at least partially removing the protecting group in a subsequent step; Reaction between 2 -base and -COOH groups; peptide chain growth.
  • Nanostructures The nanostructures used may be nanostructures of any morphology, such as: nanoparticles, nanobeads, nanotubes, nanorods, nanoprotrusions on a carrier, and the like.
  • the nanostructures preferably used in the embodiments of the present invention are nanostructures containing inorganic matrix (for example, inorganic matrix nanostructures, inorganic-containing nanostructures, and inorganic-coated nanostructures). It includes: oxide nanoparticles, nanobeads (home made, see related examples below), nanoprotrusions (home made, see related examples below).
  • the oxide nanoparticles used include: silicon oxide nanoparticles (silica nanoparticles LUDOX AS-40, average particle size 25 nm, specific surface area about 135 m 2 /g, Sigma-Aldridi), aluminum oxide nanoparticles (MC2R ⁇ - Phase nano-alumina, average particle size 60nm, specific surface area 140 m 2 /g, Zhejiang Hongsheng Materials Technology Co., Ltd. Ltd.), Titanium oxide nanoparticles (titanium oxide nanoparticles, average particle size ⁇ 80nm, specific surface area 120 m 2 /g, Zhejiang Zhoushan Mingri Nano Material Co., Ltd.).
  • Nanostructures of other inorganic materials can also be used in the methods of the following examples to prepare activated nanostructures and functionalized nanostructures.
  • the organic material nanostructures can also be used to prepare activated nanostructures and functionalized nanostructures either directly or indirectly (e.g., coated with inorganic materials) for use in the methods of the following examples.
  • Activator an activator used, including a base activator that provides at least a portion of the activating group that binds to the coupling group, and when the activating group is composed of not only the group provided by the base activator (eg, a derivative) A second activator that provides an additional portion of the activating group.
  • the activator used is a reagent for peptide synthesis, such as a peptide coupling reagent and a peptide synthesis reagent containing an amino acid group.
  • Embodiments of the invention preferably employ a polyfunctional reagent containing an amino group (-Dish 2 ) or/and a carboxyl group (-COOH).
  • the polyfunctional peptide synthesis reagents used include aminoguanidine and amino acids.
  • the present invention preferably employs a peptide linker comprising a peptide synthesis protecting group (e.g., Fmoc) and an amino acid group-containing peptide synthesis reagent such as Fmoc-aminoguanidine and Fmoc-amino acid.
  • a peptide synthesis protecting group e.g., Fmoc
  • an amino acid group-containing peptide synthesis reagent such as Fmoc-aminoguanidine and Fmoc-amino acid.
  • protecting groups play a very important role in the activity of protecting groups such as amino or carboxyl groups during the synthesis.
  • Fmoc aminoguanidine is supplied by Chengdu Kaitai New Technology Co., Ltd.
  • amino acid or Fmoc-amino acid is provided by Chengdu Taige Chemical Research Institute, including: arginine, asparagine, glutamine, glycine, lysine, Glutamine.
  • Peptide synthesis reagents containing other protecting groups can also be used in the methods of the following examples to prepare activated nanostructures and functionalized nanostructures.
  • the second activator used includes an amino group-free polyfunctional reagent (e.g., glutaraldehyde, 1, 4-butanediol diglycidyl ether), and an amino group-containing polyfunctional reagent (e.g., various amino acids as described above).
  • the coupling agent used includes a silicone coupling agent such as a silane coupling agent.
  • the silane coupling agent used includes: 3-aminopropyltrimethoxysilane (Cathay Huarong Chemical New Material Company), aminopropyltriethoxysilane (Cathay Huarong Chemical New Material Company), 3-isocyanatepropyl three Ethoxysilane (Huasheng Chemical Co., Ltd.).
  • the basic preparation method of the activated nanostructure comprises: fixing the activating group and the coupling group to the nanostructure, forming a coupling group on the nanostructure, and immobilizing the coupling group; The activated nanostructure of the group.
  • a more specific preparation method for various nanostructures is supplemented by the following examples.
  • the preparation method of the activated nanoparticles includes two methods.
  • T/CN2006/001374 An example of the first method, comprising at least: (1.1).
  • Preparation of coupled nanoparticles mixing the nanoparticles with a coupling agent solution and performing a coupling reaction.
  • the reaction conditions are as follows: nanoparticle concentration (w / v) l ° / oo -2%; coupling agent concentration (v / v) 1-3%; reaction medium is aqueous alcohol; reaction temperature from room temperature to the boiling point of the reaction medium The following 5 ° C; reaction time 0.5-5 hours.
  • Those skilled in the art can obtain the desired optimization conditions by adjusting these parameters.
  • the coupled nanoparticles are centrifuged off the suspension and then stored in DMF.
  • Preparation of activated nanoparticles The above-mentioned coupled nanoparticles are mixed with an activator solution, and an activation reaction is carried out.
  • the reaction conditions are as follows: concentration of nanoparticles (w / v) l ° / oo - 2%; activator concentration (v / v) is 0.5 - 5%; reaction temperature between room temperature and 5 ° C below the boiling point of the reaction medium; The reaction time is 0.5-15 hours; the reaction medium is DMF.
  • concentration of nanoparticles w / v) l ° / oo - 2%
  • activator concentration (v / v) is 0.5 - 5%
  • reaction temperature between room temperature and 5 ° C below the boiling point of the reaction medium
  • the reaction time is 0.5-15 hours
  • the reaction medium is DMF.
  • the activator contains a protecting group (e.g., Fmoc), these protecting groups are also removed.
  • the deprotection method is selected from a deprotection method in a known peptide synthesis method. After the reaction is completed, the activated nanoparticles are centrifuged off the suspension and stored in DMF.
  • An example of a second method comprising at least: (2.1) providing an activating group-coupling group complex: providing an activator (base activator or a base activating group - activating the second activating group complex) And reacting with a silicon germanium coupling agent (for example, 3-isocyanate propyl triethoxysilane) to prepare an activating group-coupling group complex (for example, aminodecyl-3-isocyanatepropyl three) Ethoxysilane.
  • a silicon germanium coupling agent for example, 3-isocyanate propyl triethoxysilane
  • an activating group-coupling group complex for example, aminodecyl-3-isocyanatepropyl three
  • the coupled nanoparticles are represented by coupling groups/nanoparticles.
  • the coupled nanoparticles prepared in the examples of the present invention include silane coupling groups/oxide nanoparticles.
  • the silicon germanium coupling group comprises: 3-aminopropyltrimethoxysilyl, aminopropyltriethoxysilyl, 3-isocyanatepropyltriethoxysilane
  • the oxide nanoparticles include: oxidation Silicon nanoparticles, alumina nanoparticles, and titanium oxide nanoparticles.
  • the obtained coupled nanoparticles can be calculated by elemental analysis (for example, (H, N elemental analysis), NMR analysis, etc., to calculate the density of the coupling group fixed per unit area on the surface of the nanoparticles.
  • elemental analysis for example, (H, N elemental analysis), NMR analysis, etc.
  • the above coupling group density changes greatly, for example, the nitrogen content (elemental analysis) is between 0.25-0.65 N%, which is equivalent to the fixed coupling group on the lg nanoparticle varies between 179-464 ⁇ , or the coupling group immobilized on the surface of the lm 2 nanoparticle varies between 1.3 and 3.4 ⁇ .
  • Coupling nanoparticles having the following compositional characteristics are preferred for performing the activated nanostructure or affinity nano in the following examples.
  • the coupling group immobilized on the surface of the lm 2 nanoparticles is greater than 1.85 ⁇ , preferably greater than 2.0 ⁇ >1 , more preferably greater than 2.50 ⁇ 1.
  • activated nanoparticles are represented by primary activating groups/conjugated nanoparticles.
  • the activated nanoparticles prepared in the examples of the present invention include: aminoguanidine/conjugated nanoparticles, aminoguanidine derivative groups/coupled nanoparticles, amino acid groups/coupled nanoparticles, amino acid derivative groups/coupling Nanoparticles, synthetic peptidyl/coupled nanoparticles, synthetic peptide derivative/coupled nanoparticles.
  • the coupled nanoparticles comprise coupling groups/nanoparticles contained in activated nanoparticles prepared by two different preparation methods as described above;
  • the activating group comprises a group provided by an activator as described above .
  • the activated nanoparticles obtained can be calculated by elemental analysis (for example, C, H, N elemental analysis), NMR analysis, and the like, to calculate the density of the activated groups fixed per unit area on the surface of the nanoparticles.
  • the density of the above-mentioned activating groups varies greatly depending on the coupling nanoparticles used, the activator and the selected activation reaction parameters (for example, 0.1-2.85 ⁇ ⁇ 1 / ⁇ 2 nanoparticle surface).
  • activated nanoparticles having the following compositional features for the preparation of activated nanostructures or affinity nanostructures in the following examples: the activated groups immobilized on the surface of the 1 m 2 nanoparticle are greater than 0.5 ⁇ m, preferably greater than 1 ⁇ 1, more preferably More than 1.5
  • Example 1.1.1 Preparation method of activated nanoparticle containing aminoguanidine group
  • the coupling group on the coupled nanoparticles is subjected to a carbonylation treatment, and the above activation is carried out using Fmoc-aminoguanidine as the activator.
  • Fmoc-aminoguanidine is provided, and reacted with a silane coupling agent (for example, 3-isocyanatepropyltriethoxysilane) to obtain aminoguanidino-3- Isocyanate propyl triethoxysilane, and aminoguanidino-3-isocyanate propyl triethoxysilane is immobilized on the nanoparticles.
  • the activated nanoparticles prepared in this example include aminoguanidine/conjugated nanoparticles.
  • Example 1.1.2 Preparation method of activated nanoparticle containing aminoguanidine derivative group
  • Example 1.1 Taking the first preparation method described in the above Example 1.1 as an example, a plurality of activation reactions were carried out.
  • the aminoguanidine/conjugated nanoparticles are first obtained by the method described in the above Example 1.1.1, followed by a second activator (for example, glutaraldehyde, 1, 4-butanediol diglycidyl ether, amino acid or The synthetic peptide) is subjected to a second activation, and then the second activation product is separated, and the like, and the conditions of each activation reaction are similar to those described above.
  • a second activator for example, glutaraldehyde, 1, 4-butanediol diglycidyl ether, amino acid or The synthetic peptide
  • the activated nanoparticles prepared in this embodiment include: glutaraldehyde-aminoguanidino/conjugated nanoparticles, epoxyalkyl-aminoguanidino/conjugated nanoparticles Particles, amino acids - aminoguanidino groups / coupled nanoparticles, synthetic peptides - aminoguanidino groups / coupled nanoparticles, and the like.
  • Example 1.1.3 Preparation method of activated nanoparticle containing amino acid group
  • the above activation is carried out using an amino acid or an Fmoc-amino acid as the activator.
  • One of the preferred reactions is the reaction of the -COOH group on the amino acid with a coupling agent or a -NH 2 group on the coupling group.
  • the activated nanoparticles prepared in this example include: arginine/coupled nanoparticles, asparagine/coupled nanoparticles, glutamyl/coupled nanoparticles, glycine/coupled nanoparticles Particles, lysine groups/coupled nanoparticles, glutamine groups/coupled nanoparticles.
  • Example 1.1.4 Method for preparing activated nanoparticle containing amino acid derivative activating group
  • the amino acid group/conjugated nanoparticles are first obtained by the method described in the above embodiment 1.1.2, and then the second activator (for example, glutaraldehyde, 1, 4-butanediol diglycidyl ether) is used for the second.
  • the second activator for example, glutaraldehyde, 1, 4-butanediol diglycidyl ether
  • the secondary activation, then the second activation product is separated, and the like, and the conditions of each activation reaction are similar to those described above.
  • an amino acid with a second activator to prepare an activator (e.g., an aldehyde-based amino acid) containing a basal activating group/derived activating group complex, which is then used as an activator for the above-described activation reaction.
  • an activator e.g., an aldehyde-based amino acid
  • the activated nanoparticles prepared in this example include: glutaraldehyde-arginine/coupled nanoparticles, epoxyalkyl-arginine/coupled nanoparticles, glutaraldehyde-asparagine/ Coupling nanoparticles, glutaraldehyde-glutamylamino/coupled nanoparticles, and the like.
  • Example 1.1.5 Preparation method of activated nanoparticles containing synthetic peptide activating group
  • the first preparation method described in the above embodiment 1.1 is taken as an example.
  • One method is: using a standard peptide synthesis method, that is, using the appropriate Fmoc-amino acid on the amino acid group on the activated nanoparticles prepared in the above Example 1.1.4, performing condensation, washing, deprotecting, neutralizing and washing. In a round of condensation, the amino acids are sequentially linked until the number of amino acid groups meets the requirements.
  • Another method is: peptide synthesis is carried out according to a known peptide synthesis method until a synthetic peptide having the desired number of amino acid groups is obtained, and then used as the activator and the coupled nanoparticles for the above activation reaction.
  • Example 1.1 Taking the second preparation method described in the above Example 1.1 as an example: peptide synthesis is carried out according to a known peptide synthesis method until a synthetic peptide having the desired number of amino acid groups is obtained, and then used as an activator to react with a coupling agent to prepare the synthesis. The peptidyl-coupling group complex was then prepared as described in (2.2) of Example 1.1 above.
  • the activated nanoparticles prepared in this example include synthetic peptidyl/coupled nanoparticles. among them-
  • the number of amino acids in the synthetic peptidyl group is greater than or equal to 2 (eg, 2-5); the amino acid species in the synthetic peptidyl group are of the same species (eg, arginine, asparagine, glutamylamine, etc.), or different (eg, Arginine and asparagine, asparagine and glycine, glutamine and lysine, etc.).
  • Example 1.1.6 Preparation method of activated nanoparticles containing activated peptide derivative activating group
  • a plurality of activation reactions are carried out, for example, obtaining the synthetic peptidyl group/coupled nanoparticles in the above embodiment 1.1.5, and then using a second activator (for example)
  • the glutaraldehyde, 1, 4-butanediol diglycidyl ether is subjected to a second activation, and then the second activation product is separated, and the like, and the conditions of each activation reaction are similar to those of the above reaction conditions.
  • the base activator can also be reacted with a second activator to prepare an activator (eg, an aldehyde-based polypeptide) comprising a basal activating group/derived activating group complex, and the activator is used as an activator of the above activating reaction.
  • an activator eg, an aldehyde-based polypeptide
  • the activated nanoparticles prepared in this example include: glutaraldehyde-peptidyl/coupled nanoparticles, epoxyalkyl-peptidyl/coupled nanoparticles.
  • the preparation method of the activated nanobeads of the present embodiment for example: providing activated nanoparticles (selected from the preparation of the above Example 1.1, such as amino acid groups/conjugated nanoparticles or aminoguanidine/conjugated nanoparticles), and Combination of different activated nanoparticles with mutual reactivity.
  • activated nanoparticles selected from the preparation of the above Example 1.1, such as amino acid groups/conjugated nanoparticles or aminoguanidine/conjugated nanoparticles
  • Combination of different activated nanoparticles with mutual reactivity for example, a glutamine-based/conjugated nanoparticle suspension with deprotected groups is mixed with an arginine-based/conjugated nanoparticle (or aminoguanidine/conjugated nanoparticles) suspension in equal amounts.
  • the binding of the glutamine group to the arginine group is carried out under the effective conditions according to a known peptide synthesis method, thereby generating activated nanobeads composed of two activated nanoparticles.
  • the reaction conditions were as follows: The concentration of the nanoparticles (w/v) was 1%. -2%; activator concentration (v/v) is 0.5-5%; reaction temperature is between room temperature and 5 ° C below the boiling point of the reaction medium; reaction time is 0.5-15 hours; and the reaction medium is DMF. Those skilled in the art can obtain the desired optimization conditions by adjusting these parameters.
  • activated nanobeads are represented by [activated nanoparticles] n (n is the number of activated nanoparticles).
  • the activated nanobeads prepared in this example included activated nanobeads based on activated nanoparticles prepared in Example 1.1.
  • the activated nanobeads obtained are the same as the activated nanoparticles used, which have the same density of activated groups per unit area on the surface.
  • Activated nanobeads having the following compositional characteristics are preferred for the preparation of activated nanostructures or affinity nanostructures in the following examples:
  • the activated groups immobilized on the surface of the lm 2 nanobeads are greater than 0.5 ⁇ preferably greater than 1 ⁇ 1, more preferably greater than 1.5 ⁇ 1.
  • a conventional carrier for forming an activated nanoprotrusion thereon may be It is any activated or unactivated solid phase carrier which is not itself a nanostructure carrier which can be used to hold the nanostructure thereon, and includes: a planar carrier, a granular carrier, a film carrier.
  • the planar carrier comprises a slide, an activation slide, and an ELISA porous plate;
  • the particulate carrier comprises silica gel, a chromatography gel;
  • the film carrier comprises a fiber membrane strip.
  • Activated slides include amino slides prepared according to published methods (see Schena, M., Microarray analysis, John Wiley & Sons, INC., New York) > Acid-based slides (see Schena, ⁇ , Microarray analysis) , John Wiley & Sons, INC., New York), aminoguanine slides (see Xavier Duburcq et al., Biocongate Chemistry 2002, 13: 713-720).
  • the ELISA porous plate includes a polystyrene porous plate (Shenzhen Jincanyu Co., Ltd.).
  • the silica gel includes silica particles having a particle size of 40 to 60 ⁇ m (Chemical Research Institute of the Chinese Academy of Sciences).
  • the chromatography gel includes Sephadex A 50 and CM-Shepharose CL (Pharmacia).
  • the fiber membrane strip includes a nitrocellulose membrane strip and a nylon fiber membrane strip (Fujian Quanzhou Changli Biochemical Co., Ltd.).
  • the preparation method of this embodiment is also suitable for conventional carriers made of the following materials or derivatives thereof: silicon wafers, silica gel, ceramics, metal oxides, metals, other polymer materials, and composites thereof.
  • the nano-protrusion and the minimum size of the height and the half height and the distribution density thereof are measured by a SPA-300HV scanning microscope (DFM) and analysis software.
  • DFM SPA-300HV scanning microscope
  • the activated nano-protrusion is prepared by four methods: 1) preparing the coupled nanoparticles, and then fixing the coupled nanoparticles on the surface of the conventional carrier to form a coupled nano-convex, coupled The nano-protrusion is activated to form an activated nano-convex; 2) the activated nano-particle is prepared, and the activated nano-particle is fixed on the surface of the conventional carrier to form an activated nano-protrusion; 3) the existing nano-protrusion is activated Activating the nano-convex body; and 4) adding a nanostructure to the existing nano-protrusion and forming a new activated nano-protrusion.
  • the coupled nano-protrusion is represented by a coupling group/nano-convex.
  • the coupled nanoprotrusions prepared in this embodiment include oxysilane groups/oxide nano-protrusions.
  • the silane group includes all the coupling groups contained in the above silane coupling agent;
  • the oxide nano protrusions include all of the above oxide protrusions (for example, silicon oxide nano protrusions, titanium oxide nano protrusions, aluminum oxides) Nano-convex).
  • the activated nano-protrusions prepared by the aforementioned preparation methods 1) and 2) contain a coupled nano-protrusion.
  • Coupled nanoprotrusions having the following compositional characteristics are preferred for the preparation of activated nanoprotrusions (or affinity nanoprotrusions) in the following examples: lm 2 nanoprotrusions
  • the surface-immobilized coupling group is greater than 1.85 ⁇ 1, preferably greater than 2.0 ⁇ 1, more preferably greater than 2.50 ⁇ 1.
  • the activated nano-protrusions are represented by a primary activating group/coupled nano-protrusion.
  • the prepared activated nanoprotrusions include: aminoguanidine/coupled nanoprotrusions, aminoguanidine derivative groups/coupled nanoprotrusions, amino acid groups/coupled nanoprostheses, amino acid derivatives Substrate/coupled nanoprotrusions, synthetic peptidyl/coupled nanoprotrusions, synthetic peptide derivative groups/coupled nanoprotrusions.
  • the coupled nano-protrusions include the coupling groups/nano-convex bodies contained in the activated nano-protrusions prepared by different preparation methods as described above.
  • the activated nanoprotrusions prepared by the aforementioned preparation method 2) are the same as the activated nanoparticles used, and the density of the coupling activating groups fixed per unit area on the surface thereof is the same.
  • Preferred such activated nanoprotrusions having the following compositional features are preferred for the preparation of activated nano-convex or affinity nano-protrusions in the following embodiments:
  • the activated groups immobilized on the surface of the lm 2 nano-convex are greater than 0.5 ⁇ . More than 1 ⁇ 0 1 , more preferably greater than 1.5 ⁇ 1 ⁇
  • a more specific preparation method for preparing activated nano-protrusions by the above four methods can be referred to the related methods in the following Examples 2.1-2.3.
  • the nanostructures used the coupling agent, the activator, the conventional support, and the nanostructures used in the above examples of the preparation of activated nanostructures (eg, Examples 1.1-1.3),
  • the coupling agent, activator, and conventional carrier are the same;
  • the nanostructured convex carrier used includes nanostructured slides and nanostructured silica particles.
  • the nanostructured convex carrier used is prepared by a method of coating nanoparticles into a conventional carrier. Briefly as follows: immerse slides (activated slides or unactivated slides) or silica gel particles in a suspension of nanoparticles of optimized concentration for more than 10 hours, then wash and then dry at appropriate temperature for a sufficient period of time. . It is particularly emphasized that other nano-convex carriers can also be used as the nano-protrusion support in this embodiment, such as a contiguous array of sub-micron whisker structures, and the like.
  • the activated nano-protrusion carrier can be prepared by at least four methods: 1).
  • the coupled nano-particles are fixed on the surface of a conventional carrier to form a coupled nano-convex carrier, and then activated to be activated.
  • a nanostructure is then added to the convex support and a new activated nanoprotrusion carrier is formed.
  • the activated nanostructure carrier is represented by an activating group/nano-convex/carrier.
  • the activated nanostructure carrier prepared by the embodiment of the invention comprises: an activating group/nano-convex/planar carrier (for example, an activating group/nano-convex/chip-based substrate, an activating group/nano-convex/enzyme micro-micro) Orifice-based substrate, activating group/nano-convex/granular carrier (eg, activating group/nano-convex/chromatographic particle matrix), activating group/nano-convex/membranous carrier (eg, an activating group) Cluster/nanoconvex Body/film).
  • an activating group/nano-convex/planar carrier for example, an activating group/nano-convex/chip-based substrate, an activating group/nano-convex/enzyme micro-micro) Orifice-based substrate, activating group/nano
  • the activating group/nano-convex body includes all of the activated nano-protrusions prepared in the above Example 1.3, wherein the activating group is the same as the activating group contained in the activated nanoparticles prepared in the above Example U, and includes: A group, an aminoguanidine derivative group, an amino acid group, an amino acid derivative group, a synthetic peptide group, a synthetic peptide derivative group.
  • a conventional carrier for example, an activated or unactivated slide, silica gel particles
  • a suspension of a optimized concentration of the coupled nanoparticles selected from the coupled nanoparticles prepared in the above Example 1.1
  • the reaction conditions are as follows: Conjugated nanoparticle concentration (w/v) 0.01-l%; reaction temperature is room temperature; reaction time 1-15 hours.
  • the coupled nano-protrusion is mixed with an activator solution and reacted.
  • the reaction conditions are as follows: activator concentration (v/v) 1-5% ; reaction temperature between room temperature and 5 ° C below the boiling point of the reaction medium; reaction time 0.5-15 hours.
  • the activator contains a protecting group (e.g., Fmoc-amino acid), these protecting groups are also removed.
  • the prepared activated nano-convex carrier comprises: (1) aminosulfonyl/nano-convex/slide, amino acid/nano-convex/slide, amino acid derivative/nano-convex/glass Tablets, synthetic peptidyl/nano-convex/slide, synthetic peptide derivative/nano-convex/slide; (2).
  • the reactive group is the same as the reactive group contained in the activated nanoparticles prepared in the above Example 1.1.
  • a method of preparing activated nanoparticles containing the same activating group can also be referred to in the above Examples 1.1.1-1.1.6, respectively.
  • Example 2.2 Preparation method of activated nano-convex carrier (2)
  • a conventional carrier for example, an activated or unactivated slide, a bottom of an ELISA microplate, a granular carrier, a membranous carrier
  • activated nanoparticles selected from activated nanoparticles prepared in the above Example 1.1. Soak in the liquid, react, then wash, and then dry at the appropriate temperature for a sufficient period of time.
  • the reaction conditions are as follows: Activated nanoparticle concentration (w/v) 0.01-1%; reaction temperature is room temperature; reaction time 1-15 hours. Those skilled in the art can obtain the desired optimization conditions by adjusting these parameters.
  • the prepared activated nano-convex carrier comprises: (1) aminoguanidine/nano-convex/slide, aminoguanidine derivative/nano-convex/slide, amino acid/nano-convex/ Slide, ammonia Acid-based derivative/nano-convex/slide, synthetic peptidyl/nano-convex/slide, synthetic peptide derivative/nano-convex/slide; (2).
  • aminoguanidino/nano-convex/particle Amino acid group/Nano-convex/microparticle, Amino acid derivative/Nano-convex/microparticle, Synthetic peptidyl/nano-convex/microparticle, Synthetic peptide derivative/Nano-convex/particle; (3). Aminoguanidine /Nano-convex/microplate, Amino acid group/Nano-convex/microplate, Amino acid derivative/Nano-convex/microplate, Synthetic peptidyl/nano-convex/microplate, Synthetic peptide derivative /Nano-convex/microplate; (4).
  • Aminoguanidine/nano-convex/fiber membrane strip amino acid-based/nano-convex/fiber membrane strip, amino acid derivative/nano-convex/fiber membrane strip, synthesis Peptidyl/nano-convex/fiber membrane strips, synthetic peptide derivative/nano-convex/fiber membrane strips.
  • the reactive group is the same as the reactive group contained in the activated nanoparticles prepared in the above Example 1.1.
  • a sulfuric acid/hydrogen peroxide etching on a nano-protrusion carrier for example, a nano-convex slide glass or a nano-convex silica gel particle
  • a coupling agent solution for example, a sulfuric acid/hydrogen peroxide etching on a nano-protrusion carrier (for example, a nano-convex slide glass or a nano-convex silica gel particle)
  • the reaction conditions are as follows: coupling agent concentration (v/v) 1-5%; the reaction medium is an aqueous alcohol; the reaction temperature is between room temperature and the boiling point of the reaction medium below 5 ° C; the reaction time is 0.5-5 hours.
  • the skilled person can adjust the parameters to obtain the desired optimization conditions.
  • the activation is carried out according to the following two methods:
  • the above-mentioned coupled nano-protrusion carrier is mixed and reacted with an activator solution.
  • the reaction conditions are as follows: Activator concentration (v/v) 1-5%; reaction temperature between room temperature and 5 ° C below the boiling point of the reaction medium; reaction time 0.5-15 hours. Those skilled in the art can obtain the desired optimization conditions by adjusting these parameters.
  • a synthetic peptidyl/coupled nano-convex support is prepared by the same method as in the above Example 1.1.5; synthetic peptide-derived by using the same method as in the above Example 1.1.6 Substrate/coupled nanoprotrusions.
  • the synthetic peptidyl group and the synthetic peptide derivative group are the same as the synthetic peptidyl group and the synthetic peptide derivative group in the above Examples 1.1.5 and 1.1.6, respectively.
  • the prepared activated nano-convex carrier comprises: (1) aminoguanidine/nano-convex/slide, aminoguanidine derivative/nano-convex/slide, amino acid/nano-convex/ Slide, ammonia Acid-based derivative/nano-convex/slide, synthetic peptidyl/nano-convex/slide, synthetic peptide derivative/nano-convex/slide; (2).
  • aminoguanidino/nano-convex/particle Amino acid group/nano-convex/microparticles, Amino acid derivative group/Nano-convex/microparticle, Synthetic peptidyl/nano-convex/microparticle, Synthetic peptide derivative/nano-convex/particle.
  • the reactive group is the same as the reactive group contained in the activated nanoparticles prepared in the above Example 1.1.
  • Example 2.4 Preparation method of activated nano-convex carrier (4)
  • the nanoparticles, the coupled nanoparticles, and the activated nanoparticles are respectively coated onto the nano-protrusion carrier, and then: the nanoparticles are coated with the nano-convex carrier, and activated according to the method in the above embodiment 2.3;
  • the nanoparticles were coated with a nano-convex carrier and activated as described in Example 2.1 above.
  • the coating method used therein is the same as the known method of coating nanoparticles into a conventional carrier. Briefly described as follows: The nano-convex carrier is placed in a suspension of nanoparticles of optimized concentration for more than 10 hours, then washed and then dried at the appropriate temperature for a sufficient period of time.
  • the activated nano-convex carrier (activated group/nano-convex/carrier) prepared in this embodiment comprises: (1) aminoguanidine-activated nanoparticles/nano-convex/slide, amino acid-based activated nanoparticles/nano-convex Body/slide, amino acid derivative-based activated nanoparticles/nano-convex/slide, synthetic peptidyl-activated nanoparticles/nano-convex/slide, synthetic peptide-derived-activated nanoparticles/nano-convex/slide; (2).
  • Aminoguanidine-activated nanoparticles/nano-convex/microparticles amino acid-based activated nanoparticles/nano-convex/microparticles, amino acid derivative-based activated nanoparticles/nano-convex/microparticles, synthetic peptidyl-activated nanoparticles/ Nanoprotrusions/microparticles, synthetic peptide derivative-based activated nanoparticles/nano-convex/particles.
  • the reactive group is the same as the active group contained in the activated nanoparticles prepared in the above Example 1.1.
  • Example 3 Method for preparing functionalized nanostructures
  • the nanostructure, coupling agent, and activator used are the same as the nanostructure, coupling agent, and activator used in the above Example 1; the activated nanoparticles used are the above examples.
  • the activated nanobeads used are the activated nanobeads prepared in the above Example 1.2;
  • the functional reagents used include: polypeptides, antigens, antibodies, and other functional reagents.
  • the synthetic polypeptide used includes EBV-VCA-P18 antigen (self-made, preparation method refers to Tranchand-Bunel, D., Auriault, C., Diesis, E., Gras-Masse, H. (1998) Detection of human antibodies using "Convergent” combinatorial peptide libraries or "mixotopes” designed form a nonvariable antigen: Application to the EBV viral capsid antigen pi 8, J. Peptide Res.
  • the antigens used include: Hepatitis C virus antigen (HCV) Ag), HIV antigen, syphilis antigen (both provided by the Institute of Liver Diseases, Peking University People's Hospital); antibodies used include anti-hepatitis B virus surface antibody (HBsAb, Institute of Liver Diseases, Peking University People's Hospital) and monoclonal Or polyclonal goat anti-human secondary antibody (Beijing Institute of Biological Products); other functional reagents used include protein A (Shanghai Institute of Biological Products).
  • the method of this example is also suitable for other functional agents, such as: drugs, polysaccharides, vitamins, antibiotics, biotin, avidin, functional organisms, single or multi-stranded DNA, RNA, and viruses, cells or their constituents.
  • a basic method of preparing a functionalized nanostructure comprises: preparing an activated nanostructure, and then immobilizing a functional agent onto the activated nanostructure.
  • a more specific preparation method based on different nanostructures is supplemented by the following Examples 3.1-3.3.
  • the functional reagent is brought into contact with the activated nanoparticles and reacts.
  • the reaction conditions are as follows: activated nanoparticle concentration (w/v) 0.01-3%; functional reagent concentration (w/v) 0.1-3.0 mg/ml; buffer pH 5.0-9.5; reaction temperature 20-37 ⁇ ; reaction time 0.5 -72 hours.
  • activated nanoparticle concentration w/v 0.01-3%
  • functional reagent concentration w/v
  • 0.1-3.0 mg/ml buffer pH 5.0-9.5
  • reaction temperature 20-37 ⁇ reaction time 0.5 -72 hours.
  • Those skilled in the art can obtain the required optimization conditions by adjusting these parameters. It also includes purification, or / and passivation of functionalized nanoparticles, if necessary. Commonly used purifying agents include proteins and amino acids.
  • functionalized nanoparticles are represented by functional reagents/activated nanoparticles.
  • the functionalized nanoparticles prepared in this example include: antigen/activated nanoparticles (eg, HCV Ag/activated nanoparticles, fflV Ag/activated nanoparticles, syphilis antigen/activated nanoparticles, etc.), antibody/activated nanoparticles (eg, HBs Ad/activated nanoparticles), other functional reagents/activated nanoparticles (eg Protein A/activated nanoparticles).
  • the activated nanoparticles were the activated nanoparticles prepared in the above Example 1.1.
  • the functional reagent is brought into contact with and activated by the activated nanobeads.
  • the reaction conditions are as follows: nanobead concentration (w/v) 0.01-3%; functional reagent concentration (w/v) 0.1-3.0 mg/ml; buffer pH 5.0-9.5; reaction temperature 20-37 ° C; reaction time 0.5-72 hours.
  • nanobead concentration w/v 0.01-3%
  • functional reagent concentration w/v
  • 0.1-3.0 mg/ml buffer pH 5.0-9.5
  • reaction temperature 20-37 ° C reaction time 0.5-72 hours.
  • Those skilled in the art can obtain the desired optimization conditions by adjusting these parameters.
  • purification, or / and passivation Commonly used purifying agents include proteins and amino acids.
  • functionalized nanobeads are represented by functional reagents/activated nanobeads.
  • the functionalized nanobeads prepared in this example include: antigen/activated nanobeads (eg, HCV Ag/activated nanobeads, HIV Ag/activated nanobeads, syphilis antigen/activated nanobeads, etc.), antibody/activated nanobeads (eg, HBs Ad/activated nanobeads), other functional reagents/activated nanobeads (eg Protein A/activated nanobeads).
  • the activated nanobeads are the above examples 1.2 Preparation of activated nanobeads.
  • Example 3.3 Method for preparing functionalized nanoprotrusions
  • the functionalized nanoprotrusions can be prepared by at least a method of sputum-
  • the immobilization reaction conditions are as follows: Functionalized nanoparticle concentration (w/v) 0.01-3%; Buffer pH 5.0-9.5; Reaction temperature 20-37 ° C; Reaction time 0.5-72 hours.
  • the immobilization reaction conditions were as follows: functionalized nanoparticles or/and functionalized nanobeads (w/v) 0.01-3%; functional reagent concentration (wA0O.l-3.Omg/ml; buffer pH 5.0-9.5; The reaction temperature is 20-37 ° C; the reaction time is 0.5-72 hours.
  • the functionalized nanoparticles used include all of the functionalized nanoparticles prepared in the above Example 3.1; the functionalized nanobeads used include all of the functionalized nanobeads prepared in the above Example 3.2; the activated nanoprotrusions used, selected from the foregoing implementations of the present invention
  • Activated nanoprotrusions prepared in Example 1.3 eg, activated silicon oxide nanoprotrusions, activated titanium oxide nanoprotrusions, activated aluminum oxide nanoprotrusions).
  • the functionalized nano-convex is represented by a functional reagent/activated nanoprotrusion, or a functional reagent/primary activating group/conjugated nanoprotrusion.
  • the functionalized nanoprotrusions prepared in this embodiment include: antigen/activated nanoprotrusions (eg, HCVAg/activated nanoprotrusions, HIV Ag/activated nanoprotrusions, syphilis antigens/activated nanoprotrusions, etc.), antibody/activation Nanoprotrusions (eg, HBs Ad/activated nanoprotrusions), other functional agents/activated nanoprotrusions (eg, Protein A/activated nanoprotrusions).
  • the activated structure in the activated nano-protrusion comprises the activated structure in the activated nano-protrusion prepared in the above Example 1.3.
  • Example 4 A more specific preparation method for preparing the functionalized nanoprotrusions by the above-described methods can be referred to the related methods in the following Example 4 (including Examples 4.1 to 4.3).
  • the conventional vector used in Example 1.3 is the same; the functional reagent used is the same as the functional reagent used in the above Example 3 (including: polypeptide, antigen, antibody, and Other functional reagents).
  • three basic methods are used for the preparation of the functionalized nanostructure carrier: 1) fixing the functional reagent on the activated nanoprotrusion carrier; 2) functionalizing the nanoparticle or/and functionalizing the nanobead Immobilized on a conventional support; 3).
  • the functionalized nanoparticles or/and functionalized nanobeads are immobilized on an activated nano-convex support.
  • the functionalized nanoparticles used are selected from the group consisting of all of the functionalized nanoparticles prepared in the above Example 3.1; the functionalized nanobeads used include all of the functionalized nanobeads prepared in the above Example 3.2; the activated nano-convex carriers used include all of the above implementations Activated nanoprotrusion support prepared in Example 2 (including Examples 2.1-2.4).
  • the activating group on the activated nanoprotrusion is the same as the activating group contained in the activated nanostructure prepared in the above Example 1, including an aminoguanidine group, an aminoguanidine derivative group, and an amino acid.
  • a base, an amino acid derivative group, a synthetic peptide group, a synthetic peptide derivative group A more specific preparation method is supplemented by the following Examples 4.1-4.3, and reference is made to Example 5 below.
  • the functionalized nano-convex carrier is represented by a functional reagent/activated nanoprotrusion/carrier.
  • the functionalized nanoprotrusion carriers prepared in the following examples include: antigen/activated nanoprotrusions/carriers (eg, multiple antigens/activated nanoprotrusions/chip-based matrices, antigen/activated nanoprotrusions/microplates) Base matrix, antigen/activated nanoprotrusion/membrane, antigen/activated nanoprotrusion/chromatographic particle matrix), antibody/activated nanoprotrusion/carrier (eg, antibody/activated nanoprotrusion/chip-based matrix, antibody /activated nanoprotrusion / microplate microplate base matrix, antibody / activated nanoprotrusion / chromatography particle matrix), antigen and antibody / activated nanoprotrusion / carrier (multiple antigens and HBs antibodies / activated nano-convex / Chip base matrix), other functional reagents / activated nanoprotrusion
  • one or more functional reagent solutions are spotted on the activated nano-protrusion/chip-based substrate prepared in the above Example 3 according to a known chip preparation method.
  • the reaction is carried out at 37 ° C for more than 3 hours and then passivated with a passivating agent (such as bovine serum albumin) to form a functionalized nano-convex/chip-based matrix (nanoanalysis chip).
  • a passivating agent such as bovine serum albumin
  • the functional reagent solution (functional reagent concentration between 0.1-2 mg/ml) and the activated nanoprotrusion/chromatographic particle matrix prepared in the above Example 3 (particle concentration) are prepared according to a known method for preparing affinity chromatography particles. (w/v) contact between 0.5-5%), stirring at room temperature, coating for 3-24 hours to form a functionalized nano-convex/chromatographic particle matrix (affinity chromatography nano-stationary phase).
  • the functional reagent solution (functional reagent concentration between 0.0.5-2 g/ml) is added to the activated nanoprotrusion/enzyme microparticle prepared in the above Example 3 according to a known method for preparing a microplate.
  • the plate base matrix bottom of the well
  • a passivating agent such as bovine serum albumin
  • the functional reagent solution (functional reagent concentration between 0.1-2 mg/ml) is spotted in the activated nanometer prepared in the above Example 3 according to a known method for preparing a planar affinity chromatography membrane (for example, a rapid test reagent strip).
  • a planar affinity chromatography membrane for example, a rapid test reagent strip.
  • On the convex/film react at 37 ° C for more than 3 hours, and coat to form a functionalized nano-protrusion / film (such as a rapid test strip).
  • one or more functionalized nanoparticles or/and functionalized nanobead suspensions are spotted according to well-known chip preparation methods (functional reagent concentrations between 0.1 and 2 mg/ml, nanostructure concentration (w/v) Between 0.01% and 5%) spotted on an activated chip substrate, reacted at 37 ° C for more than 3 hours, and then passivated with a passivating agent (such as bovine serum albumin) to form a functionalized nano-convex / Chip base matrix (nanoanalysis chip).
  • a passivating agent such as bovine serum albumin
  • the functionalized nanoparticles or/and functionalized nanobead suspensions are prepared according to well-known affinity chromatography particle preparation methods (functional reagent concentration is between 0.5-2 mg/ml, nanostructure concentration (w/v) Contact between the chromatographic particles (particle concentration (w/v) between 0.5 and 5%), stirring at room temperature, coating for 3-24 hours to form a functionalized nanoprotrusion / Chromatographic particle matrix (affinity chromatography nano-stationary phase).
  • the functionalized nanoparticles or/and the functionalized nanobead suspension are prepared according to a well-known method for preparing a microplate (the functional reagent concentration is between 0.1 and 2 g/ml, and the nanostructure concentration (w/v) is Between 0.001% and 0.05%) is added to the bottom of the microplate of the enzyme-labeled microplate, shaken at room temperature, coated for 3-24 hours, and then passivated with a passivating agent (such as bovine serum albumin) to form a functionalized nano-convex Body/enzyme-labeled microplate-based matrix (nano-labeled microplate).
  • a passivating agent such as bovine serum albumin
  • the functionalized nanoparticles or/and the functionalized nanobead suspension are prepared according to a known method for preparing a planar affinity chromatography membrane (for example, a rapid test strip) (the concentration of the functional reagent is between 0.1 and 2 mg/ml, The nanostructure concentration (w/v) is between 0.01% and 5%) and is applied to the fiber membrane strip, and reacted at 37 ° C for more than 3 hours to form a functionalized nanoprotrusion/membrane (for example, a rapid test strip). At this time, only the activated nanostructures are present in the functional reagent sites, and there is no non-specific adsorption associated with the nanostructures outside the functional reagent sites.
  • a planar affinity chromatography membrane for example, a rapid test strip
  • one or more functionalized nanoparticles or/and functionalized nanobead suspensions (functional reagent concentrations between 0.1 and 2 mg/ml, nanostructures) according to well known chip preparation methods
  • concentration (w/v) is between 0.01% and 5%.
  • the point is on the activated nano-protrusion/chip-based substrate prepared in the above Example 2, reacted at 37 ° C for more than 3 hours, and then used as a passivating agent (for example, cattle).
  • the serum albumin is passivated and reacts to form a functionalized nanoprotrusion/chip-based matrix (nanoanalysis chip).
  • only the activated nanostructures are present in the functional reagent sites, and there is no non-specific adsorption associated with the nanostructures outside the functional reagent sites.
  • the functionalized nanoparticles or/and functionalized nanobead suspensions are prepared according to well-known affinity chromatography particle preparation methods (functional reagent concentration is between 0.5-2 mg/ml, nanostructure concentration (w/v) Between 0.1% and 3%) contact with the activated nano-protrusion/chromatographic particle substrate (particle concentration (w/v) between 0.5 and 5%) prepared in the above Example 2, stirring at room temperature, coating 3 - 24 hours, forming a functionalized nano-convex/chromatographic particle matrix (affinity chromatography nanostationary phase).
  • affinity chromatography particle preparation methods functional reagent concentration is between 0.5-2 mg/ml, nanostructure concentration (w/v) Between 0.1% and 3%) contact with the activated nano-protrusion/chromatographic particle substrate (particle concentration (w/v) between 0.5 and 5%) prepared in the above Example 2, stirring at room temperature, coating 3 - 24 hours, forming a functionalized nano-convex/chromatographic particle matrix (affinity chromatography nanostationary phase).
  • the functionalized nanoparticles or/and the functionalized nanobead suspension are prepared according to a well-known method for preparing a microplate (the functional reagent concentration is between 0.1 and 2 g/ml, and the nanostructure concentration (w/v) is Between 0.001% and 0.05%) was added to the activated nanoprotrusion/enzyme-labeled microplate-based substrate (bottom bottom) prepared in the above Example 2, shaken at room temperature, coated for 3-24 hours, and then passivated.
  • the agent such as bovine serum albumin
  • the nanostructures, coupling agents, activators, conventional carriers, functional reagents (including: polypeptides, antigens, antibodies, and other functional reagents) used are the same as in the above-mentioned Embodiment 4
  • the preparation method used was the same as in the above Example 4.
  • the functionalized nanoparticles used, the functionalized nanobeads, and the activated nanoprotrusion carrier are the same as in the above-mentioned Embodiment 4.
  • any reaction system which may include the activated nanostructure, such as a sensor, an analytical chip, an ELISA plate, a rapid test strip, or the like, may be prepared. More specific preparation methods for certain reaction systems (or devices) are supplemented by the following Examples 5.1-5.3.
  • the preparation method and reaction conditions of the nanostructure analysis chip are the same as those of the functionalized nanoprotrusion/chip substrate based in Examples 4.1-4.3.
  • the spotting can be manual spotting or mechanical spotting (DY-2003 Biochip spotting instrument, Institute of Electrical Engineering, Chinese Academy of Sciences). 2-4 points for each solution or suspension. All functional reagent spots form an array of MxN functional reagents on the chip substrate. Where M is greater than 1, and N is greater than 1.
  • at least one functional reagent point has a functionalized nanostructure (for example, a functional reagent/activated nano-convex), which may be a functional nanostructure of all functional reagent points, or may be a partial functional test.
  • the agent sites have functionalized nanostructures (eg, the presence of non-nanostructured functional reagent sites).
  • the nanoanalysis chip only some or all of the functional reagent dots may have a nanostructure (for example, formed on an activated conventional substrate), or the entire lattice region may have a nanostructure (for example, in activating a nano-convex substrate).
  • the preferred analysis chip prepared in this embodiment has a distribution density of at least one functional reagent point (the height is greater than 3 nm, and the convex half height is at least one dimension at 1 to 500 nm) and the distribution density is greater than 5/ ⁇ 2 .
  • the method of this embodiment is of course suitable for various chips, such as single reaction cell chips, multiple reaction cell chips, flow chips, non-flow chips, and the like.
  • multi-reaction cell non-flow chip chip base refer to the embodiment 1 of our patent application "High integration analysis chip with minimum reactor height and its application” (Application No. PCT/CN2004/000169) . Then, perform the above “spotting” operation and other operations on the base pool.
  • a flow biochip reference may be made to Example 9 or 10 of our patent application "Highly Integrated Analysis Chip for Reactor Height Minimization and Its Application” (Application No. PCT/CN2004/000169).
  • the chip prepared by the method of the present embodiment comprises: a nanostructure antigen chip (for example, a nanostructure chip of the invention to which at least an HCV antigen, an HIV antigen, or/and a syphilis antigen is immobilized), an antibody chip (for example, at least an HBs antibody is immobilized) Nanostructured chips of the invention), antigens and antibody chips (eg, nanostructured chips of the invention having at least immobilized HCV antigens and HBs antibodies), other affinity chips (eg, nanostructured chips of the invention having at least protein A immobilized) .
  • a nanostructure antigen chip for example, a nanostructure chip of the invention to which at least an HCV antigen, an HIV antigen, or/and a syphilis antigen is immobilized
  • an antibody chip for example, at least an HBs antibody is immobilized
  • Nanostructured chips of the invention Nanostructured chips of the invention
  • antigens and antibody chips eg, nanostructured chips of the invention
  • the preparation method and reaction conditions of the nano-enzyme-labeled microplate are the same as those of the functionalized nano-protrusion/enzyme-labeled microplate base matrix in the examples 4.1-4.3, and the prepared nanostructured enzyme Targets, including: nanostructured antigen ELISA plate (such as HCV antigen, mv antigen, or syphilis antigen coated ELISA plate), nanostructured antibody ELISA plate (such as HBs antibody coated ELISA plate), other nanostructured pro And the ELISA plate (for example, a protein A coated ELISA plate).
  • nanostructured antigen ELISA plate such as HCV antigen, mv antigen, or syphilis antigen coated ELISA plate
  • nanostructured antibody ELISA plate such as HBs antibody coated ELISA plate
  • other nanostructured pro And the ELISA plate for example, a protein A coated ELISA plate.
  • Example 5.3 Preparation method of nano-planar affinity chromatography membrane
  • the preparation method and reaction conditions of the nano-planar affinity chromatography membrane are the same as those of the functionalized nano-protrusion/membrane preparation method in Examples 4.1-4.3, and the prepared nano-planar affinity chromatography membrane includes : Antigen rapid test strips (eg, HCV antigen, HIV antigen, or syphilis antigen coated rapid test strip) immobilized with protein A and colloidal gold-labeled goat anti-human secondary antibody.
  • Antigen rapid test strips eg, HCV antigen, HIV antigen, or syphilis antigen coated rapid test strip
  • the functionalized nanoparticles used are selected from the functionalized nanoparticles prepared in the above Example 3.1; the functionalized nanobeads used are selected from the functionalized nanobeads prepared in the above Example 3.2;
  • the functionalized nanoprotrusion is selected from the above implementation Functionalized nanoprotrusions on the affinity chromatography nanostains prepared in Example 3.3 or 4.
  • the above affinity chromatography nano-stationary phase can be used as an affinity chromatography stationary phase, including batch reaction affinity chromatography and affinity column chromatography.
  • a method of forming an affinity chromatography system from an affinity chromatography stationary phase is a well-known method.
  • the nano-affinity chromatography system prepared by the method of the present embodiment comprises a nano-affinity chromatography system in which a protein, a protein G, and an HCV antigen are respectively immobilized.
  • the functionalized nanomagnetic separation system prepared in this embodiment contains affinity magnetic particles in addition to functionalized nanoparticles or/and functionalized nanobeads.
  • the affinity magnetic particles comprise functional reagents and coated magnetic particles or coated magnetic particle derivatives.
  • the magnetic particles used include magnetic microparticles (1- ⁇ ) and magnetic nanoparticles (10-100 nm).
  • the preparation method of the affinity magnetic microparticles and the affinity magnetic nanoparticles is prepared according to a known method, in short: the magnetic particles are dispersed into water to form a suspension, and an equal volume of 5% dextran solution is added at 85-90°. The C package was taken for 1 hour. After cooling, the dextran coated magnetic particles were purified by a magnetic column, and then heated to 60 ° C to add an appropriate amount of DVS, and then an appropriate amount of triethyl amine was added to pH 10.5. After reacting at 60 ° C for 2 hours, the activated dextran coated magnetic particles were purified using a magnetic column.
  • a pairing function reagent for example, a paired hepatitis B surface antibody for sandwich method, a paired HIV antigen, a paired HCV antigen
  • a pairing function reagent is immobilized on the activated glucan-coated magnetic particles to prepare an affinity magnetic particle.
  • the nanostructures in the activated nanostructures and functionalized nanostructures of the invention comprise: nanostructures commonly used as physical labeling materials (eg, gold nanoparticles, fluorescent nanoparticles, semiconductor nanometers) Particles, magnetic nanoparticles, etc.; nanostructures commonly used as chemically labeled materials (eg gold nanoparticles in gold-silver labeling, etc.); nanostructures that are not normally used as physical or chemical labeling substances (eg oxidation) Oxides such as silicon, aluminum oxide, organic compounds, etc.).
  • molecularly labeled substances such as fluorescent substance molecules, enzymes, dyes, etc.
  • the functionalized nanoparticles used include all of the functionalized nanoparticles prepared in the above Example 3.1; the functionalized nanobeads used include all of the functionalized nanobeads prepared in the above Example 3.2; the functional reagents used include those used in Example 3 above.
  • Secondary antibody, and the antigen used, the corresponding antigen of the antibody, the antibody paired purchase, for double antigen sandwich method, double antibody sandwich method);
  • the labeling substances used include: fluorescent substances (such as rhodamine, CY3, CY4), labeling enzymes ( For example, horseradish peroxidase), a coloring agent (such as crystal violet).
  • the method of this embodiment is also suitable Other marking materials such as chemiluminescent materials, chemiluminescent catalysts, non-ferrous metal salts, dyes and pigments.
  • the preparation of the nanomarker comprises two methods: A) fixing the labeling substance on the functionalized nanoparticle or/and the functionalized nanobead and then purifying; B) fixing the labeling substance on the functional reagent
  • the purified labeling substance/functional reagent complex is then immobilized on activated nanoparticles or/and functionalized nanobeads.
  • the conditions of the immobilization reaction can refer to the reaction conditions of the functional reagent and the labeling substance in the preparation method of a known conventional label (for example, a fluorescent substance label, an enzyme label), but preferred conditions include a much longer reaction time. (eg greater than 12 hours).
  • the purification conditions can be referred to a purification method (e.g., filtration method, chromatography, and the like) of the label in the conventional method for preparing a conventional label, but a preferred method includes centrifugation.
  • a purification method e.g., filtration method, chromatography, and the like
  • the nanomarker is represented by a labeling substance/activated nanoparticle/functional reagent.
  • the partial nanomarkers prepared in this example are as follows: 1). Fluorescent substance/activated oxide nanoparticles/paired antigen, fluorescent substance/activated oxide nanoparticles/paired antibody, fluorescent substance/activated oxide nanoparticles/anti-antibody, Fluorescent/activated oxide nanoparticles/protein A; 2). Horseradish/activated oxide nanoparticles/paired antibody, horseradishase/activated oxide nanoparticles/anti-antibody; 3). Stain/activation Oxide nanoparticles / anti-antibody.
  • the kit of the embodiment of the present invention is prepared by containing at least the reaction system prepared in the above Example 5, and may further comprise the labeling system prepared in the above Example 7, or / and the separation system prepared in the above Example 6.
  • the kits of the present invention contain one, two, and three types of systems comprising the functionalized nanostructures of the present invention, respectively.
  • a more specific preparation method is supplemented by the following examples.
  • kits prepared in the following examples are listed in Tables 1, 2, and 3, respectively.
  • kits prepared in this example are: including the above-mentioned embodiment 5 (including 5.1-5.3) A kit for the prepared nanoreaction system; a kit comprising the nanoseparation system prepared in the above Example 6.2; and a kit comprising the nanolabeling system prepared in the above Example 7. Some of the kits prepared in this example are listed in Table 1.
  • kits prepared in this example are: including the above-mentioned embodiment 5 (including 5.1-5.3) a kit for preparing a nanoreaction system and the nanolabeling system prepared in the above Example 7; a kit comprising the nanoseparation system prepared in the above Example 6.2 and the nanolabeling system prepared in the above Example 7; comprising the above Example 5 (including 5.1-5.3) A kit for the prepared nanoreaction system and the nanoseparation system prepared in the above Example 6.2. Some of the kits prepared in this example are listed in Table 2.
  • the kit prepared in this embodiment contains the preparation prepared in the above Example 5 (including 5.1-5.3)
  • Some of the kits prepared in this example are listed in Table 3.
  • the samples are: HCV antibody positive serum, fflVi + 2 antibody positive human serum, HBS Ag positive serum, EBV antibody positive serum, syphilis antibody positive serum, and negative serum (HCV antibody, fflV 1 +2 antibody, HBsAg and syphilis antibodies are negative serum). All samples were pre-tested using a classical ELISA method under serum 10-fold dilution conditions.
  • a device or kit containing the reaction system of the present invention e.g., A1-A5 in Table 1 can be applied in accordance with a known method of application of the corresponding device or kit. In the following examples, only some comparative studies are given to illustrate.
  • Example 9.1 Nanostructured chip of the present invention
  • the chips used are: The nanostructured chip of the present invention, which is compared with a conventional chip and a control nanostructure chip.
  • the nanostructured chip of the present invention used is the nanostructured chip prepared in the above Example 5.1; the conventional control chip used is used on an activated slide (amino slide, aminoguanidine slide, refer to the above Example 1.3)
  • the same functional reagent as the nanostructured chip of the present invention is prepared under the same conditions (refer to the above Example 4.3), and does not contain nanostructure functional reagent dots;
  • the control nanostructured chip used, in the amino group-containing coupled nanoprotrusion/chip a base (refer to Example 2, for example, a coupled nano-protrusion prepared by using 3-aminopropyltrimethoxysilane as a coupling agent), using the same functional reagent as the nanostructured chip of the present invention under the same conditions (Refer to Example 4.3 above), without the functionalized nanostructures of the present invention.
  • the label used in the chip test is a conventional marker, for example: Rodin
  • the chip test method is as follows: (1). Test of non-flow chip: 5 ⁇ 1 of the appropriately diluted test sample is separately added to the reaction cell of the corresponding chip, reacted at 37 ° C for 30 minutes, rinsed with washing solution, and then added with 5 ⁇ l of the appropriate concentration of the mark. The mixture was reacted at 37 ° C for 30 minutes, rinsed with a washing solution, then dried and then scanned.
  • the scanner is a confocal laser scanner (Afymetrix GMS 418 chip scanner), scanning excitation light wavelength 532 nm, emission light wavelength 570 nm, laser intensity 35/50-55/70, read signal processing software (JAGUAR) II) Processing, and then taking the average to get the result.
  • (2) Test of the flow chip Heat the appropriately diluted test sample to 37 ° C, add the flow rate to the chip reactor at a flow rate of 10-50 ⁇ l / ⁇ ⁇ , add the sample time for 60 minutes, then add the wash solution, then add 5-10 ⁇ 1. The labeled label is labeled, finally washed, dried, and scanned in the same manner as the test for the non-flowing chip.
  • the nanostructured chip of the present invention is compared with the control conventional chip and the control nanostructure chip, and the same positive sample is used at a preferred functional reagent concentration (for example, a functional reagent concentration of 0.1-1.Omg/ml when spotting).
  • a preferred functional reagent concentration for example, a functional reagent concentration of 0.1-1.Omg/ml when spotting.
  • the average signal readings under the same scanning conditions were 200% higher and 100% higher, respectively.
  • the nanostructured chip containing the synthetic peptide-based or synthetic peptide-derived reactive group of the present invention has an average signal reading of more than 150% as compared with the other nanostructured chips of the present invention.
  • the results demonstrate that the nanochip of the present invention has a higher sensitivity.
  • the nanostructured chip of the present invention is detectable in comparison with a control conventional chip and a control nanostructure chip at a preferred functional reagent concentration (for example, a functional reagent concentration of 0.1-1.0 mg/ml when spotting).
  • the minimum concentration of positive samples was 10 times and 3 times lower, respectively.
  • the nanostructured chip containing the synthetic peptide-based or synthetic peptide derivative-based reactive group of the present invention has a measurable minimum of more than one time as compared with the other nanostructured chips of the present invention.
  • the measurable minimum of the positive sample is represented by dilution of the positive sample to a critical concentration of negative and positive. The results demonstrate that the nanochip of the present invention has higher sensitivity.
  • the nanostructured chip of the present invention has a lower average signal reduction rate under the same scanning condition when the same positive sample is used, after being placed at 37 ° C for 71 hours, compared with the control conventional chip and the control nanostructure chip. 50% and more than 20%. The results show that the nanochip of the present invention has higher stability.
  • the ELISA plates used are the nanostructure ELISA plate and the control nanostructure ELISA plate of the present invention, respectively.
  • the nanostructured enzyme label of the present invention used is the above embodiment 5.2 system Prepared nanostructured microplate; control nanostructured microplate used in amino-containing coupled nano-convex/enzyme plate base (refer to Example 2, for example, 3-aminopropyltrimethoxysilane) Coupling nano-protrusion/enzyme plate prepared by a coupling agent), prepared by coating the same functional reagent under the same coating conditions as the nanostructured microplate of the present invention (refer to the above Example 4.1), Contains no functionalized nanostructures of the invention.
  • the test method is the same as the classical ELISA method, for example, the appropriately diluted test sample ⁇ is separately added to the corresponding 96-well microtiter plate, reacted at 37 ° C for 0.5-1 hour, and then washed by washing solution 3 After the reaction (300 ⁇ l each time), the ⁇ label was added, and the reaction was carried out at 37 ° C for 30 minutes, and then the substrate was added, and after the reaction, colorimetric analysis was carried out using a microplate reader (Thermo Labsystems, Shanghai Leibo Analytical Instruments Co., Ltd.).
  • the nanostructured enzyme plate of the present invention is compared with the control nanostructured plate, and the same positive concentration is used at a preferred functional reagent concentration (for example, a functional reagent concentration of 0.05-0. ⁇ g/ml when coated).
  • a preferred functional reagent concentration for example, a functional reagent concentration of 0.05-0. ⁇ g/ml when coated.
  • the average signal reading of the sample under the same scanning conditions is higher than 150%, the lowest concentration of the positive sample that can be detected is more than 1 time lower, and the average reduction rate of the signal value of the same positive sample after being placed at 37 ° C for 71 hours. It should be 20% lower.
  • the results demonstrate that the nanoenzyme plate of the present invention has higher sensitivity and stability.
  • the fast test reagent strips used are the nanostructure quick test reagent strips and the control nanostructure quick test reagent strips of the present invention, respectively.
  • the nanostructure quick test reagent strip of the present invention used is the nanostructure quick test reagent strip prepared in the above embodiment 5.3
  • the control nanostructure quick test reagent strip used has an active group in the activated nanostructure as an amino group, for example, 3 -Aminopropyltrimethoxysilane is a coupled nanoparticle prepared by a coupling agent and combined with a functional reagent, and then spotted onto a membrane (for example, a nitrocellulose membrane strip) to form a nanostructure rapid detection reagent strip.
  • the test method is the same as the known quick test strip detection method.
  • the appropriately diluted sample is separately added to the above-mentioned quick test strip, and then the washing liquid is added, so that the test strip is slowly sucked to the quality control line.
  • the nanostructure quick test reagent strip of the invention has a minimum concentration of the positive sample which can be detected more than one time, and the signal value of the same positive sample does not decrease after being placed at 37 ° C for 71 hours. The results demonstrate that the nanostructured quick test strip of the present invention has higher sensitivity and stability.
  • Example 10.1 Application of the Nanoaffinity Chromatography System of the Invention
  • the affinity chromatography system used is a column containing nanostructured affinity chromatography particles.
  • the nanostructured affinity chromatography particles used were the nanostructured affinity chromatography particles of the present invention prepared in the above Examples 4.1-4.3, and the control nanostructure affinity chromatography particles, respectively.
  • the active group in the activated nanostructure of the control nanostructure affinity chromatography particle is an amino group, for example, the coupled nanoparticle-binding functional reagent prepared by using 3-aminopropyltrimethoxysilane as a coupling agent is combined with conventional A nanostructure quick test reagent strip formed on the chromatographic particles (the basic preparation method is referred to the above related examples).
  • the test method is the same as the known affinity chromatography detection method.
  • the present embodiment detects the nano-affinity chromatography system prepared by the above examples (for example, containing an affinity reagent/activated nano-convex/silica gel particles and an affinity reagent/activated nano-convex/polysaccharide-containing particles, etc., respectively).
  • the column's kinetic adsorption capacity was tested as follows: the column for filling the above medium was 0.5 cm in inner diameter and 2 cm in length, the buffer was 0.01 M Tris-HCl/pH 7.40, the flow rate was 1 ml/min, and the chromatograph used was HP 1090.
  • the affinity reagent is protein A
  • the sample used is a human antibody.
  • the nano-affinity chromatography system prepared in the above example is compared with the control nano-affinity chromatography system (containing the control nanostructure affinity chromatography particles), the kinetic adsorption The capacity is 30% higher.
  • Example 10.2 Application of the functionalized nanomagnetic separation system of the present invention
  • the functionalized nanomagnetic separation system used is the functionalized nanomagnetic separation system of the present invention (e.g., Bl-B3 in Table 1) prepared in the above Example 6.2, and the comparative functionalized nanomagnetic separation system.
  • the control functionalized nanomagnetic separation system contains the same paired affinity magnetic particles (refer to Example 6.2 above), but the reactive group in the activated nanostructure in the functionalized affinity nanostructure is an amino group, such as 3-aminopropyl.
  • the trimethoxysilane is a coupled nanostructure-binding functional reagent prepared by a coupling agent, and then combined with the functionalized nanostructure formed by the aforementioned functionalizing reagent (the basic preparation method refers to the above related embodiment:).
  • the separation method is the same as the separation method of the known functionalized nanomagnetic separation system.
  • Functionalized affinity nanostructures (particles or/and beading) in the above functionalized nanomagnetic separation system eg: hepatitis B surface antibody/activated nanoparticles, HIV antigen/activated nanoparticles, HCV antigen/activated nanoparticles
  • Hepatitis B surface antibody/activated nanobeads, HIV antigen/activated nanobeads, HCV antigen/activated nanobeads respectively
  • targets eg, human hepatitis B surface antigen, HIV antibody, HCV antibody
  • the target/functionalized nanoparticle composite is reacted under effective conditions, and then the sample containing the target/functionalized nanostructure composite is reacted with the affinity magnetic particles paired above to generate a target/functionalization under effective conditions.
  • Nanostructure/affinity magnetic particle composite and then use external magnetic field to target/work
  • the energized nanostructure/affinity magnetic particle composite is separated, washed with an appropriate buffer if necessary, and then qualitatively or quantitatively analyzed for the target in the target/functionalized nanostructure/affinitive magnetic particle composite.
  • the functionalized nano magnetic separation system of the invention has a separation efficiency more than one time higher than that of the control functionalized nano magnetic separation system (for example, the lowest concentration of the detectable positive sample is more than one time lower), and 37 ° C
  • the separation efficiency reduction rate was lower by 30% after being placed for 71 hours.
  • the results demonstrate that the functionalized nanomagnetic separation system of the present invention has higher sensitivity and stability.
  • Devices or kits containing the labeling system of the present invention can be used in accordance with the application of known corresponding devices or kits.
  • the labeling system of the present invention e.g., C1-C4 in Table 1
  • devices or kits containing the labeling system of the present invention can be used in accordance with the application of known corresponding devices or kits.
  • only some comparative studies are given to illustrate their application.
  • a more specific comparative research method is supplemented by the following examples.
  • Example 11.1 Application of the analytical chip marking system of the present invention
  • the kit used was selected from Cl-C3 in Table 1.
  • the analytical chip analysis method used was basically the same as that of the analytical chip used in the above Example 9.1, except that the chip used was a conventional chip in Table 1, and the marking system used was a nano-marking system.
  • the nanomarkers used were the nanomarkers and control nanomarkers of the present invention prepared in the above Example 7.
  • the control nanomarker contains the same labeling functionalizing agent and labeling substance (refer to Example 7 above), but wherein the reactive group in the activated nanostructure is an amino group, for example, 3-aminopropyltrimethoxysilane is coupled Coated nanostructures prepared by the agent.
  • the average signal reading under the same scanning condition is more than 150% when the same positive sample is used, and the lowest concentration of the detectable positive sample is 150% lower.
  • the average reduction rate of the signal value of the same positive sample after being left at 37 ° C for 71 hours was 25% or more lower.
  • the kit used was selected from C4 in Table 1.
  • the Elisa analysis method used was basically the same as the Elisa analysis method used in the above Example 9.2, except that the ELISA plate used was the conventional chip in Table 1, and the labeling system used was a nano-labeling system.
  • the nanomarkers used were the nanomarkers of the present invention and the control nanomarkers prepared in the above Example 7.
  • the control nanomarker contains the same labeling functionalizing agent and labeling substance (refer to Example 7 above), but wherein the reactive group in the activated nanostructure is an amino group, for example, 3-aminopropyltrimethoxysilane is coupled Coated nanostructures prepared by the agent.
  • the nanomarker of the present invention is compared with the control nanomarker, and the phase is used.
  • the average signal reading under the same colorimetric condition is more than 150% higher than the positive sample, and the lowest concentration of the positive sample that can be detected is lower than 150%, and the signal value of the same positive sample is reduced after 71 hours at 37 °C. The rate is lower by 25%.
  • the results demonstrate that the nanomarkers of the present invention have higher sensitivity and stability.
  • Example 12 Application of a kit containing a plurality of systems of the invention
  • a device or kit e.g., Table 2, Table 3 containing a plurality of systems of the present invention (nano-reaction system, nano-labeling system, nano-separation system) can be applied in accordance with the application of a known corresponding device or kit.
  • a device or kit e.g., Table 2, Table 3
  • nano-reaction system nano-labeling system
  • nano-separation system nano-separation system
  • Example 12.1 Application of a kit containing the nanoreaction system and nanolabeling system of the present invention
  • the analytical method used was substantially the same as that used in the above Examples 9.1 and 9.2, except for the kit used.
  • the reaction system is a nano-reaction system and the labeling system used is a nano-labeling system.
  • the kit of the invention is selected from D1-D4 in Table 2; the control kit used contains the control nanoreaction system described in Examples 9.1 and 9.2 and the control nanolabeling system described in Examples 11.1 and 11.2 .
  • the use of the kit of the invention is compared to the use of a control kit: when using the same positive sample, the average signal reading under the same colorimetric conditions is more than 130% higher, and the lowest detectable positive sample is 120 lower. Above %, the average reduction rate of the signal value of the same positive sample after being placed at 37 ° C for 71 hours was lower by 15% or more.
  • the results demonstrate that the kit of the present invention has higher sensitivity and stability.
  • Example 12.2 Application of kit containing the nanoseparation system and nanolabeling system of the present invention In the present example, the analytical method used was substantially the same as that used in the above Examples 9.1 and 10.2, except for the kit used.
  • the reaction system is a conventional chip
  • the label used is a nano-marker, and also contains a functionalized nano-magnetic separation system.
  • the kit of the invention is selected from E1-E3 in Table 2
  • the control kit used contains the control nanolabel described in Example 11.1 and the control functionalized nanomagnetic separation system described in Example 10.2.
  • the use of the kit of the invention compared to the use of a control kit when using the same positive sample, the average signal reading under the same colorimetric conditions is significantly improved, the lowest concentration of the detectable positive sample is significantly reduced, and 37 ⁇ is placed The average reduction rate of the signal values of the same positive samples after 71 hours was also low.
  • Example 12.3 Application of kit containing the nano-separation system and nano-reaction system of the present invention
  • the analysis method used was basically the same as that used in the above Examples 9.1 and 10.2, except for the kit used.
  • the chip kit used in this embodiment the reaction system is a nanochip, and the label used is a conventional label, and also contains a functionalized nano magnetic separation system.
  • the kit of the invention is selected from F1-F3 in Table 2; the control kit used contains the control nanochip described in Example 9.1 and the control functionalized nanomagnetic separation system described in Example 10.2.
  • the use of the kit of the invention compared to the use of a control kit when using the same positive sample, the average signal reading under the same colorimetric conditions is significantly improved, the lowest concentration of the detectable positive sample is significantly reduced, and 37 ⁇ is placed The average reduction rate of the signal values of the same positive samples after 71 hours was also low.
  • the results demonstrate that the kit of the present invention has higher sensitivity and stability.
  • Example 12.4 Application of a kit containing the nano-separation system, nano-reaction system and nano-labeling system of the present invention
  • the analysis method used was basically the same as that used in the above Examples 9.1 and 10.2, except for the kit used.
  • the reaction system is a nanochip
  • the label used is a nano-marker, and also contains a functionalized nano-magnetic separation system.
  • the kit of the invention is selected from Table 3; the control kit used contains the control nanochip described in Example 9.1, the control functionalized nanomagnetic separation system described in Example 10.2, and the control nanoparticle described in Example 11U. Mark.
  • the use of the kit of the invention compared to the use of a control kit the average signal reading under the same colorimetric conditions is significantly improved when the same positive sample is used, and the minimum concentration of the detectable positive sample is significantly reduced, while 37°
  • the average decrease rate of the signal value of the same positive sample after C was placed for 71 hours was also low.
  • the results demonstrate that the kit of the present invention has higher sensitivity and stability.

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Abstract

Composition de séparation et/ou d'analyse à nanostructure active, laquelle comprend au moins une nanostructure et une structure active liée en covalence à la nanostructure. La structure active comprend au moins des groupes actifs à combiner avec un réactif fonctionnalisé. Ces groupes actifs comprennent des groupes polyfonctionnels à groupes amino et/ou groupes dérivés correspondants, sur la base d'un réactif de synthèse polypeptidique. La nanostructure active améliore l'efficacité de réaction de la nanostructure (par exemple, la sensibilité) et/ou la stabilité du réactif fonctionnalisé immobilisé sur la nanostructure.
PCT/CN2006/001374 2006-01-25 2006-06-16 Composition de séparation ou d'analyse à nanostructure active et procédé de séparation ou d'analyse WO2007115444A1 (fr)

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