WO2007115444A1 - Seperation or analysis composition comprising active nanostructure and seperation or analysis method - Google Patents

Seperation or analysis composition comprising active nanostructure and seperation or analysis method 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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Prior art keywords
nanostructure
activated
group
functionalized
composition
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PCT/CN2006/001374
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French (fr)
Chinese (zh)
Inventor
Fanglin Zou
Chunsheng Chen
Jianxia Wang
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Chengdu Kuachang Medical Industrial Limited
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Application filed by Chengdu Kuachang Medical Industrial Limited filed Critical Chengdu Kuachang Medical Industrial Limited
Priority to PCT/CN2006/002659 priority Critical patent/WO2007085156A1/en
Publication of WO2007115444A1 publication Critical patent/WO2007115444A1/en

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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.

Abstract

A separation or/and analysis composition comprising active nanostructure is provided. The active nanostructure at least includes a nanostructure and an active structure covalently bonded onto said nanostructure. The active structure at least includes active groups to combine with funcionalized reagent. The active groups include polyfunctional groups comprising amino or/and their derivative groups based on polypeptide synthesis reagent. The active nanostructure improves the reaction efficiency of nanostructure( for example sensibility) or /and the stability of functionalized reagent immobilized on the nanostructure.

Description

含活化纳米结构的分离或分析组成及相关分离或分析方法 技术领域  Separation or analytical composition of activated nanostructures and related separation or analysis methods
本发明涉及一种含活化纳米结构的分离或分析组成， 以及一种与本 发明的组成物相关的分离或分析方法。  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.
背景技术 Background technique
本发明是国际专利申请 PCT/CN2004/000437的继续。  The present invention is a continuation of International Patent Application PCT/CN2004/000437.
含纳米结构的分离或分析组成的开发及其应用， 已经公开了越来越 多的方案， 例如： 美国专利申请 20030207296、 美国专利 6025202、 国际 专利申请 WO 0183825、 国际专利申请 WO 00/72018 Al、 美国专利申请 20030211488、 美国专利申请 20030232388、 美国专利申请 20030166297、 美国专利申请 20020142480、美国专利 6986989， 以及下述文献： Brust et al., Adv. Mater., 7， 795-797 (1995), Bain & Whitesides, Angew. Chem. Int. Ed. Engl., 28， 506-512 (1989) and Dubois & Nuzzo, Annu. Rev. Phys. Chem., 43， 437-464 (1992) 0 The development of separation or analytical compositions containing nanostructures and their use has been disclosed in an increasing number of schemes, for example: US Patent Application No. 20030207296, US Patent 60,025,202, International Patent Application WO 0183825, International Patent Application WO 00/72018 Al, U.S. Patent Application No. 20030211488, U.S. Patent Application No. 2,030, 232, 388, U.S. Patent Application No. 2,030, 166, 297, U.S. Patent Application No. 2,001, 024, 480, U.S. Patent No. 6,986, 989, and the following documents: Brust et al., Adv. Mater., 7, 795-797 (1995), Bain & Whitesides, Angew. Chem. Int. Ed. Engl., 28, 506-512 (1989) and Dubois & Nuzzo, Annu. Rev. Phys. Chem., 43, 437-464 (1992) 0
然而， 目前的纳米结构化学， 仍局限于有限的几种活化结构之内， 所制成的功能纳米结构， 在反应效率 (例如灵敏度)和稳定性 (尤其是生物 稳定性)方面， 都有尚待改进之处。  However, current nanostructure chemistry is still limited to a limited number of activated structures, and the functional nanostructures produced are still in terms of reaction efficiency (eg sensitivity) and stability (especially biostability). What to improve.
发明内容 Summary of the invention
本发明的主要目的， 是提高功能化纳米结构的反应效率包括灵敏度 或 /和功能稳定性。  The primary object of the present invention is to increase the efficiency of the functionalized nanostructures including sensitivity or/and functional stability.
本发明的第一个方面，提供一种用于分离或分析的组成， 它包含有活 化纳米结构， 该活化纳米结构至少包含有纳米结构和共价键合在该纳米 结构上的活化结构， 所述活化结构至少包含有用以结合功能试剂的活化 基团，所述活化基团包括有基于多肽合成试剂的含氨基的多官能团基团、 或 /和其衍生物基团。  In a first aspect of the invention, there is provided 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.
本发明的第二个方面， 提供一种用于分离或分析的组成， 它包含有 功能化纳米结构， 该功能化纳米结构至少包含有活化纳米结构和固定在 该活化纳米结构上的功能试剂， 其中所述活化纳米结构至少包含有纳米 结构和共价键合在该纳米结构上的活化结构， 所述该活化结构至少包含 有用以结合功能试剂的活化基团， 所述该活化基团包括有基于多肽合成 试剂的含氨基的多官能团基团、 或 /和其衍生物基团。 本发明的第三个方面， 提供一种用于分离或分析的组成， 它包含有 活化纳米结构载体， 该活化纳米结构载体至少包含有常规载体和活化纳 米结构， 所述该活化纳米结构至少包含有纳米结构和共价键合在该纳米 结构上的活化结构； 所述该活化结构至少包含有用以结合功能试剂的活 化基团， 所述该活化基团包括有基于多肽合成试剂的含氨基的多官能团 基团、 或 /和其衍生物基团。 In a second aspect of the invention, there is provided 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, Wherein 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. In a third aspect of the invention, there is provided 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.
本发明的第四个方面， 提供一种用于分离或分析的组成， 它包含功 能化纳米结构载体， 该功能化纳米结构载体至少包含有常规载体和功能 化纳米结构， 所述该功能化纳米结构至少包含活化纳米结构和固定在该 活化纳米结构上的功能试剂， 其中所述活化纳米结构至少包含纳米结构 和共价键合在所述纳米结构上的活化结构， 所述该活化结构至少包含有 用以结合功能试剂的活化基团， 所述该活化基团包括有基于多肽合成试 剂的含氨基的多官能团基团、 或 /和其衍生物基团。  In a fourth aspect of the invention, there is provided 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.
本发明的第五个方面， 提供一种含活化纳米结构的分离或分析组成 方法， 它包括提供和用于确定本发明第一至第四个方面分离或分析组成 所用的步骤。  In a fifth aspect of the invention, there is provided 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.
下面提供帮助理解本发明内容的一些术语的定义：  The definitions of some terms that help understand the context of the present invention are provided below:
本发明中所述的"分离或分析组成"， 是指用于分离或分析过程的组 成， 例如装置或试剂盒、 或它们中的一种成份； "分析 "是指在体外或体 内进行的定性或 /和定量分析； "分离"是指一种或多种组分与样品的其它 组分分离； "装置"是指具有特定功能的用品， 例如含有功能试剂的仪器， 例如： 分析芯片、 酶标板、 亲和电泳条、 亲和层析柱、平面层析试剂条、 等等。  The term "isolated or analytical composition" as used in the present invention 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.
本发明中所述的"纳米结构"， 是指在三维结构中至少有一维为纳米 尺寸的结构， 例如粒径、 管径、 线径、 等等为纳米尺寸的结构， 其优选 尺寸为 3nm-300nm; "纳米粒子"是指粒径为纳米尺寸的粒子， 优选粒径 为 3nm-300nm的粒子； "纳米串珠"是指一个以上纳米粒子以有机基团共 价连接的纳米结构复合物； "纳米凸体"是指非纳米材料表面上的任何纳 米结构， 例如： 固定化纳米粒子、 固定化纳米管、 固定化纳米纤维、 及 纳米粒子在载体上的自组装形成的纳米结构等等。 The "nanostructure" as used in the present invention 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.
本发明中所述"偶联基团"， 是指结合于纳米结构表面上、 用以固定 活化基团的基团， 例如硅垸偶联剂与纳米结构表面上羟基反应的共价键 合； "偶联化纳米结构"是指这样一种组成： 它含有纳米结构和在该纳米 结构上固定的偶联基团。 The term "coupling group" as used in the present invention refers to a group bonded to a surface of a nanostructure to immobilize an activating group, such as a covalent bond of a silicon germanium coupling agent to a hydroxyl group on a surface of a nanostructure; "Coupled nanostructure" refers to a composition that contains nanostructures and A structurally fixed coupling group.
本发明中所述 "活化结构"是指这样一种组成： 它至少含有活化基团， 还可以含有偶联基团； "活化基团"是指用以提供与功能试剂结合的基团， 例如提供氨基、羧基等的基团如氨基酸， 或复合基团例如氨基酸衍生物、 合成肽基、 合成肽衍生物基； "活化纳米结构"是指这样一种组成： 它含 有纳米结构和在其上固定的活化结构； "活化纳米粒子"是指这样一种组 成： 它含纳米粒子和在其上固定的活化结构； "活化纳米凸体"是指这样 一种组成： 它含有纳米凸体和在其上固定的活化结构； "活化纳米串珠" 是指这样一种组成： 它含有纳米串珠和在其上固定的活化结构。  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.
本发明中所述"功能试剂"， 是指为赋予所述纳米结构以反应活性的 试剂， 例如与目标物反应活性的试剂。 功能试剂通过相互作用包括亲和 作用、 离子交换、 亲油作用等捕获目标物， 它包括配基 （相当于英语中 的 Ligand)、离子交换剂等。 离子交换剂包括： 二乙氨乙基（DEAE)、 二 乙基一 （2—羟丙基） 氨乙基 （QAE)、 羧甲基 （CM)、 磺酸丙基 （SP)、 巯乙基吡啶基 （MEP)、 一 NH2、 一 RCOOH、 硅氧烷基、 硫醇基、 烷基。 配基包括： 抗原、 抗体、 配体、 配体指数增强***进化技术筛选的适配 分子、 多肽、 多糖、 共酶、 辅因子、 抗生素、 类固醇、 病毒、 细胞等。 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.
本发明中所述 "功能化纳米结构"是指这样一种组成： 它含有纳米结 构和在其上固定的功能试剂； "功能化纳米粒子"是指这样一种组成： 它 含有纳米粒子和在其上固定的功能试剂； "功能化纳米凸体"是指这样一 种组成： 它含有纳米凸体和在其上固定的功能试剂； "功能化纳米串珠" 是指这样一种组成： 它含有纳米串珠和在其上固定的功能试剂。  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.
本发明中所述"活化纳米结构载体"是指这样一种组成： 它至少含有 活化纳米结构例如活化纳米凸体和常规载体； "功能化纳米结构载体"是 指这样一种组成： 它至少含有功能化纳米结构例如功能化纳米凸体和常 规载体； "片基"是指具有固定功能、 其一面具有宏观平面的常规载体， 例如分析芯片片基、 酶标板片基、 电泳胶片、 平面层析载体等。  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.
本发明中所述"分析芯片"， 简称为 "芯片"， 包括但不限于英语中的 Biochip、 Microarray、 Bioarray, 是指定性和 /或定量分析中的一种检测装 置， 其反应器中微量功能试剂同样品中的目标分子发生反应的结果， 可 以以可寻址的方式进行识别;"纳米结构芯片"为至少含有一个纳米结构活 性区 （例如功能试剂微阵列中的一个样点） 的芯片。 一个芯片可以有多 个反应器， 一个反应器可以有多个含活性试剂的样点 （功能试剂点）， 只 要其中有一个样点为本发明的纳米结构活性区， 则芯片为本发明的纳米 结构活性载体或纳米结构芯片。 The "analytical chip" described in the present invention, abbreviated as "chip", including but not limited to Biochip, Microarray, Bioarray in English, 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.
本发明中所述 "层析 "相当于英语 "Chromatogmphy", 包括亲和层析、 反相层析、 疏水层析、 离子交换层析等等， 它分为平面层析 （例如快检 试剂条和快检试剂盒） 和柱层析等。  The "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.
本发明中所述 "多肽 "相当于英语中的" polypeptide", 包括天然或合成 蛋白质、 蛋白质片断、 合成肽等等， 免疫检测中通常的目标物和检测中 通用的配基， 例如抗原、 抗体等等都属于多肽;"分子标记物质 "是指用以 形成或参与形成检出信号、 并在标记时具有分子形态的物质， 例如芯片 检测常用标记物中的罗丹明、 CY3、 CY5等。  The "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.
如前面 "发明内容"部分所述， 本发明第一至第四方面的分析或分离 组成都包含活化纳米结构， 所述活化结构至少包含有用以结合功能试剂 的活化基团， 所述活化基团包括基于多肽合成试剂的含氨基的多官能团 基团、 或 /和其衍生物基团。  As described in the "Summary of the Invention" section above, 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.
在根据本发明第一至第四方面的分析或分离组成的所述活化纳米结 构中： 所述活化基团包括氨基肼基团或 /和氨基肼衍生物基团； 所述活化 基团包括氨基酸基团或 /和氨基酸衍生物基团； 所述活化基团包括合成肽 基团或 /和合成肽衍生物基团。 实际上，本发明源于本发明实施例的出人 意料的结果： 通常用作钝化剂的氨基酸， 居然可以提供反应基团、 特别 是活化基团。 本发明实施例中： 所述氨基酸基团包括精氨酸基、 天冬酰 氨基、 谷氨酰氨基、 甘氨酸基、 赖氨酸基、 谷氨酰胺基； 所述合成肽基 中的氨基酸数目大于或等于 2(例如 2-5)，其中的氨基酸种类相同 (例如精 氨酸、 天冬酰氨、 谷氨酰氨等等形成的单氨基酸肽基)或不同 (例如精氨 酸和天冬酰氨、 天冬酰氨和甘氨酸、 谷氨酰氨和赖氨酸等等形成的多氨 基酸肽基)。 实施例中， 活化基团一般由活化剂提供。 所用活化剂， 包括 可至少提供与偶联基团结合的部分活化基团的基础活化剂， 以及在活化 基团不仅由基础活化剂提供的基团组成时 (例如衍生物基)提供活化基团 其它部分的第二活化剂。 上述肽合成试剂 (reagent for peptide synthesis), 例如肽连接剂 (peptide coupling reagent)和含氨基酸基的肽合成试剂，既可 用作基础活化剂又可用作第二活化剂。 实施例中， 所述肽合成试剂优选 多官能团试剂， 更优选含有氨基 (-^1 或/和羧基 (-COOH)的多官能团试 剂。 此外， 还优选含有肽合成保护基团 (例如 Fmoc)的肽连接剂和含氨基 酸基的肽合成试剂， 例如 Fmoc-氨基肼和 Fmoc-氨基酸。 在根据本发明第一至第四方面的分析或分离组成的所述活化纳米结 构中： 所述活化结构还含有连接所述纳米结构和活化基团的偶联基团， 所述偶联基团包括硅烷基团。 本发明实施例中， 所用硅烷偶联剂包括：In the activated nanostructure consisting of the analysis or separation according to the first to fourth aspects of the invention: 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. In fact, the present invention stems from the surprising results of the examples of the present invention: Amino acids commonly used as passivating agents can provide reactive groups, particularly activating groups. In an embodiment of the invention, 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.). In embodiments, 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. In the embodiment, 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. In the activated nanostructure composed of the analysis or separation according to the first to fourth aspects of the invention: the activation structure further contains a coupling group linking the nanostructure and an activating group, the coupling group Includes silane groups. In the embodiment of the invention, the silane coupling agent used comprises:
3-氨丙基三甲氧基硅烷、氨丙基三乙氧基硅烷、 3-异氰酸酯丙基三乙氧基 娃烧。 3-aminopropyltrimethoxysilane, aminopropyltriethoxysilane, 3-isocyanatepropyltriethoxysilane.
在根据本发明第一至第四方面的分析或分离组成中， 所述活化纳米 结构中， 所述纳米结构包括含有无机或 /和有机材料 (例如塑料、 多糖、乳 胶、 树脂)的纳米结构。 所述无机材料包括非磁性无机材料和磁性无机材 料。所述非磁性无机材料包括金属材料 (例如金、钒、铅)和非金属无机材 料。 所述非金属无机材料包括无机氧化物。 本发明实施例中， 所用无机 氧化物包括硅氧化物、 铝氧化物、 钛氧化物。  In the analysis or separation composition according to the first to fourth aspects of the invention, in the activated nanostructure, the 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. In the examples of the present invention, the inorganic oxide used includes silicon oxide, aluminum oxide, and titanium oxide.
在根据本发明第一至第四方面的分析或分离组成的所述活化纳米结 构中， 所述纳米结构包括： 纳米粒子、 纳米串珠、 纳米凸体。  In the activated nanostructure composed of the analysis or separation according to the first to fourth aspects of the present invention, the nanostructures include: nanoparticles, nanobeads, nanoprotrusions.
在根据本发明第一方面的分析或分离组成中， 所述活化纳米结构包 括所述纳米结构和所述活化结构， 它们分别为： 活化纳米粒子、 活化纳 米串珠、 活化纳米凸体。 在本发明实施例中， 本发明第一方面的分析或 分离组成包括： 活化纳米粒子、 活化纳米串珠、 活化纳米凸体。 本发明 中的一些方法， 还可用来生成纳米结构， 例如用于计算机、 手机、 微芯 片卡等装置中的纳米结构。  In the analysis or separation composition according to the first aspect of the present invention, the activated nanostructures include the nanostructures and the activated structures, which are: activated nanoparticles, activated nanobeads, activated nanoprotrusions, respectively. In an embodiment of the invention, the analytical or separation composition of the first aspect of the invention comprises: activating nanoparticles, activating nanobeads, and activating nano-protrusions. Some of the methods of the present invention can also be used to generate nanostructures, such as nanostructures for use in devices such as computers, cell phones, microchip cards, and the like.
在根据本发明第二方面的分析或分离组成中， 所述功能化纳米结构 包含所述纳米结构、 所述活化结构和功能试剂， 它们分别为： 功能化纳 米粒子、 功能化纳米串珠、 功能化纳米凸体。 在本发明实施例中， 本发 明第二方面的分析或分离组成包括： 功能化纳米粒子、 功能化纳米串珠、 功能化纳米凸体。 本发明第二方面的分析或分离组成还包括含上述功能 化纳米结构的组合的组成， 例如一种以上的多种所述功能化纳米粒子的 复合物、 一种以上的多种所述功能化纳米粒子组成的***等等。  In an analytical or separation composition according to the second aspect of the present invention, the functionalized nanostructure comprises the nanostructure, the activated structure and a functional reagent, respectively: functionalized nanoparticles, functionalized nanobeads, functionalized Nano convex body. In an embodiment of the invention, 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.
在根据本发明第二或第四方面的分析或分离组成的所述功能化纳米 结构中， 所述功能试剂包括任何可固定在所述活化基团上、 而又不丧失 其功能的物质， 例如核酸或 /和多肽。 本发明实施例中， 所用多肽包括： 抗原、 抗体、 及其它配基。 其中， 所用抗原包括： EBV— VCA— P18抗 原、 丙肝病毒抗原 （HCVAg)、 艾滋病病毒抗原 （HIVAg)、 梅毒抗原； 所用抗体包括抗乙肝病毒表面抗体（HBs Ab)、单克隆或多克隆二抗； 所 用其它配基包括蛋白质入。 在根据本发明第三或第四方面的分析或分离组成的活化纳米结构载 体中， 所述常规载体包括由以下之一组或多组材料或其衍生物制成的、 至少二维尺寸大于 lOOOnm的载体： 玻璃、 硅片、 硅胶、 陶瓷、 金属氧 化物、 金属、 聚合物材料及它们的复合物。 所述常规载体还包括常规载 体衍生物。 所述衍生物包括结合有表面基团或 /和包被有机物的衍生物。 在本发明实施例中，所述常规载体包括下述载体之一：粒状常规载体（例 如层析凝胶、特别是微米粒子层析凝胶)，面状常规载体（例如生物芯片、 酶标板等的片基） 和膜状常规载体（例如平面层析条）。 In the functionalized nanostructure consisting of the analytical or isolated composition according to the second or fourth aspect of the present invention, 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. In the embodiments of the present invention, 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. In the activated nanostructure carrier of the analysis or separation composition according to the third or fourth aspect of the present invention, 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. In an embodiment of the invention, 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).
在根据本发明第三方面的分析或分离组成中， 所述活化纳米结构载 体包括活化纳米凸体载体。 所述活化纳米凸体载体包含所述活化纳米凸 体和所述常规载体， 例如： 活化纳米结构载体、 活化纳米粒子 /片基复合 物、 活化纳米粒子 /微米粒子复合物、 活化纳米粒子 /微米粒子 /片基复合 物等等。 本发明实施例中， 本发明第三方面的分析或分离组成包括下述 任一组： 分析芯片纳米结构片基、 纳米结构酶标、 微孔板、 平面层析纳 米结构片基、纳米结构活化层析固定相。所述分析芯片纳米结构片基中， 所述活化纳米结构分布在部分或全部片基上。而且，在至少部分片基上， 所述纳米凸体的分布密度大于 1个纳米凸体 /μπι2、 优选大于 5个纳米凸 体 /μιη2。 此外， 本发明中的一些方法， 还可用来生成纳米结构载体， 例 如用于计算机、 手机、 微芯片卡等装置中的纳米结构载体。 In an analytical or isolated composition according to the third aspect of the invention, 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. In an embodiment of the present invention, 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. In the analysis chip nanostructure substrate, 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 / μπι 2 , preferably greater than 5 nanoprotrusions / μιη 2 . In addition, 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.
在根据本发明第四方面的分析或分离组成中， 所述功能化纳米结构 载体包括功能化纳米凸体载体。 所述功能化纳米凸体载体包含所述功能 化纳米凸体和所述常规载体， 例如： 功能化纳米结构载体、 功能化纳米 粒子 /片基复合物、 功能化纳米粒子 /微米粒子复合物、 功能化纳米粒子 / 微米粒子 /片基复合物等等。本发明第四方面的分析或分离组成还包括含 多种上述功能化纳米结构和常规载体的组成， 例如： 一种以上的多种功 能化纳米粒子 /微米粒子复合物、一种以上的多种功能化纳米粒子与一种 或一种以上的多种微米粒子组成的***、 一种以上的多种功能化纳米粒 子与片基的复合物、 一种以上的多种功能化纳米粒子与亲和片基组成的 ***、 一种以上的多种功能试剂与纳米结构片基的复合物等等。  In an analytical or isolated composition according to the fourth aspect of the invention, 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.
在根据本发明第四方面的分析或分离组成中， 功能化纳米结构载体 中的一种或多种纳米结构与常规载体之间可以有一重或多重配基、 或 / 和一种或多种配基与常规载体之间有一重或多重纳米结构、或 /和所述至 少一重纳米结构与另一重纳米结构之间有一重或多重配基。 例如， 下述 方法制备的一种或多种纳米粒子与载体之间有多重配基的纳米结构活性 载体： 分别将载体包被一重配基 1形成配基 1包被载体， 并将纳米粒子 包被一重另一种配基 2 (配基 1和 2之间可发生配对反应）形成配基 2/ 纳米粒子复合物， 再将配基 2/纳米粒子复合物包被或点样至配基 1包被 载体上， 形成配基 2—纳米粒子一配基 2—配基 1一载体形式的复合物。 当配基层数大于 2时以此类推。 一重纳米粒子与另一层纳米粒子之间有 多重配基的纳米结构活性载体有很多种类， 例如配基 3—纳米粒子一配 基 3—配基 2—纳米粒子一配基 2—配基 1一载体。 一种或多种配基与载 体之间有多重纳米粒子的纳米结构活性载体可如下制备： 先将一种或一 种以上所述纳米粒子与多种所述配基结合在一起形成多种活性纳米粒子 (例如配基 2—纳米粒子一配基 2、 配基 3—纳米粒子一配基 2、 配基 1 一纳米粒子一配基 1等），再将这些活性纳米粒子先后或同时结合在所述 载体上， 形成诸如配基 2—纳米粒子一配基 2—配基 1一纳米粒子一配基 1一载体、配基 3—纳米粒子一配基 2—配基 1一纳米粒子一配基 1一载体 等形式的纳米结构活性载体。 In an analytical or isolated composition according to the fourth aspect of the invention, 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. For example, the following 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. Forming a complex of ligand 2 - nanoparticle-ligand 2 - ligand 1 - in the form of a carrier. When the number of base layers is greater than 2, and so on. There are many kinds of 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.), and then these active nanoparticles are combined sequentially or simultaneously On the support, 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 A nanostructured active carrier in the form of a carrier or the like.
本发明第二或第四方面的分析或分离组成， 包括含所述功能化纳米 结构或 /和功能化纳米结构载体的分离***。 本发明实施例中， 所述功能 化纳米结构分别为： 功能化纳米粒子 (例如功能化纳米磁分离***中的功 能化纳米粒子)、功能化纳米串珠 (例如功能化纳米磁分离***中的功能化 纳米串珠)、功能化纳米凸体 (例如纳米亲和层析***中的亲和层析纳米固 定相上的功能化纳米凸体)。  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. In an embodiment of the invention, 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).
本发明第二或第四方面的分析或分离组成， 包括含所述功能化纳米 结构或 /和功能化纳米结构载体的标记***。 本发明实施例中， 所述功能 化纳米结构分别为： 功能化纳米粒子 (例如纳米标记物中的功能化纳米粒 子)、 功能化纳米串珠 (例如纳米标记物中的功能化纳米串珠)。  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. In an embodiment of the invention, the functionalized nanostructures are: functionalized nanoparticles (eg, functionalized nanoparticles in nanomarkers), functionalized nanobeads (eg, functionalized nanobeads in nanomarkers).
本发明第二或第四方面的分析或分离组成， 包括含功能化纳米结构 或 /和功能化纳米结构载体的反应***。 本发明实施例中， 所述功能化纳 米结构为功能化纳米凸体。 本发明第四方面的分析或分离组成， 包括含 所述反应***的装置， 例如传感器、 分析芯片、 ELISA酶标板、 快检试 剂条、 等等。 本发明实施例中， 所述装置包括： 纳米结构分析芯片、 纳 米结构酶标板、 纳米结构平面层析试剂条。 所述纳米结构分析芯片中， 所述活化纳米结构仅分布在部分或全部功能试剂点上。 而且， 在至少部 分含活化纳米结构的所述功能试剂点中，所述纳米凸体的分布密度大于 1 个纳米凸体 /μιη2、 优选大于 5个纳米凸体 /μπι2。 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. In an embodiment of the invention, 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. In the embodiment of the present invention, the device comprises: a nanostructure analysis chip, a nanostructure ELISA plate, and a nanostructure planar chromatography reagent strip. In the nanostructure analysis chip, the activated nanostructures are only distributed on some or all of the functional reagent points. Moreover, in the functional reagent point at least partially containing the activated nanostructure, the nano-protrusion has a distribution density greater than 1 Nanoprotrusions / μιη 2 , preferably greater than 5 nanoprotrusions / μπι 2 .
本发明第二或第四方面的分析或分离组成， 包括含下述之一种、 二 种或三种***的试剂盒： 所述纳米反应***、 所述纳米标记***、 所述 纳米分离***。 本发明实施例中， 所述试剂盒包括下述组之一： 纳米结 构分析芯片试剂盒、 纳米结构酶标板试剂盒、 纳米结构平面层析试剂条 试剂盒。  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. In the embodiment of the present invention, 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.
本发明第一至第四方面的分析或分离组成，其中所述分离或 /和分析 的目标物包括多肽或 /和与多肽相互作用的药物， 或 /和核酸或 /和与核酸 相互作用的药物。  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 .
在根据本发明第五方面的分析或分离方法中， 其包括提供和应用本 发明第一至第四方面的分析或分离组成的步骤。  In the analysis or separation method according to the fifth aspect of the present invention, it comprises the steps of providing and applying the analysis or separation composition of the first to fourth aspects of the invention.
在根据本发明第五方面的分析或分离方法中， 所述分离或分析组成 的提供， 包括提供所述纳米结构， 并将所述活化结构共价固定到所述纳 米结构上形成所述活化纳米结构。 本发明实施例中， 所述活化结构中活 化基团的形成或 /和引入的方法之一， 是合成肽方法。 本发明实施例中， 所述合成肽方法包括下述组之一种或多种步骤： 提供含保护基团的反应 物并在其后的步骤中至少部分脱去所述保护基团； -ΝΗ2基和- COOH基 之间的反应； 肽链增长。 In an analysis or separation method according to the fifth aspect of the present 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. In one embodiment of the invention, one of the methods of forming or/and introducing an activating group in the activated structure is a method of synthesizing a peptide. In an embodiment of the present invention, 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.
具体实施例 Specific embodiment
在以下实施例中， 已知的芯片制备方法、 应用方法参考 Schena, M.， Microarray Analysis , 2003， John Wiley &Sons， Inc., New York：。  In the following examples, known chip preparation methods and methods of application are described in Schena, M., Microarray Analysis, 2003, John Wiley & Sons, Inc., New York:.
实施例 1: 活化纳米结构的制备方法 Example 1: Preparation method of activated nanostructure
在以下所有关于活化纳米结构的制备的实施例中， 所提供用于所述 制备方法的试剂和材料， 除个别情况外， 均可在市场上购得。  In all of the following examples regarding the preparation of activated nanostructures, the reagents and materials provided for the preparation process, unless otherwise available, are commercially available.
1).纳米结构：所用纳米结构可以是任何形态的纳米结构，例如： 纳 米粒子、 纳米串珠、 纳米管、 纳米棒、 载体上的纳米凸体等等。 本发明 实施例优选使用的纳米结构， 为含无机基质的纳米结构 (例如， 无机基质 纳米结构、 包容无机物的纳米结构、 和无机物包被纳米结构)。 它包括： 氧化物纳米粒子、纳米串珠 (自制， 见以下相关实施例)、纳米凸体 (自制， 见以下相关实施例)。所用氧化物纳米粒子包括： 硅氧化物纳米粒子（氧 化硅纳米粒子 LUDOX AS— 40， 粒子平均尺寸 25nm， 比表面积约 135m2/g, Sigma-Aldridi公司）， 铝氧化物纳米粒子 (MC2R γ -相纳米氧 化铝， 粒子平均尺寸 60nm， 比表面积 140 m2/g， 浙江弘晟材料科技股份 有限公司)， 钛氧化物纳米粒子 (氧化钛纳米粒子， 粒子平均尺寸 <80nm， 比表面积 120 m2/g，浙江舟山明日纳米材料有限公司)。其它无机材料 (例 如其它无机氧化物、 金属、 等等)的纳米结构， 也可用于以下实施例的方 法制备活化纳米结构及功能化纳米结构。 有机材料纳米结构， 也可直接 或间接 (例如包被无机质)用于以下实施例的方法制备活化纳米结构及功 能化纳米结构。 1). 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 γ - 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 (e.g., other inorganic oxides, metals, and the like) 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.
2) . 活化剂:所用活化剂， 包括可至少提供与偶联基团结合的活化基团 部分的基础活化剂， 和在活化基团不仅由基础活化剂提供的基团组成时 (例如衍生物基)提供活化基团其它部分的第二活化剂。所用活化剂为肽合 成试齐 (reagent for peptide synthesis ) ，例如月太连接齐 !j (peptide coupling reagent)和含氨基酸基的肽合成试剂。 本发明实施例优选使用含有氨基 (-皿2)或/和羧基 (-COOH)的多官能团试剂。 所用多官能团肽合成试剂包 括氨基肼和氨基酸。 本发明实施例优选使用含有肽合成保护基团 (例如 Fmoc)的肽连接剂和含氨基酸基的肽合成试剂， 例如 Fmoc-氨基肼和 Fmoc-氨基酸。 众所周知， 保护基团对在合成过程中保护基团 (例如氨基 或羧基)活性具有非常重要的作用。 Fmoc氨基肼由成都凯泰新技术有限 责任公司提供， 氨基酸或 Fmoc-氨基酸由成都泰格化工研究所提供， 包 括： 精氨酸、 天冬酰氨、 谷氨酰氨、 甘氨酸、 赖氨酸、 谷氨酰胺。 含其 它保护基团 (：例如 Boc -、 CBZ -、 等等)的肽合成试剂， 也可用于以下实施 例的方法制备活化纳米结构及功能化纳米结构。 所用第二活化剂包括不 含氨基的多官能团试剂 (例如戊二醛、 1， 4-丁二醇二缩水甘油醚)， 和含 氨基的多官能团试剂 (例如上述各种氨基酸)。 2) 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. It is well known that 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., and 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 (e.g., Boc -, CBZ -, etc.) 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).
3) . 偶联剂  3) . Coupling agent
所用偶联剂， 包括有机硅偶联剂,例如硅烷偶联剂。所用硅烷偶联剂 包括： 3-氨丙基三甲氧基硅烷 (国泰华荣化工新材料公司)、氨丙基三乙氧 基硅烷 (国泰华荣化工新材料公司)、 3-异氰酸酯丙基三乙氧基硅垸 (华盛 化学有限公司)。  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.).
在本发明实施例中， 活化纳米结构的基本制备方法包括： 将活化基 团、 偶联基团固定到纳米结构上， 形成纳米结构上固定有偶联基团、 偶 联基团上固定有活化基团的活化纳米结构。 各种纳米结构的更具体的制 备方法由以下实施例补充。  In the embodiment of the present invention, 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.
实施例 1.1: 活化纳米粒子的制备方法 Example 1.1: Preparation method of activated nanoparticles
本发明实施例中,活化纳米粒子的制备方法， 包括两种方法。 T/CN2006/001374 第一种方法的一个例子， 至少包括： （1.1).制备偶联化纳米粒子： 将 纳米粒子与偶联剂溶液混合， 并进行偶联反应。 其反应条件如下： 纳米 粒子浓度 (w/v) l°/oo -2%； 偶联剂浓度 (v/v) 1-3%； 反应介质为含水的醇； 反应温度在室温至反应介质沸点以下 5°C之间； 反应时间 0.5-5小时。 本 专业的技术人员通过调节这些参数可获得所需优化条件。 反应完成后， 对悬浮液离心离出偶联化纳米粒子， 然后保存在 DMF 中。 （1.2).制备活 化纳米粒子： 将上述偶联化纳米粒子与活化剂溶液混合， 并进行活化反 应。 反应条件如下： 纳米粒子的浓度 (w/v) l°/oo-2%; 活化剂浓度 (v/v)在 0.5-5%； 反应温度在室温至反应介质沸点以下 5°C之间； 反应时间 0.5-15 小时； 反应介质为 DMF。 本专业的技术人员通过调节这些参数可获得所 需的优化条件。 若活化剂含保护基团 (例如 Fmoc)， 还要脱去这些保护基 团。 脱保护方法选自已知的肽合成方法中的脱保护方法。 反应完成后， 对悬浮液离心离出活化纳米粒子， 保存在 DMF中。 In the embodiment of the invention, 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. After the reaction is completed, the coupled nanoparticles are centrifuged off the suspension and then stored in DMF. (1.2). 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. Those skilled in the art can obtain the desired optimization conditions by adjusting these parameters. If 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.
第二种方法的一个例子， 至少包括： （2.1).提供活化基团 -偶联基团复 合物：提供活化剂 (基础活化剂或上述基础活化基团-第二活化基团复合物 的活化剂)， 并和硅垸偶联剂（例如 3-异氰酸酯丙基三乙氧基硅垸)反应， 制得活化基团-偶联基团复合物 (例如氨基肼基 -3-异氰酸酯丙基三乙氧基 硅烷。 这类复合物的制备方法为已知方法。 （2.2).制备活化纳米粒子： 如 同本实施例上述 (1.1)中的 "制备偶联化纳米粒子"， 只是在其使用偶联剂 的地方， 这里使用活化基团 -偶联基团复合物。  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. The preparation method of such a complex is a known method. (2.2). Preparation of activated nanoparticles: As in the above example (1.1), "Preparation of coupled nanoparticles" is only used in its use. Where the agent is used, an activating group-coupling group complex is used herein.
本发明中， 以偶联基团 /纳米粒子来表示偶联化纳米粒子。 本发明实 施例制备的偶联化纳米粒子包括硅烷偶联基团 /氧化物纳米粒子。 其中： 硅垸偶联基团包括： 3-氨丙基三甲氧基硅烷基、 氨丙基三乙氧基硅烷基、 3-异氰酸酯丙基三乙氧基硅烷基； 氧化物纳米粒子包括： 氧化硅纳米粒 子、 氧化铝纳米粒子、 氧化钛纳米粒子。 所获偶联化纳米粒子， 可通过 元素分析 (例如（、 H、 N元素分析)、 NMR分析、等等， 计算出纳米粒子 表面上单位面积所固定的偶联基团的密度。 偶联反应参数不同， 上述偶 联基团密度的变化较大， 例如氮含量 (元素分析)在 0.25-0.65 N%之间， 相 当于 lg纳米粒子上固定的偶联基团在 179-464 μπιοΐ之间变动， 或 lm2 纳米粒子表面上固定的偶联基团在 1.3-3.4 μπιοΐ之间变动。优选具有下述 组成特征的偶联化纳米粒子， 用以进行以下实施例中活化纳米结构或亲 和纳米结构的制备： lm2纳米粒子表面上固定的偶联基团大于 1.85 μπιοΚ 优选大于 2.0 μπ >1 、 更优选大于 2.50μιηο1。 本发明中， 以主要活化基团 /偶联化纳米粒子来表示活化纳米粒子。 本发明实施例制备的活化纳米粒子包括： 氨基肼基 /偶联化纳米粒子、 氨 基肼衍生物基 /偶联化纳米粒子、氨基酸基 /偶联化纳米粒子、氨基酸衍生 物基 /偶联化纳米粒子、 合成肽基 /偶联化纳米粒子、 合成肽衍生物基 /偶 联化纳米粒子。 其中:偶联化纳米粒子包括如前所述的两种不同制备方法 制备的活化纳米粒子中所含的偶联基团 /纳米粒子;活化基团包括如前所 述的活化剂提供的基团。 所获活化纳米粒子， 可通过元素分析 (例如 C、 H、 N元素分析)、 NMR分析、 等等， 计算出纳米粒子表面上单位面积所 固定的活化基团的密度。 按所用偶联化纳米粒子、 活化剂和所选活化反 应参数的不同，上述活化基团密度的变化较大 (例如 0.1-2.85μηιΟ1/ηι2纳米 粒子表面)。 优选具有下述组成特征的活化纳米粒子， 用以进行以下实施 例中活化纳米结构或亲和纳米结构的制备：1m2纳米粒子表面上固定的活 化基团大于 0.5μιηο1、 优选大于 1μπιο1、 更优选大于 1.5|amol。 In the present invention, 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. Wherein: 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. Different parameters, 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 μπιοΐ , or the coupling group immobilized on the surface of the lm 2 nanoparticle varies between 1.3 and 3.4 μπιο. Coupling nanoparticles having the following compositional characteristics are preferred for performing the activated nanostructure or affinity nano in the following examples. Preparation of the structure: The coupling group immobilized on the surface of the lm 2 nanoparticles is greater than 1.85 μπιο, preferably greater than 2.0 μπ >1 , more preferably greater than 2.50 μιηο1. In the present invention, 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. Wherein: 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 μηι Ο 1 / ηι 2 nanoparticle surface). Preferred are 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 μm, preferably greater than 1 μπι 1, more preferably More than 1.5|amol.
含不同活化基团的活化纳米粒子的更具体的制备方法， 由以下实施 例 1.1.1-1.1.6补充。  A more specific preparation method of activated nanoparticles containing different activating groups is supplemented by the following Examples 1.1.1-1.1.6.
实施例 1.1.1: 含氨基肼基的活化纳米粒子的制备方法 Example 1.1.1: Preparation method of activated nanoparticle containing aminoguanidine group
以上述实施例 1.1所述第一种制备方法为例， 先对偶联化纳米粒子 上的偶联基团进行羰基化处理， 再以 Fmoc-氨基肼作为所述活化剂进行 上述活化。以上述实施例 1.1所述第二种制备方法为例，提供 Fmoc-氨基 肼， 并和硅烷偶联剂（例如 3-异氰酸酯丙基三乙氧基硅烷)反应， 制得氨 基肼基 -3-异氰酸酯丙基三乙氧基硅烷，再将氨基肼基 -3-异氰酸酯丙基三 乙氧基硅烷固定在纳米粒子上。 本实施例制备的活化纳米粒子包括氨基 肼基 /偶联化纳米粒子。  Taking the first preparation method described in the above Example 1.1 as an example, 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. Taking the second preparation method described in the above embodiment 1.1 as an example, 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.
实施例 1.1.2: 含氨基肼衍生物基的活化纳米粒子的制备方法 Example 1.1.2: Preparation method of activated nanoparticle containing aminoguanidine derivative group
以上述实施例 1.1所述第一种制备方法为例，进行多次活化反应。例 如，先以上述实施例 1.1.1所述方法获得氨基肼基 /偶联化纳米粒子，再以 第二活化剂 (例如戊二醛、 1， 4-丁二醇二缩水甘油醚、 氨基酸或合成肽) 进行第二次活化、 然后分离出第二次活化产物、 等等， 每次活化反应的 条件与上述反应条件相似。 也可以将氨基肼与第二活化剂反应， 制备含 基础活化基团 /衍生活化基团复合物的活化剂， 再将此活化剂用作上述活 化反应的活化剂。 还可以按上述实施例 1.1所述第二种方法制备。  Taking the first preparation method described in the above Example 1.1 as an example, a plurality of activation reactions were carried out. For example, 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. It is also possible to react an aminoguanidine with a second activator to prepare an activator containing a base activating group/derived activating group complex, which is then used as an activator for the above-mentioned activation reaction. It can also be prepared by the second method described in the above Example 1.1.
本实施例制备的活化纳米粒子 (氨基肼衍生物基 /偶联化纳米粒子)包 括： 戊二醛-氨基肼基 /偶联化纳米粒子、 环氧烷基-氨基肼基 /偶联化纳米 粒子、 氨基酸-氨基肼基 /偶联化纳米粒子、 合成肽-氨基肼基 /偶联化纳米 粒子、 等等。 The activated nanoparticles prepared in this embodiment (aminoguanidine derivative groups/conjugated nanoparticles) 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.
实施例 1.1.3: 含氨基酸基的活化纳米粒子的制备方法 Example 1.1.3: Preparation method of activated nanoparticle containing amino acid group
以上述实施例 1.1所述第一种制备方法为例，以氨基酸或 Fmoc-氨基 酸作为所述活化剂进行上述活化。 优选的反应之一是氨基酸上的 -COOH 基与偶联剂或偶联基团上的 -NH2基反应。 本实施例制备的活化纳米粒子 包括： 精氨酸基 /偶联化纳米粒子、天冬酰氨基 /偶联化纳米粒子、谷氨酰 氨基 /偶联化纳米粒子、 甘氨酸基 /偶联化纳米粒子、 赖氨酸基 /偶联化纳 米粒子、 谷氨酰胺基 /偶联化纳米粒子。 Taking the first preparation method described in the above Example 1.1 as an example, 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.
实施例 1.1.4: 含氨基酸衍生物活化基团的活化纳米粒子的制备方法 Example 1.1.4: Method for preparing activated nanoparticle containing amino acid derivative activating group
以上述实施例 1.1所述第一种制备方法为例，进行多次活化反应。例 如，先以上述实施例 1.1.2所述方法获得氨基酸基 /偶联化纳米粒子，再以 第二活化剂 (例如戊二醛、 1， 4-丁二醇二缩水甘油醚)进行第二次活化、 然后分离出第二次活化产物、 等等， 每次活化反应的条件与上述反应条 件相似。 也可以将氨基酸与第二活化剂反应， 制备含基础活化基团 /衍生 活化基团复合物的活化剂 (例如醛基化氨基酸)，再将此活化剂用作上述活 化反应的活化剂。  Taking the first preparation method described in the above Example 1.1 as an example, a plurality of activation reactions were carried out. For example, 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 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. It is also possible to react 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.
本实施例制备的活化纳米粒子包括：戊二醛-精氨酸基 /偶联化纳米粒 子、 环氧烷基-精氨酸基 /偶联化纳米粒子、 戊二醛 -天冬酰氨基 /偶联化纳 米粒子、 戊二醛 -谷氨酰氨基 /偶联化纳米粒子、 等等。  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.
实施例 1.1.5: 含合成肽活化基团的活化纳米粒子的制备方法 Example 1.1.5: Preparation method of activated nanoparticles containing synthetic peptide activating group
以上述实施例 1.1所述第一种制备方法为例。 一种方法是： 使用标准 的多肽合成方法， 即以上述实施例 1.1.4制备的活化纳米粒子上氨基酸基 团上， 选用适当 Fmoc-氨基酸,进行缩合一洗涤一去保护一中和和洗涤一 下一轮缩合的方式， 将氨基酸依次连接上去， 直到氨基酸基数目满足要 求。 另一种方法是： 按公知的肽合成方法进行肽合成， 直到获得所需氨 基酸基数目的合成肽， 然后用作所述活化剂与偶联化纳米粒子进行上述 活化反应。  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.
以上述实施例 1.1所述第二种制备方法为例：按公知的肽合成方法进 行肽合成， 直到获得所需氨基酸基数目的合成肽， 然后用作活化剂与偶 联剂反应，制备所述合成肽基 -偶联基团复合物，然后再按上述实施例 1.1 中 (2.2)所述方法制备活化纳米粒子。  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.
本实施例制备的活化纳米粒子包括合成肽基 /偶联化纳米粒子。其中- 合成肽基中的氨基酸数目大于或等于 2(例如 2-5); 合成肽基中的氨基酸 种类相同 (例如精氨酸、 天冬酰氨、 谷氨酰氨、 等等)， 或不同 (例如精氨 酸和天冬酰氨、 天冬酰氨和甘氨酸、 谷氨酰氨和赖氨酸、 等等)。 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.).
实施例 1.1.6: 含合成肽衍生物活化基团的活化纳米粒子的制备方法 Example 1.1.6: Preparation method of activated nanoparticles containing activated peptide derivative activating group
以上述实施例 1.1所述第一种制备方法为例，进行多次活化反应，例 如先获得上述实施例 1.1.5中的合成肽基 /偶联化纳米粒子，再以第二活化 剂 (例如戊二醛、 1， 4-丁二醇二缩水甘油醚)进行第二次活化、 然后分离 出第二次活化产物、 等等， 每次活化反应的条件与上述反应条件相似。 也可以将基础活化剂与第二活化剂反应， 制备含基础活化基团 /衍生活化 基团复合物的活化剂 (例如醛基化多肽)，再将此活化剂用作上述活化反应 的活化剂。本实施例制备的活化纳米粒子包括： 戊二醛 -肽基 /偶联化纳米 粒子、 环氧烷基 -肽基 /偶联化纳米粒子。  Taking the first preparation method described in the above embodiment 1.1 as an example, 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. . The activated nanoparticles prepared in this example include: glutaraldehyde-peptidyl/coupled nanoparticles, epoxyalkyl-peptidyl/coupled nanoparticles.
实施例 1.2: 活化纳米串珠的制备方法 Example 1.2: Preparation method of activated nanobeads
本实施例活化纳米串珠的制备方法， 例如： 提供活化纳米粒子 (选自 以上实施例 1.1的制备物， 例如氨基酸基 /偶联化纳米粒子或氨基肼基 /偶 联化纳米粒子)，并使具有相互反应能力的不同活化纳米粒子结合。例如， 将脱去保护基团的谷氨酰胺基 /偶联化纳米粒子悬浮液，与精氨酸基 /偶联 化纳米粒子 (或氨基肼基 /偶联化纳米粒子)悬浮液等量混合， 按公知的肽 合成方法在有效条件下进行谷氨酰胺基与精氨酸基的结合， 即可生成由 两种活化纳米粒子构成的活化纳米串珠。 反应条件如下： 纳米粒子的浓 度 (w/v) 1%。-2%；活化剂浓度 (v/v)在 0.5-5%;反应温度在室温至反应介质 沸点以下 5°C之间； 反应时间 0.5-15小时； 反应介质为 DMF。 本专业的 技术人员通过调节这些参数可获得所需的优化条件。  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. 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.
本发明中， 以 [活化纳米粒子] n(n为活化纳米粒子重数)来表示活化纳 米串珠。本实施例中制备的活化纳米串珠包括基于实施例 1.1制备的活化 纳米粒子的活化纳米串珠。本实施例中制备的活化纳米串珠 ([活化纳米粒 子] η)中，活化纳米粒子重数为 2-4(η=2-4)之间。所获活化纳米串珠与所用 活化纳米粒子， 它们的表面上单位面积所固定的活化基团的密度相同。 优选具有下述组成特征的活化纳米串珠， 用以进行以下实施例中活化纳 米结构或亲和纳米结构的制备： lm2纳米串珠表面上固定的活化基团大于 0.5 μηιοΚ 优选大于 1μπιο1、 更优选大于 1.5μπιο1。 In the present invention, 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. In the activated nanobeads ([activated nanoparticles] η) prepared in the present embodiment, the activated nanoparticles have a weight of between 2-4 (η=2-4). 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 μηιοΚ preferably greater than 1 μπιο1, more preferably greater than 1.5μπιο1.
实施例 1.3: 活化纳米凸体的制备方法 Example 1.3: Preparation method of activated nano-protrusion
本发明实施例中， 用来在其上形成活化纳米凸体的常规载体， 可以 是任何可用于保持其上的纳米结构的、 本身并非纳米结构载体的活化或 未活化固相载体， 包括： 平面载体、 粒状载体、 膜状载体。 平面载体包 括载玻片、活化载玻片、和 ELISA多孔板； 粒状载体包括硅胶、层析胶； 膜状载体包括纤维膜条。 活化载玻片包括按照己公开的方法制备的氨基 玻片 (参考 Schena, M.， Microarray analysis, John Wiley & Sons, INC., New York) > 酸基玻片 (参考 Schena, Μ·， Microarray analysis, John Wiley & Sons, INC., New York)、 氨基肼玻片 (参考 Xavier Duburcq et al.， Biocongate Chemistry 2002, 13： 713-720)。 ELISA多孔板包括聚苯乙烯 多孔板 (深圳金灿烨有限责任公司)。硅胶包括粒径 40— 60μπι的氧化硅粒 子 (中国科学院化学研究所)。层析胶包括 Sephadex A 50和 CM-Shepharose CL(Pharmacia公司)。 纤维膜条包括硝基纤维膜条和尼龙纤维膜条 (福建 泉州长立生化有限公司)。 本实施例的制备方法同样适于由以下材料或其 衍生物制成的常规载体： 硅片、 硅胶、 陶瓷、 金属氧化物、 金属、 其它 聚合物材料及它们的复合物。 本发明实施例中， 纳米凸体及其高度、 半 高处的最小尺寸及其分布密度的测定， 利用 SPA— 300HV型扫描显微镜 (DFM)及分析软件进行。 In the embodiment of the present invention, 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; and 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, Μ·, 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 μm (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. In the embodiment of the present invention, 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.
本发明实施例中， 活化纳米凸体通过 4种方法制备： 1).制备偶联化 纳米粒子， 再将偶联化纳米粒子固定在上述常规载体表面上形成偶联化 纳米凸体， 偶联化纳米凸体经活化制成活化纳米凸体； 2)制备活化纳米 粒子，再将活化纳米粒子固定在上述常规载体表面上形成活化纳米凸体； 3).对已有纳米凸体进行活化制成活化纳米凸体； 和 4).对已有纳米凸体上 再加入纳米结构并制成新的活化纳米凸体。  In the embodiment of the present invention, 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.
本发明中， 以偶联基团 /纳米凸体来表示偶联化纳米凸体。 本实施例 所制备的偶联化纳米凸体包括氧硅垸基团 /氧化物纳米凸体。 其中， 硅烷 基团包括所有上述硅烷偶联剂包含的偶联基团； 氧化物纳米凸体包括所 有上述氧化物凸体 (例如硅氧化物纳米凸体、 钛氧化物纳米凸体、 铝氧化 物纳米凸体)。 前述制备方法 1)和 2)制备的活化纳米凸体中， 含偶联化 纳米凸体。 它们与所用偶联化纳米粒子或活化纳米粒子的表面上单位面 积所固定的偶联活化基团的密度相同。 优选具有下述组成特征的这类偶 联化纳米凸体 (或活化纳米凸体)， 用以进行以下实施例中活化纳米凸体 (或亲和纳米凸体)的制备： lm2纳米凸体表面上固定的偶联基团大于 1.85μπιο1、 优选大于 2.0μπιο1、 更优选大于 2.50μιιιο1。 In the present invention, 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. Wherein, 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. They are the same density as the coupling activation groups immobilized per unit area on the surface of the coupled nanoparticles or activated nanoparticles used. Such coupled nanoprotrusions (or activated 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 μπιο1, preferably greater than 2.0 μπιο1, more preferably greater than 2.50 μιιι1.
本发明中， 以主要活化基团 /偶联化纳米凸体来表示活化纳米凸体。 本实施例中， 所制备的活化纳米凸体包括： 氨基肼基 /偶联化纳米凸体、 氨基肼衍生物基 /偶联化纳米凸体、氨基酸基 /偶联化纳米凸体、氨基酸衍 生物基 /偶联化纳米凸体、 合成肽基 /偶联化纳米凸体、 合成肽衍生物基 / 偶联化纳米凸体。 其中偶联化纳米凸体包括如前所述的叁种不同制备方 法制备的活化纳米凸体中所含的偶联基团 /纳米凸体。前述制备方法 2)制 备的活化纳米凸体与所用活化纳米粒子， 它们的表面上单位面积所固定 的偶联活化基团的密度相同。 优选具有下述组成特征的这类活化纳米凸 体， 用以进行以下实施例中活化纳米凸体或亲和纳米凸体的制备： lm2 纳米凸体表面上固定的活化基团大于 0.5 μπιοΚ优选大于 1μπι01、更优选 大于 1.5μιτιο1ο In the present invention, the activated nano-protrusions are represented by a primary activating group/coupled nano-protrusion. In this embodiment, 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 μπιοΚ. More than 1μπι 0 1 , more preferably greater than 1.5μιτιο1ο
通过上述 4种方法制备活化纳米凸体的更具体的制备方法， 可参考 下述实施例 2.1-2.3中的相关方法。  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.
实施例 2: 活化纳米结构载体的制备 Example 2: Preparation of activated nanostructure carrier
本发明有关活化纳米结构载体的制备的实施例中：所用纳米结构、 偶 联剂、 活化剂、 常规载体与上述有关活化纳米结构的制备的实施例 (例如 实施例 1.1-1.3)所用纳米结构、偶联剂、活化剂、常规载体相同；所用纳米 结构凸体载体， 包括纳米结构玻片和纳米结构硅胶粒子。 所用纳米结构 凸体载体， 其制备方法为以知的将纳米粒子包被至常规载体的方法。 简 述如下：将载玻片 (活化玻片或未活化玻片)或硅胶粒子放入优化浓度的纳 米粒子悬浮液中浸泡 10小时以上， 然后洗涤， 再在适当温度下烘干足够 长的时间。 特别要强调的是， 其它纳米凸体载体， 也可以用作本实施例 中的纳米凸体载体， 例如定向排列的不连续的亚微米须晶结构、 等等。  In an embodiment of the invention relating to the preparation of an activated nanostructure carrier: 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.
本发明实施例中， 活化纳米凸体载体至少可通过 4种方法制备： 1). 将偶联化纳米粒子固定在常规载体表面上， 形成偶联化纳米凸体载体， 再经活化制成活化纳米凸体载体； 2)将活化纳米粒子固定在常规载体表 面上形成活化纳米凸体载体； 3).对纳米凸体载体进行活化制成活化纳米 凸体载体； 和 4).对已有纳米凸体载体上再加入纳米结构并制成新的活化 纳米凸体载体。  In the embodiment of the present invention, 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. Nano-convex carrier; 2) immobilized activated nanoparticles on the surface of a conventional carrier to form an activated nano-convex carrier; 3) activation of the nano-convex carrier to form an activated nano-convex carrier; and 4). A nanostructure is then added to the convex support and a new activated nanoprotrusion carrier is formed.
本发明中， 以活化基团 /纳米凸体 /载体来表示活化纳米结构载体。本 发明实施例所制备的活化纳米结构载体包括：活化基团 /纳米凸体 /平面载 体 (例如， 活化基团 /纳米凸体 /芯片片基基质、 活化基团 /纳米凸体 /酶标微 孔板片基基质)、活化基团 /纳米凸体 /粒状载体 (例如， 活化基团 /纳米凸体 /层析粒子基质)、 活化基团 /纳米凸体 /膜状载体（例如， 活化基团 /纳米凸 体 /膜)。其中的活化基团 /纳米凸体包括所有上述实施例 1.3制备的活化纳 米凸体，其中的活化基团与上述实施例 U的制备的活化纳米粒子中所含 活化基团相同， 包括： 氨基肼基、 氨基肼衍生物基、 氨基酸基、 氨基酸 衍生物基、 合成肽基、 合成肽衍生物基。 In the present invention, 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). 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.
更具体的制备方法由以下实施例 2.1-2.4补充。  A more specific preparation method is supplemented by the following Examples 2.1-2.4.
实施例 2.1: 活化纳米凸体载体的制备方法 (1) Example 2.1: Preparation method of activated nano-convex carrier (1)
例如：将常规载体 (例如活化或未活化玻片、硅胶粒子)放入优化浓度 的偶联化纳米粒子 (选自上述实施例 1.1制备的偶联化纳米粒子)的悬浮液 中浸泡、 反应， 然后洗涤， 再在适当温度下烘干足够长的时间。 反应条 件如下： 偶联化纳米粒子浓度 (w/v)0.01-l%; 反应温度为室温； 反应时间 1-15小时。 然后,再将此偶联化纳米凸体与活化剂溶液混合、 反应。 反应 条件如下： 活化剂浓度 (v/v)l-5%; 反应温度在室温至反应介质沸点以下 5°C之间； 反应时间 0.5-15小时。本专业的技术人员通过调节这些参数可 获得所需的偶联化和活化优化条件。 若活化剂含保护基团 (例如 Fmoc-氨 基酸)， 还要脱去这些保护基团。 For example, a conventional carrier (for example, an activated or unactivated slide, silica gel particles) is placed in a suspension of a optimized concentration of the coupled nanoparticles (selected from the coupled nanoparticles prepared in the above Example 1.1), and reacted. It is then washed and dried at the appropriate temperature for a sufficient period of time. The reaction conditions are as follows: Conjugated nanoparticle concentration (w/v) 0.01-l%; reaction temperature is room temperature; reaction time 1-15 hours. Then, 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. Those skilled in the art can obtain the desired coupling and activation optimization conditions by adjusting these parameters. If the activator contains a protecting group (e.g., Fmoc-amino acid), these protecting groups are also removed.
本实施例中，所制备的活化纳米凸体载体包括：（1).氨基肼基 /纳米凸 体 /玻片、 氨基酸基 /纳米凸体 /玻片、 氨基酸衍生物基 /纳米凸体 /玻片、 合 成肽基 /纳米凸体 /玻片、 合成肽衍生物基 /纳米凸体 /玻片； （2).氨基肼基 / 纳米凸体 /微粒、 氨基酸基 /纳米凸体 /微粒、 氨基酸衍生物基 /纳米凸体 /微 粒、 合成肽基 /纳米凸体 /微粒、 合成肽衍生物基 /纳米凸体 /微粒。 其中所 述活性基团与上述实施例 1.1 中制备的活化纳米粒子中所含的活性基团 相同。 制备含不同活性基团的活化纳米凸体载体的更详细的方法， 也可 分别参考上述实施例 1.1.1-1.1.6 中制备含相同活化基团的活化纳米粒子 的方法。  In this embodiment, 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). aminoguanidino/nano-convex/particle, amino acid/nano-convex/particle, amino acid Derivative/nano-convex/microparticles, synthetic peptidyl/nano-convex/microparticles, synthetic peptide derivative/nano-convex/microparticles. Wherein the reactive group is the same as the reactive group contained in the activated nanoparticles prepared in the above Example 1.1. For a more detailed method of preparing an activated nano-convex support containing different reactive groups, 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.
实施例 2.2: 活化纳米凸体载体的制备方法 (2) Example 2.2: Preparation method of activated nano-convex carrier (2)
例如：将常规载体 (例如，活化或未活化玻片、 ELISA微孔板的孔底、 粒状载体、膜状载体)放入活化纳米粒子 (选自上述实施例 1.1制备的活化 纳米粒子)的悬浮液中浸泡、 反应， 然后洗涤， 再在适当温度下烘干足够 长的时间。 反应条件如下： 活化纳米粒子浓度 (w/v) 0.01-1%； 反应温度 为室温； 反应时间 1-15小时。 本专业的技术人员通过调节这些参数可获 得所需的优化条件。  For example, a conventional carrier (for example, an activated or unactivated slide, a bottom of an ELISA microplate, a granular carrier, a membranous carrier) is placed in suspension of 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.
本实施例中，所制备的活化纳米凸体载体包括：（1).氨基肼基 /纳米凸 体 /玻片、 氨基肼衍生物基 /纳米凸体 /玻片、 氨基酸基 /纳米凸体 /玻片、 氨 基酸衍生物基 /纳米凸体 /玻片、 合成肽基 /纳米凸体 /玻片、 合成肽衍生物 基 /纳米凸体 /玻片； （2). 氨基肼基 /纳米凸体 /微粒、 氨基酸基 /纳米凸体 / 微粒、 氨基酸衍生物基 /纳米凸体 /微粒、 合成肽基 /纳米凸体 /微粒、 合成 肽衍生物基 /纳米凸体 /微粒； （3). 氨基肼基 /纳米凸体 /微孔板、 氨基酸基 / 纳米凸体 /微孔板、 氨基酸衍生物基 /纳米凸体 /微孔板、 合成肽基 /纳米凸 体 /微孔板、 合成肽衍生物基 /纳米凸体 /微孔板； (4). 氨基肼基 /纳米凸体 / 纤维膜条、 氨基酸基 /纳米凸体 /纤维膜条、 氨基酸衍生物基 /纳米凸体 /纤 维膜条、 合成肽基 /纳米凸体 /纤维膜条、 合成肽衍生物基 /纳米凸体 /纤维 膜条。其中所述活性基团与上述实施例 1.1中制备的活化纳米粒子中所含 的活性基团相同。 In this embodiment, 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. Wherein the reactive group is the same as the reactive group contained in the activated nanoparticles prepared in the above Example 1.1.
实施例 2.3: 活化纳米凸体载体的制备方法 (3) Example 2.3: Preparation method of activated nano-convex carrier (3)
例如： 先对纳米凸体载体 (例如纳米凸体玻片、 纳米凸体硅胶粒子) 进行的硫酸 /过氧化氢蚀刻， 烛刻后的纳米凸体载体与偶联剂溶液混合、 反应。 反应条件如下： 偶联剂浓度 (v/v)l-5%; 反应介质为含水的醇； 反 应温度在室温至反应介质沸点以下 5°C之间； 反应时间 0.5-5小时。 本专 业的技术人员通过调节这些参数可获得所需的优化条件。 活化则分别按 下述 2种方法进行：  For example: first, 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), and the nano-protrusion carrier after the candle is mixed and reacted with a coupling agent solution. 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:
(1) .将上述偶联化纳米凸体载体与活化剂溶液混合、 反应。 反应条件 如下： 活化剂浓度 (v/v)l-5%; 反应温度在室温至反应介质沸点以下 5°C 之间； 反应时间 0.5-15小时。 本专业的技术人员通过调节这些参数可获 得所需的优化条件。 要强调的是， 通过使用与上述实施例 1.1.5相同的方 法， 制得合成肽基 /偶联化纳米凸体载体； 通过使用与上述实施例 1.1.6 相同的方法， 制得合成肽衍生物基 /偶联化纳米凸体。 其中， 合成肽基和 合成肽衍生物基分别与上述实施例 1.1.5和 1.1.6中的合成肽基和合成肽 衍生物基相同。  (1) 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. It is emphasized that 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. Among them, 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.
(2) . 将上述偶联化纳米凸体载体与选自上述实施例 1.1 制备的适当 活化纳米粒子 (例如含有可反应的 -COOH基的氨基酸基 /偶联化纳米粒子) 的悬浮液混合、反应。反应条件如下：活化纳米粒子浓度 (w/v)在 0.01-3%; 反应温度 20-37Ό ; 反应时间 0.5-15小时。本专业的技术人员通过调节这 些参数可获得所需的优化条件。 按此方法重复进行， 可获得具多重 (例如 2-4重)纳米粒子的活化纳米凸体。  (2) mixing the above-mentioned coupled nano-protrusion carrier with a suspension of a suitable activated nanoparticle (for example, an amino acid group/coupling nanoparticle containing a reactive -COOH group) prepared in the above Example 1.1, reaction. The reaction conditions are as follows: the activated nanoparticle concentration (w/v) is 0.01-3%; the reaction temperature is 20-37 Torr; and the reaction time is 0.5-15 hours. Those skilled in the art can obtain the desired optimization conditions by adjusting these parameters. Repeated in this manner, activated nano-protrusions with multiple (e.g., 2-4 heavy) nanoparticles can be obtained.
本实施例中，所制备的活化纳米凸体载体包括： （1).氨基肼基 /纳米凸 体 /玻片、 氨基肼衍生物基 /纳米凸体 /玻片、 氨基酸基 /纳米凸体 /玻片、 氨 基酸衍生物基 /纳米凸体 /玻片、 合成肽基 /纳米凸体 /玻片、 合成肽衍生物 基 /纳米凸体 /玻片； （2). 氨基肼基 /纳米凸体 /微粒、 氨基酸基 /纳米凸体 / 微粒、 氨基酸衍生物基 /纳米凸体 /微粒、 合成肽基 /纳米凸体 /微粒、 合成 肽衍生物基 /纳米凸体 /微粒。 其中所述活性基团与上述实施例 1.1中制备 的活化纳米粒子中所含的活性基团相同。 In this embodiment, 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. Wherein the reactive group is the same as the reactive group contained in the activated nanoparticles prepared in the above Example 1.1.
实施例 2.4: 活化纳米凸体载体的制备方法 (4) Example 2.4: Preparation method of activated nano-convex carrier (4)
例如： 将纳米粒子、 偶联化纳米粒子、 活化纳米粒子分别包被至纳 米凸体载体上， 然后： 对纳米粒子包被纳米凸体载体， 按上述实施例 2.3 中的方法进行活化； 对偶联化纳米粒子包被纳米凸体载体， 按上述实施 例 2.1中的方法进行活化。其中所用包被方法与以知的将纳米粒子包被至 常规载体的方法相同。 简述如下： 将纳米凸体载体放入优化浓度的纳米 粒子悬浮液中浸泡 10小时以上， 然后洗涤， 再在适当温度下烘干足够长 的时间。  For example: 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.
本实施例所制备的活化纳米凸体载体 (活化基团 /纳米凸体 /载体)包 括： （1).氨基肼基活化纳米粒子 /纳米凸体 /玻片、 氨基酸基活化纳米粒子 / 纳米凸体 /玻片、 氨基酸衍生物基活化纳米粒子 /纳米凸体 /玻片、 合成肽 基活化纳米粒子 /纳米凸体 /玻片、 合成肽衍生物基活化纳米粒子 /纳米凸 体 /玻片； (2).氨基肼基活化纳米粒子 /纳米凸体 /微粒、氨基酸基活化纳米 粒子 /纳米凸体 /微粒、 氨基酸衍生物基活化纳米粒子 /纳米凸体 /微粒、 合 成肽基活化纳米粒子 /纳米凸体 /微粒、 合成肽衍生物基活化纳米粒子 /纳 米凸体 /微粒。 其中所述活性基团与上述实施例 1.1 中制备的活化纳米粒 子中所含的活性基团相同。  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. Wherein the reactive group is the same as the active group contained in the activated nanoparticles prepared in the above Example 1.1.
实施例 3: 功能化纳米结构的制备方法 Example 3: Method for preparing functionalized nanostructures
在本发明关于功能化纳米结构的制备的实施例中： 所用纳米结构、 偶联剂、 活化剂与上述实施例 1所用纳米结构、 偶联剂、 活化剂相同； 所用活化纳米粒子为以上实施例 1.1制备的活化纳米粒子； 所用活化纳 米串珠为以上实施例 1.2制备的活化纳米串珠； 所用功能试剂包括： 多 肽、抗原、抗体、及其它功能试剂。其中：所用合成多肽包括 EBV—VCA — P18抗原（自制， 制备方法参考 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. 52, 1998, 495— 508); 所用抗原包括： 丙肝病毒抗原（HCV Ag), 爱滋病毒抗原 （HIV Ag), 梅毒抗原 (均为北京大学人民医院肝病 研究所提供)； 所用抗体包括抗乙肝病毒表面抗体（HBsAb， 北京大学人 民医院肝病研究所提供）和单克隆或多克隆羊抗人二抗 (北京生物制品研 究所)； 所用其它功能试剂包括蛋白质 A (上海生物制品研究所)。 本实施 例的方法也适于其它功能试剂， 例如： 药物、 多糖、 维生素、 抗生素、 生物素、 亲和素、 功能有机物、 单链或多链 DNA、 RNA、 以及病毒、 细 胞或它们的组成。 In the embodiment of the present invention regarding the preparation of the functionalized nanostructure: 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. 1.1 Activated Nanoparticles Prepared; 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. Among them: 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. 52, 1998, 495-508); 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.
在本发明实施例中， 制备功能化纳米结构的基本方法包括： 制备活 化纳米结构， 再将功能试剂固定到活化纳米结构上。 基于不同纳米结构 的更具体的制备方法由以下实施例 3.1-3.3补充。  In an embodiment of the invention, 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.
实施例 3.1: 功能化纳米粒子的制备方法 Example 3.1: Preparation of Functionalized Nanoparticles
例如： 使功能试剂与活化纳米粒子接触并发生反应。 反应条件如下： 活化纳米粒子浓度 (w/v)0.01-3%; 功能试剂浓度 (w/v)0.1-3.0mg/ml; 缓冲 液 pH5.0-9.5; 反应温度 20-37Ό ; 反应时间 0.5-72小时。 本专业的技术 人员通过调节这些参数可获得所需的优化条件。 必要时还包括功能化纳 米粒子的纯化、 或 /和钝化。 常用纯化剂包括蛋白质和氨基酸。  For example: 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 Ό; 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.
本发明中， 以功能试剂 /活化纳米粒子来表示功能化纳米粒子。 本实 施例制备的功能化纳米粒子包括：抗原 /活化纳米粒子 (例如 HCV Ag/活化 纳米粒子、 fflV Ag/活化纳米粒子、 梅毒抗原 /活化纳米粒子、 等等)、 抗 体 /活化纳米粒子 (例如 HBs Ad/活化纳米粒子)、 其它功能试剂 /活化纳米 粒子 (例如 Protein A/活化纳米粒子)。 其中， 活化纳米粒子为上述实施例 1.1制备的活化纳米粒子。  In the present invention, 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). Among them, the activated nanoparticles were the activated nanoparticles prepared in the above Example 1.1.
实施例 3.2: 功能化纳米串珠的制备方法 Example 3.2: Preparation method of functionalized nanobeads
例如： 使功能试剂与活化纳米串珠接触并发生反应。 反应条件如下： 纳米串珠浓度 (w/v)0.01-3% ; 功能试剂浓度 (w/v)0.1-3.0mg/ml; 缓冲液 PH5.0-9.5; 反应温度 20-37°C ; 反应时间 0.5-72小时。本专业的技术人员 通过调节这些参数可获得所需的优化条件。 必要时还包括纯化、 或 /和钝 化。 常用纯化剂包括蛋白质和氨基酸。  For example: 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. Those skilled in the art can obtain the desired optimization conditions by adjusting these parameters. Also included, if necessary, purification, or / and passivation. Commonly used purifying agents include proteins and amino acids.
本发明中， 以功能试剂 /活化纳米串珠来表示功能化纳米串珠。 本实 施例制备的功能化纳米串珠包括：抗原 /活化纳米串珠 (例如 HCV Ag/活化 纳米串珠、 HIV Ag/活化纳米串珠、 梅毒抗原 /活化纳米串珠、 等等)、 抗 体 /活化纳米串珠（例如 HBs Ad/活化纳米串珠)、其它功能试剂 /活化纳米 串珠（例如 Protein A/活化纳米串珠)。其中， 活化纳米串珠为上述实施例 1.2制备的活化纳米串珠。 In the present invention, 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). Wherein, the activated nanobeads are the above examples 1.2 Preparation of activated nanobeads.
实施例 3.3: 功能化纳米凸体的制备方法 Example 3.3: Method for preparing functionalized nanoprotrusions
本实施例中， 功能化纳米凸体至少可通过叁种方法制备- In this embodiment, the functionalized nanoprotrusions can be prepared by at least a method of sputum-
1) .将功能试剂固定在活化纳米凸体上。 固定化反应条件如下： 功能 试剂浓度 (w/v) 0.1-3.0mg/ml; 缓冲液 pH5.0-9.5; 反应温度 20-37°C ; 反应 时间 0.5-72小时。 1) Fix the functional reagent on the activated nanoprotrusion. The immobilization reaction conditions were as follows: 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.
2) .将功能化纳米粒子或 /和功能化纳米串珠固定在常规载体上。 固定 化反应条件如下： 功能化纳米粒子浓度 (w/v)0.01-3%; 缓冲液 pH5.0-9.5; 反应温度 20-37°C； 反应时间 0.5-72小时。  2) Fix the functionalized nanoparticles or / and functionalized nanobeads on a conventional support. 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.
3) . 将功能化纳米粒子或 /和功能化纳米串珠固定在活化纳米凸体 上。 固定化反应条件如下： 功能化纳米粒子或 /和功能化纳米串珠浓度 (w/v)0.01-3%; 功能试剂浓度 (wA0O.l-3.Omg/ml; 缓冲液 pH5.0-9.5 ; 反应 温度 20-37°C； 反应时间 0.5-72小时。  3). Fix the functionalized nanoparticles or / and functionalized nanobeads on the activated nanoprotrusions. 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.
本专业的技术人员通过调节反应参数可获得所需的优化条件。 必要 时还包括纯化、 或 /和钝化。 常用纯化剂包括蛋白质和氨基酸。 其中： 所 用功能化纳米粒子包括所有上述实施例 3.1制备的功能化纳米粒子;所用 功能化纳米串珠包括所有上述实施例 3.2制备的功能化纳米串珠；所用活 化纳米凸体， 选自本发明前述实施例 1.3制备的活化纳米凸体 (例如活化 硅氧化物纳米凸体、 活化钛氧化物纳米凸体、 活化铝氧化物纳米凸体)。  Those skilled in the art can obtain the desired optimization conditions by adjusting the reaction parameters. Also included, if necessary, purification, or / and passivation. Commonly used purifying agents include proteins and amino acids. Wherein: 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).
本发明中， 以功能试剂 /活化纳米凸体、 或功能试剂 /主要活化基团 / 偶联化纳米凸体作记号， 来表示功能化纳米凸体。 本实施例制备的功能 化纳米凸体包括： 抗原 /活化纳米凸体 (例如 HCVAg/活化纳米凸体、 HIV Ag/活化纳米凸体、 梅毒抗原 /活化纳米凸体、 等等)、 抗体 /活化纳米凸体 (例如 HBs Ad/活化纳米凸体)、其它功能试剂 /活化纳米凸体（例如 Protein A/活化纳米凸体)。其中，活化纳米凸体中的活化结构包括上述实施例 1.3 制备的活化纳米凸体中的活化结构。  In the present invention, 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). Wherein the activated structure in the activated nano-protrusion comprises the activated structure in the activated nano-protrusion prepared in the above Example 1.3.
通过上述叁种方法制备功能化纳米凸体的更具体的制备方法， 可参 考下述实施例 4(包括实施例 4.1-4.3)中的相关方法。  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).
实施例 4: 功能化纳米结构载体的制备 Example 4: Preparation of a Functionalized Nanostructure Carrier
在本发明关于功能化纳米结构载体的制备的实施例中：所用纳米结 构、 偶联剂、 活化剂， 与上述实施例 1所用纳米结构、 偶联剂、 活化剂 相同； 所用常规载体与上述实施例 1.3 中所用常规载体相同； 所用功能 试剂与前述实施例 3中所用功能试剂相同 (包括： 多肽、 抗原、 抗体、及 其它功能试剂)。 In the embodiment of the present invention regarding the preparation of the functionalized nanostructure carrier: the nanostructure, coupling agent, activator used, and the nanostructure, coupling agent, and activator used in the above Example 1; conventional carrier used and the above implementation 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).
本发明实施例中,功能化纳米结构载体的制备用了 3种基本方法： 1). 将功能试剂固定在活化纳米凸体载体上； 2).将功能化纳米粒子或 /和功能 化纳米串珠固定在常规载体上； 3). 将功能化纳米粒子或 /和功能化纳米 串珠固定在活化纳米凸体载体上。 其中： 所用功能化纳米粒子选自包括 所有上述实施例 3.1制备的功能化纳米粒子；所用功能化纳米串珠包括所 有上述实施例 3.2制备的功能化纳米串珠;所用活化纳米凸体载体包括所 有上述实施例 2(包括实施例 2.1-2.4)制备的活化纳米凸体载体。其中的活 化纳米凸体 /载体中， 活化纳米凸体上的活化基团与上述实施例 1的制备 的活化纳米结构中所含活化基团相同， 包括氨基肼基、 氨基肼衍生物基、 氨基酸基、 氨基酸衍生物基、 合成肽基、 合成肽衍生物基。 更具体的制 备方法由以下实施例 4.1-4.3补充、 及参考以下实施例 5。  In the embodiments of the present invention, 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. Wherein: 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). In the activated nanoprotrusion/carrier, 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.
本发明中， 以功能试剂 /活化纳米凸体 /载体来表示功能化纳米凸体载 体。 以下实施例制备的功能化纳米凸体载体包括： 抗原 /活化纳米凸体 / 载体 (例如， 多种抗原 /活化纳米凸体 /芯片片基基质、 抗原 /活化纳米凸体 / 酶标微孔板片基基质、 抗原 /活化纳米凸体 /膜、 抗原 /活化纳米凸体 /层析 粒子基质)、抗体 /活化纳米凸体 /载体 (例如，抗体 /活化纳米凸体 /芯片片基 基质、 抗体 /活化纳米凸体 /酶标微孔板片基基质、 抗体 /活化纳米凸体 /层 析粒子基质)、抗原和抗体 /活化纳米凸体 /载体 (多种抗原和 HBs抗体 /活化 纳米凸体 /芯片片基基质)、其它功能试剂 /活化纳米凸体 /载体 (：例如 Protein A/活化纳米凸体 /芯片片基基质、 ProteinA/活化纳米凸体 /层析粒子基质)。 实施例 4.1: 功能化纳米凸体载体的制备方法 (1) '  In the present invention, 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 nanoprotrusions / carriers (eg, Protein A / activated nano-convex / chip-based matrix, Protein A / activated nano-convex / chromatography particle matrix). Example 4.1: Preparation of functionalized nano-convex carrier (1) '
例如， 按公知的芯片制备方法， 将一种或多种功能试剂溶液 (功能试 剂浓度在 0.1-2mg/ml之间)点在上述实施例 3制备的活化纳米凸体 /芯片片 基基质上， 37°C反应 3小时以上，然后用钝化剂 (：例如牛血清白蛋白)钝化， 形成功能化纳米凸体 /芯片片基基质 (纳米分析芯片)。 这时， 功能试剂点 内、 外都有活化纳米结构。  For example, one or more functional reagent solutions (functional reagent concentrations between 0.1-2 mg/ml) 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). At this time, the functional reagents have activated nanostructures inside and outside.
又例如， 按公知的亲和层析粒子制备方法， 使功能试剂溶液 (功能试 剂浓度在 0.1-2mg/ml之间)与上述实施例 3制备的活化纳米凸体 /层析粒 子基质 (粒子浓度 (w/v)在 0.5-5%之间)接触，室温下搅拌，包被 3-24小时， 形成功能化纳米凸体 /层析粒子基质 (亲和层析纳米固定相)。  For another example, 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).
又例如， 按公知的酶标板制备方法， 将上述功能试剂溶液 (功能试剂 浓度在 0.0.5-2 g/ml之间)加在上述实施例 3制备的活化纳米凸体 /酶标微 孔板片基基质 (孔底)上， 室温下摇动， 包被 3-24小时， 然后用钝化剂 (例 如牛血清白蛋白)钝化， 形成功能化纳米凸体 /酶标微孔板片基基质 (纳米 酶标微孔板)。 For another example, 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) is 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/enzyme-labeled microplate. Base matrix (nano-labeled microplate).
又例如， 按公知的平面亲和层析膜 (例如快检试剂条)制备方法， 将 上述功能试剂溶液 (功能试剂浓度在 0.1-2mg/ml之间)点在上述实施例 3 制备的活化纳米凸体 /膜上， 37°C反应 3小时以上， 包被形成功能化纳米 凸体 /膜 (例如快检试剂条)。  For another example, 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). 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).
实施例 4.2: 功能化纳米凸体载体的制备方法 (2) Example 4.2: Preparation of functionalized nano-convex carrier (2)
例如， 按公知的芯片制备方法， 将一种或多种功能化纳米粒子或 / 和功能化纳米串珠悬浮液点 (功能试剂浓度在 0.1-2mg/ml之间，纳米结构 浓度 (w/v)在 0.01%-5%之间)点样在活化芯片片基上， 37°C反应 3小时以 上， 然后用钝化剂 (例如牛血清白蛋白)钝化， 反应后形成功能化纳米凸 体 /芯片片基基质 (纳米分析芯片)。 这时， 仅功能试剂点内有活化纳米结 构， 在功能试剂点外不存在与纳米结构有关的非特异性吸附。  For example, 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). 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.
又例如， 按公知的亲和层析粒子制备方法， 将上述功能化纳米粒子 或 /和功能化纳米串珠悬浮液 (功能试剂浓度在 0.5-2mg/ml之间， 纳米结 构浓度 (w/v)在 0·1%-3%之间）与层析粒子 (粒子浓度 (w/v)在 0.5-5%之间) 接触，室温下搅拌，包被 3-24小时，形成功能化纳米凸体 /层析粒子基质 (亲和层析纳米固定相)。  For another example, 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).
又例如， 按公知的酶标板制备方法， 将上述功能化纳米粒子或 /和功 能化纳米串珠悬浮液 (功能试剂浓度在 0.1-2 g/ml之间， 纳米结构浓度 (w/v)在 0.001%-0.05%之间)加在酶标微孔板孔底上， 室温下摇动， 包被 3-24小时， 然后用钝化剂 (例如牛血清白蛋白)钝化， 形成功能化纳米凸 体 /酶标微孔板片基基质 (纳米酶标微孔板)。  For another example, 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).
又例如， 按公知的平面亲和层析膜 (例如快检试剂条)制备方法， 将 上述功能化纳米粒子或 /和功能化纳米串珠悬浮液 (功能试剂浓度在 0.1-2mg/ml之间，纳米结构浓度 (w/v)在 0.01%-5%之间)加在纤维膜条上， 37°C反应 3小时以上， 包被形成功能化纳米凸体 /膜 (例如快检试剂条)。 这时， 仅功能试剂点内有活化纳米结构， 在功能试剂点外不存在与纳米 结构有关的非特异性吸附。  For another example, 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.
实施例 4.3: 功能化纳米凸体载体的制备方法 (3) Example 4.3: Preparation of functionalized nano-convex carrier (3)
例如， 按公知的芯片制备方法， 将一种或多种功能化纳米粒子或 / 和功能化纳米串珠悬浮液（功能试剂浓度在 0.1-2mg/ml之间， 纳米结构 浓度 (w/v)在 0.01%-5%之间） 点在上述实施例 2制备的活化纳米凸体 /芯 片片基基质上， 37°C反应 3小时以上，然后用钝化剂 (例如牛血清白蛋白） 钝化，反应后形成功能化纳米凸体 /芯片片基基质 (纳米分析芯片)。这时， 仅功能试剂点内有活化纳米结构， 在功能试剂点外不存在与纳米结构有 关的非特异性吸附。 For example, 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 The 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). 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.
又例如， 按公知的亲和层析粒子制备方法， 将上述功能化纳米粒子 或 /和功能化纳米串珠悬浮液 (功能试剂浓度在 0.5-2mg/ml之间， 纳米结 构浓度 (w/v)在 0.1%-3%之间）与上述实施例 2制备的活化纳米凸体 /层析 粒子基质 (粒子浓度 (w/v)在 0.5-5%之间)接触， 室温下搅拌， 包被 3-24小 时， 形成功能化纳米凸体 /层析粒子基质 (亲和层析纳米固定相)。  For another example, 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).
又例如， 按公知的酶标板制备方法， 将上述功能化纳米粒子或 /和功 能化纳米串珠悬浮液 (功能试剂浓度在 0.1-2 g/ml之间， 纳米结构浓度 (w/v)在 0.001%-0.05%之间)加在上述实施例 2制备的活化纳米凸体 /酶标 微孔板片基基质 (孔底)上，室温下摇动，包被 3-24小时，然后用钝化剂 (例 如牛血清白蛋白 )钝化， 形成功能化纳米凸体 /酶标微孔板片基基质 (纳米 酶标微孔板)。  For another example, 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) is inactivated to form a functionalized nano-convex/enzyme-labeled microplate base matrix (nano-labeled microplate).
实施例 5: 纳米反应***的制备方法 Example 5: Preparation method of nano reaction system
本发明关于纳米反应***的制备的实施例中： 所用纳米结构、 偶联 剂、 活化剂、 常规载体、 功能试剂 (包括： 多肽、 抗原、 抗体、 及其它功 能试剂)， 与上述实施例 4相同； 所用制备方法与上述实施例 4相同。 其 中： 所用功能化纳米粒子、 功能化纳米串珠、 活化纳米凸体载体， 与上 述实施例 4相同。 本发明的实施例中的方法， 可以制备任何可包含所述 活化纳米结构的反应*** (或装置)，例如传感器、分析芯片、 ELISA酶标 板、快检试剂条、等等。某些反应*** (或装置)更具体的制备方法由以下 实施例 5.1-5.3补充。  In the examples of the preparation of the nanoreaction system of the present invention: 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. Among them: the functionalized nanoparticles used, the functionalized nanobeads, and the activated nanoprotrusion carrier are the same as in the above-mentioned Embodiment 4. In the method of the embodiment of the present invention, any reaction system (or apparatus) 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.
实施例 5.1: 纳米分析芯片的制备方法 Example 5.1: Preparation method of nanometer analysis chip
本实施例中， 纳米结构分析芯片的制备方法及反应条件与实施例 4.1-4.3中的功能化纳米凸体 /芯片片基基质的制备方法相同。 其中， 点样 可为手工点样或机械点样 (DY— 2003 生物芯片点样仪， 中国科学院电工 研究所)。 每种溶液或悬浮液点 2-4个点。 所有功能试剂点在芯片片基上 形成 MxN功能试剂阵列。 其中 M大于 1， N大于 1。 纳米分析芯片上， 至少有一个功能试剂点具有功能化纳米结构 (例如功能试剂 /活化纳米凸 体)， 可以是全部功能试剂点具有功能化纳米结构， 也可以是部分功能试 剂点具有功能化纳米结构 (例如存在非纳米结构功能试剂点)。纳米分析芯 片上， 可以是仅仅部分或全部功能试剂点具有纳米结构 (例如在活化常规 片基上形成者)，也可以是整个点阵区都具有具有纳米结构 (例如在活化纳 米凸体片基上形成者)。 本实施例制备的优选的分析芯片， 至少有一个功 能试剂点中纳米凸体 (高度大于 3 nm、 且凸出半高处至少一维尺寸在 1一 500 nm)分布密度大于 5个 /μπι2。 In this embodiment, 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. Among them, 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. On the nano-analysis chip, 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). On 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). On the formation). 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/μπι 2 .
本实施例的方法当然适于各种芯片， 例如单反应池芯片、 多反应池 芯片、 流动芯片、 非流动芯片、 等等。 多反应池非流动芯片片基的制备 方法，可参考我们的另一专利申请《反应器高度最小化的高集成度分析芯 片及其应用》 (申请号 PCT/CN2004/000169)中的实施例 1。 然后， 再对片 基池进行上述 "点样 "操作和其它操作。流动生物芯片的制备方法， 可参 考我们的另一专利申请《反应器高度最小化的高集成度分析芯片及其应 用》 (申请号 PCT/CN2004/000169)中的实施例 9或 10。  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. For the preparation method of the 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. For the preparation of 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).
本实施例的方法制备的芯片， 包括： 纳米结构抗原芯片 (例如至少固 定有 HCV抗原、 HIV抗原、 或 /和梅毒抗原的本发明的纳米结构芯片)、 抗体芯片 (例如至少固定有 HBs抗体的本发明的纳米结构芯片)、 抗原和 抗体芯片 (例如至少固定有 HCV抗原和 HBs抗体的本发明的纳米结构芯 片)、其它亲和芯片 (例如至少固定有蛋白质 A的本发明的纳米结构芯片)。 实施例 5.2: 纳米酶标微孔板的制备方法  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) . Example 5.2: Preparation method of nanometer-labeled microplate
本实施例中， 纳米酶标微孔板的制备方法及反应条件与实施例 4.1-4.3中的功能化纳米凸体 /酶标微孔板片基基质的制备方法相同， 所制 备的纳米结构酶标板， 包括： 纳米结构抗原酶标板 (例如 HCV抗原、 mv 抗原、 或梅毒抗原包被酶标板)、 纳米结构抗体酶标板 (例如 HBs抗体包 被酶标板)、其它纳米结构亲和酶标板 (例如固定有蛋白质 A包被酶标板)。 实施例 5.3: 纳米平面亲和层析膜的制备方法  In this embodiment, 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). Example 5.3: Preparation method of nano-planar affinity chromatography membrane
本实施例中， 纳米平面亲和层析膜的制备方法及反应条件与实施例 4.1-4.3中的功能化纳米凸体 /膜的制备方法相同， 所制备的纳米平面亲和 层析膜， 包括： 固定有蛋白质 A和胶体金标记羊抗人二抗的抗原快检试 剂条 (例如 HCV抗原、 HIV抗原、 或梅毒抗原包被快检试剂条)。  In this embodiment, 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.
实施例 6: 纳米分离***的制备方法 Example 6: Preparation method of nano separation system
本发明关于纳米分离***的制备的实施例中： 所用功能化纳米粒子 选自上述实施例 3.1制备的功能化纳米粒子；所用功能化纳米串珠选自上 述实施例 3.2制备的功能化纳米串珠;所用功能化纳米凸体选自上述实施 例 3.3或 4制备的亲和层析纳米固定相上的功能化纳米凸体。不同纳米分 离***的更具体的制备方法， 由以下实施例补充。 In an embodiment of the invention relating to the preparation of the nanoseparation system: 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. A more specific preparation method for different nano-separation systems is supplemented by the following examples.
实施例 6.1: 纳米亲和层析*** Example 6.1: Nanoaffinity chromatography system
上述亲和层析纳米固定相， 可用作亲和层析固定相， 包括批反应亲 和层析和亲和柱层析。 由亲和层析固定相构成亲和层析***的方法， 为 公知的方法。 本实施例的方法制备的纳米亲和层析***， 包括分别固定 有蛋白质 、 蛋白质 G、 HCV抗原的纳米亲和层析***。  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.
实施例 6.2: 功能化纳米磁分离*** Example 6.2: Functionalized Nano Magnetic Separation System
本实施例制备的功能化纳米磁分离***中， 除含功能化纳米粒子或 / 和功能化纳米串珠外， 还含亲和磁粒子。 亲和磁粒子包含功能试剂和包 被磁粒子或包被磁粒子衍生物。 所用磁粒子包括磁微米粒子 (l-ΙΟμηι)和 磁纳米粒子 (10-100nm)。  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-μμηι) and magnetic nanoparticles (10-100 nm).
亲和磁微米粒子和亲和磁纳米粒子的制备方法，根据公知方法制得， 简言之：将磁粒子分散至水中形成悬浮液，再等体积加入 5%葡聚糖溶液， 在 85-90°C包被 1小时。冷却后通过磁柱纯化葡聚糖包被磁粒子， 然后加 热至 60°C加入适量 DVS， 再加入适量 triethyl amine至 pH 10.5。 在 60 °C 反应 2小时后， 活化葡聚糖包被磁粒子用磁柱纯化。 然后， 再将配对功 能试剂 (例如用于夹心法的配对乙肝表面抗体、配对 HIV抗原、配对 HCV 抗原） 固定在活化葡聚糖包被磁粒子上， 制成亲和磁粒子。  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. Then, a pairing function reagent (for example, a paired hepatitis B surface antibody for sandwich method, a paired HIV antigen, a paired HCV antigen) is immobilized on the activated glucan-coated magnetic particles to prepare an affinity magnetic particle.
实施例 7: 纳米标记***的制备方法 Example 7: Preparation method of nano-labeling system
本发明关于纳米标记***的制备的实施例中， 本发明活化纳米结构 及功能化纳米结构中的纳米结构包括： 通常用作物理标记物质的纳米结 构 (例如金纳米粒子、 荧光纳米粒子、 半导体纳米粒子、 磁纳米粒子、 等 等)； 通常用作化学标记物质的纳米结构 (例如金-银标记法中的金纳米粒 子、 等等)； 通常不用作物理或化学标记物质的纳米结构 (例如氧化硅、 氧化铝等氧化物、 有机物、 等等)。 不具有物理化学以纳米结构为载体， 在纳米结构上固定的分子标记物质 (例如荧光物质分子、酶、染料、等等)。  In an embodiment of the invention relating to the preparation of a nanolabeling system, 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.). There are no molecularly labeled substances (such as fluorescent substance molecules, enzymes, dyes, etc.) that are immobilized on the nanostructures by physicochemicals using nanostructures as carriers.
本实施例中： 所用功能化纳米粒子包括所有上述实施例 3.1制备的 功能化纳米粒子； 所用功能化纳米串珠包括所有上述实施例 3.2制备的 功能化纳米串珠； 所用功能试剂包括上述实施例 3所用二抗， 和所用抗 原、 抗体的对应抗原、 抗体 (配对购买， 供双抗原夹心法、 双抗体夹心法 使用)； 所用标记物质包括： 荧光物质 (例如罗丹明、 CY3、 CY4), 标记 酶 (例如辣根过氧化物酶)， 染色剂 (例如结晶紫)。本实施例的方法也适于 其它标记物质， 例如化学发光物质、 化学发光催化剂、 有色金属盐、 染 料和颜料。 In this embodiment: 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.
本实施例中， 纳米标记物的制备包括两种方法： A).将标记物质固定 在功能化纳米粒子或 /和功能化纳米串珠上再进行纯化； B).将标记物质固 定在功能试剂上，再将纯化的标记物质 /功能试剂复合物固定在活化纳米 粒子或 /和功能化纳米串珠上。 其中， 固定化反应的条件可参考公知的常 规标记物 (例如荧光物质标记物、 酶标记物、 ）制备方法中功能试剂与标 记物质的反应条件，但优选的条件包括一个长得多的反应时间 (例如大于 12小时)。所用标记方法和纯化方法，采用公知的制备常规标记物的标记 方法和纯化方法。 纯化条件可参考公知的常规标记物制备方法中标记物 的纯化方法 (例如过滤法、 层析法、 等等)， 但优选的方法中包括离心法。  In this embodiment, 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. Wherein, 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). As the labeling method and purification method used, a well-known labeling method and purification method for preparing a conventional label are employed. 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.
本发明中， 以标记物质 /活化纳米粒子 /功能试剂为记号来表示纳米标 记物。 本实施例制备的部分纳米标记物如下： 1).荧光物质 /活化氧化物纳 米粒子 /配对抗原、 荧光物质 /活化氧化物纳米粒子 /配对抗体、 荧光物质 / 活化氧化物纳米粒子 /抗抗体、 荧光物质 /活化氧化物纳米粒子 /蛋白质 A; 2).辣根化酶 /活化氧化物纳米粒子 /配对抗体、 辣根化酶 /活化氧化物纳米 粒子 /抗抗体； 3).染色剂 /活化氧化物纳米粒子 /抗抗体。  In the present invention, 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.
实施例 8: 本发明的试剂盒的制备方法 Example 8: Preparation method of kit of the present invention
本发明的实施例中的试剂盒的制备， 是使其至少含上述实施例 5制 备的反应***， 还可含上述实施例 7制备的标记***， 或 /和上述实施例 6制备的分离***。实际上， 本发明的试剂盒， 分别含一类、二类和三类 包含有本发明的功能化纳米结构的***。 更具体的制备方法由以下实施 例补充。  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. In fact, 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.
以下实施例中所制备的部分试剂盒分别列于表 1、 2、 3中。 其中的 标记： 1： 选自实施例 5.1制备的本发明的纳米结构芯片； 2： 选自实施例 5.2制备的本发明的纳米结构酶标板； 3： 选自实施例 5.3制备的本发明的 纳米结构抗原快检试剂条； 4： 常规芯片， 例如使用相同的亲和物质 (末固 定在纳米结构上)和活化玻片， 按公知的芯片制备方法， 在相同条件下制 备的、 作为本发明的纳米结构芯片对照物的对照常规芯片； 5： 选自实施 例 6制备的本发明的功能化纳米过滤*** (实施例 6.1)或功能化纳米磁分 离*** (实施例 6.2); 6： 选自实施例 7制备的本发明的纳米标记物。 Some of the kits prepared in the following examples are listed in Tables 1, 2, and 3, respectively. Wherein the label: 1 : a nanostructured chip of the invention selected from the preparation of Example 5.1; 2 : a nanostructured enzyme target of the invention selected from the preparation of Example 5.2; 3 : selected from the invention of the invention prepared in Example 5.3 Nanostructure antigen rapid test reagent strip; 4 : conventional chip, for example using the same affinity substance (finally immobilized on the nanostructure) and activated slide, prepared according to the known chip preparation method under the same conditions, as the present invention Controlled conventional chip of nanostructured chip control; 5 : Functionalized nanofiltration system of the invention (Example 6.1) or functionalized nanomagnetic separation system (Example 6.2) selected from Example 6 ; 6 : selected from The nanomarker of the invention prepared in Example 7.
实施例 8.1: 本发明的试剂盒的制备方法 (1) Example 8.1: Preparation method of kit of the present invention (1)
本实施例中所制备的试剂盒， 分别为： 含上述实施例 5(包括 5.1-5.3) 制备的纳米反应***的试剂盒；含上述实施例 6.2制备的纳米分离***的 试剂盒；含上述实施例 7制备的纳米标记***的试剂盒。本实施例制备的 部分试剂盒列于表 1。 The 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.
表 1  Table 1
Figure imgf000029_0001
Figure imgf000029_0001
实施例 8.2: 本发明的试剂盒的制备方法 (2)  Example 8.2: Preparation method of kit of the present invention (2)
本实施例中所制备的试剂盒， 分别为： 含上述实施例 5(包括 5.1-5.3) 制备的纳米反应***和上述实施例 7制备的纳米标记***的试剂盒； 含 上述实施例 6.2制备的纳米分离***和上述实施例 7制备的纳米标记*** 的试剂盒；含上述实施例 5(包括 5.1-5.3)制备的纳米反应***和上述实施 例 6.2制备的纳米分离***的试剂盒。本实施例制备的部分试剂盒列于表 2中。 The 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.
表 2  Table 2
Figure imgf000030_0001
实施例 8.3: 本发明的试剂盒的制备方法 (3)
Figure imgf000030_0001
Example 8.3: Preparation method of kit of the present invention (3)
本实施例中所制备的试剂盒，含上述实施例 5(包括 5.1-5.3)制备的纳 米反应***、上述实施例 6.2制备的纳米分离***、和上述实施例 7制备 的纳米标记***的试剂盒。 本实施例制备的部分试剂盒列于表 3。 The kit prepared in this embodiment contains the preparation prepared in the above Example 5 (including 5.1-5.3) The rice reaction system, the nano-separation system prepared in the above Example 6.2, and the kit of the nano-labeling system prepared in the above Example 7. Some of the kits prepared in this example are listed in Table 3.
表 3  table 3
Figure imgf000031_0001
实施例 9: 本发明的反应***的应用
Figure imgf000031_0001
Example 9: Application of the reaction system of the present invention
本发明有关分析的实施例中，样品分别为： HCV抗体阳性血清， fflVi +2抗体阳性人血清， HBS Ag阳性血清， EBV抗体阳性血清， 梅毒抗体 阳性血清， 和阴性血清 （HCV抗体、 fflV1+2抗体、 HBs Ag和梅毒抗体 都为阴性的血清）。 所有的样品均经使用经典的 ELISA方法在血清 10倍 稀释反应条件下预先检测。 含本发明的反应***的装置或试剂盒 (例如 表 1中的 A1-A5), 可以按公知的相应装置或试剂盒的应用方法去应用。 以下实施例中， 仅给出一些对比研究来加以说明。 In the examples of the analysis of the present invention, 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.
实施例 9.1: 本发明的纳米结构芯片  Example 9.1: Nanostructured chip of the present invention
本实施例中， 所用芯片分别为： 本发明的纳米结构芯片,对照常规芯 片和对照纳米结构芯片。其中:所用本发明的纳米结构芯片,为上述实施例 5.1制备的纳米结构芯片；所用对照常规芯片，在活化载玻片 (氨基玻片、 氨基肼玻片，参考上述实施例 1.3)上,使用与本发明的纳米结构芯片相同功 能试剂在相同条件下制得 (参考上述实施例 4.3),不含纳米结构功能试剂 点； 所用对照纳米结构芯片,在含氨基的偶联化纳米凸体 /芯片片基 (参考 实施例 2，例如以 3-氨丙基三甲氧基硅烷为偶联剂制备的偶联化纳米凸体) 上，使用与本发明的纳米结构芯片相同功能试剂在相同条件下制得 (参考 上述实施例 4.3),不含本发明的功能化纳米结构。  In this embodiment, the chips used are: The nanostructured chip of the present invention, which is compared with a conventional chip and a control nanostructure chip. Wherein: 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.
本实施例中， 芯片测试时使用的标记物为常规标记物， 例如： 罗丹 明标记二抗、 罗丹明标记对应抗原、 罗丹明标记对应抗体、 罗丹明标记 对应抗原和对应抗体。 芯片测试方法如下： （1).非流动芯片的测试： 将 适当稀释的测试样品 5μ1分别加入相应芯片的反应池中, 37°C反应 30分钟 后用洗涤液冲洗，再加入 5μ1适当浓度的标记物，在 37°C反应 30分钟后用 洗涤液冲洗，然后干燥再进行扫描。 扫描仪为共聚焦激光扫描仪 (Afymetrix公司 GMS 418芯片扫描仪）， 扫描激发光波长 532 nm, 发射 光波长 570 nm,激光强度 35/50-55/70，读取的信号经处理软件（JAGUAR II )处理， 然后取平均值后得到结果。 （2) 流动芯片的测试： 将适当稀释 的测试样品加热至 37°C， 以流速 10-50μ1/πώι加入芯片反应器,加样时间 60分钟,然后加入洗液洗涤，再加入 5-10μ1适当浓度的标记物进行标记，最 后洗涤、 干燥,再按与非流动芯片的测试相同的方法进行扫描。 In this embodiment, the label used in the chip test is a conventional marker, for example: Rodin The labeled secondary antibody, the rhodamine labeled corresponding antigen, the rhodamine labeled corresponding antibody, the rhodamine labeled corresponding antigen, and the corresponding antibody. The chip test method is as follows: (1). Test of non-flow chip: 5μ1 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 μl 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 μl / π ώ, add the sample time for 60 minutes, then add the wash solution, then add 5-10μ1. The labeled label is labeled, finally washed, dried, and scanned in the same manner as the test for the non-flowing chip.
本实施例中， 本发明的纳米结构芯片与对照常规芯片和对照纳米结 构芯片比较， 在优选的功能试剂浓度（例如点样时功能试剂浓度 0.1-1.Omg/ml)下，使用相同阳性样品时在相同扫描条件下的平均信号读数 分别要高 200%和 100%以上。 其中， 本发明的含合成肽基或合成肽衍生 物基活性基团的纳米结构芯片,与本发明的其它纳米结构芯片相比， 上述 平均信号读数要高 150%以上。结果说明本发明的纳米芯片具有更高的灵 敏度。  In this embodiment, 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). The average signal readings under the same scanning conditions were 200% higher and 100% higher, respectively. Among them, 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.
本实施例中， 本发明的纳米结构芯片与对照常规芯片和对照纳米结 构芯片比较， 在优选的功能试剂浓度（例如点样时功能试剂浓度 0.1-l.Omg/ml)下，可检出的阳性样品最低浓度分别要低 10倍和 3倍以上。 其中， 本发明的含合成肽基或合成肽衍生物基活性基团的纳米结构芯片, 与本发明的其它纳米结构芯片相比， 阳性样品的可测最低限度低 1 倍以 上。 本发明实施例中， 阳性样品的可测最低限度,通过阳性样品稀释至判 定阴、 阳性的临界浓度来表示。 结果说明本发明的纳米芯片具有更高的 灵敏度。  In this embodiment, 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. Among them, 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. In the examples 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.
本实施例中， 本发明的纳米结构芯片与对照常规芯片和对照纳米结 构芯片比较， 37°C放置 71小时后， 使用相同阳性样品时在相同扫描条件 下的信号值平均减小率分别要低 50%和 20%以上。 结果说明本发明的纳 米芯片具有更高的稳定性。  In this embodiment, 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.
实施例 9.2本发明的纳米结构酶标板的比较研究 EXAMPLES 9.2 Comparative Study of Nanostructured Enzyme Plates of the Invention
本实施例中， 所用酶标板分别为本发明的纳米结构酶标板和对照纳 米结构酶标板。 其中:所用本发明的纳米结构酶标板,为上述实施例 5.2制 备的纳米结构酶标板； 所用对照纳米结构酶标板,在含氨基的偶联化纳米 凸体 /酶标板片基 (参考实施例 2，例如以 3-氨丙基三甲氧基硅烷为偶联剂 制备的偶联化纳米凸体 /酶标板片基）上，在与本发明的纳米结构酶标板 相同包被条件下包被相同功能试剂制得 (参考上述实施例 4.1),不含本发明 的功能化纳米结构。 In this embodiment, the ELISA plates used are the nanostructure ELISA plate and the control nanostructure ELISA plate of the present invention, respectively. Wherein: 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.
本实施例中，测试方法与经典的 ELISA法相同，例如：将适当稀释的 测试样品 ΙΟΟμΙ分别加入相应的上述 96孔酶标板中， 37°C反应 0.5-1小 时,再加入洗涤液洗涤 3次 (每次 300μ1),然后加入 ΙΟΟμΙ标记物， 37°C反应 30分钟,再加入底物,反应后利用酶标仪（Thermo Labsystems, 上海雷勃 分析仪器有限公司）进行比色分析。  In this embodiment, the test method is the same as the classical ELISA method, for example, the appropriately diluted test sample ΙΟΟμΙ 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 μl each time), the ΙΟΟμΙ 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.).
本实施例中，本发明的纳米结构酶标板与对照纳米结构酶标板比较， 在优选的功能试剂浓度 (例如包被时功能试剂浓度 0.05-0.^g/ml)下，使用 相同阳性样品时在相同扫描条件下的平均信号读数分别要高 150%以上, 可检出的阳性样品最低浓度要低 1倍以上，而 37°C放置 71小时后相同阳 性样品的信号值平均减小率要低 20%以上。 结果说明本发明的纳米酶标 板具有更高的灵敏度和稳定性。  In this embodiment, 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). 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.
实施例 9.3本发明的纳米结构快检试剂条的比较研究 EXAMPLE 9.3 Comparative Study of Nanostructure Rapid Test Reagent Strips of the Invention
本实施例中， 所用快检试剂条分别为本发明的纳米结构快检试剂条 和对照纳米结构快检试剂条。 其中:所用本发明的纳米结构快检试剂条, 为上述实施例 5.3制备的纳米结构快检试剂条；所用对照纳米结构快检试 剂条,其活化纳米结构中的活性基团为氨基,例如 3-氨丙基三甲氧基硅烷 为偶联剂制备的偶联化纳米粒子结合功能试剂后点样至膜 (例如硝基酸 纤维膜条)上,形成的纳米结构快检试剂条。  In this embodiment, 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. Wherein: 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.
本实施例中， 测试方法与已知的快检试剂条检测方法相同。 例如,将 适当稀释的样品分别加入上述快检试纸条，再加入洗涤液， 使试纸条缓慢 吸至质控线出现。 本发明的纳米结构快检试剂条与对照纳米结构快检试 剂条比较， 可检出的阳性样品最低浓度要低 1倍以上，而 37°C放置 71小 时后相同阳性样品的信号值不降低。 结果说明本发明的纳米结构快检试 剂条具有更高的灵敏度和稳定性。  In this embodiment, the test method is the same as the known quick test strip detection method. For example, 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. Compared with the control nanostructure fast test strip, 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.
实施例 10: 本发明的分离***的应用 Example 10: Application of the separation system of the present invention
含本发明的分离***的装置或试剂盒， 可以按公知的相应装置或试 剂盒的应用而应用。 以下实施例中， 仅给出一些对比研究， 来说明其应 用。 实施例 10.1: 本发明的纳米亲和层析***的应用 The device or kit containing the separation system of the present invention can be applied in accordance with the application of known corresponding devices or kits. In the following examples, only some comparative studies are given to illustrate their application. Example 10.1: Application of the Nanoaffinity Chromatography System of the Invention
本实施例中， 所用亲和层析***为含纳米结构亲和层析粒子的柱。 所用纳米结构亲和层析粒子，分别为上述实施例 4.1-4.3 制备的本发明的 纳米结构亲和层析粒子,和对照纳米结构亲和层析粒子。 对照纳米结构亲 和层析粒子的活化纳米结构中的活性基团为氨基，例如 3-氨丙基三甲氧基 硅烷为偶联剂制备的偶联化纳米粒子结合功能试剂后，再结合至常规层析 粒子上形成的纳米结构快检试剂条 (基本制备方法参考上述相关实施例)。  In this embodiment, 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).
本实施例中， 测试方法与已知的亲和层析检测方法相同。 例如，本实 施例检测了上述实施例制备的纳米亲和层析*** (例如分别含亲和试剂 / 活化纳米凸体 /硅胶粒子和亲和试剂 /活化纳米凸体 /含聚多糖的粒子、 等 等的柱)的动力学吸附容量。动力学吸附容量检测条件如下:用于填充上述 介质的柱子内径 0.5 cm和长度 2 cm,缓冲液为 0.01M Tris— HCl/pH 7.40, 流速为 1 ml/min， 所用层析仪为 HP 1090。 以亲和试剂为蛋白质 A时为 例， 所用样品为人抗体。按照公知的亲和层析动力学吸附容量测定方法， 上述实施例制备的纳米亲和层析***与对照纳米亲和层析*** (含对照 纳米结构亲和层析粒子)相比， 动力学吸附容量要高 30%以上。  In this embodiment, the test method is the same as the known affinity chromatography detection method. For example, 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. The 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. For example, when the affinity reagent is protein A, the sample used is a human antibody. According to the well-known affinity chromatography kinetic adsorption capacity determination method, 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.
实施例 10.2: 本发明的功能化纳米磁分离***的应用 Example 10.2: Application of the functionalized nanomagnetic separation system of the present invention
本实施例中， 所用功能化纳米磁分离***，分别为上述实施例 6.2制 备的本发明的功能化纳米磁分离*** (例如表 1 中的 Bl-B3)，和对照功能 化纳米磁分离***。 对照功能化纳米磁分离***含相同的配对亲和磁粒 子 (参考上述实施例 6.2),但其功能化亲和纳米结构中的活化纳米结构中的 活性基团为氨基,例如 3-氨丙基三甲氧基硅烷为偶联剂制备的偶联化纳米 结构结合功能试剂后，再结合前述功能化试剂形成的功能化纳米结构 （基 本制备方法参考上述相关实施例：)。  In this embodiment, 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:).
本实施例中， 分离方法与已知的功能化纳米磁分离***的分离方法 相同。例如,： 将上述功能化纳米磁分离***中的功能化亲和纳米结构 (粒 子或 /和串珠） (例如： 乙肝表面抗体 /活化纳米粒子、 HIV抗原 /活化纳米粒 子、 HCV抗原 /活化纳米粒子、 乙肝表面抗体 /活化纳米串珠、 HIV抗原 / 活化纳米串珠、 HCV抗原 /活化纳米串珠)， 分别与样品中可能存在的目 标物 (例如人乙肝表面抗原、 HIV抗体、 HCV抗体)接触， 并在有效条件 下反应生成目标物 /功能化纳米粒子复合物，然后将含此目标物 /功能化纳 米结构复合物的样品与上述相配对的亲和磁粒子在有效条件下反应生成 目标物 /功能化纳米结构 /亲和磁粒子复合物， 再利用外磁场将目标物 /功 能化纳米结构 /亲和磁粒子复合物分离出来， 如有必要可用适当缓冲液洗 涤，然后再对目标物 /功能化纳米结构 /亲和磁粒子复合物中的目标物进行 定性或定量分析。 In this embodiment, the separation method is the same as the separation method of the known functionalized nanomagnetic separation system. For example: 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, in contact with possible targets (eg, human hepatitis B surface antigen, HIV antibody, HCV antibody) in the sample, and 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.
本发明的功能化纳米磁分离***， 与对照功能化纳米磁分离***相 比，其分离效率要高 1倍以上 (例如可检出的阳性样品最低浓度要低 1倍 以上)，而 37°C放置 71小时后分离效率减小率要低 30%以上。 结果说明本 发明的功能化纳米磁分离***具有更高的灵敏度和稳定性。  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.
实施例 11: 本发明的标记***的应用 Example 11: Application of the marking system of the present invention
含本发明的标记***的装置或试剂盒 (例如表 1中的 C1-C4),可按公 知的相应装置或试剂盒的应用来应用。 本实施例中， 仅给出一些对比研 究， 来说明其应用。 更具体的对比研究方法由以下实施例补充。  Devices or kits containing the labeling system of the present invention (e.g., C1-C4 in Table 1) can be used in accordance with the application of known corresponding devices or kits. In this embodiment, only some comparative studies are given to illustrate their application. A more specific comparative research method is supplemented by the following examples.
实施例 11.1: 本发明的分析芯片标记***的应用 Example 11.1: Application of the analytical chip marking system of the present invention
本实施例中,所用试剂盒选自表 1中的 Cl-C3。 所用分析芯片分析方 法与上述实施例 9.1所用分析芯片分析方法基本相同， 不同之处在于所 用芯片为表 1中的常规芯片,而所用标记***为纳米标记***。  In this example, 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.
本实施例中，所用纳米标记物,分别为上述实施例 7制备的本发明的 纳米标记物和对照纳米标记物。 对照纳米标记物含相同的标记用功能化 试剂和标记物质 (参考上述实施例 7),但其中的活化纳米结构中的活性基 团为氨基,例如 3-氨丙基三甲氧基硅烷为偶联剂制备的偶联化纳米结构。  In the present embodiment, 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.
本实施例中， 本发明的纳米标记物与对照纳米标记物比较， 使用相 同阳性样品时在相同扫描条件下的平均信号读数要高 150%以上，可检出 的阳性样品最低浓度要低 150%以上,而 37°C放置 71小时后相同阳性样品 的信号值平均减小率要低 25%以上。 结果说明本发明的纳米标记物具有 更高的灵敏度和稳定性。  In this embodiment, when the nanomarker of the present invention is compared with the control nanomarker, 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. Above, 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 results demonstrate that the nanomarkers of the present invention have higher sensitivity and stability.
实施例 11.2: 本发明的酶标记***的应用 Example 11.2: Application of the enzyme labeling system of the invention
本实施例中,所用试剂盒选自表 1中的 C4。 所用 Elisa分析方法与上 述实施例 9.2所用 Elisa分析方法基本相同， 不同之处在于所用酶标板为 表 1中的常规芯片,而所用标记***为纳米标记***。  In this example, 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.
本实施例中， 所用纳米标记物,分别为上述实施例 7制备的本发明的 纳米标记物和对照纳米标记物。 对照纳米标记物含相同的标记用功能化 试剂和标记物质 (参考上述实施例 7)，但其中的活化纳米结构中的活性基 团为氨基,例如 3-氨丙基三甲氧基硅烷为偶联剂制备的偶联化纳米结构。  In the present embodiment, 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.
本实施例中， 本发明的纳米标记物与对照纳米标记物比较， 使用相 同阳性样品时在相同比色条件下的平均信号读数要高 150%以上,可检出 的阳性样品最低浓度要低 150%以上，而 37°C放置 71小时后相同阳性样品 的信号值平均减小率要低 25%以上。 结果说明本发明的纳米标记物具有 更高的灵敏度和稳定性。 In this embodiment, 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.
实施例 12: 含多种本发明的***的试剂盒的应用 Example 12: Application of a kit containing a plurality of systems of the invention
含多种本发明的*** (纳米反应***、纳米标记***、纳米分离***) 的装置或试剂盒 (例如表 2、 表 3)， 可以按公知的相应装置或试剂盒的应 用而应用。 本实施例中， 仅给出一些对比研究， 来说明其应用。 更具体 的对比研究方法由以下实施例补充。  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. In this embodiment, only some comparative studies are given to illustrate its application. A more specific comparative research method is supplemented by the following examples.
实施例 12.1: 含本发明的纳米反应***和纳米标记***的试剂盒的应用 本实施例中,所用分析方法与上述实施例 9.1和 9.2所用分析方法基 本相同， 不同之处在于所用试剂盒。 本实施例所用试剂盒中,反应***为 纳米反应***和所用标记***为纳米标记***。 其中:本发明试剂盒选自 表 2中的 D1-D4;对,所用对照试剂盒含实施例 9.1和 9.2中所述的对照纳 米反应***和实施例 11.1和 11.2中所述的对照纳米标记***。一般而言， 使用本发明的试剂盒与使用对照试剂盒相比： 使用相同阳性样品时在相 同比色条件下的平均信号读数要高 130%以上，可检出的阳性样品最低浓 度要低 120%以上,而 37°C放置 71小时后相同阳性样品的信号值平均减小 率要低 15%以上。结果说明本发明的试剂盒具有更高的灵敏度和稳定性。 实施例 12.2: 含本发明的纳米分离***和纳米标记***的试剂盒的应用 本实施例中，所用分析方法与上述实施例 9.1和 10.2所用分析方法基 本相同， 不同之处在于所用试剂盒。 本实施例所用芯片试剂盒中，反应系 统为常规芯片，所用标记物为纳米标记物，还含功能化纳米磁分离***。 其中:本发明试剂盒选自表 2中的 E1-E3;所用对照试剂盒含实施例 11.1中 所述的对照纳米标记物和实施例 10.2所述的对照功能化纳米磁分离系 统。 一般而言， 使用本发明的试剂盒与使用对照试剂盒相比： 使用相同 阳性样品时在相同比色条件下的平均信号读数明显提高，可检出的阳性样 品最低浓度明显降低，而 37Ό放置 71小时后相同阳性样品的信号值平均 减小率也较低。 结果说明本发明的试剂盒具有更高的灵敏度和稳定性。 实施例 12.3: 含本发明的纳米分离***和纳米反应***的试剂盒的应用 本实施例中,所用分析方法与上述实施例 9.1和 10.2所用分析方法基 本相同， 不同之处在于所用试剂盒。 本实施例所用芯片试剂盒中，反应系 统为纳米芯片，所用标记物为常规标记物,还含功能化纳米磁分离***。 其中:本发明试剂盒选自表 2中的 F1-F3;所用对照试剂盒含实施例 9.1中 所述的对照纳米芯片和实施例 10.2所述的对照功能化纳米磁分离***。 一般而言， 使用本发明的试剂盒与使用对照试剂盒相比： 使用相同阳性 样品时在相同比色条件下的平均信号读数明显提高，可检出的阳性样品最 低浓度明显降低,而 37Ό放置 71小时后相同阳性样品的信号值平均减小 率也较低。 结果说明本发明的试剂盒具有更高的灵敏度和稳定性。 Example 12.1: Application of a kit containing the nanoreaction 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 9.2, except for the kit used. In the kit used in this embodiment, the reaction system is a nano-reaction system and the labeling system used is a nano-labeling system. Wherein: 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 . In general, 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. In the chip kit used in this embodiment, the reaction system is a conventional chip, and the label used is a nano-marker, and also contains a functionalized nano-magnetic separation system. Wherein: 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. In general, 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 Ό 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 12.3: Application of kit containing the nano-separation system and nano-reaction system of the present invention In the present example, 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. In 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. Wherein: 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. In general, 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 Ό 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.
实施例 12.4: 含本发明的纳米分离***、 纳米反应***和纳米标记*** 的试剂盒的应用 Example 12.4: Application of a kit containing the nano-separation system, nano-reaction system and nano-labeling system of the present invention
本实施例中,所用分析方法与上述实施例 9.1和 10.2所用分析方法基 本相同， 不同之处在于所用试剂盒。 本实施例所用芯片试剂盒中，反应系 统为纳米芯片,所用标记物为纳米标记物，还含功能化纳米磁分离***。 其中:本发明试剂盒选自表 3;所用对照试剂盒含实施例 9.1中所述的对照 纳米芯片、实施例 10.2所述的对照功能化纳米磁分离***、和实施例 11U 所述的对照纳米标记物。 一般而言， 使用本发明的试剂盒与使用对照试 剂盒相比： 使用相同阳性样品时在相同比色条件下的平均信号读数明显 提高，可检出的阳性样品最低浓度明显降低，而 37°C放置 71小时后相同阳 性样品的信号值平均减小率也较低。 结果说明本发明的试剂盒具有更高 的灵敏度和稳定性。  In the present example, 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. In the chip kit used in this embodiment, the reaction system is a nanochip, and the label used is a nano-marker, and also contains a functionalized nano-magnetic separation system. Wherein: 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. In general, 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.

Claims

权 利 要 求 书 Claim
1. 一种用于分离或分析的组成，其包含活化纳米结构，其中所述活化纳 米结构至少包含纳米结构和共价键合在所述纳米结构上的活化结构， 所述活化结构至少包含用以结合功能试剂的活化基团，所述活化基团 包括基于多肽合成试剂的含氨基的多官能团基团、 或 /和其衍生物基 团。 CLAIMS 1. A composition for separation or analysis comprising an 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 In combination with an activating group of a functional agent, the activating group includes an amino group-containing polyfunctional group based on a polypeptide synthesis reagent, or/and a derivative group thereof.
2. —种用于分离或分析的组成，其包含功能化纳米结构，所述功能化纳 米结构至少包含活化纳米结构和固定在所述活化纳米结构上的功能 试剂，其中所述活化纳米结构至少包含纳米结构和共价键合在所述纳 米结构上的活化结构，所述活化结构至少包含用以结合功能试剂的活 化基团，所述活化基团包括基于多肽合成试剂的含氨基的多官能团基 团、 或 /和其衍生物基团。  2. 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, wherein the activated nanostructure is at least An activation structure comprising a nanostructure and an covalently bonded structure on the nanostructure, the activation structure comprising at least an activating group for binding to a functional reagent, the activating group comprising an amino group-containing polyfunctional group based on a polypeptide synthesis reagent a group, or/and a derivative thereof.
3. 一种用于分离或分析的组成，其包含活化纳米结构载体，其中所述活 化纳米结构载体至少包含常规载体和活化纳米结构，其中所述活化纳 米结构至少包含纳米结构和共价键合在所述纳米结构上的活化结构， 所述活化结构至少包含用以结合功能试剂的活化基团，所述活化基团 包括基于多肽合成试剂的含氨基的多官能团基团、 或 /和其衍生物基 团。  3. A composition for separation or analysis comprising an activated nanostructure support, wherein the activated nanostructure support comprises at least a conventional support and an activated nanostructure, wherein the activated nanostructure comprises at least a nanostructure and a covalent bond An activating structure on the nanostructure, the activating structure comprising at least an activating group for binding to a functional reagent, the activating group comprising an amino group-containing polyfunctional group based on a polypeptide synthesis reagent, or/and a derivative thereof Substance group.
4. 一种用于分离或分析的组成，其包含功能化纳米结构载体，所述功能 化纳米结构载体至少包含常规载体和功能化纳米结构，所述功能化纳 米结构至少包含活化纳米结构和固定在所述活化纳米结构上的功能 试剂，其中所述活化纳米结构至少包含纳米结构和共价键合在所述纳 米结构上的活化结构，所述活化结构至少包含用以结合功能试剂的活 化基团，所述活化基团包括基于多肽合成试剂的含氨基的多官能团基 团、 或 /和其衍生物基团。  4. A composition for separation or analysis comprising a functionalized nanostructure carrier comprising at least a conventional support and a functionalized nanostructure, the functionalized nanostructure comprising at least activated nanostructures and immobilization a functional agent 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 an activating group for binding to a functional reagent The activating group includes an amino group-containing polyfunctional group based on a polypeptide synthesis reagent, or/and a derivative group thereof.
5. 权利要求 1-4之一所述的组成，其中所述活化基团包括氨基肼基团或 /和氨基肼衍生物基团。  5. The composition of any one of claims 1 to 4, wherein the activating group comprises an aminoguanidine group or/and an aminoguanidine derivative group.
6. 权利要求 1-4之一所述的组成，其中所述活化基团包括氨基酸基团或 /和氨基酸衍生物基团。  6. The composition of any one of claims 1 to 4, wherein the activating group comprises an amino acid group or/and an amino acid derivative group.
7. 权利要求 1-4之一所述的组成，其中所述活化基团包括合成肽基团或 /和合成肽衍生物基团。  7. The composition of any one of claims 1 to 4, wherein the activating group comprises a synthetic peptide group or/and a synthetic peptide derivative group.
8. 权利要求 1-7之一所述的组成，其中所述活化结构还含连接所述纳米 结构和活化基团的偶联基团。 8. The composition of any one of claims 1-7, wherein the activated structure further comprises a link to the nano a coupling group of a structure and an activating group.
9. 权利要求 8所述的组成， 其中所述偶联基团包括硅烷基团。  9. The composition of claim 8 wherein the coupling group comprises a silane group.
10.权利要求 1-9之一所述的组成，其中所述纳米结构包括含无机材料的 纳米结构。  10. The composition of any of claims 1-9, wherein the nanostructures comprise nanostructures comprising inorganic materials.
11.权利要求 3-10之一所述的组成，其中所述常规载体包括下述组之一： 粒状常规载体， 面状常规载体， 和膜状常规载体。  The composition according to any one of claims 3 to 10, wherein the conventional carrier comprises one of the following groups: a granulated conventional carrier, a planar conventional carrier, and a membranous conventional carrier.
12.权利要求 1-11之一所述的组成， 其中所述纳米结构包括： 纳米粒子、 纳米串珠、 或 /和纳米凸体。  12. The composition of any of claims 1-11, wherein the nanostructures comprise: nanoparticles, nanobeads, or/and nanoprotrusions.
13.权利要求 12所述的组成， 其中所述活化纳米结构包括： 活化纳米粒 子、 活化纳米串珠、 或 /和活化纳米凸体。  13. The composition of claim 12, wherein the activated nanostructures comprise: activated nanoparticles, activated nanobeads, or/and activated nanoprotrusions.
14.权利要求 12所述的组成， 其中所述活化纳米结构载体包括活化纳米 凸体载体。  14. The composition of claim 12 wherein the activated nanostructure support comprises an activated nano-convex support.
15.权利要求 14所述的组成， 其包括下述组之一： 分析芯片纳米结构片 基、 纳米结构酶标、微孔板、平面层析纳米结构片基、纳米结构活化 层析固定相。  15. The composition of claim 14 comprising one of the group consisting of: an assay chip nanostructure substrate, a nanostructure enzyme label, a microplate, a planar tomographic nanostructure sheet, a nanostructure activation chromatography stationary phase.
16.权利要求 12所述的组成， 其中所述功能化纳米结构包括： 功能化纳 米粒子、 功能化纳米串珠、 或 /和功能化纳米凸体。  16. The composition of claim 12, wherein the functionalized nanostructures comprise: functionalized nanoparticles, functionalized nanobeads, or/and functionalized nanoprotrusions.
17.权利要求 12所述的组成， 其中所述功能化纳米结构载体包括功能化 纳米凸体载体。  17. The composition of claim 12, wherein the functionalized nanostructure carrier comprises a functionalized nano-convex support.
18.权利要求 16或 17所述的组成， 其包括含所述功能化纳米结构或 /和 功能化纳米结构载体的分离***。  18. The composition of claim 16 or 17, comprising a separation system comprising the functionalized nanostructures or/and functionalized nanostructure supports.
19.权利要求 16或 17所述的组成， 其包括含所述功能化纳米结构或 /和 功能化纳米结构载体的标记***。  19. The composition of claim 16 or 17, comprising a labeling system comprising the functionalized nanostructures or/and a functionalized nanostructure carrier.
20.权利要求 16或 17所述的组成， 其包括含功能化纳米结构或 /和功能 化纳米结构载体的反应***。  20. The composition of claim 16 or 17, comprising a reaction system comprising a functionalized nanostructure or/and a functionalized nanostructure carrier.
21.权利要求 20所述的组成， 其包括含所述反应***的下述组之一的装 置：纳米结构分析芯片、纳米结构酶标板、纳米结构平面层析试剂条。 21. The composition of claim 20 comprising means comprising one of the following: a nanostructure analysis chip, a nanostructure ELISA plate, a nanostructure planar chromatography reagent strip.
22.权利要求 16-21之一所述的组成， 其包括含下述之一种、二种或三种 ***的试剂盒： 所述纳米反应***、所述纳米标记***、所述纳米分 离***。 22. The composition of any one of claims 16-21, comprising a kit comprising one, two or three systems: the nanoreactor system, the nanolabeling system, the nanoseparation system .
23.权利要求 22所述的组成， 其中所述试剂盒包括下述组之一： 纳米结 构分析芯片试剂盒、纳米结构酶标试剂盒、纳米结构平面层析试剂条 试剂盒。 23. The composition of claim 22, wherein the kit comprises one of the group consisting of: a nanostructure analysis chip kit, a nanostructured enzyme labeling kit, a nanostructured planar chromatography reagent strip kit.
24.权利要求 1-23之一所述的组成， 其中所述分离或 /和分析的目标物包 括： 1).多肽或 /和与多肽相互作用的药物； 或 /和 2).核酸或 /和与核酸 相互作用的药物。 24. The composition of any one of claims 1 to 23, wherein the target of isolation or/and analysis comprises: 1) a polypeptide or/and a drug that interacts with the polypeptide; or/and 2) a nucleic acid or / And drugs that interact with nucleic acids.
25.一种分离或分析方法，其包括提供和应用权利要求 1一 24之一所述的 用于分离或分析的组成的步骤。  25. A method of separation or analysis comprising the steps of providing and applying a composition for separation or analysis as described in one of claims 1-24.
26.根据权利要求 25所述的方法， 其中所述分离或分析组成的提供， 包 括提供所述纳米结构，并将所述活化结构共价固定到所述纳米结构上 形成所述活化纳米结构。  26. The method of claim 25, wherein the providing or analyzing the composition of the composition comprises providing the nanostructure and covalently immobilizing the activated structure onto the nanostructure to form the activated nanostructure.
27.根据权利要求 26所述的方法， 其中所述活化结构中活化基团的形成 或 /和引入， 使用合成肽方法。  27. The method of claim 26, wherein the formation or/and introduction of an activating group in the activated structure uses a synthetic peptide method.
28.根据权利要求 27所述的方法， 其中所述活化基团的形成或 /和引入， 包括下述组之一种或多种步骤：提供含保护基团的反应物并在其后的 步骤中至少部分脱去所述保护基团; -NH2基和 -COOH基之间的反应; 肽链增长。 28. The method of claim 27, wherein the forming or/and introducing of the activating group comprises one or more of the steps of: providing a protecting group-containing reactant and subsequent steps At least partially removing the protecting group; reacting between -NH 2 group and -COOH group; peptide chain growth.
PCT/CN2006/001374 2006-01-25 2006-06-16 Seperation or analysis composition comprising active nanostructure and seperation or analysis method WO2007115444A1 (en)

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