WO2004081571A1

WO2004081571A1 - The detecting method and device of polypeptide, and the ligand compound comprising nanoparticles

Info

Publication number: WO2004081571A1
Application number: PCT/CN2004/000077
Authority: WO
Inventors: Fanglin Zou; Chunsheng Chen; Ning Chen; Jianxia Wang
Original assignee: Chengdu Kuachang Medical Industrial Limited; Chengdu Kuachang Science & Technology Co., Ltd
Priority date: 2003-03-13
Filing date: 2004-01-20
Publication date: 2004-09-23

Abstract

The invention relates to a quantitative and/or qualitative detecting method of polypeptides, where the objects react with affinity nanocarriers and / or ligands / nanoparticles marked molecule compounds respectively. The invention also relates to the detecting device which relates to affinity nanocarriers and / or ligands / nanoparticles / marked molecule compounds, especially relates to chips, enzyme marked plates, chromatographic bands. Moreover, the invention relates to affinity nanocarriers used as detecting and isolating medium, and the method of preparing such carriers. Also, the invention relates to ligands / nanoparticles / marked molecule compounds and the method of preparing such compounds. The invention provides chips, kits, detecting method and device which comprise magnetic nanoparticles.

Description

Polypeptide pick-up and detection method, detection device and ligand complex containing nanometer particles

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. For immobilized ligands used in separation, chromatographic gels can be taken as an example. An example of an immobilized ligand for detection is an analysis chip. In addition, in qualitative and / or quantitative detection analysis, 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. In bioassay analysis, the target of root samples can be divided into nucleic acid detection, peptide detection, and so on. In the following, 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. Among them, glass and its derivatives, such as amino glass slides, aldehyde groups, etc. Slides, epoxy-based slides, and polyamino acid-coated slides are the main substrates currently used. At present, another type of 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. For example, reactors that are at least partially film-based have reduced sensitivity due to their high background noise. In addition, reactors based on membranes, microparticles, etc. have other practical problems (such as cleaning of membranes, adhesion of microparticles, etc.). As a result, 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. At present, 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. At present, other detection devices containing a substrate, such as microplate readers, have the same problems as biochips.

In addition, immobilized ligands such as affinity chromatography stationary phases are also widely used in separation devices such as chromatography devices. Although the 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 μηι and a pore size of 0.05-0.5 nm. In a commercially available film-slide base, a porous membrane having a pore size distribution from nm to μm is used as a ligand carrier. However, in International Patent Application Publication WO 0183825, 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. To process chips with uniform probe points. In addition, in U.S. Patent Application No. 20030207296, nanoparticles are used only as a ligand carrier for nucleic acid detection. In addition, in the analysis chip detection, a gene chip is used for nucleic acid detection, and a polypeptide chip is used for polypeptide detection. Although recognized advances have been made in nucleic acid detection using gene chips (such as nucleic acid detection in the human genome sequencing project), chip peptide detection is still in development. Summary of the Invention

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. As a result of research on immobilized ligands, another object of the present invention is to improve the separation efficiency of the separation medium.

According to one aspect of the present invention, a method for quantitative or / and qualitative detection of a polypeptide is provided, which at least It includes the following steps:

(a) An affinity nanostructure carrier is provided. 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. A structure constructed on the surface that retains the main nanophenomenon characteristics and thus has a higher reaction efficiency than ligands, the affinity nanoparticles include nanoparticles and one or more ligands immobilized thereon, 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 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;

(b) contacting the sample to be detected with the affinity nanostructure carrier and performing a reaction between the complex and the target polypeptide in the sample;

(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-coated derivatives containing derivative groups on the surface.

According to another aspect of the present invention, it provides a polypeptide detection device comprising the affinity nanostructure carrier and the ligand / nanoparticle / molecular labeling substance complex as described above.

According to still another aspect of the present invention, it 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:

(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 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;

(b) preparing 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;

(c) binding the colloid or / and the nanoparticle to the carrier, wherein the combination of the nanoparticle and the carrier includes one or more of the following methods: coating on the coating 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.

According to another aspect of the present invention, it provides 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.

According to another aspect of the present invention, it provides a method for preparing an affinity nanostructure carrier for polypeptide detection or component separation, which includes the following steps:

(a) preparing a carrier, one or more ligands, and one or more nanoparticles, said 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;

(b) mixing the ligand with the highly-diluted suspension of the nanoparticles to make one or more nanoparticles having a volume concentration of between 5% and 1 / 100,000; 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;

(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.

According to another aspect of the present invention, it provides an affinity nanostructure carrier for polypeptide detection or component separation, wherein 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 rnn and less than Non-magnetic particles of 100 legs, preferably greater than 1 nm and less than 50 nm.

According to another aspect of the present invention, it provides a method for preparing a ligand / nanoparticle / molecular labeling substance complex, which includes the following steps:

(a) preparing one or more ligands, one or more nanoparticles, and one or more of said molecular marker substances, wherein said nanoparticles / ligands / molecule marker substances are mixtures or purified substances, 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

(b) combining the one or more ligands, one or more nanoparticles, and one or more of the molecular markers in one of the following ways: combining the ligand with the nanometer Particles are then bound to the molecular labeling substance, the nanoparticle is bound to the molecular labeling substance and then to the ligand, the ligand is bound to the molecular labeling substance and then to the nanoparticle , Simultaneously combining the ligand with the molecularly labeled substance and the nanoparticle, and combinations based on these methods.

According to another aspect of the present invention, it provides 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.

According to yet another aspect of the present invention, it provides a polypeptide detection device comprising the affinity nanostructure carrier and / or the ligand / nanoparticle / molecular labeling substance complex as described above.

According to another aspect of the present invention, 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.

According to another aspect of the present invention, 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.

According to another aspect of the present invention, it provides a chip detection method for quantitative or / and qualitative polypeptides, which includes at least one, two, three or four steps as follows:

(a) mixing the sample with magnetic particles or / and magnetic flakes;

(b) mixing the sample with a ligand / magnetic nanoparticle, said 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;

(c) contacting and reacting the sample with the ligand / magnetic nanoparticle / platelet complex, and optionally an external magnetic field may be present during the reaction, 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 There is a heavy or multiple magnetic nanoparticle in between, or / and at least one heavy magnetic nanoparticle and another heavy magnetic nanoparticle have a heavy or multiple ligand, 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 nm. 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;

(d) 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; 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 Base selected Substances capable of interacting with peptides in the following groups: peptides, polysaccharides, vitamins, antibiotics, viruses, cells, and functional organic matter, the magnetic nanoparticles have at least one dimension in a three-dimensional space of 1-200 nm, preferably 1-100 nm, More preferably, it is 1 to 50 nm and is not itself a molecular marker substance enhancer.

According to another aspect of the present invention, it provides 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 nm, more preferably 1-50 nm ferrite and its derivatives including ferric tetroxide and ferric oxide, the derivatives including surface-modified or functional organic-coated derivatives containing derivatizing groups on the surface; Said sheet substrate selected from the following group of materials and their derivatives: glass, silicon, ceramics, metal oxides, metals, polymeric materials and composite materials thereof.

According to another aspect of the present invention, it provides a method for preparing a polypeptide detection chip as described above, which includes the following steps:

(a) preparing the substrate, one or more of the ligands, and one or more of the magnetic nanoparticles;

(b) combining the magnetic nanoparticles with the ligand to form one or more affinity magnetic nanoparticles, the affinity magnetic nanoparticles including a mixture and a purified substance;

(c) binding the affinity magnetic nanoparticles prepared in step (b) to the carrier, and applying an external magnetic field when binding.

According to another aspect of the present invention, it provides 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.

According to yet another aspect of the present invention, it provides 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, and 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.

According to another aspect of the present invention, there is provided 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. detailed description

Definition of Terms

The term "detection device" according to the present invention 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. -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.

The term "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. Examples of the separation device include a chromatography device, and examples of a substance having a separation function include a chromatographic carrier.

The term "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.

The term "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.

The term "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. 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.

The term "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.

The term "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.

The term “carrier” in the present invention, referred to as the carrier, 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.

The term “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.

The term “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. For example: Ligand-nanoparticles, Ligand-nanoparticles, Ligands, Ligands Nanoparticles-ligands-nanoparticles-based, ligand-nanoparticles-ligands-nanoparticles-ligand-based-groups, etc.

The term "affinity nanoparticle" in the present invention refers to a complex formed by the ligand and the nanoparticle by covalent or / and non-covalent bonding.

The term “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 The reaction cell in the biochip of the reactor, the related isolation structure, the structure of the inlet and outlet liquid, the wells in a 96-well microtiter plate, the reagent strips of the rapid detection kit, etc.

The term "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. There can be one or more substrate pools on the substrate. Monolithic substrates usually have no isolation structure on them. In this case, the substrate is both a substrate (such as a commercially available amino slide). The multi-chip base substrate has an isolation structure. At this time, 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.

The term “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 , and the more preferred solution is greater than 40 points / cm ² , and the area of each ligand point is not more than 1 mm ² o

The term "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. The term “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. In our research, we were surprised to find that when ligands were combined with inorganic particles with a size of 1 to 90 nm to form affinity nanoparticles and used to make peptide detection chips, they showed unusual detection sensitivity and detection time. advantage. The present invention is not intended to enter the theoretical discussion, it is only assumed that the affinity nanoparticle itself is completely different from the classic affinity carrier at this time. In fact, some special "nano phenomena" revealed by nanoscience may partially explain the specificity of the present invention:

1) Surface effect: As the particle size decreases, the percentage of surface atoms in the total number of atoms increases, the surface activity is high, and it is easy to bind with ligand molecules; especially its large specific surface area, in a unit volume carrier More ligands can be immobilized, and more favorable reaction kinetic conditions are provided for the ligands immobilized thereon. As a result, it is observed that the reaction time is significantly reduced and the detection sensitivity is improved (Reference Examples).

2) Small size effect: With the reduction of particle size, some macroscopic properties change, especially the change in optical properties, which can make certain affinity particles fixed on the substrate, not only Obviously, it is also possible to reduce the background noise of the substrate, which opens a new way to improve the detection sensitivity. In fact, the superabsorbency and translucency of some nanomaterials have been used in other fields.

3) 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.

4) 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.

Similarly, 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. 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.

In particular, we are even more surprised to find that in the detection of peptides, although the use of 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. Similarly, 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:

(a) providing an affinity nanostructure support, the 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;

(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 derivatives containing derivative groups on the surface. 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. It is particularly emphasized that 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). Some high-density nanoparticles (for example, a volume concentration greater than 1%) / ligand mixed solution spotted high-density aggregation of affinity nanoparticles formed on the chip substrate ₅ is difficult to observe an increase in sensitivity. In summary, 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%. .

In the polypeptide quantitative or / and qualitative detection method of the present invention, 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.

In the polypeptide quantitative or / and qualitative detection method of the present invention, 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. Although 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.

In the polypeptide quantitative or / and qualitative detection 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.

In the polypeptide quantitative or / and qualitative detection method of the present invention, 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.

In the present invention, especially due to the preparation of the coated derivative, the range of active organic groups that can be introduced on the surface of the nanoparticle is large. For example, in the embodiment of the present invention, a nanoparticle derivative such as superhydrophobic silica (CS7) has a fluorenyl group, and the surface of the coated derivative has an organic coating group, for example, a polyamino acid has an amino group and an aminohydrazine-polyamino acid has Amino and aminohydrazine, DEAE-Dextran has diethylaminoethyl, and so on. On the other hand, 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. Complex and ligand / nanoparticle / molecular labeling substance complex. In fact, 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. For example, silica particles coated with a dispersant or / and dispersion stabilizer (one coat) 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.

In the polypeptide quantitative or / and qualitative detection method of the present invention, 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. Specifically, 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. Specifically, 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;

(b) preparing 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;

In the method for preparing a microcarrier-coated carrier for polypeptide detection or component separation according to the present invention, 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.

In the method for preparing a microcarrier-coated carrier for polypeptide detection or component separation of the present invention, 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.

The 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. Substrate, microcarrier-coated microtiter plate substrate, microcarrier-coated planar chromatography reagent strip, and nanoparticle-coated chromatography carrier. 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:

(a) preparing a carrier, one or more ligands, and one or more nanoparticles, 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;

(b) mixing the ligand with the highly-diluted suspension of the nanoparticles to make one or more nanoparticles having a volume concentration of between 5% and 1 / 100,000; 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) Moreover, 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;

(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. For example, 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. Alternatively, 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.

The same method can also be used to prepare affinity nanostructure carriers with multiple ligands between at least one heavy nanoparticle and another layer of nanoparticles. 2—ligand 1—carrier or ligand 2—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.

In the method for preparing an affinity nanostructured carrier of the present invention, it 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.

In the method for preparing an affinity nanostructure carrier for polypeptide detection or component separation of the present invention, the volume concentration of the nanoparticles in the affinity nanoparticle liquid is between 1/1000 and 20,000.

In the method for preparing an affinity nanostructured carrier for polypeptide detection or component separation of the present invention, 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.

Method for preparing affinity nanostructure carrier for polypeptide detection or component separation in the present invention Wherein, 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.

Preferably, 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:

(a) preparing one or more ligands, one or more nanoparticles, and one or more of said molecular marker substances, and said nanoparticles / ligands / molecular marker substances are mixtures or purified substances, 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 ways: combining the ligand with the nanometer Particles are then bound to the molecular labeling substance, the nanoparticle is bound to the molecular labeling substance and then to the ligand, the ligand is bound to the molecular labeling substance and then to the nanoparticle , Simultaneously combining the ligand with the molecularly labeled substance and the nanoparticle, and combinations based on these methods. For example, 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 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.).

In the method for preparing a ligand / nanoparticle / molecular labeling substance complex of the present invention, the inorganic non-metal particles include non-magnetic oxide particles, and the non-magnetic oxide particles include silicon oxide, titanium oxide, and aluminum oxide . For 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.

In the method for preparing a ligand / nanoparticle / molecular labeling substance complex of the present invention, 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. Specifically, 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 be referred to those described in the above quantitative or / and qualitative detection methods for polypeptides.

In this aspect, 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.

Preferably, 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 Substrate plate kits for substance complexes, or flat chromatography reagent strip kits containing affinity nanostructure carriers and / or ligands / nanoparticles / molecularly labeled substance complexes as described above. At the same time, 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:

(a) mixing the sample with magnetic particles or / and magnetic microchips;

(b) mixing the sample with a ligand / magnetic nanoparticle, said 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;

(c) contacting and reacting the sample with the ligand / magnetic nanoparticle / sheet-based composite, and optionally an external magnetic field may be present during the reaction, 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 There is a heavy or multiple magnetic nanoparticle in between, or / and at least one heavy magnetic nanoparticle and another heavy magnetic nanoparticle have a heavy or multiple ligand, 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, the sheet 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;

(d) 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.

In the above-mentioned quantitative or / and qualitative chip detection method of the present invention, 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 nm, and more preferably 1 to 50 nm. 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. A structure constructed for a unit on the surface, which retains the main nano-phenomenon characteristics and thus has a higher reaction efficiency than a ligand, the affinity magnetic nanoparticles include the above-mentioned magnetic nanoparticles and one or more immobilized thereon or A variety of the aforementioned ligands.

The detection chip can be prepared as follows:

(a) preparing the substrate, one or more of the ligands, and one or more of the magnetic nano-particles;

(c) binding the affinity magnetic nanoparticles prepared in step (b) to the carrier, and providing an external magnetic field when binding.

In the polypeptide detection chip, 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 enhancers. In the kit, the magnetic particles or / and microchips are used to generate a reaction medium mixture, and the ligand / magnetic nanoparticle is used to concentrate a sample target, the ligand is

/ Magnetic nanoparticle / Molecular labeling substance complex is used to label the reaction.

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 A particle / molecular labeling substance complex, 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, and 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.

In the above kit, 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.

Finally, 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.

Advantages of the method of the present invention for preparing a microcarrier-coated carrier for polypeptide detection or component separation It is the coating that does not need to be heated that makes the surface of the macroscopic solid phase carrier undergo less change.

The advantage of the 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.

The advantages of the 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.

Hereinafter, the present invention will be described in more detail through examples. Specific embodiment

The purchased inorganic non-magnetic nanoparticles and their derivatives with a particle size of 1-100 nm used in the following examples are shown in Table 1 below. Table 1

The substrates used in the following examples are shown in Table 2 below.

Table 2: Film base (conventional)

*: For the preparation method, refer to Melnyk 0, etc., Peptide arrays for highly sensitive and special antibody-binding fluorescence arrays, Bioconjug Cliem. 13: 713-20.2002.

For the production method, please refer to Chinese Patent Application No. 03135618.4 Example 1: Preparation of Nanoparticle-Coated Derivatives

The nanoparticle-coated derivatives prepared in this example, the nanoparticles used 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).

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).

The nanoparticle-coated derivatives prepared by the present invention are also listed in Table 3 below. table 3

Example 2: Preparation and application of microcarrier-coated chip substrate and chip containing microcarrier-coated chip substrate

1) Preparation of microcarrier-coated chip substrate

The nanoparticles, colloids, and substrates used in this example are listed in Table 4. (1) Preparation of colloid-coated chip substrate

After the slide was treated with 10% NaOH and HN0 ₃ and washed, Q-glucan colloid (provided by Chenguang Chemical Research Institute) (w / v concentration 1/5000) was coated on the surface, and then dried. dry. Colloidal Q-dextran colloid coated glass slides, followed by the method disclosed by one of the inventors for cross-linking of dextran (refer to FL ZHOU et al., COATED SILICA SUPPORTS FOR HIGH-PERFORMANCE AFFINFTY CHROMATOGRAPHY OF PROTEINS, Journal of Chromatography, 476 (1989) 195-203). The obtained gel-coated chip base was designated as base 1 (Table 4).

(2) Preparation of nanoparticle-coated chip substrate

The nanoparticle liquid (Table 4) with a concentration of 1/5000 to 1/10000 (v / v) was brought into contact with the substrate, reacted at room temperature for 15 hours, and then repeatedly washed with distilled water. After drying, it was detected by an electron microscope. Nanoparticles distributed on the surface of the glass substrate (detected by the Analysis and Testing Center of Sichuan University), the density of nanoparticle distribution on the surface of the glass slide is greater than 10 particles / mm ² . The prepared nanoparticle-coated substrates are denoted as substrates 2, 3, 4, and 5 (Table 4).

(3) Preparation of nanoparticle / colloid coated chip substrate

After the slide was treated with 10% NaOH and HN0 ₃ and washed, 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).

2) Preparation of multi-chip substrates containing microcarrier-coated chip substrates

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). There are 8 substrate cells on each substrate, and the size of each substrate cell is 4.5 mm X 4.5 mm. 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). In a substrate pool of the substrate prepared in 2) above, HCV antigen (1.0 mg / ml) and HIV _{1 + 2} antigen solution (1.0 mg / ml) were spotted on the substrate according to a general spotting method. In the pool, two kinds of antigens each have three points with a diameter of 200 μm, and the distance between them is 600-700 μm, 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.

4) Application of nanoparticle-coated multi-reaction cell chip

In this example, sample 1 is HCV antibody-positive serum, sample 2 is HIV _{1 + 2} antibody-positive human serum, and 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).

During the experiment, the aforementioned three samples were respectively added to the reaction cell of the chip described in Table 4. Among them, the photos are the amino slides in Table 1, and the amount of sample added was 15 μ1. After 30 minutes of reaction, the product was washed five times, and the washing solution was added in an amount of 25 μ1 each time. The labeling amount was 15 μΐ, and the reaction was washed 5 times. The washing solution was added 25 μ 1 each time. After drying, 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.

Table 4

Example 3: Preparation and application of nanoparticle-coated enzyme-labeled plate substrate

1) Preparation of nanoparticle-coated microplates

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). ).

In this embodiment, 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.

2) Preparation of microcarrier coated microplate

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.

3) Application of microcarrier coated microplate

In this embodiment, 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.

During the experiment, two kinds of samples were added to the control plate and three microcarrier-coated plates, and each sample was added to four reaction cells. The sample was appropriately diluted during sample loading, the sample volume was 100 μΐ, and the reaction temperature was 37 ° C. The washing solution was added 300 μl 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 μl, 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.

table 5

It can be seen from the results in Table 5 that the 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

1) Preparation 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.

2) Preparation of nanoparticle-coated rapid detection reagent strip

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. Band analysis and two kinds of spotting (detection line) on the basis of photos, and then point the rabbit anti-goat IgG quality control line according to conventional methods, then block with bovine serum albumin solution, reassemble the sample area, colloidal gold mark Sheep anti-human marker area and water absorption area are combined (Table 7).

The control quick test reagent strip is a quick test reagent strip prepared based on the photo.

3) Application of Nanoparticle Coated Quick Test Reagent Strip

In this example, 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.

During the experiment, two kinds of samples were added to the six kinds of quick test strips prepared above. The sample was appropriately diluted during sample loading, and the sample volume was 50 μ1. After the sample was added, the washing solution test strip was slowly sucked until the quality control line appeared. The results are shown in Table 6. Table 6

From the results in Table 6 above, it can be seen that the nanoparticle-coated rapid detection reagent strip takes only twice as much time as the control reagent strip. Example 5: Preparation and application of ligand / nanoparticles / chip-chip composites

1) Preparation of Ligand / Nanoparticle / Chip Chip Complex

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).

(1) Preparation of multi-chip base substrate

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.

(2) Preparation of affinity nanoparticles

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.

(3) Preparation of ligand-nanoparticle-ligand one-sheet complex

In this embodiment, 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.

(4) Preparation of ligand-nanoparticle-ligand-nanoparticle one-chip composite

In this embodiment, 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.

2) Application of nanoparticle-coated multi-reaction cell chip

In this example, sample 1 is HCV antibody-positive serum, sample 2 is HW _{1 + 2} antibody-positive human serum, and 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).

During the experiment, the aforementioned four samples were respectively added to the reaction cell of the chip described in Table 7. The sample volume was 15 μ1. After 30 minutes of reaction, the product was washed 5 times, and the washing solution was added in an amount of 25 μ1 each time. The labeling amount was 15 μ1, and the reaction was washed 5 times. The washing solution was added 25 μl each time. After drying, 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

(JAGUAR II) processing, and then take the average value to determine the yin (―) yang (+) according to the Cut-off value. The results are shown in Table 7.

Table 7

mouth

Test results of Cardiac Ligand / Nanoparticles / Substrate Complexes Substrate Ligand or Affinity Nanoparticles Sample 1 Sample 2 Sample 3 "Sample dilution vs. photo aldehyde-based slide ligand + +-100 times Ligands —— ——, Hydrobenes, Nanoparticles, and ¾ One-sheet Complex

201 Aldehyde glass with colloidal gold-+-500 times

202 Aldehyde slides Ligand amine based colloidal gold (2020) + + ― 500 times

203 Aldehyde slide Ligand DEAE—dextran coating + 500 times Nanoparticles

204 Aldehyde slide Ligand polyvinylpyrrolidone coating + 500 times Nanoparticles

205 Aldehyde slide Ligand aminohydrazine-polylysine package + 500 times by nanoparticles

206 Aldehyde glass slide Ligand hydrophobic silica nanoparticles + 500 times

(CDS1)

207 Epoxy glass slide Ligand silica nanoparticles (STN + 500 times

-3)

208 Epoxy based glass slide with silica (LUDOX AS + + 500 times

-40)

209 Epoxy glass slide Lithium porous silica + 500 times

(AEROSIL 200)

210 Epoxy glass slide Ligand titanium oxide nanoparticles + + ― 500 times

211 Epoxy glass slide ligand DEAE—dextran coating + 500 times nanoparticle

212 Epoxy glass slide Ligand polyvinylpyrrolidone coating + 500 times Nanoparticles

213 Epoxy glass slide Ligidine aminohydrazine-polylysine package + 500 times by nanoparticles

214 Epoxy glass slide Ligand hydrophobic silica nanoparticles + 500 times

(CDS1)

215 Aminohydrazine slide Ligand hydrophobic silica nanoparticles + 500 times

(CDS1) 216 Aminohydrazine slides with silica nanoparticles (STN + 500 times

-3)

217 Aminohydrazine slide Ligand silica (LUD0X AS + + 500 times a 40)

218 Aminohydrazine slide Ligand aminohydrazine-polylysine package + 500 times by nanoparticles

219 PVP Coated Glass Ligand Hydrophobic Silica Nanoparticles + 500 times (CDS1)

220 PVP coated vitreous silica nanoparticles (STN + + 500 times tablets one 3)

221 PVP Coated Glass Lithium Silica (LUDOX AS + + 500 times -40 pieces)

222 PVP Coated with Hydrazine-Polylysine + 500 times Tablets Nanoparticles

Ligand-nanoparticles-ligand-nanoparticles

223 substrate 2 ligand silicon oxide (LUD0X AS— + + 500 times

40)

224 Tablets 2 Ligidine aminohydrazine-polylysine coating + 500 times Nanoparticles Example 6: Preparation and application of multiple ligand / nanoparticles / tablets composites

In this embodiment, 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.

1) Preparation of multiple ligand / nanoparticle / chip-based composite chips

(1) Preparation of affinity nanoparticles

The preparation method is the same as in Example 5 for the preparation of ψ affinity nanoparticles.

(2) Ligand 2-nanoparticles-ligands 2-ligands 1-nanoparticles-ligands 1-sheet composites

In this embodiment, 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. Two spots of each affinity nanoparticle were formed to form a 3 × 2 array, and then blocked with a bovine serum albumin solution before use. The obtained chip is referred to as a chip 601. The HBsAb concentration was 3 mg / ml when spotted.

(3) Preparation of ligand 2-nanoparticles-ligand 2-ligand 1 one-sheet complex

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

2) Application of multiple ligands / nanoparticles / chip-based composite chips

In this example, 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. Using the detection chip of this embodiment and the detection method of preparing a multi-ligand / nanoparticle / chip-based composite chip, 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 base with or without affinity nanoparticle No. 1 sample No. 2 control chip amino slide no-1

Chip 601 amino slides have + one

Chip 602 amino slides have + ― Example 7: Preparation and application of ligand / nanoparticle / sheet-based complex microplate

1) Preparation of Ligand / Nanoparticle / Substrate Complex Plate

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 μg / 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.

2) Application of Ligand / Nanoparticle / Substrate Complex Plate

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. As a result, when the anti-EBV-VCA-P18 IgA-positive serum was diluted 1000-fold, 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

1) Preparation of Ligand / Nanoparticle / Molecular Marker 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.). .

The 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.

(1) Preparation of affinity nanoparticles

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.

(2) Preparation of nanoparticle-molecular labeling substance

In this embodiment, 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.

(3) Preparation of a ligand-labeled substance

The preparation method of the ligand-molecular labeling substance complex in this embodiment is a well-known preparation method of rhodamine-labeled antibody.

(4) Preparation of ligand / nanoparticle / molecular labeling substance complex

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.

2) Application of Ligand / Nanoparticle / Molecular Labeling Substance Complex

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.

The sample and detection method used in this example are the same as those in Example 5. The obtained results are shown in Table 9. Table 9

Example 9: Preparation and application of a kit containing a ligand / nanomicrochip-based complex and a ligand / nanomicron molecular marker substance complex

1) Preparation of a kit containing a ligand / nanoparticle / tablet complex and a ligand / nanoparticle / molecular labeling substance complex

In this embodiment, the kit is a chip kit, which includes a ligand / nanoparticle / tablet complex (chip) and a ligand / nanoparticle / molecular labeling substance complex (label). Wherein, the preparation of the ligand / nanoparticle / tablet complex was the same as in Examples 5 and 6, and 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.

2) Application of a kit containing a ligand / nanoparticle / sheet complex and a ligand / nanoparticle / molecular labeling substance complex

In the application of the kit of this embodiment, only the reagent composed of the chip 207 prepared in Example 5 and the marker 3 prepared in Example 8 is taken as an example. The pair of photos used is the control chip in Example 5, and the control used The marker was the control marker in Example 8.

In this embodiment, the samples and detection methods used are the same as those in Example 8. The results obtained are shown in Table 10. Table 10

Example 10: Preparation and application of magnetic chip and magnetic chip kit

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.

1) Preparation of magnetic nanoparticle chip

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. The 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.

2) Preparation of magnetic nano-labels

The magnetic nanomarker in this embodiment is a ligand / magnetic nanoparticle / molecular labeling substance complex. Among them, hexyl is a goat anti-human secondary antibody (Jackson ImmnoResearch Laboratories).

3) Preparation of magnetic chip kit

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.

4) Application of magnetic chip kit

'' In this example, 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 μ 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 μ 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

Among them: 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.

1) Preparation of ligand / nanoparticle / carrier complex separation media

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 μm 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. Actual In addition, due to the introduction of nano-dextran, it can also be used to derive a variety of chromatographic stationary phases using classical dextran-derived methods.

As a comparative method for preparing the DEAE-dextran / silica gel particle composite, refer to the method published by one inventor of the present invention (ibid.).

2) Application of ligand / nanoparticle / carrier complex separation media

In this example, 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. In the kinetic adsorption capacity test, 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. According to the known kinetic adsorption capacity of human albumin, 1.2 mg / m, 7.8 mg / ml and 13.5 mg / ml, respectively, of which the DEAE-dextran / nanoparticle / silica particle complex has the highest kinetic adsorption.

Claims

Claims A method for quantitative or / and qualitative peptide detection, which includes at least the following steps.-

(a) An affinity nanostructure carrier is provided. The affinity nanostructure carrier includes a solid phase carrier 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, which retains the main nanophenomenon characteristics and 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 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 50 nm ;

(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 that can interact with the target polypeptide. · Polypeptides, 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 Non-magnetic inorganic non-metallic microparticles or derivatives thereof that are less than 10 urn and that are not themselves labeling substance enhancers, the derivatives include surface-modified or functional organic-coated derivatives containing derivative groups on the surface.

2. The method according to claim 1, wherein the non-magnetic particles include non-magnetic inorganic particles, non-magnetic organic particles, and derivatives thereof, and the non-magnetic inorganic particles include non-magnetic inorganic non-metal particles and non-magnetic metals Microparticles, the derivatives include surface-modified or functional organic-coated derivatives containing derivatizing groups on the surface.

3. The method according to claim 2, wherein the non-magnetic inorganic non-metal particles include oxygen Oxide particles including silicon oxide, titanium oxide, and aluminum oxide, and the metal particles include gold, silver, copper, and aluminum particles.

4. The method according to any one of claims 1 to 3, wherein the solid phase support comprises a base and carrier particles made of the following materials or derivatives thereof: glass, silicon wafer, silica gel, ceramic, metal oxide , Metal, 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.

5. The method according to any one of claims 1 to 4, wherein the detection device containing the affinity nanostructure carrier comprises an analysis chip, a microplate, and a flat chromatography reagent strip, and the ligand / nanoparticle / The labels of the molecular labeling substance complex include an analysis chip label, an enzyme plate label, and a planar chromatography reagent strip label.

6. The method according to any one of claims 1 to 5, wherein the derivatizing group comprises one or more of the following organic groups: amino, aldehyde, epoxy, aminohydrazine, diethylaminoethyl Diethyl one

(2-hydroxypropyl) aminoethyl, carboxymethyl, sulfopropyl, mercaptoethylpyridyl, siloxanyl, thiol

Volume, ¾ o

7. The method according to any one of claims 1 to 6, wherein the functional organic substance comprises one or more of the following organic substances: a surfactant including polyvinylpyrrolidone, a Tween-type surfactant, Polyelectrolytes including polyamino acids, lipophilic organics including polysiloxane, ion exchange polymers including dextran derivatives, agarose derivatives, cellulose derivatives, polyacrylamides, and Affinity substances including heparin sodium, biotin, avidin.

8. The method according to any one of claims 1 to 7, wherein 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.

9. The method according to claim 8, wherein the molecularly labeled substance comprises one or more of the following: fluorescein, rhodamine, seaweed protein, silver salt, enzyme, basic black, basic violet, amine group Black, Coomassie brilliant blue, crystal violet.

10. A polypeptide detection device comprising the affinity nanostructure carrier according to any one of claims 1 to 9 and a ligand / nanoparticle / molecular labeling substance complex.

11. The device according to claim 10, comprising an analysis chip reagent containing the affinity nanostructure carrier according to any one of claims 1 to 7 and a ligand / nanoparticle / molecular labeling substance complex Kit, microplate reader, and planar chromatography kit.

12. A method for preparing a microcarrier-coated carrier for polypeptide detection or component separation, comprising 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 mil and less than 100 legs, 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;-

13. The method according to claim 12, wherein the microcarrier-coated carrier comprises an analysis chip base for peptide detection, a microplate reader, a planar chromatography reagent strip base, and component separation. The chromatography stationary phase matrix.

14. The method according to claim 12 or 13, wherein the colloids include polymer colloids including polysaccharides, nitrocellulose, and derivatives thereof, and the derivatives include surfaces having derivatizing groups on the surface Modified or / and functional organic coated derivatives.

15. The method according to any one of claims 12 to 14, wherein the non-magnetic particles include non-magnetic inorganic particles, non-magnetic organic particles and derivatives thereof, and the non-magnetic inorganic particles include non-magnetic inorganic non-metal particles And non-magnetic metal particles, the derivatives include surface-modified or functional organic-coated derivatives containing derivatizing groups on the surface.

16. The method according to claim 15, wherein the oxide particles include silicon oxide, titanium oxide, and aluminum oxide particles, and the metal particles include gold, silver, copper, and aluminum particles.

17. The method according to any one of claims 12 to 16, wherein the derivatizing group comprises one or more of the following organic groups: amino, aldehyde, epoxy, aminohydrazine, diethylaminoethyl , Second (1-hydroxypropyl) aminoethyl, carboxymethyl, sulfopropyl, mercaptoethylpyridyl, siloxane, thiol, alkyl.

18. The method according to any one of claims 12 to 17, wherein the functional organic substance comprises one or more of the following organic substances: a surfactant including polyvinylpyrrolidone and a Tween-based surfactant, including a polymer Polyelectrolytes including amino acids, lipophilic organics including polysiloxanes, ion-exchange polymers including dextran organisms, agarose derivatives, cellulose derivatives, polyacrylamide, and heparin sodium , Biotin, avidin and other affinity substances.

19. A microcarrier-coated carrier prepared according to the method of any one of claims 12-18.

20. The microcarrier coated carrier according to claim 19, comprising a microcarrier coated chip base, a microcarrier coated enzyme plate base, a microcarrier coated planar chromatography reagent strip, and a nanoparticle coating layer. Analysis carrier.

21. A detection device or separation medium comprising the microcarrier-coated carrier according to claim 19 or 20 and a ligand fixed on the microcarrier-coated carrier.

22. The detection device or separation medium according to claim 21, comprising a chip containing a microcarrier-coated carrier according to claim 19 or 20, a microplate, and a flat chromatography reagent strip.

23. A method for preparing an affinity nanostructured carrier for polypeptide detection or component separation, comprising the following steps:

(a) preparing a carrier, one or more ligands, and one or more nanoparticles, said ligands being selected from the group consisting of substances capable of interacting with the target polypeptide: polypeptides, polysaccharides, vitamins, antibiotics, viruses, Cells and functional organic matter, the nano-particles are non-magnetic particles with 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;

(c) fixing the affinity nano-particles prepared in step (b) to the carrier in a liquid or solid state to form the affinity nano-structured carrier, wherein there is one or more layers 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.

24. The method according to claim 23, wherein the volume concentration of the nanoparticles in the affinity nanoparticle liquid is between 1/1000 and 20,000.

25. The method according to claim 23 or 24, wherein the inorganic fine particles are selected from the group consisting of oxide fine particles, metal fine particles, and derivatives thereof, and the derivatives include surface modification or / and Functional organic coated derivatives.

26. The method according to claim 25, wherein the non-magnetic inorganic non-metal particles include oxide particles including silicon oxide, titanium oxide, and aluminum oxide, and the metal particles include gold, silver, copper, and Aluminum particles.

27. The method according to any one of claims 23 to 26, wherein the carrier comprises a base and carrier particles made of the following materials or derivatives thereof: glass, silicon wafer, silica gel, ceramic, metal oxide, metal Polymer materials and their composites, the derivatives include surface-modified or functional organic-coated derivatives containing derivative groups on the surface, and the microcarrier-coated carrier according to claim 19 or 20.

28. The method according to any one of claims 23 to 27, wherein the derivatizing group comprises one or more of the following organic groups: amino, aldehyde, epoxy, aminohydrazine, diethylaminoethyl , Diethylmono (2-hydroxypropyl) aminoethyl, carboxymethyl, sulfopropyl, mercaptoethylpyridyl, siloxanyl, thiol, alkyl.

29. The method according to any one of claims 23-28, wherein the functional organic substance comprises one or more of the following organic substances: a surfactant including polyvinylpyrrolidone and a Tween-type surfactant, Polyelectrolytes including polyamino acids, lipophilic organics including polysiloxanes, ion-exchange polymers including dextran derivatives, agarose derivatives, cellulose derivatives, polyacrylamides, and Affinity substances including protein, protein G, heparin sodium, biotin, avidin.

30. An affinity nanostructure support for polypeptide detection or component separation, wherein the affinity nanostructure support includes a solid phase support and an affinity nanostructure distributed on a surface thereof, wherein the affinity nanostructure is A structure constructed on the surface by using 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 an immobilized on the surface. One or more ligands, and one or more nanoparticles in the affinity nanostructure solid phase carrier and the solid phase carrier have one or more ligands, or / and one or more ligands and There is a heavy or multiple nanoparticle between solid phase carriers, 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 consisting of Substances: peptides, polysaccharides, vitamins, antibiotics, viruses, cells, and functional organics. The nanoparticles are non-magnetic particles with 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 50 nm.

31. The complex according to claim 30, which is an affinity nanostructure carrier containing the affinity nanoparticles for polypeptide detection or component separation prepared according to the method of any one of claims 23-29. .

32. The complex according to claim 30 or 31, comprising an analysis chip for detection, a microplate, a flat chromatography reagent strip, and a chromatography stationary phase for separation.

33. A method for preparing a ligand / nano-powder molecularly labeled substance complex, comprising the following steps:

(a) preparing one or more ligands, one or more nanoparticles, and one or more of said molecular marker substances, and said nanoparticles / ligands / molecular marker substances are mixtures or purified substances, 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 more than 1 nm and less than 10 nm, non-magnetic inorganic non-metal particles that are not labeling substance enhancers per se;

34. The method according to claim 33, wherein the inorganic non-metal particles include oxide particles or derivatives thereof, the oxide particles include silica, titanium oxide, and alumina, and the derivatives include surface-containing derivatives Surface modified or functional organic coated derivatives of the group.

35. The method according to claim 33 or 34, wherein the functional organic substance comprises one or more of the following organic substances: surface activity including polyvinylpyrrolidone and Tween-type surfactants Agents, polyelectrolytes including polyamino acids, lipophilic organics including polysiloxanes, ion-exchange polymers including dextran derivatives, agarose derivatives, cellulose derivatives, polyacrylamide , And affinity substances including heparin sodium, biotin, avidin.

36. The method according to any one of claims 33 to 35, wherein 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.

37. The method according to claim 36, wherein the molecular labeling substance comprises one or more of the following: fluorescein, rhodamine, seaweed protein, silver salt, enzyme, basic black, basic violet, amine Base black, Coomassie brilliant blue and crystal violet.

38. A ligand / nanoparticle / molecular labeling substance complex for polypeptide detection, comprising one or more molecular labeling substances, one or more nanoparticles, and one or more ligands, said 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 group: polypeptide, polysaccharide, vitamin, antibiotic, virus, cell, and functional organic substance, said Nanoparticles are non-magnetic inorganic non-metallic particles having 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.

39. The complex according to claim 38, which is a ligand / nanoparticle / molecular labeling substance complex for polypeptide detection prepared according to the method of any one of claims 33 to 37.

40. The complex according to claim 39, which is a label for one of the following detections: chip detection, enzyme label detection, and planar chromatography reagent strip detection.

41. A polypeptide detection device comprising the affinity nanostructure carrier according to any one of claims 30 to 32 and / or the ligand / nanoparticle / molecular labeling substance complex according to any one of claims 38 to 40.

42. The polypeptide detection device according to claim 41, which comprises the affinity nanostructure carrier according to any one of claims 30 to 32 and the ligand / nanoparticle / Chip kit for molecularly labeled substance complexes.

43. The polypeptide detection device according to claim 41, which comprises an affinity nanostructure carrier according to one of claims 30-32 or a ligand / nanoparticle / Chip kit for molecularly labeled substance complexes.

44. The polypeptide detection device according to claim 41, which contains the polypeptide detection device according to claims 30-32. One of the affinity nanostructure carrier and / or the ligand / nanoparticle / molecular labeling substance complex enzyme plate plate kit according to any one of claims 38-40.

45. The polypeptide detection device according to claim 41, which comprises an affinity nanostructure carrier according to one of claims 30-32 and / or a ligand / nanoparticle according to one of claims 38-40 / Molecularly labeled substance complex for flat chromatography reagent strip kit.

46. A method for quantitative or qualitative detection of a polypeptide, comprising using an affinity nanostructured carrier according to any one of claims 28 to 30 to capture a target polypeptide in a sample.

47. A method for quantitative or qualitative detection of a polypeptide, comprising delabeling with a ligand / nanoparticle / molecular labeling substance complex according to one of claims 38-40.

48. A chip detection method for quantitative or / and qualitative peptides, which includes at least one, two, three or four steps as follows:

(a) mixing the sample with magnetic particles or / and magnetic flakes;

(b) mixing the sample with a ligand / magnetic nanoparticle, said 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 in at least one dimension in the three-dimensional space are 1 to 200 nm, preferably 1 to 100 nm, and more preferably 1 to 50 nm;

(c) contacting and reacting the sample with the ligand / magnetic nanoparticle / sheet-based composite, and optionally an external magnetic field may be present during the reaction, 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 There is a heavy or multiple magnetic nanoparticle in between, or / and at least one heavy magnetic nanoparticle and another heavy magnetic nanoparticle have a heavy or multiple ligand, 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 nm. 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 modification or / And functional organic coating derivatives;

(d) a ligand / magnetic nanoparticle / molecular labeling substance complex is used for the labeling reaction, and an external magnetic field may optionally be present during the labeling; the ligand / magnetic nanoparticle / molecular labeling substance complex contains one or A plurality of molecularly labeled substances, one or more magnetic nanoparticles, one or more ligands, and optionally a blocking agent, wherein the ligands / magnetic nanoparticles / sub-labeled substances are a mixture or a purified substance, said The ligand is selected from the group of substances capable of interacting with polypeptides: polypeptides, polysaccharides, vitamins, antibiotics, viruses, cells, and functional organics. The magnetic nanoparticles have at least one dimension of 1-200 nm in a three-dimensional space, preferably 1 -100 nm, more preferably 1-50 nm, and is not itself a molecularly labeled substance enhancer.

49. The method of claim 48, wherein the applied magnetic field is pulsed.

50. A polypeptide detection chip, comprising at least one or more ligand / magnetic nanoparticle / sheet complexes, said ligand / magnetic nanoparticle / sheet complexes comprising 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 there is one between the one or more ligands and the carrier A heavy or multiple magnetic nanoparticle, or / and at least one heavy magnetic nanoparticle and another heavy magnetic nanoparticle has a heavy or multiple ligand, said ligand is selected from the group of substances that can interact with the target polypeptide: polypeptide, polysaccharide , Vitamins, antibiotics, viruses, cells, and functional organics; the magnetic nanoparticles are selected from the group consisting of tetraoxide in at least one dimension in a three-dimensional space of 1-200 nm, preferably 1-100 nm, more preferably 1-50 nm Ferrite and its derivatives including ferric oxide and ferric oxide, the derivatives including surface-modified or functional organic-coated derivatives containing derivatized groups on the surface; the base is selected from the following materials Groups and their derivatives : Glass, silicon, ceramics, metal oxides, metals, polymeric materials and their composites.

51. The detection chip according to claim 50, wherein the ligand / magnetic nanoparticle / sheet composite is an affinity magnetic nanostructure chip, and the affinity magnetic nanostructure chip includes a substrate and is distributed on a surface thereof. Affinity magnetic nanostructures, wherein the affinity magnetic nanostructures are constructed on the surface in units of affinity magnetic nanoparticles, and retain the main nano-phenomenon characteristics and have a higher reaction efficiency than ligands The affinity magnetic nanoparticles include the magnetic nanoparticles and one or more of the ligands fixed on the magnetic nanoparticles.

52. A method for preparing a detection chip according to claim 50 or 51, comprising the following steps:

(b) combining the magnetic nanoparticles with the ligand to form one or more affinity magnetic nanoparticles, the affinity magnetic nanoparticles including a mixture and a purified substance; (c) binding the affinity magnetic nanoparticles prepared in step (b) to the carrier, and providing an external magnetic field during binding.

53. A peptide detection chip kit, wherein the chip is the chip according to claim 22, 32, 50 or 51, further comprising at least one of the following magnetic substances: magnetic particles or / and microchips, affinity magnetic nanometers Microparticles, 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 organics The magnetic nanoparticles in at least one dimension in the three-dimensional space are 1 to 200, preferably 1 to 100, more preferably 1 to 50 legs, and the magnetic particles themselves in the ligand / nanoparticle / molecular labeling substance complex Not a molecular marker substance enhancer.

54. The kit according to claim 53, wherein the magnetic microparticles and / or magnetic microchips are used to generate a reaction medium mixture, and affinity magnetic nanoparticles are used to concentrate sample targets, ligands / magnetic nanoparticles, and molecular markers. The substance complex is used to mark the reaction.

55. The kit according to claim 53 or 54, wherein the magnetic particles are selected from the group consisting of iron oxides including iron trioxide, iron oxide, and derivatives thereof, and the derivatives include surface-derived derivatives Surface modified or functional organic coated derivatives of the group.

56. A peptide detection chip kit, comprising at least one of the following magnetic nanoparticle-containing substances: magnetic nanoparticle, affinity magnetic nanoparticle, and ligand / magnetic nanoparticle / molecular labeling substance complex, The ligand is selected from the group of substances capable of interacting with the target polypeptide in the following groups: polypeptides, polysaccharides, vitamins, antibiotics, viruses, cells, and functional organics. The magnetic nanoparticles have at least one dimension of 1 to 200 nm in a three-dimensional space, preferably 1-100 nm, more preferably 1-50 nm, and the microparticles in the ligand / nanoparticle / molecular labeling substance complex are not themselves molecular labeling substance enhancers. '

57. The kit according to claim 56, wherein the magnetic particles are selected from the group consisting of ferrite and ferric oxide, and derivatives thereof, and the derivatives include a derivative group on the surface. Surface-modified or functional organic-coated derivatives of the group.

58. A chip detector for performing detection using the polypeptide detection chip kit according to any one of claims 52 to 57 comprising means for providing a magnetic field, preferably a pulsed magnetic field, during a reaction or / and a label during detection.