WO2004081571A1 - Procede et dispositif de detection de polypeptides, et compose ligand comprenant des nanoparticules - Google Patents

Procede et dispositif de detection de polypeptides, et compose ligand comprenant des nanoparticules Download PDF

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Publication number
WO2004081571A1
WO2004081571A1 PCT/CN2004/000077 CN2004000077W WO2004081571A1 WO 2004081571 A1 WO2004081571 A1 WO 2004081571A1 CN 2004000077 W CN2004000077 W CN 2004000077W WO 2004081571 A1 WO2004081571 A1 WO 2004081571A1
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Prior art keywords
ligand
magnetic
nanoparticle
nanoparticles
affinity
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PCT/CN2004/000077
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English (en)
Chinese (zh)
Inventor
Fanglin Zou
Chunsheng Chen
Ning Chen
Jianxia Wang
Original Assignee
Chengdu Kuachang Medical Industrial Limited
Chengdu Kuachang Science & Technology Co., Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Priority claimed from CN 03117446 external-priority patent/CN1250969C/zh
Priority claimed from CNA031177875A external-priority patent/CN1514243A/zh
Application filed by Chengdu Kuachang Medical Industrial Limited, Chengdu Kuachang Science & Technology Co., Ltd filed Critical Chengdu Kuachang Medical Industrial Limited
Priority to PCT/CN2004/000203 priority Critical patent/WO2004090548A1/fr
Priority to JP2006529552A priority patent/JP2007502998A/ja
Priority to PCT/CN2004/000437 priority patent/WO2004102196A1/fr
Priority to EP04730459A priority patent/EP1624306A4/fr
Publication of WO2004081571A1 publication Critical patent/WO2004081571A1/fr
Priority to US11/258,996 priority patent/US7842515B2/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
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/00029—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
    • G01N2035/00099—Characterised by type of test elements
    • G01N2035/00158—Elements containing microarrays, i.e. "biochip"

Definitions

  • the invention relates to a method and a device for quantitative or / and qualitative detection of a polypeptide, in particular an analysis chip, a microplate, a flat chromatography strip, and a kit thereof.
  • the invention also relates to an immobilized ligand in a detection device and a separation device and a preparation method thereof.
  • the present invention also relates to a ligand / nanoparticle / molecularly labeled substance complex and a method for preparing the same.
  • the invention also relates to a microcarrier-coated carrier and a preparation method thereof. Background technique
  • the immobilized ligand with selective reactivity has been widely used in many aspects, especially in the qualitative and / or quantitative detection, analysis and separation of target substances in samples.
  • chromatographic gels can be taken as an example.
  • An example of an immobilized ligand for detection is an analysis chip.
  • labeling systems containing ligands are the most widely used labeling systems. Due to the different immobilized ligands and / or ligand labeling systems, different analytical methods can be used.
  • bioassay analysis the target of root samples can be divided into nucleic acid detection, peptide detection, and so on.
  • biochip detection is used as an example to briefly explain the problems that need to be solved with existing peptide detection methods and devices.
  • a biochip is a detection device formed by fixing two or more kinds of microligands in an addressable manner on a substrate. Because of its high throughput and miniaturization, biochips have a wide range of applications, including gene expression detection, gene screening, drug screening, disease diagnosis and treatment, environmental monitoring and governance, and judicial identification.
  • One of the main indicators of biochip detection is sensitivity.
  • the immobilized ligand is one of the keys to determine the sensitivity.
  • the current chip-immobilized ligands are mainly formed by directly fixing the ligands on a flat substrate.
  • the substrate used is mainly a rectangular, circular or other shape flat carrier containing active derivatization groups made of glass, metal, plastic and other materials and their derivatives.
  • chip-immobilized ligand is a substrate having a high specific surface, such as a film, a glass substrate, a particle-adhesive, a glass substrate, etc. On the formation.
  • the first type of chip-immobilized ligand due to the smaller surface area of the immobilized ligand, etc., its reaction kinetics conditions need to be optimized, and its performance is still to be improved, or / and The response time has yet to be shortened.
  • the second kind of chip immobilized ligand theoretically speaking, a higher specific surface can improve sensitivity, but the actual situation is not always the case.
  • reactors that are at least partially film-based have reduced sensitivity due to their high background noise.
  • reactors based on membranes, microparticles, etc. have other practical problems (such as cleaning of membranes, adhesion of microparticles, etc.).
  • their current applications are not even as good as those of the first method.
  • Another key factor in determining sensitivity and detection time is the marking system.
  • the labeling system used in the detection of chip peptides is mainly a molecular dispersion labeling system, and its sensitivity needs to be improved. Since the sensitivity needs to be improved, the degree of freedom in selecting the chip substrate also needs to be improved, and there are not many types of chip substrates available.
  • other detection devices containing a substrate such as microplate readers, have the same problems as biochips.
  • immobilized ligands such as affinity chromatography stationary phases are also widely used in separation devices such as chromatography devices.
  • separation devices such as chromatography devices.
  • microparticles used as the matrix of the chromatography stationary phase have a larger specific surface area than the substrate, there is still room for improvement in terms of chromatography time and chromatography yield.
  • One of the most commonly used reaction media in detection and separation is the immobilized ligand in the form of particles recognizable by the naked eye.
  • the most typical example is affinity chromatography, where the ligand carrier is a gel particle with a size of 5-100 ⁇ and a pore size of 0.05-0.5 nm.
  • a porous membrane having a pore size distribution from nm to ⁇ m is used as a ligand carrier.
  • colloids especially organic particles having a size of 100 nm to 500 nm, are used as a ligand carrier, and the purpose and effect to be achieved is only to reduce the processing labor of the chip, such as processing steps.
  • the main objective of the present invention is to improve the efficiency of ligand reaction in peptide detection, especially in peptide chip detection and enzyme-labeled detection devices, thereby increasing detection sensitivity or / and reducing detection time or / and providing more species with sufficiently high sensitivity Film base.
  • the object of the present invention is achieved by the development of immobilized ligands and labeling systems.
  • another object of the present invention is to improve the separation efficiency of the separation medium.
  • a method for quantitative or / and qualitative detection of a polypeptide includes the following steps:
  • the affinity nanostructure carrier includes a solid phase carrier and an affinity nanostructure distributed on a surface thereof, wherein the affinity nanostructure uses affinity nanoparticles as a unit.
  • step (c) providing a ligand / nanoparticle / molecular labeling substance complex and labeling the reaction result in step (b), said ligand / nanoparticle / molecular labeling substance complex containing one or more molecules A labeling substance, one or more nanoparticles, one or more ligands, and an optional blocking agent, the nanoparticle / ligand / molecular labeling substance is a mixture or a purified substance, the ligand is selected from the following group Substances capable of interacting with the target polypeptide: peptides, polysaccharides, vitamins, antibiotics, viruses, cells, and functional organics, the nanoparticles are at least one dimension in a three-dimensional space greater than 1 nm and less than 100 nm, preferably greater than 1 nm and Non-magnetic inorganic non-metallic microparticles or derivatives thereof that are smaller than 10 nm and are not themselves labeling substance enhancers, the derivatives including surface-modified or functional organic-
  • polypeptide detection device comprising the affinity nanostructure carrier and the ligand / nanoparticle / molecular labeling substance complex as described above.
  • the present invention provides a method for preparing a microcarrier-coated carrier for polypeptide detection or component separation, and a microcarrier-coated carrier prepared by the method.
  • the method includes the following steps:
  • microcarriers comprising nanoparticles or colloids, said nanoparticles being at least one dimension in a three-dimensional space greater than lnm and less than 100nm, preferably greater than lnm And non-magnetic inorganic particles smaller than 10 nm, the colloid is an organic matter dispersion system of 100-600 nm;
  • a carrier which comprises a base and carrier particles made of the following materials or their derivatives: glass, silicon wafers, silica gel, ceramics, metal oxides, metals, polymer materials, and composites thereof,
  • the derivatives include surface-modified or functional organic-coated derivatives containing derivatizing groups on the surface;
  • the colloidal particles of the carrier are coated on the carrier by physicochemical adsorption at room temperature, chemical bonding at room temperature, cross-linking of nanoparticles, or a combination thereof.
  • a detection device or a separation medium which includes the microcarrier-coated carrier as described above and a probe fixed on the microcarrier-coated carrier.
  • an affinity nanostructure carrier for polypeptide detection or component separation which includes the following steps:
  • ligands being selected from the group consisting of substances capable of interacting with the target polypeptide: polypeptides, polysaccharides, vitamins, antibiotics, viruses, Cells, and functional organics
  • the nanoparticles are non-magnetic particles in at least one dimension in a three-dimensional space of greater than 1 nm and less than 100 nm, preferably greater than 1 nm and less than 10 nm;
  • the affinity nanoparticle liquid includes a mixture and a purified substance, and one or more kinds of ligands are immobilized on a nanoparticle in the purified substance;
  • step (c) fixing the affinity nanoparticles prepared in step (b) to the carrier in a liquid or solid state to form the affinity nanostructure carrier, wherein there is a double or multiple between the one or more nanoparticles and the carrier; A ligand, or / and one or more nanoparticles between the one or more ligands and the carrier, or / and one or more ligands between at least one heavy nanoparticle and another heavy nanoparticle.
  • the affinity nanostructure carrier for polypeptide detection or component separation
  • the affinity nanostructure carrier includes a solid phase carrier and an affinity nanostructure distributed on a surface thereof, wherein the affinity nanostructure is constructed on the surface with affinity nanoparticles as a unit, and retains the main nano-phenomenon characteristics and has a higher reaction efficiency than the ligand
  • the affinity nanoparticle includes a nanoparticle and one or more ligands fixed thereon, and there is a heavy or Multiple ligands, or / and one or more nanoparticles between one or more ligands and the solid support, or / and one or more ligands between the at least one heavy nanoparticle and another heavy nanoparticle
  • the ligand is selected from the group of substances that can interact with the target polypeptide in the following groups: polypeptides, polysaccharides, vitamins, antibiotics, viruses, cells, and functional organics, and the nanoparticles are at least one dimension in a three-dimensional space greater than 1
  • a method for preparing a ligand / nanoparticle / molecular labeling substance complex which includes the following steps:
  • the ligand is selected from the group consisting of substances capable of interacting with the target polypeptide in the following groups: polypeptides, polysaccharides, vitamins, antibiotics, viruses, cells, and functional organics, and the nanoparticles are at least one dimension in a three-dimensional space greater than 1 nm and less than 100 ran, preferably greater than 1 nm and less than 10 nm, and non-magnetic inorganic non-metallic microparticles or derivatives thereof that are not labeling substance enhancers, the derivatives include surface modification or / and functional organic coatings containing derivatizing groups on the surface Derivative
  • a ligand / nanoparticle / molecular labeling substance complex for polypeptide detection which contains one or more molecular labeling substances, one or more nanoparticles, and a Or a plurality of ligands
  • said nanoparticle / ligand / molecular labeling substance is a mixture or a purified substance
  • said ligand is selected from the group of substances which can interact with the target polypeptide in the following group: polypeptide, polysaccharide, vitamin, antibiotic, virus, Cells, and functional organic matter
  • the nanoparticle is a non-magnetic inorganic non-metal particle or a derivative thereof in at least one dimension in a three-dimensional space of greater than 1 nm and less than 100 mn, preferably greater than 1 nm and less than 10 nm, said derivative Surface modification or functional organic Physically coated derivatives.
  • polypeptide detection device comprising the affinity nanostructure carrier and / or the ligand / nanoparticle / molecular labeling substance complex as described above.
  • it provides a method for quantitative or qualitative detection of a polypeptide, which comprises using an affinity nanostructure carrier as described above to capture a target polypeptide in a sample.
  • it provides a method for quantitative or qualitative detection of a polypeptide, which includes delabeling with a ligand / nanoparticle / molecular labeling substance complex as described above.
  • a chip detection method for quantitative or / and qualitative polypeptides which includes at least one, two, three or four steps as follows:
  • ligand being selected from the group consisting of substances capable of interacting with the target polypeptide in the following group: peptides, polysaccharides, vitamins, antibiotics, viruses, cells, and functional organics; said magnetic Nanoparticles have at least one dimension in the three-dimensional space of 1 to 200 nm, preferably 1 to 100 nm, and more preferably 1 to 50 nm;
  • the ligand / magnetic nanoparticle / platelet complex containing a carrier, one or A plurality of magnetic nanoparticles and one or more ligands, and there is a heavy or multiple ligand between the one or more magnetic nanoparticles and a carrier, or / and the one or more ligands and a carrier
  • the ligand is selected from the group of substances that can interact with the target polypeptide: Peptides, polysaccharides, vitamins, antibiotics, viruses, cells, and functional organic matter
  • the magnetic nanoparticles have at least one dimension in a three-dimensional space of 1 to 200 nm, preferably 1 to 100 nm, and more preferably 1 to 50
  • the base is selected from the following group of materials and their derivatives: glass, silicon wafers, ceramics, metal oxides, metals, polymer materials and their composites, said derivatives including surface modifications with derivatizing groups on the surface / Functional organic coating and derivatives thereof;
  • a ligand / magnetic nanoparticle / molecular labeling substance complex is used for the labeling reaction, and an external magnetic field may optionally be present during labeling;
  • a polypeptide detection chip which at least includes one or more ligands / magnetic nanoparticles / sheet-based complexes, and the ligands / magnetic nanoparticles / sheet-based complexes contain A carrier, one or more magnetic nanoparticles, and one or more ligands, and there is a heavy or multiple ligand between the one or more magnetic nanoparticles and the carrier, or / and the one or more There is a heavy or multiple magnetic nanoparticle between the ligand and the carrier, and / or there is a heavy or multiple magnetic ligand between at least one heavy magnetic nanoparticle and another heavy magnetic nanoparticle, the ligand is selected from the group consisting of Substances acting on peptides: peptides, polysaccharides, vitamins, antibiotics, viruses, cells, and functional organics; the magnetic nanoparticles are selected from at least one dimension in a three-dimensional space of 1 to 200 nm, preferably 1 to 100
  • step (c) binding the affinity magnetic nanoparticles prepared in step (b) to the carrier, and applying an external magnetic field when binding.
  • a polypeptide detection chip kit which includes the affinity nanostructure carrier as described above, or the polypeptide detection chip as described above, and further includes at least one of the following magnetic substances: magnetic Microparticles and / or microchips, affinity magnetic nanoparticles, and ligands / magnetic nanoparticles / molecular labeling substance complexes, said ligands being selected from the group of substances that can interact with the target polypeptide: peptides, polysaccharides, vitamins, Antibiotics, viruses, cells, and functional organic matter, the magnetic nanoparticles are magnetic particles having at least one dimension of 1 to 200 legs in a three-dimensional space, preferably 1 to 100 nm, and more preferably 1 to 50 nm. Microparticles and their derivatives, and the magnetic microparticles in the ligand / nanoparticle / molecular labeling substance complex are not themselves molecular labeling substance enhancers.
  • a peptide detection chip kit which includes at least one of the following magnetic nanoparticle-containing substances: magnetic nanoparticle, affinity magnetic nanoparticle, and ligand / magnetic nanoparticle / A molecularly labeled substance complex
  • the ligand is selected from the group of substances capable of interacting with the target polypeptide in the following group: polypeptides, polysaccharides, vitamins, antibiotics, viruses, cells, and functional organics
  • the magnetic nanoparticles are at least in a three-dimensional space Magnetic particles and their derivatives having a dimension of 1 to 200 nm, preferably 1 to 100 mn, more preferably 1 to 50 nm, and said particles in the ligand / nanoparticle / molecular labeling substance complex are not themselves molecular markers Substance enhancer.
  • a chip detector for performing detection using the polypeptide detection chip reagent kit as described above, further comprising a device for supplying a magnetic field during a reaction or / and a label during detection.
  • detection device is an article containing a ligand useful for interacting with a sample target, such as a device containing a capture ligand, a consumable, and a reagent containing a capture ligand and a labeling reagent, used in a specified amount or / and qualitative detection process.
  • a sample target such as a device containing a capture ligand, a consumable, and a reagent containing a capture ligand and a labeling reagent, used in a specified amount or / and qualitative detection process.
  • -Based labeling kit examples include analysis chips, microplates, affinity electrophoresis strips, affinity columns, planar chromatography reagent strips, analysis chip kits, microplate readers, affinity electrophoresis kits, and the like. Quantitative or / and qualitative testing can be performed in vitro or in vivo.
  • separation device used in the present invention refers to a separation product used in a separation process and containing a substance having a separation function.
  • the separation process is a process in which all or part of the components of a sample are obtained by a separation method.
  • the separation device include a chromatography device, and examples of a substance having a separation function include a chromatographic carrier.
  • affinity nanostructure carrier in the present invention refers to a complex containing at least a ligand, a nanoparticle, and a carrier, which includes a solid phase carrier and an affinity nanostructure distributed on a surface thereof, wherein the affinity Nanostructures are constructed on the surface with affinity nanoparticles as a unit, which retains the main nanophenomenon characteristics and have a higher reaction efficiency than ligands.
  • the affinity nanoparticles include nanoparticles and immobilized on them.
  • One or more ligands, and the binding between the ligands, nanoparticles, and carriers can have different forms, including direct binding (such as direct binding of nanoparticles to ligands) and Indirect binding (for example, in the embodiment, the nanoparticle and the chip substrate are bonded by a ligand fixed on the nanoparticle), for example: ligand-nanoparticle-carrier, ligand-nanoparticle-ligand-carrier, ligand A nanoparticle, a ligand, a nanoparticle, a carrier, a ligand 2—a microparticle—a ligand 1—a carrier, a ligand 2—a microparticle—a ligand 2—a ligand 1—a microparticle—a ligand 1—a carrier, and so on.
  • direct binding such as direct binding of nanoparticles to ligands
  • Indirect binding for example, in the embodiment, the nanoparticle and the chip substrate are bonded by a ligand fixed on the
  • ligand / magnetic nanoparticle / solid phase support in the present invention refers to a complex containing at least a ligand, a magnetic nanoparticle, and a carrier, wherein the binding between the ligand, the magnetic nanoparticle, and the carrier may be different
  • the forms include direct binding and indirect binding.
  • ligand in the present invention is equivalent to Ligand in English, and refers to a substance for capturing its ligand (equivalent to Ligate in English) through interaction (including affinity, ion exchange, lipophilicity, etc.), It can be combined with a carrier, such as a substrate, to form a reactor, or it can be combined with a molecular labeling substance to form a label.
  • ligand there are many substances that can be used as a ligand, such as one or more of the following: diethylaminoethyl (DEAE), diethylmono (2-hydroxypropyl) aminoethyl (QAE), carboxymethyl (CM), sulfopropyl (SP), mercaptoethylpyridyl (MEP), one NR 3+ , one RCOOH, siloxane group, thiol group, alkyl group, antigen, antibody, ligand, ligand Exponential enhancement of phylogenetic technology for screening of adaptive molecules, ligands, peptides, polysaccharides, coenzymes, cofactors, antibiotics, steroids, viruses, cells, biotin, avidin, etc.
  • DEAE diethylaminoethyl
  • QAE diethylmono (2-hydroxypropyl) aminoethyl
  • CM carboxymethyl
  • SP sulfopropyl
  • polypeptide in the present invention is equivalent to "polypeptide" in English, and includes natural or synthetic proteins, protein fragments, synthetic peptides, and the like. Common targets in immunoassays and commonly used ligands in tests, such as antigens, antibodies, And so on are all peptides.
  • nanoparticle in the present invention refers to a solid-phase carrier particle having a dimension of less than 500 nm, preferably 1 to 90 nm, and more preferably less than 50 nm in at least one dimension in a three-dimensional space.
  • carrier in the present invention, refers to a carrier having a solid phase morphology and having a size larger than the above-mentioned nanoparticles, for example, an analysis chip substrate, an enzyme-labeled plate substrate, an enzyme-labeled bead carrier, a chromatography carrier, and electrophoresis Glue, biosensor ligand carrier, etc.
  • film substrate in the present invention refers to a solid-phase carrier having a macroscopic plane on one side of its fixed function, for example, an analysis chip substrate, an enzyme-labeled plate substrate, an electrophoresis film, and a planar chromatography carrier.
  • ligand / nanoparticle / tablet complex in the present invention refers to a composite composition comprising a ligand, a nanoparticle, and a tablet, and the binding between the ligand, the nanoparticle, and the tablet may have different forms.
  • affinity nanoparticle in the present invention refers to a complex formed by the ligand and the nanoparticle by covalent or / and non-covalent bonding.
  • reactor in the present invention refers to a ligand in which a specific reaction with a target molecule in a sample is immobilized, a place where the ligand reacts specifically with the target molecule, and other related structures communicating with it, such as an open
  • substrate in the present invention refers to a product based on a substrate and combined with the presence or absence of other structures (such as an isolation structure) to form a chip after fixing the substrate.
  • Monolithic substrates usually have no isolation structure on them.
  • the substrate is both a substrate (such as a commercially available amino slide).
  • the multi-chip base substrate has an isolation structure.
  • the substrate includes a base and an isolation structure. After the substrate base is fixed on the substrate, a reactor is formed, and the multiple substrate bases form a multi-reactor chip.
  • the substrate is a substrate for fixing the ligand and other auxiliary agents (if any), and its surface chemical and optical properties are important factors affecting chip performance and cost.
  • film base pool in the present invention refers to a structure formed by a film base and its isolation structure.
  • the term "analysis chip” in the present invention is referred to as "chip” for short, and includes, but is not limited to, Bi 0 chip, Microarray, and Bioarray in English. It is a detection device in designated and / or quantitative analysis. The results of specific reactions of target molecules in the same product can be identified in an addressable manner.
  • the core of the chip is the reactor therein, and the core of the reactor is the chip substrate and the ligand fixed on the chip substrate.
  • the chip includes a microchannel chip (equivalent to Microchamel Bioc ip in English) and a microarray chip (equivalent to Biochip, Microarray, Bioarray in English), but it is well known that it does not include existing rapid test reagent strips.
  • the chip of the present invention contains a single reactor or multiple reactors and has a labeling system.
  • the distribution density of the ligands on the substrate in the reactor is greater than 10 points / cm 2
  • the preferred solution is greater than 20 points / cm 2
  • the more preferred solution is greater than 40 points / cm 2
  • the area of each ligand point is not more than 1 mm 2 o
  • chromatography in the present invention is equivalent to "Chromatogmpliy” in English, and includes affinity chromatography, reversed phase chromatography, hydrophobic chromatography, ion exchange chromatography, and the like, which are divided into planar chromatography (such as quick detection reagent strips and Quick test kits> and column chromatography.
  • molecularly labeled substance in the present invention refers to a substance that is used to form or participate in the formation of a detection signal and has a molecular form at the time of labeling, such as rhodamine, CY3, CY5, etc., which are commonly used in chip detection.
  • Fixing stability As the particle size of the particles decreases, the fixing conditions on the carrier (for example, the substrate) develop in a direction favorable to the fixing and the fixing stability.
  • Particle uniformity Compared with organic particles, especially colloidal particles, inorganic particles with a size of 1 to 90 nm are easier to prepare more uniformly.
  • microparticle-containing labels have attracted widespread attention. But mainly organic particles (such as the aminated organosilicon sodium rice particles in Chinese Patent Application Publication No. 1392097), and nano-labels for nucleic acid detection.
  • organic particles such as the aminated organosilicon sodium rice particles in Chinese Patent Application Publication No. 1392097
  • nano-labels for nucleic acid detection In a recent work, some complexes of metal nanoparticles with ligand-enhancing properties and ligands and labeling substances were successfully used for labeling (Surface enhanced raman scattering from metal nanoparticle-analyte-noble metal substrate sandwiches, US Patent No. 6,149,868). In our research, we were surprised to find that the application of the complex of non-metallic nanoparticles without ligand-enhancing properties with ligands and labeling substances can improve High detection sensitivity, especially chip detection sensitivity.
  • affinity nanostructure carriers and ligand / nanoparticle / molecular labeling substance complexes independently has greatly improved detection sensitivity, they have been used in combination.
  • the detection sensitivity can be further improved.
  • the present invention does not intend to enter the theoretical discussion, it is only assumed that the formation of the nanostructure (such as the affinity nanostructure carrier-target molecule-ligand / nanoparticle / molecular labeling substance complex) at this time has its own characteristics.
  • a subject of the present invention is a method for quantitative or / and qualitative peptide detection, which includes at least the following steps:
  • affinity nanostructure support comprising a solid phase support and an affinity nanostructure distributed on a surface thereof, wherein the affinity nanostructure is made of affinity nanoparticles as a unit; A structure constructed on the surface that retains the main nanophenomenon characteristics and thus has a higher reaction efficiency than a ligand, the affinity nanoparticle includes a nanoparticle and one or more ligands immobilized thereon, and There is a heavy or multiple ligand between one or more nanoparticles in the affinity nanostructured solid phase support and the solid phase support, or / and a heavy or multiple between one or more ligands and the solid phase support.
  • Nanoparticles or / and a heavy or multiple ligand between the at least one heavy nanoparticle and another heavy nanoparticle
  • the ligand is selected from the group of substances that can interact with the target polypeptide: polypeptide, polysaccharide, vitamin, antibiotic Viruses, cells, and functional organic matter
  • the nanoparticles are non-magnetic particles in at least one dimension in three-dimensional space of greater than 1 nm and less than 100 nm, preferably greater than 1 nm and less than 50 nm;
  • step (c) providing a ligand / nanoparticle / molecular labeling substance complex and labeling the reaction result in step (b), said ligand / nanoparticle / molecular labeling substance complex containing one or more molecules A labeling substance, one or more nanoparticles, one or more ligands, and an optional blocking agent, the nanoparticle / ligand / molecular labeling substance is a mixture or a purified substance, the ligand is selected from the group Substances capable of interacting with the target polypeptide: peptides, polysaccharides, vitamins, antibiotics, viruses, cells and functional organics, the nanoparticles are at least one dimension in a three-dimensional space greater than 1 nm and less than 100 nm, preferably greater than 1 nm and Non-magnetic inorganic non-metallic microparticles or derivatives thereof that are smaller than 10 nm and are not themselves labeling substance enhancers, the derivatives including surface-modified or functional organic-coated
  • the detection method of the present invention is a double-nanoparticle composite sandwich detection method, which includes a target in a sample being captured by an affinity nanostructure and labeled with a ligand / nanoparticle / labeling substance complex.
  • the affinity nanostructure carrier in the present invention is different from a carrier in which an affinity carrier is immobilized in a general sense. It is critical that the affinity nanoparticles form nanostructures on a solid support (eg, independently distributed affinity nanoparticles and / or loose aggregates of affinity nanoparticles observed under a microscope).
  • a substrate to which affinity nanoparticles are immobilized is considered to be an affinity nanostructure carrier only when its detection sensitivity is higher than that of the substrate to which only the same ligand is immobilized, preferably 25%, more preferably 50%. .
  • the non-magnetic particles include non-magnetic inorganic particles, non-magnetic organic particles and their derivatives, and the non-magnetic inorganic particles include non-magnetic inorganic non-metal particles and Non-magnetic metal microparticles, the derivatives include surface-modified or functional organic-coated derivatives containing derivatizing groups on the surface.
  • the non-magnetic inorganic non-metal particles include oxide particles including silicon oxide, titanium oxide, and alumina, and the metal particles include gold, silver, and copper , And aluminum particles.
  • metal particles such as microparticle gold have been used for rapid detection reagent strips, they are used as microparticle molecular markers, not as solid phase carriers in the method of the present invention.
  • the detection device containing the affinity nanostructure carrier includes an analysis chip, a microplate, and a planar chromatography reagent strip, and the ligand / nanoparticle / molecule is contained.
  • the label of the labeling substance complex includes an analysis chip label, an enzyme plate label, and a planar chromatography reagent strip label.
  • the solid-phase carrier includes a substrate and carrier particles made of the following materials or derivatives thereof: glass, silicon wafer, silica gel, ceramic, metal oxide, Metals, polymer materials, and their composites, the derivatives include surface-modified or / and functional organic-coated derivatives containing derivative groups on the surface, and microcarrier-coated carriers.
  • the derivatizing group includes a long-arm derivatizing group R— (CH 2 ) X— , where R is a derivatizing group, and X is equal to or greater than 2, preferably greater than 4, and more preferably greater than 6.
  • the derived group includes one or more of the following organic groups: amino, aldehyde, epoxy, aminohydrazine, diethylaminoethyl (DEAE), diethylmono (2-hydroxypropyl) ammonia Ethyl (QAE), carboxymethyl (CM), sulfopropyl (SP), mercaptoethylpyridine Pyridyl (MEP), siloxane, thiol, fluorenyl.
  • the functional organics include one or more of the following: surfactants such as polyvinylpyrrolidone, Tween-type surfactants, polyelectrolytes such as polyamino acids, lipophilic organics such as polysiloxanes, ion exchange polymers Such as dextran derivatives, agarose derivatives, cellulose derivatives, polyacrylamides, and affinity substances such as protein A (SPA), protein G (SPG), heparin sodium, biotin, avidin.
  • surfactants such as polyvinylpyrrolidone, Tween-type surfactants, polyelectrolytes such as polyamino acids, lipophilic organics such as polysiloxanes, ion exchange polymers
  • dextran derivatives agarose derivatives, cellulose derivatives, polyacrylamides
  • affinity substances such as protein A (SPA), protein G (SPG), heparin sodium, biotin, avidin.
  • a nanoparticle derivative such as superhydrophobic silica (CS7) has a fluorenyl group
  • the surface of the coated derivative has an organic coating group
  • a polyamino acid has an amino group and an aminohydrazine-polyamino acid has Amino and aminohydrazine
  • DEAE-Dextran has diethylaminoethyl, and so on.
  • surfactants such as polyvinylpyrrolidone, polyelectrolytes such as polyamino acids, ion-exchange polymers such as DEAE-Dextran, and affinity substances such as protein A are used in the method of the present invention to prepare ligands / nanoparticles / tablets.
  • an important aspect of microparticle derivatives is the coated derivative.
  • the coated derivative in the method of the present invention may be a single or multiple coated derivative.
  • silica particles coated with a dispersant or / and dispersion stabilizer can also be coated with PVP (double coat), and can continue to be coated with protein A (triple coat), etc. . Therefore, the range of organic coatings that can be selected is very large.
  • the molecularly labeled substance is selected from the group consisting of a fluorescent substance, a chemiluminescent substance, a chemiluminescent catalyst, a non-ferrous metal salt, a dye, and a pigment.
  • these molecularly labeled substances include one or more of the following: fluorescein, rhodamine, seaweed protein, silver salts, enzymes, basic black, basic violet, amino black, Coomassie brilliant blue, crystals purple.
  • the invention also relates to a polypeptide detection device, which comprises an affinity nanostructure carrier and a ligand / nanoparticle / molecular labeling substance complex as described above.
  • the polypeptide detection device includes a chip kit, a microplate reader, and a planar chromatography kit containing the affinity nanostructure carrier and the ligand / nanoparticle / molecular labeling substance complex.
  • the invention also relates to a method for preparing a microcarrier-coated carrier for polypeptide detection or component separation, which comprises the following steps: (a) preparing one or more microcarriers, said microcarriers comprising nanoparticles or colloids, said nanoparticles being at least one dimension in a three-dimensional space greater than 1 nm and less than 100 nm, preferably greater than 1 nm and Non-magnetic inorganic particles smaller than 10 nm, the colloid is an organic matter dispersion system of 100-600 nm;
  • a carrier which includes a base and carrier particles made of the following materials or derivatives thereof: glass, silicon wafer, silica gel, ceramic, metal oxide, metal, polymer material, and composites thereof,
  • the derivatives include surface-modified or functional organic-coated derivatives containing derivatizing groups on the surface;
  • the colloidal particles of the carrier are coated on the carrier by physicochemical adsorption at room temperature, chemical bonding at room temperature, cross-linking of nanoparticles, or a combination thereof.
  • the microcarrier-coated carrier includes an analysis chip substrate, an enzyme-labeled plate substrate, and a planar layer for polypeptide detection. Analytical reagent strips, and chromatographic stationary phase matrix for component separation.
  • the colloid includes a polymer colloid including polysaccharide and nitrocellulose and derivatives thereof, and the derivative Substances include surface-modified or functional organic-coated derivatives containing derivatizing groups on the surface.
  • the non-magnetic fine particles include non-magnetic inorganic fine particles, non-magnetic organic fine particles, and derivatives thereof.
  • the non-magnetic inorganic fine particles include non-magnetic inorganic non-metal fine particles and non-magnetic metal fine particles, and the derivatives include derivative groups on the surface.
  • Surface-modified or functional organic-coated derivatives of the group For example, wherein the oxide particles include silicon oxide, titanium oxide, and aluminum oxide particles, and the metal particles include gold, silver, copper, and aluminum particles.
  • derivatizing groups and functional organics described herein can be referred to those described in the above-mentioned polypeptide quantitative or / and qualitative detection methods.
  • the present invention also relates to a microcarrier-coated carrier, which is prepared by the above-mentioned method for preparing a microcarrier-coated carrier for polypeptide detection or component separation.
  • the microcarrier-coated carrier includes a microcarrier-coated chip.
  • the present invention also relates to a detection device or a separation medium, which includes the microcarrier-coated carrier as described above and a probe fixed on the microcarrier-coated carrier.
  • the detection device or separation medium includes a chip containing the microcarrier-coated carrier, a microplate, and a flat chromatography reagent strip.
  • the invention also relates to a method for preparing an affinity nanostructured carrier for polypeptide detection or component separation, which comprises the following steps:
  • the ligands being selected from the group of substances that can interact with the target polypeptide: polypeptides, polysaccharides, vitamins, antibiotics, viruses, Cells, and functional organic matter
  • the nanoparticles are non-magnetic particles with at least one dimension in a three-dimensional space of greater than 1 rnn and less than 100 nm, preferably greater than 1 nm and less than 10 nm.
  • the nanoparticles and the ligand may be prepared as a mixture with other substances, such as a nanoparticle suspension containing a coloring agent and / or a binder, a ligand solution containing a stabilizer, and the like.
  • the carrier according to the present invention may also be prepared in a manner coexisting with other structures, such as a substrate in a multi-chip base substrate;
  • the affinity nanoparticle liquid includes a mixture (for example, an unpurified substance after the nanoparticle and the ligand are mixed and reacted) and a purified substance (for example, the nanoparticle and the ligand are subjected to centrifugal separation to remove a purified substance with a free ligand)
  • a purified substance for example, the nanoparticle and the ligand are subjected to centrifugal separation to remove a purified substance with a free ligand
  • one or more kinds of ligands are immobilized on a nanoparticle in the purified product. It should be particularly emphasized that when the nanoparticles are not sufficiently diluted or over-diluted, no increase in sensitivity is observed for the complexes prepared using certain nanoparticles;
  • step (c) fixing the affinity nanoparticles prepared in step (b) to the carrier in a liquid or solid state to form the affinity nanostructure carrier, wherein there is a heavy or multiple between the one or more nanoparticles and the carrier; A ligand, or / and one or more nanoparticles between the one or more ligands and the carrier, or / and one or more ligands between at least one heavy nanoparticle and another heavy nanoparticle.
  • the nanoparticle suspension is mixed with the ligand solution to form affinity nanoparticles, and the mixed solution is spotted on the chip substrate to perform a binding reaction.
  • the ligand is combined with the nanoparticles and then with one or more nanoparticles and then with the substrate, the ligand is combined with the nanoparticles and then with the substrate and then is compounded with the one or more nanoparticles or the ligand-nanoparticle Combination of things, etc.
  • Affinity nanostructured carriers having multiple ligands between one or more nanoparticles and a carrier are prepared, for example, as follows: the carrier is coated with a reassortant 1 to form a ligand 1 coated carrier, and the nanoparticles are prepared. Coated with another ligand 2 (pairing reaction can occur between ligands 1 and 2) to form a ligand 2 / nanoparticle complex, and then coat or spot the ligand 2 / nanoparticle complex to the ligand 1 coats the carrier to form a complex in the form of ligand 2-nanoparticles-ligand 2-ligand 1-carrier. When the number of ligand layers is greater than 2, and so on.
  • affinity nanostructure carriers with multiple ligands between at least one heavy nanoparticle and another layer of nanoparticles.
  • ligand 2 ligand 1—nanoparticles—ligand 1—carrier, etc.
  • An affinity nanostructured carrier having multiple nanoparticles between one or more ligands and a carrier can be prepared as follows: First, one or more of the nanoparticles are combined with a plurality of the ligands to form a plurality of Affinity nanoparticles (for example, ligand 2—nanoparticles—ligand 2, ligand 3—nanoparticles—ligand 2, ligand 1—nanoparticles—ligand 1), and then the affinity nanoparticles are successively or At the same time, it is bound to the carrier to form, for example, ligand 2-nanoparticles-ligand 2-ligand 1-nanoparticles-ligand 1-carrier, ligand 3-nanoparticles-ligand 2-ligand 1-nano Affinity nanostructure carriers in the form of microparticles-ligand 1-carriers and the like.
  • Affinity nanoparticles for example, ligand 2—nanoparticles—ligand 2, ligand 3—
  • an affinity nanostructured carrier of the present invention further includes firstly cold-binding one or more of the nanoparticles and the carrier at 38 ° C or lower based on adsorption, or including adding chemical cross-linking.
  • the cross-linking and binding of the agent forms a nanoparticle / solid phase support, and then one or more ligands are bound thereto.
  • the volume concentration of the nanoparticles in the affinity nanoparticle liquid is between 1/1000 and 20,000.
  • the inorganic fine particles are selected from oxide fine particles, metal fine particles, and derivatives thereof, and the derivatives include a surface containing Derivative groups are surface modified or / and functional organic coated derivatives.
  • the oxide particles include silicon oxide, titanium oxide, and aluminum oxide particles, and the metal particles include gold, silver, copper, and aluminum particles.
  • the derivatizing groups and functional organics described herein can be referred to the description in the above-mentioned method for quantitative or / and qualitative detection of polypeptides.
  • the carrier includes a base and carrier particles made of the following materials or derivatives thereof: glass, silicon wafer, silica gel, ceramic, metal oxide, metal, polymer material, and composites thereof, and the derivative
  • the substance includes a surface-modified or / and functional organic-coated derivative containing a derivative group on the surface, and the above-mentioned microcarrier-coated carrier.
  • the invention also relates to an affinity nanostructure carrier for polypeptide detection or component separation, wherein the affinity nanostructure carrier comprises a solid phase carrier and an affinity nanostructure distributed on its surface. 5 wherein the affinity nanostructure The structure is constructed on the surface with affinity nanoparticles as a unit, which retains the main nano-phenomenon characteristics and has a higher reaction efficiency than ligands.
  • the affinity nanoparticles include nanoparticles and immobilized thereon.
  • One or more ligands and there is a heavy or multiple ligand between one or more nanoparticles in the affinity nanostructure solid phase support and the solid phase support, or / and one or more ligands There is a heavy or multiple nanoparticle between the solid phase carrier and / or a heavy or multiple ligand between the at least one heavy nanoparticle and another heavy nanoparticle.
  • the ligand is selected from the group consisting of capable of interacting with the target polypeptide.
  • Substances peptides, polysaccharides, vitamins, antibiotics, viruses, cells, and functional organics, the nanoparticles are at least one dimension in a three-dimensional space greater than 1 nm and less than 100 nm, preferably greater than 1 nm and less than 50 nm.
  • the derivatizing groups and functional organics described herein can be referred to the description in the above quantitative or / and qualitative detection method for polypeptides.
  • the affinity nanostructure carrier for polypeptide detection or component separation is an affinity nanoparticle containing the affinity nanoparticle prepared by the above method for preparing an affinity nanostructure carrier for polypeptide detection or component separation.
  • Affinity nanostructure carrier and includes an analysis chip for detection, a microplate, a flat chromatography reagent strip, and a chromatography stationary phase for separation.
  • the invention also relates to a method for preparing a ligand / nanoparticle / molecularly labeled substance complex, which comprises the following steps:
  • the ligand is selected from the group consisting of substances capable of interacting with the target polypeptide in the following groups: polypeptides, polysaccharides, vitamins, antibiotics, viruses, cells, and functional organics, and the nanoparticles are at least one dimension in a three-dimensional space greater than 1 nm and less than 100 nm, preferably greater than 1 nm and less than 10 nm, non-magnetic inorganic non-metallic microparticles or derivatives thereof that are not themselves labeling substance enhancers, said derivatives including surface modification or / and functional organic coatings containing derivatizing groups on the surface derivative; (b) combining the one or more ligands, one or more nanoparticles, and one or more of the molecular markers in one of the following
  • a nanoparticle suspension is mixed with a ligand solution to form an affinity nanoparticle, and the mixed solution is then mixed with a molecular labeling substance to combine the nanoparticle with the molecular labeling substance and then with the ligand (for example, rhodamine-labeled
  • a molecular labeling substance for example, rhodamine-labeled
  • the anti-antibody solution is mixed with the nanoparticle suspension, etc.), and the ligand is combined with the molecular labeling substance and the nanoparticle at the same time (for example, the nanoparticle suspension, the ligand solution is mixed with the molecular labeling substance solution, etc.).
  • the inorganic non-metal particles include non-magnetic oxide particles, and the non-magnetic oxide particles include silicon oxide, titanium oxide, and aluminum oxide .
  • the derivatizing groups and functional organics in the derivatives reference may be made to the description in the above-mentioned polypeptide quantitative or / and qualitative detection method.
  • the molecular labeling substance is selected from the group consisting of a fluorescent substance, a chemiluminescent substance, a chemiluminescent catalyst, a non-ferrous metal salt, a dye, and a pigment.
  • the molecular labeling substance includes one or more of the following: fluorescein, rhodamine, seaweed protein, silver salt, enzyme, basic black, basic violet, amino black, Coomassie brilliant blue, crystal purple.
  • the invention also relates to a ligand / nanoparticle / molecular labeling substance complex for polypeptide detection, which contains at least one or more molecular labeling substances, one or more nanoparticles, and one or more ligands.
  • the nanoparticle / ligand / molecular labeling substance is a mixture or a purified substance, and the ligand is selected from substances that can interact with the target polypeptide in the following groups: polypeptides, polysaccharides, vitamins, antibiotics, viruses, cells, and functional organics
  • the nano-particles are non-magnetic inorganic non-metallic particles or derivatives thereof in at least one dimension of more than 1 nm and less than 100 nm, preferably more than 1 nm and less than 10 nm in a three-dimensional space, and the derivatives include a surface containing a derivative group Surface-modified or / and functional organic-coated derivatives.
  • the derivatized groups and functional organics described herein can
  • the ligand / nanoparticle / molecular labeling substance complex of the present invention is different from the ligand- Nanoparticle complexes, such as ligand-microparticle ligands (documents CN02137418.X, CN02115771.5 and CN01133527.0), ligand-nanomagnetic particles (document CN02103867.8), molecular marker substances 1 Microparticle ligands (document No. CN0211411903) and the like.
  • the present invention combines a molecular labeling substance with a ligand and a nanoparticle to form a ligand / nanoparticle / molecular labeling substance complex, which is also different from a ligand-microsphere-fluorescent microparticle complex (document No. CN02121391.7) because
  • the labeling substance in the composite of the present invention is a molecular labeling substance (such as rhodamine) rather than a microparticle, and the carrier in the complex of the present invention is a nanoparticle (such as a colloid or a nano-silica) instead of a larger-sized microsphere.
  • the preparation method of the complex of the invention is simple, the preparation has good water solubility and high sensitivity.
  • the nanoparticles in the present invention are non-magnetic particles or / and ultrafine rods, and in a document published after the priority date of the present invention (Nanoparticles having oligonucleotides attached disclosure and uses theefore, 'US Patent Application No. 20030207296) Use magnetic oxide.
  • the nanoparticles in the present invention are non-magnetic inorganic non-metal particles or superfine rods, and in a document disclosed after the priority date of the present invention (Surface enhanced raman scattering from metal nanoparticle-analyte-noble metal substrate sandwiches , US Patent Ij6, 149,868) Be! J uses metal nanoparticles that enhance molecular markers.
  • the ligand / nanoparticle / molecular labeling substance complex for polypeptide detection of the present invention is prepared by the above method for preparing a ligand / nanoparticle / molecular labeling substance complex.
  • the ligand / nanoparticle / molecular labeling substance complex for peptide detection can be a marker for one of the following detections: chip detection, enzyme-labeled detection, and planar chromatography reagent strip detection.
  • the present invention also relates to a polypeptide detection device comprising the affinity nanostructure carrier as described above and / or the ligand / nanoparticle / molecular labeling substance complex as described above.
  • the polypeptide detection device may be a chip kit containing the affinity nanostructure carrier as described above and the ligand / nanoparticle / molecular labeling substance complex as described above, or a kit containing the affinity nanostructure carrier as described above.
  • the chip kit or the chip kit containing the ligand / nanoparticle / molecular labeling substance complex as described above, or the affinity nanostructure carrier and / or the ligand / nanoparticle / molecular label as described above
  • the present invention also relates to a method for quantitative or / qualitative peptide detection using the peptide detection device.
  • the present invention also relates to a method for quantitative or qualitative detection of a polypeptide, which includes And nanostructured carriers to capture the target polypeptide in the sample.
  • the present invention also relates to a method for quantitative or qualitative detection of a polypeptide, which comprises labeling a target polypeptide with the ligand / nanoparticle / molecular labeling substance complex as described above.
  • the present invention also relates to a chip detection method for quantitative or / and qualitative polypeptides, which includes at least one, two, three or four steps as follows:
  • ligand being selected from the group consisting of substances that can interact with the target polypeptide in the following group: polypeptides, polysaccharides, vitamins, antibiotics, viruses, cells, and functional organics; said magnetic Nanoparticles have at least one dimension in the three-dimensional space of 1-200 nm, preferably 1-100 nm, and more preferably 1-50 nm;
  • the ligand / magnetic nanoparticle / sheet-based composite containing a carrier, one or A plurality of magnetic nanoparticles and one or more ligands, and there is a heavy or multiple ligand between the one or more magnetic nanoparticles and a carrier, or / and the one or more ligands and a carrier
  • the ligand is selected from the group of substances that can interact with the target polypeptide: Peptides, polysaccharides, vitamins, antibiotics, viruses, cells, and functional organics
  • the magnetic nanoparticles have at least one dimension in a three-dimensional space of 1-200 nm, preferably 1-100 nm, more preferably 1-50 nm
  • a ligand / magnetic nanoparticle / molecular labeling substance complex is used for the labeling reaction, and an external magnetic field may optionally be present during labeling, and the ligand / magnetic nanoparticle / molecular labeling substance complex contains one or more Molecular labeling substance, one or more magnetic nanoparticles, one or more ligands, and optionally a blocking agent, said ligand / magnetic nanoparticles / sub-labeling substance is a mixture or a purified substance, said ligand
  • the base is selected from substances capable of interacting with polypeptides in the following groups: polypeptides, polysaccharides, vitamins, antibiotics, viruses, cells, and functional organics, and the magnetic nanoparticles have at least one dimension of 1-200 nm in a three-dimensional space, preferably 1- 100 nm, more preferably 1 to 50 nm, and is not itself a molecularly labeled substance enhancer.
  • the external magnetic field is preferably Pulsed magnetic field.
  • the invention also relates to a polypeptide detection chip, which at least comprises one or more ligands / magnetic nanoparticles / sheet-based complexes, and the ligands / magnetic nanoparticles / sheet-based complexes contain a carrier, one or more A magnetic nanoparticle and one or more ligands, and there is a heavy or multiple ligand between the one or more magnetic nanoparticles and a carrier, or / and between the one or more ligands and the carrier There is a heavy or multiple magnetic nanoparticle in between, and / or at least one heavy magnetic nanoparticle and another heavy magnetic nanoparticle has a heavy or multiple ligand, the ligand is selected from the group of substances that can interact with the target polypeptide: peptide , Polysaccharides, vitamins, antibiotics, viruses, cells, and functional organics; the magnetic nanoparticles are selected from the group consisting of at least one dimension in a three-dimensional space of 1 to 200 nm, preferably 1 to 100
  • Ferrites including ferric oxide and ferric oxide, and derivatives thereof, the derivatives including surface-modified or functional organic-coated derivatives containing derivatized groups on the surface;
  • the base is selected from the following Material group Their derivatives: glass, silicon, ceramics, metal oxides, metals, polymeric materials and their composites.
  • the detection chip may be an affinity magnetic nanostructure chip.
  • the affinity magnetic nanostructure chip includes a substrate and an affinity magnetic nanostructure distributed on a surface thereof, wherein the affinity magnetic nanostructure is an affinity magnetic nanoparticle.
  • the detection chip can be prepared as follows:
  • step (c) binding the affinity magnetic nanoparticles prepared in step (b) to the carrier, and providing an external magnetic field when binding.
  • the magnetic nanoparticles are selected from ferrite including ferric oxide, ferric oxide, and derivatives thereof, and the derivatives include surface modification or / And functional organic coating derivatives.
  • the derivatizing groups, functional organics, and microcarrier-coated carriers described herein can be referred to the description above.
  • the invention also relates to a peptide detection chip kit, wherein the chip is a chip containing no nano particles, or a detection chip as described above, and further includes at least one of the following magnetic substances: magnetic particles or / and microchips, And a magnetic nanoparticle, and a ligand / magnetic nanoparticle / molecular labeling substance complex, the ligand is selected from the group of substances that can interact with the target polypeptide in the following group: polypeptide, polysaccharide, vitamin, antibiotic, virus, cell, and function Organic matter, the magnetic nanoparticle has at least one dimension in the three-dimensional space of 1 to 200 nm, preferably at least 100 nm, more preferably 1 to 50 nm, and the magnetic substance in the ligand / nanoparticle / molecular labeling substance complex
  • the microparticles themselves are not molecular marker substance enhancer
  • the present invention also relates to a polypeptide detection chip kit, and a polypeptide detection chip kit, which includes at least one of the following magnetic nanoparticle-containing substances: magnetic nanoparticle, affinity magnetic nanoparticle, and ligand / magnetic nanoparticle
  • the ligand is selected from the group of substances that can interact with the target polypeptide in the following group: polypeptides, polysaccharides, vitamins, antibiotics, viruses, cells, and functional organics
  • the magnetic nanoparticles are in three dimensions Magnetic particles and their derivatives in at least one dimension of 1 to 200 nm, preferably 1 to 100 nm, more preferably 1 to 50 nm, and the particles themselves in the ligand / nanoparticle / molecular labeling substance complex are not Molecular marker substance enhancer.
  • the magnetic particles are selected from the group consisting of ferrite and ferric oxide, and derivatives thereof, and the derivatives include surface modification or / and Functional organics coated derivatives, and the derivatizing groups and functional organics can be referred to the description herein.
  • the present invention relates to a chip detector for performing detection using a polypeptide detection chip kit as described above, which includes a device that provides a magnetic field, preferably a pulsed magnetic field, during a reaction or / and a label during detection.
  • the advantage of the polypeptide quantitative or / and qualitative detection method of the present invention is that not only the target polypeptide in the sample is captured by the affinity nanostructure carrier, but also the ligand / nanoparticle / molecular labeling substance complex is used for labeling, which greatly improves the detection sensitivity or / And greatly improved the detection speed.
  • the advantage of the polypeptide detection device of the present invention is that it contains an affinity nanostructure carrier to capture the target polypeptide in the sample, and also contains a ligand / nanoparticle / molecular labeling substance complex for labeling, which greatly improves detection sensitivity, or / and greatly improves The detection speed.
  • microcarrier-coated carrier of the present invention is that it is easier to prepare various active carriers with low cost and sufficient sensitivity.
  • the method for preparing the affinity nanostructure carrier of the present invention has the advantage of being simple and effective.
  • affinity nanostructured carrier for polypeptide detection or component separation of the present invention are higher reaction efficiency with sample target, lower concentration limit of detectable target, and higher reaction speed.
  • the method for preparing the ligand / nanoparticle / molecular labeling substance complex of the present invention has the advantage of being simple and effective.
  • the advantage of the ligand / nanoparticle / molecular labeling substance complex for peptide detection of the present invention is that the reaction efficiency with the labeled substance is higher.
  • the detection device or separation device containing the affinity nanostructure carrier of the invention has the advantages of higher reaction efficiency with the target substance of the sample, lower concentration limit of the detectable target substance, and higher reaction speed.
  • the detection device of the ligand-containing / nanoparticle / molecular labeling substance complex of the present invention has the advantage of containing the ligand / nanoparticle / molecular-labeling substance complex for labeling, which improves detection sensitivity or / and increases detection speed.
  • An advantage of the method for quantitative or qualitative detection of a polypeptide of the present invention is that at least an affinity nanostructure carrier is used to capture a target polypeptide in a sample, thereby increasing detection sensitivity or / and increasing detection speed.
  • An advantage of the method for quantitative or qualitative detection of a polypeptide of the present invention is that at least the ligand / nanoparticle / molecular labeling substance complex is used for labeling, which improves detection sensitivity or / and increases detection speed.
  • the advantages of the chip quantitative and / or qualitative chip detection method of the present invention are fast and high sensitivity.
  • the peptide detection chip and kit of the present invention have the advantages of fastness and high sensitivity.
  • the advantages of the chip detector of the present invention are fast and high sensitivity.
  • nanoparticle-coated derivatives prepared in this example are included in Table 1, and the coatings used are listed in Table 3 below, including polyionic organic substances (polylysine), ions Derived polymers (DEAE-dextran, QAE-cellulose, aminohydrazine-polylysine), polymer surfactants (polyvinylpyrrolidone).
  • polyionic organic substances polylysine
  • ions Derived polymers DEAE-cellulose
  • aminohydrazine-polylysine aminohydrazine-polylysine
  • polymer surfactants polyvinylpyrrolidone
  • the method for preparing organic-coated nanoparticles is: dispersing the nanoparticles under ultrasonic oscillation to prepare a nanoparticle solution with a concentration of 1 / 2000—1 / 4000 (v / v), and then equalizing the volume with a concentration of 1/2000 (w / v)
  • the organic solution was mixed and reacted at 37 ° C for 1 hour under ultrasonic vibration.
  • the reaction product was dropped into a rotating tube containing gel, centrifuged at 4000 r / min, and the liquid in the collection tube was reserved for use (after all conditions are optimized, the centrifugation step can be omitted).
  • Example 2 Preparation and application of microcarrier-coated chip substrate and chip containing microcarrier-coated chip substrate
  • the prepared nanoparticle-coated substrates are denoted as substrates 2, 3, 4, and 5 (Table 4).
  • the Q-glucan-containing colloid (w / v concentration 1/2000) and the nanoparticles in Table 4 (v / v concentration 1 / 5000) mixture was coated on the surface, then dried, and then dextran cross-linking was performed according to the dextran cross-linking method described above.
  • the resulting coated chip base was designated as base 6 (Table 4).
  • the multi-basin substrate in this embodiment is prepared as follows: a high-hydrophobic silicone coating (Chengdu Chenguang Chemical Research Institute) is applied to the substrate, and then dried to form a film (film thickness less than 0.05 mm).
  • the isolation structure of the substrate pool is formed on the substrate (refer to our another invention: "A biochip with a highly minimized isolation structure of a reaction cell and a preparation method", Chinese Patent Application No. 03117397.7).
  • the width of the isolation structure between the substrate cells is 4.5 mm.
  • 3) Preparation of a multi-reaction cell chip containing a microcarrier-coated chip substrate
  • the ligands used in this example are HCV antigen (Institute of Liver Diseases, Beijing People's Hospital, China) and HIV 1 + 2 antigen (Institute of Liver Diseases, Beijing People's Hospital, China).
  • HCV antigen 1.0 mg / ml
  • HIV 1 + 2 antigen solution 1.0 mg / ml
  • two kinds of antigens each have three points with a diameter of 200 ⁇ m, and the distance between them is 600-700 ⁇ m, forming a 2X3 array.
  • the density of the ligand on the substrate of the reaction cell is greater than 96 points / cm 2 .
  • the prepared chips are listed in Table 4.
  • sample 1 is HCV antibody-positive serum
  • sample 2 is HIV 1 + 2 antibody-positive human serum
  • sample 3 is a negative control (both HCV antibody and HIV 1 + 2 antibody are negative serum controls Thing). All samples were pre-tested using the classic ELISA method under serum 20-fold dilution reaction conditions.
  • the label in this example is a rhodamine-labeled goat anti-human secondary antibody (American Jackson ImmunoRresearch Laboratories).
  • the aforementioned three samples were respectively added to the reaction cell of the chip described in Table 4.
  • the photos are the amino slides in Table 1, and the amount of sample added was 15 ⁇ 1.
  • the product was washed five times, and the washing solution was added in an amount of 25 ⁇ 1 each time.
  • the labeling amount was 15 ⁇ , and the reaction was washed 5 times.
  • the washing solution was added 25 ⁇ 1 each time.
  • scanning was performed at 35/50.
  • the scanner is a confocal laser scanner (Afymetrix GMS 418 chip scanner), which scans the excitation light wavelength of 532 nm and the emission light wavelength of 570 nm.
  • the read signal is processed by the processing software (JAGUARII), and then the average value is calculated according to Cut -off value judges the yin (one) yang (+) sex.
  • the results are shown in Table 4.
  • the substrate used in this embodiment is a 96-well polystyrene plate (Shenzhen Jincanhua Industrial Co., Ltd.), and the nanoparticles used are colloidal gold, silicon oxide nanoparticles (STN-3), and silicon oxide (LUDOX AS-40) (Table 1). ).
  • a method for preparing a nanoparticle-coated microwell plate is as follows: a nanoparticle liquid having a concentration of 1/5000 to 1/10000 (v / v) is contacted with the bottom of a microwell of a polystyrene plate, and reacted at room temperature for 15 hours , And then repeatedly washed with distilled water.
  • the prepared nanoparticle-coated tablets are listed in Table 5.
  • the ligand used in this example is a synthetic peptide, which is an EBV-VCA-P18 antigen prepared according to the methods disclosed in the following documents: Tranchand-Bunel, D., Auriault, C, Diesis, E., Gras-Masse, H. (1998) Detection of human antibodies using "convergent" combinatorial peptide libraries or "mixotopes 55 designed form a nonvariable antigen: Application to the EBV viral capsid antigen pi 8, J. Peptide Res. 52, 1998, 495-508.
  • the microcarrier-coated microtiter plate used in this embodiment is formed by immobilizing a ligand on the bottom of the microwells of the nanoparticle-coated microwell plate.
  • the synthetic peptides with a concentration of 0.3 g / ml were coated onto the above nanoparticle-coated microwell plates (colloidal gold-coated microwell plates, silica-coated microwell plates, and silica-coated plates, respectively) according to the usual method of making microplates.
  • Microplates) and control microplates 96-well polystyrene plates, each well was coated with 8 wells. After coating, block with bovine serum albumin solution before use.
  • the prepared microcarrier-coated microtiter plate is listed in Table 6.
  • the control microtiter plate in this example is a microtiter plate coated with the EBV-VCA-P18 antigen using the 96-well plate as the base and coated according to the above coating method. board.
  • the samples used are EBV-VCA-P18 anti-IgA positive serum and EBV-VCA-P18 anti-IgA negative serum. All samples were pre-tested using the classic ELISA method under serum 20-fold dilution conditions.
  • the sample was appropriately diluted during sample loading, the sample volume was 100 ⁇ , and the reaction temperature was 37 ° C.
  • the washing solution was added 300 ⁇ l each time and washed 3 times.
  • the marker was an enzyme-labeled goat anti-human IgA (Beijing Tiantan Biological Products Co., Ltd.), the amount was 100 ⁇ l, the reaction temperature was 37 ° C, and the reaction time was 30 minutes.
  • the reaction conditions for adding the substrate were the same as those of the classical ELISA method.
  • a microplate reader (Thermo Labsystems, Shanghai Leibo Analytical Instrument Co., Ltd.) was used for colorimetric analysis. The average results of 8 wells were used to determine the yin (one) and yang (+) characteristics according to the Cut-off value. The results are shown in Table 5.
  • microcarrier-coated microplate has at least higher sensitivity and a faster reaction speed than the control microplate.
  • Example 4 Preparation and application of microcarrier-coated planar chromatography test strips
  • the substrates used in this embodiment are nitrocellulose membrane strips (Fujian Quanzhou Changli Biochemical Co., Ltd.) and nylon fiber membrane strips (Fujian Quanzhou Changli Biochemical Co., Ltd.).
  • the nanoparticles used are silicon oxide nanoparticles (STN-3 ) (Zhejiang Zhoushan Mingri Nano Materials Co., Ltd.) and silicon oxide (LUDOX AS-40) (Sigma-Aldrich).
  • the preparation method of the nanoparticle / tablet composite used in this embodiment is: contacting the nanoparticle liquid with a concentration of 1/5000 to 1/10000 (v / v) with the substrate, reacting at room temperature for 5 hours, and then Wash repeatedly with distilled water.
  • the prepared microcarrier-coated planar chromatography bands are shown in Table 6.
  • the photographs are based on nitrocellulose membrane strips and nylon fiber membrane strips not treated with nanoparticle-containing buffer.
  • the ligand used in this example is HCV antigen (Institute of Liver Diseases, Beijing People's Hospital, China).
  • the HCV antigen at a concentration of 0.5 mg / ml was coated on the planar layers of the four kinds of microcarriers prepared above.
  • the control quick test reagent strip is a quick test reagent strip prepared based on the photo.
  • samples 1 and 2 are HCV antibody-positive serum and HCV antibody-negative serum, respectively, and all samples were tested for yin and positive by a classic ELISA method in advance.
  • the ligand / nanoparticle / tablet composite prepared in this embodiment is a multi-reaction cell chip.
  • the ligands of this example are the HCV antigen and HIV 1 + 2 antigen in Example 2.
  • the nanoparticles in this embodiment include metal nanoparticles, oxide nanoparticles, nanoparticle hydrophobic derivatives, nanoparticle surface modified derivatives, polyionic organic-coated nanoparticles, ion-derived polymer-coated nanoparticles, and Molecular surfactant coated nanoparticles (see Tables 1, 3).
  • the film base of this embodiment includes a glass slide without surface modification, a surface modified glass slide, and a surface-coated organic glass slide (see Table 2).
  • the substrate containing the substrate in this embodiment is a multi-chip base substrate.
  • the preparation method is the same as that of the multi-chip base substrate in Example 2.
  • the method for preparing affinity nanoparticles in this embodiment is as follows: the nanoparticles are dispersed and formulated into a nanoparticle solution with a concentration of 1/2500 (v / v) under ultrasonic oscillation, and the ligand solution with a concentration of 2 mg / ml is separately Mix 1: 1, and react for 1 hour at room temperature. Except for two kinds of affinity nanoparticles (HIV antigen silicon monoxide nanoparticles (STN-3) and HCV antigen silicon monoxide nanoparticles (STN-3)) (Table 8) are purified, the products of other mixed reactions are mixture.
  • the purification method is as follows: The mixed product is dropped into a rotating tube containing a gel, centrifuged at 4000 r / rnin, and the liquid in the collection tube is taken. The obtained affinity nanoparticles are shown in Table 7.
  • the preparation method of the ligand-nanoparticle-ligand one-chip composite is as follows: the above-mentioned affinity nanoparticles are spotted according to the usual spotting method to the multi-chip base substrate (Table 7) prepared above. In the cell, 3 spots of each affinity nanoparticle were formed to form a 3 ⁇ 2 ligand-nanoparticle array. After coating with ligand, block with bovine serum albumin solution before use. The chips obtained are chips 201 to 222 in Table 7.
  • the preparation method of the ligand-nanoparticle-ligand-nanoparticle one-chip composite is as follows: The above-mentioned affinity nanoparticles were spotted in the usual spotting method onto the nanoparticle-coated chip substrate (the substrate 2) prepared in Example 2. Each affinity nanoparticle was spotted at 3 points to form a 3 X 2 complex. Based-nanoparticle array. After coating with ligand, block with bovine serum albumin solution before use. The chips obtained are shown in Table 7 as chips 223 to 224.
  • sample 1 is HCV antibody-positive serum
  • sample 2 is HW 1 + 2 antibody-positive human serum
  • sample 3 is a negative control (HCV antibody, HIV 1 + 2 antibody-negative serum control) . All samples were pre-tested using the classic ELISA method under serum 20-fold dilution reaction conditions.
  • the label in this example is a rhodamine-labeled goat anti-human secondary antibody (American Jackson ImmunoRresearch Laboratories).
  • the aforementioned four samples were respectively added to the reaction cell of the chip described in Table 7.
  • the sample volume was 15 ⁇ 1.
  • the product was washed 5 times, and the washing solution was added in an amount of 25 ⁇ 1 each time.
  • the labeling amount was 15 ⁇ 1, and the reaction was washed 5 times.
  • the washing solution was added 25 ⁇ l each time.
  • the scanning was performed at 35/50.
  • the scanner is a confocal laser scanner (Afymetrix's GMS 418 chip scanner), scanning the excitation light wavelength 532 nm, the emission light wavelength 570 nm, and the read signal is processed by software
  • the example of Ligand 1 is HBsAg (Institute of Liver Diseases, Beijing People's Hospital, China), and the example of Ligand 2 is HBsAb (Institute of Liver Diseases, Beijing People's Hospital, China).
  • the base in this example is the amino slide in Table 2.
  • the preparation method is the same as in Example 5 for the preparation of ⁇ affinity nanoparticles.
  • Ligand 2-Nanoparticles-Ligand 2-Ligand 1-Nanoparticles-Ligand 1 The preparation method of the compound is as follows: according to the above-mentioned preparation method of the affinity nano-particles, ligands 2-nano-particles-ligand 2 and ligand 1-nanoparticles-ligand 1 are prepared respectively. Among them, the ligand 1 can perform a pairing reaction with the ligand 2, and the ligand 1 is easier to bind to the base than the ligand 2. After mixing Ligand 2-Nanoparticles-Ligand 2 and Ligand 1-Nanoparticles-Ligand 1 as 1: 1, spotting onto the substrate according to the usual spotting method.
  • the obtained chip is referred to as a chip 601.
  • the HBsAb concentration was 3 mg / ml when spotted.
  • the preparation method is as follows: after mixing the above-mentioned ligand 2—nanoparticles-ligand 2 and ligand 1 as 1: 1, spotting onto the substrate according to the usual spotting method. Two spots of each affinity nanoparticle were formed to form a 3 ⁇ 2 array, and then blocked with a bovine serum albumin solution for later use. The obtained chip is referred to as a chip 602. HBsAb concentration at the spot is 3 mg / mlo
  • sample No. 1 is hepatitis B surface antigen (HBsAg) positive serum
  • sample No. 2 is hepatitis B surface antigen (HBsAg) negative human serum. All samples were pre-tested using the classic ELISA method under serum 20-fold dilution reaction conditions.
  • the control chip used in this implementation was a chip prepared by spotting HBsAb (3 mg / ml) on the amino slide in Table 2 in the same manner as in the spotting method.
  • the sample is first diluted 500 times, and the remaining steps are the same as in Example 5 using the ligand / nanoparticle / chip-based composite chip
  • the detection method is the same, but the label used in this example is a rhodamine-labeled murine monoclonal antibody made according to a known method. The results are shown in Table 8. Table 8
  • Chip 601 amino slides have + one
  • Chip 602 amino slides have + ⁇
  • Example 7 Preparation and application of ligand / nanoparticle / sheet-based complex microplate
  • the ligand used in this example is a synthetic peptide (EBV-VCA-P18 antigen, and the preparation method is the same as in Example 3).
  • the preparation method of the affinity nanoparticles containing the EBV-VCA-P18 antigen and the silicon oxide nanoparticles (STN-3) is the same as the method for preparing the affinity nanoparticles in Example 5.
  • the nanoparticle concentration in the affinity nanoparticle with an EBV-VCA-P18 antigen concentration of 0.1 ⁇ g / ml was 1: 4000, and the microwells of the 96-well plate in Table 2 were coated according to the well-known microplate coating method. Inside, 8 wells were coated, and after being coated, they were blocked with bovine serum albumin solution and used.
  • the serum sample and detection method used in this example are the same as those in Example 3.
  • the control enzyme plate used is an EBV-VCA-P18 antigen enzyme plate coated with the same concentration and the same method.
  • the detection result using the control enzyme plate was negative, and the ligand / nanoparticle / platelet complex enzyme plate prepared in this example was negative.
  • the test result is still positive.
  • Example 8 Preparation and application of a ligand / nanoparticle / molecularly labeled substance complex
  • the ligand / nanoparticle / molecular labeling substance complex prepared in this example is a nanoparticle labeling substance for a chip.
  • the nanoparticles in this example include oxide nanoparticles and their derivatives (Table 9).
  • the molecular marker used is rhodamine (Sigma), and the ligand used is goat anti-human secondary antibody (Beijing Tiantan Biological Products Co., Ltd.). .
  • control marker in this example is a conventional marker (rhodamine-labeled goat anti-human secondary antibody, Jackson ImmunoRresearch Laboratories, USA) that has not been treated with a nanoparticle-containing liquid.
  • the method for preparing the affinity nanoparticles used in this embodiment is: dispersing the nanoparticles under ultrasonic oscillation to prepare a nanoparticle solution with a concentration of 1/2500 (v / v), and then preparing a ligand with a concentration of 2 mg / ml
  • the solutions were mixed 1: 1 with them and reacted for 1 hour at room temperature.
  • the purification method of the mixture is as follows: Drop into a rotating tube containing the gel, centrifuge at 4000 r / min, and take the liquid from the collection tube.
  • a nanoparticle-molecular labeling substance complex is prepared by dispersing the nanoparticle under ultrasonic vibration to prepare a nanoparticle liquid with a concentration of 1/2500 (v / v), and then dissolving the molecule with a concentration of 2 mg / ml
  • the labeling substance solution was mixed 1: 1 with it, and reacted at room temperature for 1 hour. If necessary, purify the mixture by dripping the mixed product into a gel-filled spin tube, centrifuge at 4000 r / min, and take the liquid from the collection tube.
  • the preparation method of the ligand-molecular labeling substance complex in this embodiment is a well-known preparation method of rhodamine-labeled antibody.
  • the preparation method of this embodiment, wherein the combination of the ligand, the molecularly labeled substance and the nanoparticle includes one or more of the following methods: combining the above-mentioned affinity nanoparticle with the molecularly labeled substance (A), and combining the above-mentioned nanoparticle- Binding of a molecularly labeled substance to a ligand (B), binding of a molecularly labeled substance labeled ligand prepared with a known method to a nanoparticle (C), simultaneous binding of a ligand with a molecularly labeled substance and nanoparticle (D), wherein The product can be combined as a mixture and a purified product.
  • the chip used in this example is a chip made according to a known chip preparation method using the HCV antigen and HIV antigen in Example 2 as ligands, and the aldehyde-based glass slide in Table 2 as a base.
  • Example 9 Preparation and application of a kit containing a ligand / nanomicrochip-based complex and a ligand / nanomicron molecular marker substance complex
  • the kit is a chip kit, which includes a ligand / nanoparticle / tablet complex (chip) and a ligand / nanoparticle / molecular labeling substance complex (label).
  • chip a ligand / nanoparticle / tablet complex
  • label a ligand / nanoparticle / molecular labeling substance complex
  • the preparation of the ligand / nanoparticle / tablet complex was the same as in Examples 5 and 6
  • the ligand / nanoparticle / molecular labeling substance complex was the same as in Example 8. Therefore, this embodiment can form a large number of different kits.
  • the magnetic nanoparticles used in this example are the water-based magnetic fluids (NG-21A) in Table 1.
  • the sheet used is an aldehyde-based glass slide in the surfaceization.
  • the ligand used is the ligand HCV antigen and HIV 1 + 2 antigen.
  • the magnetic nanoparticle chip in this embodiment is a ligand / magnetic nanoparticle / sheet-based composite.
  • the magnetic nanochip in this embodiment is a multi-reactor chip, and the preparation of the multi-chip base substrate is the same as the preparation of the multi-chip base substrate in the embodiment.
  • the preparation and implementation of affinity magnetic nanoparticles in this embodiment are as follows.
  • the prepared affinity magnetic nanoparticles containing HCV antigen (1 mg / ml) and HIV 1 + 2 antigen (1 mg / ml) were spotted into a multi-substrate base substrate, and And magnetic nanoparticle dots to form a 2 X2 display. Then reacted at 37 ° C for one hour under the external magnetic field.
  • the role of the external magnetic field is to facilitate the movement and fixation of the affinity magnetic nanoparticles to the substrate. After the color is completed, block with medium serum protein solution and set aside.
  • control chip in this example is based on an aldehyde group, and according to a known spotting method, HCV antigen (l mg / ml) without magnetic nanoparticles and HIV 1 + 2 antigen (l mg / ml) are spotted. Preparation of chips.
  • the magnetic nanomarker in this embodiment is a ligand / magnetic nanoparticle / molecular labeling substance complex.
  • hexyl is a goat anti-human secondary antibody (Jackson ImmnoResearch Laboratories).
  • the magnetic chip kit in this embodiment may contain one, two, three, or four magnetic particle components with a water content.
  • the magnetic particle-containing components are: the magnetic nanoparticle chip prepared above, the magnetic nanoparticle label prepared above, the affinity magnetic nanoparticle (closed with calf serum albumin) and magnetic nanoparticle ( Has been blocked with calf serum albumin).
  • the different kits for different combinations are shown in Table 11.
  • the magnetic chip kit is used to detect the negative (a) and positive (+) of HIV 1 + 2 antibody and HCV antibody in the serum of the sample.
  • the serum sample used was the same as the serum sample in Example 2.
  • the difference between the detection method and the known chip detection method is that when the kit contains magnetic particle components (such as magnetic nanoparticles) in addition to the magnetic chip, the detection reaction is performed under the condition of an external magnetic field at the bottom of the chip. . Especially the fixed pulse magnetic field. Under the action of an external magnetic field, it is beneficial to the movement of the magnetic nanoparticle label and the target captured by the affinity magnetic nanoparticle to the bottom of the reactor.
  • the magnetic nanoparticles added to the sample are conducive to the flow inside the liquid sample added to the chip reactor under the action of the pulsating magnetic field to facilitate the mixing of the sample. Since there are many types of kits, one kit is taken as an example to illustrate its use method, and other kits can be used according to this example.
  • the composition of the kit used in this example is: control chip, magnetic nanoparticles, affinity magnetic nanoparticles and four serum samples were mixed [the magnetic nanoparticle concentration after mixing is 1/2000 (W / V), affinity magnetic Nanoparticle concentration is 1/4000 (W / V)]; 15 ⁇ 1 serum sample is added to the chip reaction cell and reacted at 37 ° C for 10 minutes under the action of a pulsating magnetic field; washing; 15 ⁇ 1 magnetic nano-marker and At 37 under a pulsating magnetic field. C was reacted for 10 minutes; washed and dried; and then scanned and analyzed according to the same scanning and analysis method as in Example 2. The results are shown in Table 11. Table 11
  • I control chip
  • II magnetic nanoparticle chip
  • III magnetic nanoparticle
  • IV affinity magnetic nanoparticle
  • V magnetic nanoparticle label
  • VI control label
  • Example 11 Ligand / nanoparticle And application of carrier / carrier composite separation medium
  • the complex prepared in this embodiment is a chromatographic stationary phase.
  • the ligand used in this example is DEAE-dextran (Pharmacia), the nanoparticles used are silicon oxide nanoparticles (STN-3) in Table 1, and the carrier used is an average particle size of 60 ⁇ m for chromatography.
  • Silica particles Institute of Chemistry, Chinese Academy of Sciences).
  • a DEAE-dextran solution at a concentration of 1/2500 (w / v) was mixed with silica nanoparticles (STN-3) at a concentration of 1/2500 (w / v) and stirred at room temperature for 4 hours.
  • Dried silica gel particles were immersed in it, and then according to the method published by one of the inventors (refer to the article of one of the inventors (COATED SILICA SUPPORTS FOR HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY OF PROTEIN, Journal of Chromatography, 476, (1989 ) 195-203) Dextran cross-linking to obtain DEAE-dextran / nanoparticles / silica gel particle composites.
  • it can also be used to derive a variety of chromatographic stationary phases using classical dextran-derived methods.
  • the kinetic adsorption capacity of the above silica gel particles, DEAE-dextran / silica gel particle complex, and DEAE-dextran / nanoparticles / silica gel particle complex were tested.
  • the column used to fill the above media has an inner diameter of 0.5 cm and a length of 2 cm, a buffer solution of 0.01 M Tris-HCl / pH 7.40, a flow rate of 1 ml / min, a chromatography instrument used for HP 1090, and the sample used For human albumin.

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Abstract

L'invention concerne un procédé de détection quantitative et/ou qualitative de polypeptides, dans lequel les objets réagissent, respectivement, avec des nanoporteurs d'affinité et/ou des ligands, des nanoparticules, des composés à molécules marquées. L'invention concerne également un dispositif de détection ayant trait à des nanoporteurs d'affinité et/ou des ligands, des nanoparticules, des composés à molécules marquées, notamment des puces, plaques marquées aux enzymes, bandes chromatographiques. En outre, l'invention concerne des nanoporteurs d'affinité utilisés comme supports de détection et d'isolation, ainsi que le procédé de préparation de ces porteurs. En outre, l'invention concerne des ligands, nanoparticules, composés à molécules marquées, et le procédé de préparation de ces composés. L'invention concerne des puces, des kits, un procédé de détection et un dispositif comprenant des nanoparticules magnétiques.
PCT/CN2004/000077 2003-03-13 2004-01-20 Procede et dispositif de detection de polypeptides, et compose ligand comprenant des nanoparticules WO2004081571A1 (fr)

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PCT/CN2004/000203 WO2004090548A1 (fr) 2003-03-13 2004-03-15 Dispositif d'analyse ou de separation contenant un support nanostructure, son procede de preparation et ses applications
JP2006529552A JP2007502998A (ja) 2003-04-30 2004-04-30 ナノメートル構造の分析または分離用装置、及びその製作方法と応用
PCT/CN2004/000437 WO2004102196A1 (fr) 2003-04-30 2004-04-30 Dispositif comprenant des nanostructures destine a une separation ou une analyse, et preparation et mise en oeuvre de ce dispositif
EP04730459A EP1624306A4 (fr) 2003-04-30 2004-04-30 Dispositif comprenant des nanostructures destine a une separation ou une analyse, et preparation et mise en oeuvre de ce dispositif
US11/258,996 US7842515B2 (en) 2003-04-30 2005-10-27 Nano-structured device for analysis or separation, and its preparation and application

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CN 03117446 CN1250969C (zh) 2003-03-13 2003-03-13 一种对目标物进行定性和/或定量分析的检测装置及其检测方法
CN03117787.5 2003-04-30
CNA031177875A CN1514243A (zh) 2003-04-30 2003-04-30 对目标物进行定性和/或定量分析的方法、装置和标记物及检测试剂盒

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WO2006072306A1 (fr) * 2004-12-24 2006-07-13 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Supports tridimensionnels a nanostructure et microstructure
WO2010040278A1 (fr) * 2008-10-10 2010-04-15 Hai Kang Life Corporation Limited Procédé de détection d’un analyte dans un micro-réseau d’échantillons et appareil destiné à la réalisation de ce procédé
CN102243180A (zh) * 2010-05-13 2011-11-16 南京神州英诺华医疗科技有限公司 一种细菌明胶液化反应显色鉴定法

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WO2003056336A2 (fr) * 2001-12-28 2003-07-10 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Matrice de liaison a structure fonctionnelle amelioree pour biomolecules

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US6025202A (en) * 1995-02-09 2000-02-15 The Penn State Research Foundation Self-assembled metal colloid monolayers and detection methods therewith
WO1999037814A1 (fr) * 1998-01-22 1999-07-29 Luminex Corporation Microparticules emettant des signaux fluorescents multiples
WO2000000808A2 (fr) * 1998-06-09 2000-01-06 California Institute Of Technology Particules colloidales utilisees dans une mosaique de capteurs
WO2003056336A2 (fr) * 2001-12-28 2003-07-10 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Matrice de liaison a structure fonctionnelle amelioree pour biomolecules

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006072306A1 (fr) * 2004-12-24 2006-07-13 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Supports tridimensionnels a nanostructure et microstructure
WO2010040278A1 (fr) * 2008-10-10 2010-04-15 Hai Kang Life Corporation Limited Procédé de détection d’un analyte dans un micro-réseau d’échantillons et appareil destiné à la réalisation de ce procédé
CN102171568A (zh) * 2008-10-10 2011-08-31 海康生命科技有限公司 检测样品微阵列中的分析物的方法和实施所述方法的装置
CN102243180A (zh) * 2010-05-13 2011-11-16 南京神州英诺华医疗科技有限公司 一种细菌明胶液化反应显色鉴定法

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