WO2004090548A1 - Device for analysis or separation containing an active nanostructured carrier, its preparation method and applications - Google Patents

Device for analysis or separation containing an active nanostructured carrier, its preparation method and applications Download PDF

Info

Publication number
WO2004090548A1
WO2004090548A1 PCT/CN2004/000203 CN2004000203W WO2004090548A1 WO 2004090548 A1 WO2004090548 A1 WO 2004090548A1 CN 2004000203 W CN2004000203 W CN 2004000203W WO 2004090548 A1 WO2004090548 A1 WO 2004090548A1
Authority
WO
WIPO (PCT)
Prior art keywords
active
nanoparticles
carrier
nanoparticle
nanostructured
Prior art date
Application number
PCT/CN2004/000203
Other languages
French (fr)
Chinese (zh)
Inventor
Fanglin Zou
Chunsheng Chen
Ning Chen
Jianxia Wang
Original Assignee
Chengdu Kuachang Medical Industrial Limited
Chengdu Kuachang Science & Technology Co., Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from CN 03117446 external-priority patent/CN1250969C/en
Priority claimed from CNA031177875A external-priority patent/CN1514243A/en
Priority claimed from PCT/CN2004/000077 external-priority patent/WO2004081571A1/en
Application filed by Chengdu Kuachang Medical Industrial Limited, Chengdu Kuachang Science & Technology Co., Ltd filed Critical Chengdu Kuachang Medical Industrial Limited
Priority to JP2006529552A priority Critical patent/JP2007502998A/en
Priority to PCT/CN2004/000437 priority patent/WO2004102196A1/en
Priority to EP04730459A priority patent/EP1624306A4/en
Publication of WO2004090548A1 publication Critical patent/WO2004090548A1/en
Priority to US11/258,996 priority patent/US7842515B2/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2/00—Peptides of undefined number of amino acids; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the invention relates to a high-sensitivity nanostructure active carrier for separation or / and analysis, a preparation method thereof, a nanolabel and a label method thereof.
  • the invention also relates to a biochip containing a high-sensitivity nanostructure active carrier and / or a nano-label and its application, a polypeptide detection device containing a high-sensitivity nano-structure active carrier and / or a nano-label, especially an analysis chip, and an enzyme labeling plate. , Plane chromatography strip and its application. Background technique
  • Active carriers with selective reactivity have been widely used in many aspects, especially in the qualitative and / or quantitative analysis or / and separation of target substances in samples.
  • Examples of active carriers for separation are affinity chromatography gels.
  • Examples of active carriers for detection include antigen or / and antibody analysis chips. In biological detection analysis, it can be divided into nucleic acid detection and peptide detection according to the different target objects of the sample. In the following, biochip detection is used as an example to briefly explain the problems that need to be solved with existing detection methods and devices.
  • biochips Because of its high throughput and miniaturization, biochips have a wide range of applications, including gene expression testing, gene screening, drug screening, disease diagnosis and treatment, environmental monitoring and governance, and judicial identification.
  • One of the main quality indicators of biochip detection is sensitivity.
  • the active carrier is one of the keys to determining sensitivity.
  • Current chip active carriers are mainly formed by immobilizing active reagents (such as ligands) directly on the surface of solid-phase carriers.
  • the solid phase carriers used are mainly flat substrates made of glass, metal, plastic and other materials and their derivatives. Among them, slide derivatives, such as amine-based slides, aldehyde-based slides, epoxy-based slides, and polyamino acid-coated slides, etc., are the main slide substrates currently used.
  • the second kind of chip active carrier is to fix the ligand on a film substrate (such as a film-glass substrate) with a high specific surface area, such as a film.
  • the fixation of the active reagent on the substrate is one of the key factors that affect the detection sensitivity of the biochip.
  • the immobilization methods mainly include: covalent bond immobilization method, physicochemical adsorption method, embedding method and cross-linking method.
  • Physicochemical adsorption methods include specific adsorption methods and non-specific adsorption methods. Examples of non-specific adsorption are ion adsorption.
  • An example of specific adsorption is affinity adsorption.
  • Antigen-antibody reaction, nucleotide pairing reaction, lipophilic reaction, etc. are all affinity adsorption.
  • the first kind of chip active carrier due to the smaller surface area of the active carrier, etc., its reaction kinetics conditions need to be optimized, and the performance is still to be improved, or the reaction time To be shortened.
  • the second kind of chip active carrier in theory, a higher specific surface can improve sensitivity, but the actual situation is not always the case.
  • reactors that are at least partially film-based have reduced sensitivity due to their high background noise.
  • reactors based on membranes, microparticles, etc. have other practical problems (such as cleaning of membranes, adhesion of microparticles, etc.). 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.
  • the labeling system used in chip detection 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.
  • active carriers such as affinity chromatography stationary phases are also widely used in separation devices such as chromatography devices.
  • active carriers such as affinity chromatography stationary phases
  • the microparticles used as the matrix of the chromatographic stationary phase have a larger specific surface area than the substrate, there is still room for improvement in terms of analysis time and chromatographic yield.
  • a labeling system containing a ligand is the most widely used labeling system.
  • certain nanoparticles in the current biochip labeling systems to improve detection sensitivity such as WO 00/72018 A1, US Patent Application No. 20030211488, Chinese Patent Application Nos. 02333538.9, 02137418.X 01133527.0, they use colloids Gold is used as a marker, plus a silver enhancement system), but they are either the marking substance itself (for example, Chinese patent applications No. 02141718.0, 01109551.2, which uses fluorescent rare earth complex nanoparticle markers), or a marking substance enhancer ( For example, U.S. Patent Nos. 20030232388, 20030166297, 20020142480, 20030211488, using metals with Raman enhancement effect). Summary of the invention
  • the main purpose of the present invention is to improve the reaction efficiency of the active carrier (or the immobilized active reagent) in the analysis and separation, thereby improving the detection sensitivity or / and reducing the detection time or / and providing more kinds of substrates with sufficient high sensitivity.
  • the object of the present invention is achieved by the development of vectors, active vectors and labeling systems.
  • Another object of the present invention is to improve the separation efficiency of the separation medium.
  • the device includes an active carrier containing active nanoparticles, the active carrier comprises a solid phase carrier and is fixed on a part or the entire surface of the solid phase carrier.
  • Active nanoparticles comprising nanoparticles and an active agent fixed on the nanoparticles, the active agent is selected from the group consisting of an ion exchanger, a drug, a polypeptide, a polysaccharide, a vitamin, an antibiotic, Functional organisms, antigens, and viruses, cells or their composition.
  • the active carrier can be obtained through a variety of media, but the preparation of the active carrier of the present invention is a preferred method of preparation of the active carrier of the present invention.
  • the active carrier of the present invention has a detection sensitivity 25%, even 50%, or even 50% higher than that of an active carrier that does not introduce nanoparticles and has only the same ligand immobilized on a conventional substrate.
  • a nanoparticle / ligand mixed solution spotted on an active carrier formed on a chip substrate has a very limited increase in sensitivity.
  • the active carrier includes a single active carrier and a multi-active carrier, wherein the single active carrier has only active nanoparticles containing one active agent immobilized on the same solid-phase carrier, and the Multi-active carriers have active nanoparticles containing two or more active agents fixed on the same solid-phase carrier.
  • the single active carrier has only active nanoparticles containing one active agent immobilized on the same solid-phase carrier
  • the Multi-active carriers have active nanoparticles containing two or more active agents fixed on the same solid-phase carrier.
  • a device containing a multi-active carrier is, for example, a biochip and a planar chromatography reagent strip, and a device containing a single-active carrier is, for example, an enzyme plate.
  • the active nano-particles and the solid-phase carrier are not bound through nucleotide pairing, but are bound by one or more of the following methods: covalent bonding, non-specific physical chemistry Adsorption, antigen-antibody adsorption, and affinity adsorption, and the binding is achieved through a solid phase support or / and a linker on the surface of the nanoparticle, the linker including surface groups or / and coating organic matter or / And the active agent.
  • the binding between the nanoparticles and the substrate must be performed by the pairing of nucleic acids. It is not suitable for the detection and separation of substances other than nucleic acids such as polypeptides.
  • the nanoparticles have an average particle diameter of 1 to 100 nm, preferably 1 to 50 nm.
  • the nanoparticles include inorganic nanoparticles or / and organic nanoparticles and their derivatives
  • the inorganic nanoparticles include non-magnetic inorganic nanoparticles and magnetic inorganic nanoparticles
  • the non-magnetic inorganic The nanoparticles include non-magnetic metal nanoparticles and non-magnetic non-metal nanoparticles
  • the derivative includes a derivative having a surface group bound thereto and / or a coated organic substance.
  • the inorganic non-magnetic non-metal nanoparticles include silicon oxide, titanium oxide, and alumina nanoparticles, and the non-magnetic metal nanoparticles include gold, vanadium, lead, silver, iron, and oxides thereof.
  • Nano particles, the organic nano particles include plastic, polysaccharide, latex, resin nano particles.
  • the surface group includes one or more of the following groups: amino, aldehyde, epoxy, aminohydrazine, diethylaminoethyl, diethylmono (2-hydroxy Propyl) aminoethyl, carboxymethyl, sulfonic acid propyl, mercaptoethylpyridyl, siloxane, thiol, fluorenyl;
  • the coated organics include one or more of the following: Surfactants including polyvinylpyrrolidone and Tween-based surfactants, polyelectrolytes including polyamino acids, lipophilic organics including polysiloxanes, including dextran derivatives, agarose derivatives, Ion exchange polymers including cellulose derivatives, polyacrylamide, and affinity substances including heparin sodium, biotin, actives, antigens, and antibodies.
  • the surface group may also be a long-arm derived group R— (CH 2 ) x— , where R is a derived group, and X is equal to or greater than 2, preferably greater than 4, and more preferably greater than 6.
  • R is a derived group
  • X is equal to or greater than 2, preferably greater than 4, and more preferably greater than 6.
  • a nanoparticle derivative such as superhydrophobic silica (CS7) has an alkyl group, and the surface of the coated derivative has an organic-coated group, for example, a polyamino acid has an amino group, and an aminohydrazine-polyamino acid Has amino and aminohydrazine, DEAE-Dextran has diethylaminoethyl, and the like.
  • CS7 superhydrophobic silica
  • 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 / Tablet complex and ligand / nanoparticle / molecular labeling substance complex.
  • an important aspect of microparticle derivatives is the coated derivative.
  • the coated derivative in the method of the invention may be a single or multiple coated derivative.
  • coated with a dispersant or / And dispersion stabilizer silicon oxide particles can also be coated with PVP (two-layer coating), continue to be, ⁇ ⁇ with coating—coated with a ⁇ protein A ⁇ (three- ⁇ -heavy »_ Package — be) .. etc. — »wait one. Therefore, the range of organic coatings that can be selected is very large.
  • the active agent is selected from the group consisting of an ion exchanger, a drug, a polypeptide, a polysaccharide, a vitamin, an antibiotic, a functional organic substance, an antigen, and a virus, a cell, or a composition thereof. These substances are all known substances which can interact with the target polypeptide.
  • the solid-phase carrier includes a conventional carrier and a nanostructured carrier
  • the conventional carrier includes a solid-phase substance made of the following materials or derivatives thereof: glass, silicon wafer, silica gel, ceramic, Metal oxides, metals, polymer materials and their composites
  • the nanostructured carrier includes a carrier that forms a nano-sized structure on the surface of the conventional carrier.
  • a nanostructure active support for separation or / and analysis, the carrier comprising a solid phase support and an active nanostructure on a surface of the solid phase support, the active nanostructure ⁇ "Nanostructures and the active agents on said nanostructures.
  • active areas and inactive areas are divided according to the presence or absence of active agents on the surface. Inactive areas are optional.
  • active nanostructures are mainly divided into nanostructure active regions and non-nanostructure active regions according to whether the surface is mainly distributed with active nanostructures.
  • Non-nanostructure active regions are optionally present (when substantially no active nanostructures exist in the active region Distribution region, it does not exist), and the active region has at least one or more of the following characteristics: (a) the ratio of the surface area of the active region to the surface area of the solid phase support of greater than 1.5; (b) said active nanostructures comprise projecting height greater than 3 nm, and the half-height projecting at least one dimension between 1 nanometer convex body of a 500 nm, and the active region in the nanostructure Said distribution density of nano-convex body in a projection plane perpendicular to the solid support is greater than 1 / ⁇ ⁇ 2; perpendicular (c) the nanostructured active region and the non-active nano-structured region on a solid phase support The ratio of the projected areas is greater than 1.
  • the active region refers to a region where the active agent is distributed on the surface of the nanostructured active support, and the inactive region refers to the surface except the active region.
  • the nanostructured active region refers to a region where the active nanostructure is mainly distributed on the surface
  • the non-nanostructured active region refers to a region other than the nanostructured active region in the active region.
  • the active nanostructure formed on the solid support is the key to the nanostructure active support of the present invention.
  • the nanostructure active support in the present invention is different from the active support containing nanoparticles in general.
  • 20030207296 is an active carrier including a solid carrier and nanoparticles on the solid carrier.
  • different active carrier surface topography, its activity (e.g., sensitivity) can be very different from the present invention.
  • the lucid nanostructured active support has a defined surface morphology and is therefore highly active (eg, detection sensitivity).
  • the nanoparticles attached to the surface of the carrier do not necessarily form structural regions with nanometer size.
  • the detection sensitivity of the chip is lower.
  • the active nanoparticle-containing active support formed without strict structural design is different from the nanostructure active support of the present invention. Only the active carrier having the above nanostructure parameters is the nanostructure active carrier of the present invention.
  • the ratio of the surface area of the active region to the solid-phase support is greater than 3, preferably greater than 6.
  • the distribution density of the convex body is more than 5 pieces / ⁇ m 2 , preferably more than 10 pieces / m 2 .
  • a ratio of a projected area of the nanostructured active area to the non-nanostructured active area on a solid support is greater than 2, preferably greater than 4.
  • the target of the separation or / and analysis includes a peptide, a nucleic acid, and a drug interacting with them.
  • the nanostructured active carrier includes: a biochip or an active carrier thereof, an enzyme-labeled plate, a planar chromatography reagent strip, and a chromatography active gel.
  • the active agent is selected from the group consisting of an ion exchanger, a drug, a polypeptide, a polysaccharide, a vitamin, an antibiotic, a functional organic substance, an antigen, a single- or multi-stranded DNA, RNA, Nucleotides, as well as viruses, cells or their composition.
  • the active nanostructure contains an active nanoparticle, and the active nanoparticle includes a nanoparticle and one or more of the active agents or optional links fixed thereto.
  • An agent, wherein the nanoparticle is a particle 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 50 nm.
  • the active nanoparticle and the solid-phase support are bound by one or more of the following methods: covalent bonding, non-specific physicochemical adsorption, anti- Proto-antibody adsorption, affinity adsorption, and nucleotide pairing.
  • the binding is achieved by a solid-phase support or / and a linker on the surface of the nanoparticle, the linker comprising surface groups or / and coating organic matter.
  • the nanoparticles include inorganic nanoparticles or / and organic nanoparticles and their derivatives
  • the inorganic nanoparticles include non-magnetic inorganic nanoparticles and magnetic inorganic nanoparticles
  • Non-magnetic inorganic nanoparticles include non-magnetic metal nanoparticles and non-magnetic non-metal nanoparticles
  • the derivative includes a derivative having a surface group bound thereto and / or a coated organic substance.
  • the inorganic non-magnetic non-metallic nanoparticles include silicon oxide particles, titanium oxide particles, alumina particles, iron oxide particles, and the non-magnetic metal nanoparticles include gold particles, vanadium particles, Lead particles, the non-metal nanoparticles include organic particles including plastic, polysaccharide, latex, and resin particles.
  • the surface group includes one or more of the following groups: amino, aldehyde, epoxy, aminohydrazine, diethylaminoethyl, diethyl- ( 2-hydroxypropyl) aminoethyl, carboxymethyl, sulfopropyl, mercaptoethylpyridyl, siloxane, thiol, alkyl;
  • the coated organics include one or more of the following Organics: Surfactants including polyvinylpyrrolidone, Tween-based surfactants, polyelectrolytes including polyamino acids, lipophilic organics including polysiloxanes, including dextran derivatives, agarose Derivatives, cellulose derivatives, polyacrylamide, ion exchange polymers, and affinity substances including sodium heparin, biotin, actives, antigens, and antibodies.
  • the derivatizing groups and functional organics described herein can be referred to the description in the aforementioned active support
  • the solid phase support includes a conventional support and a nanostructure support, wherein: the conventional support includes a solid phase substance made of the following materials or derivatives thereof: glass, silicon wafer, Silica gel, ceramics, metal oxides, metals, polymer materials and their composites; the nanostructured carrier is a carrier with a nanometer-sized structure distributed on the surface.
  • an analysis or / and separation device containing the above-mentioned active carrier.
  • active carriers such as biochips and planar chromatography reagent strips
  • devices containing single active carriers such as microplates.
  • an active carrier including active nanoparticles and an active carrier according to the present invention which are included in a device according to the present invention, including the following steps: (a) preparing the An active reagent, a carrier, a nanoparticle, and an optional linking agent; (b) fixing the active reagent to the nanoparticle to prepare an active nanoparticle, and if necessary, performing a pre-treatment on the nanoparticle with the linker; Facilitates the activation of the active agent; (c) Step (b) The active nanoparticle suspension prepared in contact with the carrier and undergoes an immobilization reaction, if necessary, the carrier is activated in advance with the linker to facilitate the immobilization, and the active nanoparticle is immobilized during the immobilization reaction.
  • the concentration of the nano-particles in the micro-particle suspension is: a nano-particle weight / volume concentration of two-hundredths to sixty thousandths, or a nano-particle molar concentration of 0.12 to 37.4 nM.
  • the active nanoparticle may be a nanoparticle and a ligand prepared as a mixture with other substances, such as a nanoparticle suspension containing a colorant or / and a binder, a ligand solution containing a stabilizer, and the like.
  • An example of the method of the present invention is: after the nanoparticle suspension is mixed and reacted with the ligand solution to form affinity nanoparticles, the mixed solution is spotted on the chip substrate to perform a binding reaction by spotting.
  • the carrier according to the present invention may also be prepared in a manner coexisting with other structures, such as a substrate in a multi-chip base substrate.
  • the affinity nanoparticle suspension 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 centrifugation to remove a purified substance that is free of the ligand), Furthermore, one or more kinds of ligands are immobilized on a nanoparticle in the purified product. It needs to be particularly emphasized that when the nanoparticles are not sufficiently diluted or over-diluted, no increase in sensitivity is observed for the composites prepared using certain nanoparticles;
  • a nanostructured active carrier having multiple ligands between one or more nanoparticles and a carrier is prepared, for example, as follows: the carrier is coated with a reguiding group 1 to form a ligand 1 coated carrier, and the nanoparticles are coated with a Another ligand 2 (pairing reaction between ligands 1 and 2 can take place) to form a ligand 2 / nanoparticle complex, and then coat or spot the ligand 2 / nanoparticle complex to ligand 1 On the carrier, a complex in the form of ligand 2-nanoparticle-ligand 2-ligand 1-carrier is formed. When the number of ligand layers is greater than 2, and so on.
  • the same method can also be used to prepare at least one heavy nanoparticle and another layer of nanostructured nanostructure active carrier, such as ligand 3—nanoparticles—ligand 3—ligand 2—nanoparticles—ligand 2 -Ligand 1-carrier or ligand 2-nanoparticle-ligand 2-ligand 1-nanoparticle-ligand 1-carrier, etc.
  • a nanostructured active 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 ⁇ s to form a plurality of affinity groups. And nanoparticles (for example, ligand 2—nanoparticles—ligand 2, ligand 3—nanoparticles—ligand 2, ligand 1—nanoparticles—ligand 1, etc.), and then these affinity nanoparticles are successively or simultaneously Binding 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-Nanoparticles A nanostructured active support in the form of a ligand, a support, and the like. ⁇
  • the weight / volume concentration of the nanoparticles is from one thousandth to six thousandths.
  • One ten-thousandth, preferably one ten-thousandth to one forty-thousandth, or the molar concentration of the nanoparticles is 0.12-3.74 nM, preferably 0.19-0.75 nM.
  • an active carrier including an active nanoparticle included in a device according to the present invention and an active carrier according to the present invention, which includes the following steps: (a) preparing an The active reagent, the carrier, the nanoparticle, and an optional linker; (b) contacting the suspension of the nanoparticle with the carrier and performing an immobilization reaction to form a nanostructured carrier, and if necessary, use the linker in advance
  • the agent performs activation of the nanoparticles or / and the carrier in favor of the immobilization.
  • the concentration of the nanoparticles in the suspension during the immobilization reaction is: nanoparticle weight / volume concentration of two to six percent 1 / 10,000, or a molar concentration of nanoparticles of 0.12 to 37.4 nM ; (c) immobilizing the active agent on the nanostructure carrier.
  • the nanoparticle has a weight / volume concentration of one thousandth to sixty thousandth, preferably one ten thousandth to one forty thousandth, or the molar concentration of the nanoparticle. 0.12 to 3.74 nM. It is preferably 0.19-0.75 nM.
  • a nanostructure support for separation or / and analysis which includes a solid phase support and nanostructures on the solid phase support.
  • the nanostructure support includes a nanostructure region and An optional non-nano-structured region, and the nano-structured region has at least one or more of the following characteristics: (a) the ratio of the surface area of the nano-structured region to the surface area of the solid phase support of greater than 1.5; (b) The nanostructure includes a nanoconvex having a protrusion height greater than 3 nm and a half-height at least one-dimensional dimension between 1 and 500 nm, and the nanocarrier in the nanostructure region is The distribution density on the vertical projection surface of the solid-phase support is greater than 1 / ⁇ ⁇ ⁇ 2 ; (c) the ratio of the vertical projection area of the nanostructured region to the non-nanostructured region on the solid-phase support is greater than 1 .
  • the nanostructured region refers to a region where the nanostructures are mainly distributed on the surface of the nanostructured carrier, and the non-nanostructured region refers to a region other than the nanostructured region.
  • the nanostructured support of the present invention and the current nanoparticle-containing support have very different exact meanings.
  • a ratio of the nanostructure region to the surface area of the solid-phase carrier is greater than 2, or / and the nanostructure distribution density is greater than 1/10 m 2 , or / and The ratio of the projected area of the nanostructured region to the non-nanostructured region on the solid support is greater than two.
  • the nanoparticle in the nanostructure is as described above, and the connection between the nanoparticle and the solid phase carrier can also be performed in the manner described above.
  • the nanostructure carrier according to the present invention can be used as an analysis chip substrate, an analysis chip channel, and an enzyme label. Plate base, planar chromatography reagent strip base, and carrier for chromatography gel.
  • an analysis or / and a separation device containing the above-mentioned nanocrusted carrier.
  • examples of such devices are: chromatographic columns containing nanostructured gels, chips containing nanostructured channels (e.g., a laboratory on a slice), and the like.
  • the present invention provides a method for preparing a nanostructured carrier as described above, which comprises the following steps: (a) preparing the carrier, nanoparticles, and optional linker; (b) if If necessary, use the linker to activate the carrier, or nanoparticles, and (c) contact the suspension of the nanoparticles with the carrier and perform an immobilization reaction.
  • the nanoparticle has a weight / volume concentration of one to two hundredths to sixty thousandths, preferably one to two thousandth to sixty thousandths, and more preferably one to ten thousandth to one forty thousandths. , Or the molar concentration of nanoparticles is
  • the determined nanoparticle concentration is the key.
  • the present invention provides a labeling system containing an active agent / nanostructure / molecular labeling substance complex, wherein the active agent / nanostructure / molecular labeling substance complex contains an active agent and a molecular labeling substance , Nano-particles, and a mixture or purification of optional blocking agents, wherein the active agent is a substance that imparts reactivity to the complex, and the nano-particles have a particle size of 1-100 nm and are not themselves labeled substances.
  • Agent of non-magnetic inorganic non-metal particles is containing an active agent and a molecular labeling substance , Nano-particles, and a mixture or purification of optional blocking agents, wherein the active agent is a substance that imparts reactivity to the complex, and the nano-particles have a particle size of 1-100 nm and are not themselves labeled substances.
  • the ligand / nanoparticle / molecular labeling substance complex contains one or more molecular labeling substances, one or more nanoparticles, one or more ligands, and an optional blocking agent.
  • the ligand / molecularly labeled substance is a mixture or a purified product.
  • the ligand / nanoparticle / molecular labeling substance complex of the present invention is different from the ligand-nanoparticle complex, for example, ligand-microparticle ligand (No. 02137418.X, 02115771.5, and 01133527.0 Chinese patent applications) Ligand-nano magnetic particles (Chinese Patent Application No.
  • 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 particle complex (Chinese Patent Application No.
  • the labeling substance in the composite of the invention is a molecular labeling substance (such as rhodamine) rather than a microparticle, and the carrier in the composite of the present invention is a nanoparticle (such as nano-silica) instead of a microsphere with a larger size.
  • the non-magnetic inorganic non-metallic nanoparticles contained in the composite include oxide particles having a size of 110 nm and derivatives thereof, and the oxide particles include silicon oxide, Titanium oxide and aluminum oxide, and the derivatives include derivatives containing derivatizing groups on the surface or / and coating functional organic matter.
  • the derived groups include the following: one or more types of groups: amino, aldehyde, epoxy, aminohydrazine, diethylaminoethyl, diethylmono (2-hydroxypropyl) aminoethyl, Carboxymethyl, sulfopropyl, mercaptoethylpyridyl, siloxane, thiol, alkyl;
  • the functional organics include one or more of the following organics: including polyvinylpyrrolidone, Tween-like surfaces
  • Surfactants including surfactants, polyelectrolytes including polyamino acids, lipophilic organics including polysiloxanes, including dextran derivatives, agarose derivatives, cellulose derivatives, polyacrylamides Ion exchange polymers, and active substances including heparin sodium, biotin, and active substance; wherein the microcarriers in the microcarrier-coated derivative include the nanoparticles.
  • the active agents contained in the complex include antigens, antibodies, avidin, biotin, single- or multi-stranded DNA, RNA, nucleotides, and viruses, cells, or their components .
  • the molecular labeling substance contained in the composite includes one or more of the following substances: a fluorescent substance, a chemiluminescent substance, a chemiluminescent catalyst, a non-ferrous metal salt, a dye, and a pigment.
  • these molecularly labeled substances include one or more of the following: fluorescein, rhodamine, seaweed protein, silver salts, enzymes, basic black, basic violet, amino black, Coomassie brilliant blue, crystals purple.
  • the preparation method of the active reagent / nanostructure / molecular labeling substance complex is-combining the one or more ligands, one or more nanoparticles, and one or more of the molecules
  • the labeling substance is combined in one of the following ways: combining the ligand with the nanoparticle and then with the molecular labeling substance, combining the nanoparticle with the molecular labeling substance and then with the ligand, The ligand is combined with the molecular labeling substance and then with the nanoparticle, the ligand is simultaneously bonded with the molecular labeling substance and the nanoparticle, and combinations based on these methods.
  • a labeling method which includes at least some steps: (a) preparing the above-mentioned labeling system containing an active reagent / nanostructure / molecular labeling substance complex; (b) using said activity
  • the reagent / nanostructure / molecular labeling substance complex is used for labeling, and the weight / volume concentration of the nanoparticle in the active reagent / nanostructure / molecular labeling substance complex is greater than 1 / 40,000 or the mole of the nanoparticle during labeling.
  • the concentration is greater than 0.19 nM.
  • the preparation method of the complex of the invention is simple, the preparation has good water solubility and high sensitivity.
  • the detection sensitivity of the composite increases as the concentration of the nanoparticles increases.
  • the weight / volume concentration of the nanoparticles is greater than one thousandth, preferably greater than one hundredth, and the molar concentration of the nanoparticles is greater than 7.48 nM, preferably greater than 74.8 nM.
  • the present invention provides a method for detecting an analysis chip, which comprises (a) providing an active carrier or an analysis chip containing active nanoparticles according to the present invention and contacting and reacting a sample to be detected with them, (b) ) Labeling is performed using the labeling method according to the present invention, wherein the active carrier is a multi-active carrier.
  • an analysis chip kit comprising an active carrier containing active nanoparticles or an analysis chip containing a nanostructured active carrier, and / or an active reagent / nanostructure / molecular labeling substance complex .
  • an analysis chip which includes (a) providing an active carrier containing active nanoparticles or an analysis chip containing a nanostructure active carrier according to the present invention, (b) detecting The samples are contacted and reacted with them, wherein the active carrier is a multi-active carrier.
  • the present invention provides a method for detecting an analysis chip, which includes using a labeling method according to the present invention for labeling.
  • an analysis chip kit comprising the active reagent / nanostructure / molecular labeling substance complex as described above.
  • a detection method for an analysis chip which includes at least the following steps: (a) providing a nanostructured microfluidic chip, wherein the nanostructured microfluidic chip includes a nanostructured microchannel or / and A nanostructure separation medium containing a nanostructure carrier according to the present invention, and the nanostructure separation medium is a nanostructure carrier according to the present invention or an active carrier or a nanostructure active carrier comprising active nanoparticles; (b ) The sample is added to the microfluidic chip and separated and analyzed.
  • the nanostructured microchannel or / and nanostructured separation medium includes one or more of the following: a nanostructured molecular sieve, a nanochannel-containing microchannel coating, a nanoparticle-containing stationary phase, Nanoparticles mobile phase.
  • an analysis chip kit which contains the nanostructured microchannel or / and the nanostructured separation medium as described above.
  • an analysis chip detection method for polypeptide analysis which includes at least one, two, three or four steps as follows:
  • the active reagent / magnetic nanoparticle / molecular labeling substance complex is used for the labeling reaction, and an external magnetic field may optionally be present during labeling;
  • the active reagent / magnetic nanoparticle / molecular labeling substance complex contains one or A mixture or purification of multiple molecularly labeled substances, one or more magnetic nanoparticles, one or more active agents, and optionally a blocking agent;
  • the magnetic nanoparticle has at least one dimension in the three-dimensional space of 1-200 nm, preferably 1-100 ran, more preferably 1-50 nm, and is not itself a magnetic particle and a derivative of a molecular marker substance enhancer;
  • the active agent is selected from the group of substances that can interact with polypeptides: peptides, polysaccharides, vitamins, antibiotics, viruses, cells, and functional organic matter, the active agent / magnetic nanoparticle / molecular labeling substance complex at the time of labeling magnetic nanometer
  • the weight / volume concentration of the particles is greater than 1 / 30,000, preferably greater than 1 / 3,000, more preferably greater than 5%, or the molar concentration of the nanoparticles is greater than 0.24 nM, preferably greater than 2.4 nM, more preferably greater than 14.4 nM .
  • the magnetic particles are selected from the group consisting of ferrite and ferric oxide, and derivatives thereof, and the derivatives include a surface modification or / and a function containing a derivative group on the surface.
  • Organic-coated derivatives are selected from the group consisting of ferrite and ferric oxide, and derivatives thereof, and the derivatives include a surface modification or / and a function containing a derivative group on the surface.
  • the applied magnetic field is pulsed.
  • the molecularly labeled substance includes one or more of the following substances: a fluorescent substance, a chemiluminescent substance, a chemiluminescent catalyst, a non-ferrous metal salt, a dye, and a pigment.
  • an analysis chip kit which includes at least one of the following components: magnetic particles or / and magnetic microchips, active reagent / magnetic nanoparticle complex, magnetic nanostructure-containing activity
  • the carrier chip, active reagent / magnetic nanoparticle / molecular labeling substance complex can be described in detail in the above paragraphs.
  • the magnetic particles or / and microchips are used to generate a reaction medium mixture
  • the ligand / magnetic nanoparticle is used to concentrate the sample target
  • the ligand / magnetic nanoparticle / molecular labeling substance complex is used to label the reaction. .
  • the present invention provides a method for quantitative or / and qualitative detection of polypeptides, which comprises at least the following steps: (a) providing an active carrier or active agent / nanostructure / molecule comprising active nanoparticles according to the present invention A labeling substance complex, (b) contacting a sample to be detected with the active carrier and reacting; and / or (c) performing a labeling reaction with the labeling system, wherein the active agent is selected from the group consisting of a reagent capable of interacting with a target polypeptide Substances that act: Peptides, polysaccharides, vitamins, antibiotics, functional organics, viruses and cells and their natural or synthetic composition.
  • the method can be used for chip detection, enzyme label detection, and planar chromatography detection.
  • it provides a polypeptide quantitative or / and qualitative detection kit, which comprises the active carrier comprising active nanoparticles according to the present invention, and / or the active reagent / nanostructure according to claim 47. / Molecularly labeled substance complex.
  • the kit can be used as an analytical chip kit, an enzyme labeling kit, and a planar chromatography kit.
  • the present invention provides a separation method in which one or more of an active carrier, a nanostructure carrier, and a nanostructure active carrier including active nanoparticles according to the present invention are used as a separation medium.
  • the advantage of the quantitative or / and qualitative detection method of the present invention, especially the quantitative or / and qualitative detection method of polypeptide, is that both a nanostructured active carrier is used to capture a target polypeptide in a sample, and a ligand / nanoparticle / molecular labeling substance complex is used. Marking greatly improves detection sensitivity or / and greatly improves detection speed.
  • the detection device of the present invention includes a polypeptide detection device, which has the advantage of containing both a nanostructured active carrier to capture a target polypeptide in a sample and a ligand / nanoparticle / molecular labeling substance complex for labeling, which greatly improves detection sensitivity, or And greatly improved the detection speed.
  • An advantage of the method for preparing a nanostructure-coated carrier for polypeptide detection or component separation of the present invention is that coatings that do not require heating cause less change in the surface of the macroscopic solid-phase carrier.
  • the nanostructure coating carrier of the invention has the advantage that it is easier to prepare various active carriers with low cost and sufficient sensitivity.
  • the method for preparing a nanostructure active carrier of the present invention has the advantage of being simple and effective.
  • nanostructured active carrier used for polypeptide detection or component separation of the present invention are higher reaction efficiency with the target substance of the sample, lower concentration limit of detectable target substance, 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 of the nanostructure-containing active support of the present invention has the advantages of higher reaction efficiency with the target substance of the sample, lower concentration limit of detectable target substance, and higher reaction speed.
  • the detection device of the ligand / 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 it uses at least a nanostructured active carrier Capturing a target polypeptide in a sample increases detection sensitivity or / and speeds up detection.
  • 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.
  • FIG. 1 is a DFM plan view of a nanostructured carrier according to the present invention.
  • Figure 2A is a DFM plan view of a nanostructured active support according to the present invention.
  • FIG. 2B is a perspective view of the DFM shown in FIG. 2A.
  • detection device is an article containing a ligand useful for interacting with a sample target, such as a device containing a capture ligand, a consumable, and a reagent containing a capture ligand and a labeling reagent, used in a specified amount or / and qualitative detection process.
  • a sample target such as a device containing a capture ligand, a consumable, and a reagent containing a capture ligand and a labeling reagent, used in a specified amount or / and qualitative detection process.
  • -Based labeling kit examples are analysis chips, microplates, affinity electrophoresis strips, affinity chromatography columns, planar chromatography reagent strips, analysis chip kits, microplate readers, affinity electrophoresis kits, and the like. Quantitative or / and qualitative detection can be performed in vitro or in vivo.
  • separation device refers to a separation product used in a separation process and containing a substance having a separation function.
  • the separation process is a process in which all or part of the components of a sample are obtained by a separation method.
  • the separation device include a chromatography device, and examples of a substance having a separation function include a chromatographic carrier.
  • 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, and the binding between the ligand, the magnetic nanoparticle, and the carrier may be different.
  • the forms include direct binding and indirect binding.
  • active agent in the present invention refers to a substance for capturing a target substance through interaction (including affinity, ion exchange, lipophilicity, etc.).
  • active reagents include one or more of the following: diethylaminoethyl (DEAE), diethylmono (2-hydroxypropyl) aminoethyl (QAE), carboxymethyl (CM), sulfopropyl (SP), mercaptoethylpyridyl (MEP), one NR 3+ , one RCOOE siloxane group, thiol group, fluorenyl group, antigen, Antibodies, ligands, ligand index enhancement phylogenetic techniques, adaptation molecules, ligands, peptides, polysaccharides, coenzymes, cofactors, antibiotics, steroids, viruses, cells, biotin, avidin, etc.
  • Active reagents include ligands (equivalent to Ligand in English
  • polypeptide in the present invention is equivalent to "polypeptide" in English, and includes natural or synthetic proteins, protein fragments, synthetic peptides, etc., common targets in immunoassays and commonly used ligands in tests, such as antigens, antibodies, And so on are all peptides.
  • nanoparticles in the present invention refers to solid particles having a dimension of less than 100 nm, preferably less than 50 nm in at least one dimension in a three-dimensional space.
  • solid phase carrier in the present invention, referred to as carrier for short, refers to a carrier having a solid phase morphology and a size much larger than the above-mentioned nanoparticles, for example, an analysis chip substrate, an enzyme plate substrate, an enzyme-labeled bead carrier, and chromatography. Carrier, electrophoresis gel, chromatography gel, etc.
  • film substrate in the present invention refers to a solid-phase carrier having a fixed function and a macroscopic plane on one side, such as an analysis chip substrate, an enzyme-labeled plate substrate, an electrophoresis film, and a planar chromatography carrier.
  • ligand / nanoparticle / tablet complex in the present invention refers to a composite composition containing a ligand, a nanoparticle, and a tablet, and the binding between the ligand, the nanoparticle, and the tablet may have different forms.
  • active nanoparticle in the present invention refers to a complex formed by the active agent and the nanoparticle by covalent or / and non-covalent bonding.
  • reactor in the present invention refers to a place in which an active carrier is immobilized to specifically react with a target molecule and other related structures communicating with it, such as a reaction cell and related isolation structures in and out of an open multi-reactor biochip Liquid structure, wells in 96-well microtiter plates, reagent strips for rapid detection kits, etc.
  • 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 the substrate is fixed.
  • Monolithic substrates usually do not have an 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.
  • 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 base is used to fix the ligand and For the substrate of other additives (if any), the surface chemical and optical properties are important factors affecting chip performance and cost.
  • film base pool in the present invention refers to a structure formed by a film base and its isolation structure.
  • analysis chip in the present invention is referred to as "chip” for short, and includes, but is not limited to, Bioc ip, Microarray> Bioarray in English, which is a detection device in designated and / or quantitative analysis.
  • the result of the specific reaction of the target molecule in the 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 MicroChannel Biochip in English) and a microarray chip (equivalent to Bioc ip, 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 a labeling system.
  • the distribution density of the ligand on the substrate in the reactor is greater than 10 points / cm 2 , and the preferred solution is greater than 20 points / cm 2 .
  • the area of the base point is not more than 1 mm 2 .
  • chromatography in the present invention is equivalent to "Chromatography” 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 Kit) and column chromatography.
  • molecularly labeled substance in the present invention refers to a substance used to form or participate in the formation of a detection signal and has a molecular form at the time of labeling.
  • nanostructure in the present invention refers to a structure having a nanometer size, such as a random dendrimer, a deep mound, a network, a pore, a regular sphere, and the like having a nanometer size.
  • the dendrimer may have an upward or parallel direction to the surface. Nanostructures usually reflect some or all of the nano-effects (such as surface effects, size effects, etc.).
  • convex in the present invention refers to a three-dimensional geometric shape with a protrusion on the surface, which has only one top, such as a branchless "branch” of the above dendritic structure, a "peak” of a deep mound-like structure, etc. Wait.
  • projecting distance in the present invention refers to the distance from the top of the convex body to the bottom of the convex body.
  • cross-section at a half-distance refers to the surface of the above-mentioned convex body which is perpendicular to this distance at half of the projection distance.
  • single active carrier in the present invention means an active carrier to which only one of the above active agents is immobilized.
  • multi-active carrier in the present invention means an active carrier to which one or more of the above-mentioned active agents are immobilized.
  • the term “molar concentration of nanoparticles” in the present invention refers to the molar concentration of nanoparticles as molecules, and reflects the number of nanoparticles in a unit volume of liquid.
  • the molecular weight of the nanoparticles in the present invention is A carbon atom has a mass of 1/12 as a standard. The value obtained by comparing a nanoparticle with it. This carbon atom refers to a carbon atom with 6 protons and 6 neutrons in the nucleus. The specific calculation is as follows: p V 2.65 X 4/3 X ⁇ x (20xl0 _7 ) 3 xl0 " 3 kg
  • P is the density
  • Si0 2 is 2.65 / cm 3
  • V is the volume of the nanoparticle
  • the weight of 1/12 of the mass of the carbon atom is 1.66 x l0_ 27 kg.
  • the meaning of nM used is nanomolar.
  • the nanoparticle derivatives prepared in this embodiment are derivatives coated with functional polymers.
  • the nanoparticles used are included in Table 1.
  • the functional polymers used are listed in Table 3 below, including polyionic organic compounds. (Polylysine), ionic 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 / 5000-1 / 10000 (w / v), and then equalizing the volume with a concentration of 1/5000 (w / v)
  • the organic solution was mixed and reacted at 37 ° C for 1 hour under ultrasonic vibration.
  • the reaction product was added dropwise to a rotary tube with gel, centrifugation, standby liquid collection tube at 4000 rpm / mi n conditions (when all conditions are optimized, the separation step may be omitted centrifugation).
  • nanoparticle-coated derivatives prepared by the present invention are listed in Table 4 below.
  • the active nanoparticles prepared in this example include nanoparticles selected from Table 1 and the nanoparticle derivatives prepared in Example 1.
  • the ligand used is selected from the ligands listed in Table 3.
  • the method for preparing active nanoparticles in this embodiment is: dispersing nano particles or nano particle polymers under ultrasonic oscillation to prepare a nano particle liquid with a concentration of 1/5000 (w / v), and then adding 2 mg / ml The ligand solution was mixed with each other 1: 1, and reacted at room temperature for 1 hour.
  • the purification method as follows: The product was added dropwise to a gel with the rotating tube, centrifuged at 4000 rpm / mi n conditions for liquid collection tube. See Table 5 for the obtained active nanoparticles. table 5
  • EBV antigen-STN-3 silicon oxide nanoparticles ST -3)
  • EBV-VCA-P18 antigen HCV antigen -STN-3 silicon oxide nanoparticles (STN-3) HCV antigen
  • EBV Antigen-LUDOX Silicon Oxide (LuDOX As-40)
  • EBV-VCA-P18 Antigen HCV Antigen-LuDOX Silicon Oxide (LuDOX As-40)
  • HCV Antigen HCV Antigen
  • EBV antigen-colloidal gold colloidal gold EBV VCA-P18 antigen
  • EBV pro-amine-based colloidal goldamine-based colloidal gold EBV VCA-P18 antigen
  • HCV antigen-DEAE particles DEAE-dextran coated nanoparticles HCV antigen
  • HCV antigen-QAE particles QAE-dextran coated nanoparticles HCV antigen
  • HIV antigen-QAE particles QAE-dextran coated nanoparticles HIV antigen
  • HCV antigen-polylysine particles Polylysine-coated nanoparticles HCV antigen
  • HIV antigen-polylysine particles Polylysine-coated nanoparticles HIV antigen
  • HIV antigen-aminohydrazine lysine particles Aminohydrazine polylysine coated nanoparticles HIV antigen Example 3: Preparation of nanostructured carrier
  • the nanoparticles used in this embodiment include the silicon oxide (LuDOX AS-40) in Table 1 and the nanoparticle derivatives prepared in Example 1.
  • the substrate used is the glass slide in Table 2.
  • the slides were first treated with 10% NaOH and HN0 3 and washed. After drying, the slides were immersed in a suspension with a nanoparticle concentration of 1/25000 (w / v) for 15 hours, then washed and dried.
  • the resulting nanoparticle-coated chip substrates are listed in Table 6.
  • the substrate used in this example is a 96-well polystyrene plate (Shenzhen Jincanhua Industrial Co., Ltd.), and the nanoparticles used are colloidal gold, silicon oxide nanoparticles (STN-3), and silicon oxide (LUDOX AS-40) (Table 1). ).
  • a method for preparing a nanoparticle-coated microwell plate is as follows: a nanoparticle liquid with a nanoparticle concentration of 1/25000 (w / v) is brought into contact with the bottom of a microwell of a polystyrene plate, reacted at room temperature for 15 hours, and then Wash repeatedly with distilled water.
  • the prepared nanoparticle-coated tablets are listed in Table 6. 3) Preparation of nanostructured microchannels
  • the substrate used in this embodiment is the slide glass in Table 2 (International Application No .: PCT / CN03 / 01091). Apply a suspension of silicon oxide nanoparticles (STN-3) at a concentration of 1/25000 (w / v) to the substrate to form a line with a width of 0.3-lO nrni and a length of 3n, and then leave it at room temperature for 15 hours. Rinse and dry.
  • the nanostructures prepared on the glass substrate are channels, and they have been verified to have capillary-like water transport performance.
  • the nanoparticles used were: DEAE-dextran-coated nanoparticles, QAE-cellulose-coated nanoparticles, and polyvinylpyrrolidone-coated nanoparticles.
  • microchannels of the present invention due to the effect of the nanostructures on them (for example, higher surface area, more micro-concavities), are the reasons for this type of "capillary phenomenon" Or part of the reason.
  • the microchannels of the present invention are very simple, professionals know that in combination with the microchannels of the present invention, it is easy to make various types of microchannel chips by using other known microchannel chip preparation techniques.
  • Example 4 Preparation of a chip containing a nanostructured active carrier (1)
  • the substrate used in this test is the nanostructured chip substrate prepared in Example 3, and the active agent used is a ligand selected from Table 3.
  • the nanostructured active carrier prepared in this embodiment is a nanostructured active carrier containing only one kind of ligand at one point, including a ligand-nanoparticle-coated tablet base and a ligand-nanostructure-ligand-nanoparticle coating Film base.
  • the ligands described in this example are used as both reagents and linkers.
  • the multi-basin substrate in this embodiment is prepared as follows: a highly hydrophobic organic silicon coating (Chengdu Chenguang Chemical Research Institute) is applied to the isolation structure on the substrate, and then dried to form a film (film thickness less than 0.05 mm), Thus, the isolation structure of the substrate pool is formed on the substrate (refer to Chinese Patent Application No. 03117397.7). Every 1 There are 8 substrate pools on each substrate, and the size of each substrate pool is 4.5 nunX4.5 mm, and the width of the isolation structure between the substrate pools is 4.5 mm.
  • the ligands used in this example are HCV antigen (Institute of Liver Diseases, Beijing People's Hospital, China) and HIV 1 + 2 antigen (Institute of Liver Diseases, Beijing People's Hospital, China).
  • HCV antigen Institute of Liver Diseases, Beijing People's Hospital, China
  • HIV 1 + 2 antigen Institute of Liver Diseases, Beijing People's Hospital, China
  • HCV antigen 10 mg / ml
  • HIV 1 + 2 antigen solution 10 mg / ml
  • each of the two antigens has three dots with a diameter of 200 ⁇ m, and the distance between the dots is 600-700 ⁇ m, forming a 2 ⁇ 3 array.
  • the density of the ligand on the substrate of the reaction cell is greater than 30 dots / cm 2 .
  • the prepared chips are listed in chips 101-104 in Table 7.
  • the base used in this embodiment is selected from the chip base in Table 2, and the ligands in Table 3 of the active reagent used.
  • the nanostructured active support prepared in this embodiment is a nanostructured active support that contains only one kind of ligand at one point: a ligand, a nanoparticle, and a ligand. ..
  • the ligands described in this example are used both as reagents and as linkers.
  • the chip prepared in this embodiment is a multi-reaction cell chip.
  • the ligands of this example are HCV antigen and HIV 1 + 2 antigen.
  • 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 substrate in this embodiment includes a surface-modified glass substrate, a surface-coated organic glass substrate (see Table 2), and a nanostructured substrate (see Table 6).
  • the method for preparing the multi-basin-cell substrate used in this embodiment is the same as the method for preparing the multi-basin-cell substrate in Example 3.
  • the active nanoparticles used in this example are the active nanoparticles prepared in Example 2.
  • the preparation method of this embodiment is as follows: the above active nanoparticles with a nanoparticle concentration of 1/25000 (W / V) are respectively spotted into the above-mentioned prepared multi-substrate base substrate base pool according to a general spotting method, Three dots of each active nanoparticle were formed to form a 3 X2 ligand-nanoparticle array, and then blocked with a bovine serum albumin solution for later use.
  • the chips obtained are chips 201 to 222 in Table 8.
  • DFM electron probe microscopy
  • Scanning electron microscopy was performed at the University of Analytical Testing Center of D.11. With a fully automatic helium adsorption specific surface analyzer, the specific surface area of the solid phase support and the nanostructured support can be detected and their surface area ratios calculated.
  • Table 8 shows the preparation method of the nanostructured active carrier according to the above 1), but the concentration of the nanoparticles is 1/200, 1/25000 1/125000 (w / v) Test results of three active carriers. Table 8
  • Example 8 Preparation of a chip containing a nanostructured active carrier (3)
  • the nanostructured active support prepared in this embodiment is a nanostructured active support containing more than one ligand at one point, and includes two types of nanostructured active support: Ligand 2—Nanoparticles—Ligand 2—Ligand 1 — The nanoparticle has one ligand and one ligand and two ligands—nanoparticles—ligand 2—ligand 1. Among them, Ligand 1 can be paired with Ligand 2 and Ligand 1 is easier to bind to the base than Ligand 2. In this embodiment, one kind of ligand (ligand 2) is used as both a reagent and a linker, and the other kind of ligand is used only as a linker (ligand 1).
  • 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 substrate in this example is the amino slide in Table 2.
  • the method for preparing the multi-basin-cell substrate used in this embodiment is the same as the method for preparing the multi-basin-cell substrate in Embodiment 2.
  • the active nanoparticles and the active nanoparticle composite used in this embodiment are the active nanoparticle and the active nanoparticle composite prepared in Example 2.
  • Ligand 2 nanoparticles—ligand 2—ligand 1—nanoparticles—ligand 1—one base is prepared as follows:
  • Ligand 2 nanoparticles—compounds with a nanoparticle concentration of 1/10000 (w / v)
  • a nanoparticle and a ligand 1 suspension as 1: 1, spot the sample onto the substrate according to the usual spotting method.
  • Each active nanoparticle was spotted at 2 spots to form a 3 X2 array, and then blocked with bovine serum albumin solution before use.
  • the obtained chip is referred to as a chip 223.
  • the HBsAb concentration was 3 mg / ml when spotted.
  • Ligand 2 Naparticles—Legand 2—Legand 1
  • Ligand 2 Naparticles—Ligand 2 and Ligand—with a nanoparticle concentration of 1/5000 (w / v)
  • Ligand 2 Naparticles—Ligand 2 and Ligand—with a nanoparticle concentration of 1/5000 (w / v)
  • w / v w / v
  • HBsAb concentration was 3 mg / mL when spotting
  • Example 9 Preparation of ligand / nanoparticle / molecular labeling substance complex
  • the ligand / nanoparticle / molecular labeling substance complex prepared in this embodiment is a nanoparticle labeling substance for a chip.
  • the nanoparticles in this embodiment include oxide nanoparticles and their derivatives (Table 9).
  • the molecular marker used is rhodamine (Molecular probes), and the ligand used is goat anti-human secondary antibody (Beijing Tiantan Biological Products Co., Ltd.). ).
  • control label in this example is a conventional label (rhodamine-labeled goat anti-human secondary antibody, Jackson ImmunoRresearch Laboratories, USA) that has not been treated with a nanoparticle-containing liquid.
  • the preparation method of the active nano-particles used in this embodiment is: dispersing the nano-particles under ultrasonic oscillation to prepare a nano-particle liquid with a concentration of 1/1000 (w / v), and then preparing a ligand solution with a concentration of 2 mg / ml They were mixed 1: 1 with each other and reacted at room temperature for 2 hours.
  • the purification method of the mixture is that the mixed product is dropped into a gel-filled rotating tube, centrifuged at 4000 r / min, and the collection tube liquid is taken.
  • a nanoparticle-molecular labeling substance complex is prepared by dispersing the nanoparticle under ultrasonic oscillation to prepare a nanoparticle liquid with a concentration of 1/1000 (w / v), and then dispersing 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, the purification method of the mixture is as follows: The mixed product is dropped into a gel-filled rotating tube at 4000 r / min. Centrifuge and take the liquid from the collection tube.
  • the preparation method of the ligand-molecular labeling substance complex in this embodiment is a well-known preparation method of rhodamine-labeled anti-antibody.
  • the preparation method of this embodiment, wherein the combination of the ligand, the molecularly labeled substance, and the nanoparticle includes one or more of the following methods: combining the active nanoparticle with the molecularly labeled substance (A), and combining the above nanoparticle with a molecule Binding of a labeling substance to a ligand (B), binding of a molecular labeling substance-labeled ligand prepared according to a known method with a nanoparticle (C), simultaneous binding of a ligand with a molecular-labeling substance and a nanoparticle (D), wherein said binding
  • the product can be a mixture or a purified product.
  • the chip used in this embodiment is prepared according to a known chip preparation method (the film base is an epoxy-based glass slide, and the ligands are HCV antigen and HIV antigen, respectively).
  • the identification method is as follows: Refer to the application method of the chip in Example 10. The identification results are listed in Table 9. Table 9
  • Grain Example 10 Preparation and application of chip kit (1).
  • the kit of this embodiment is an analysis chip kit containing the active carrier of the present invention.
  • the analysis chip (Table 10) containing the active carrier of the present invention in this example is the analysis chip prepared in Examples 7 and 8.
  • sample 1 is HCV antibody-positive serum
  • sample 2 is HIV 1 + 2 antibody-positive human serum
  • sample 3 is a negative control (both HCV antibody and HIV 1 + 2 antibody are negative serum controls Thing). All samples were pre-tested using the classic ELISA method under serum 20-fold dilution reaction conditions.
  • the label in this example is a rhodamine-labeled goat anti-human secondary antibody (American Jackson ImmunoRresearch Laboratories).
  • the photo is a chip prepared by using the epoxy glass slide in Table 1 with the same ligand fixed by a conventional spotting method.
  • the sample volume was 15 ⁇ 1.
  • the washing solution was added 25 ⁇ 1 each time.
  • the labeling amount was 15 ⁇ 1, and the reaction was washed 5 times.
  • the washing solution was added 25 ⁇ each time.
  • scanning was performed at 35/50.
  • the scanner is a confocal laser scanner (Afymetrix's GMS 418 chip scanner). It scans the excitation light wavelength of 532 nm and the emission light wavelength of 570 nni.
  • the read signal is processed by the processing software (JAGUAR II).
  • the cut-off value judges the yin (one) and yang (+) sex.
  • the results are shown in Table 10.
  • the other chips (chips 222-223) prepared in Example 8 have detection results with similar sensitivity.
  • Other chips (chips 101-108) prepared in Example 7 also showed results of improved sensitivity.
  • the kit of this embodiment is an analysis chip kit containing a ligand / nanoparticle / molecular labeling substance complex.
  • the ligand / nanoparticle / molecular labeling substance complex used in this example was prepared in Example 8 (Table 9).
  • the chip contained in the kit of this example is the pair of photos described in Example 10. Therefore, this embodiment can form a large number of different kits. 2) Application of the kit
  • Example 12 Preparation and application of chip kit (3)
  • the kit of this embodiment includes an analysis chip containing an active nanostructure carrier and a labeling system containing a ligand / nanoparticle / molecular labeling substance complex.
  • the analysis chip containing the active nanostructure carrier in this embodiment is the same as that in Embodiment 8.
  • the kit of this embodiment includes a magnetic chip.
  • the magnetic nanoparticles used in this example are the water-based magnetic fluid (NG-21A) in Table 1.
  • the substrate used is the epoxy glass slide in Table 2.
  • the ligand used is the HCV antigen and the ligand used in Example 2. HIV 1 + 2 antigen.
  • the magnetic nanoparticle chip in this embodiment is a ligand / magnetic nanoparticle / sheet-based composite.
  • the magnetic nanochip in this embodiment is a multi-reactor chip, in which 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 example are the preparation of active nanoparticles.
  • 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 substrate substrate, and And magnetic nanoparticle dots to form a 2 ⁇ 2 array. Then reacted at 37 ° F 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 coating was completed, it was blocked with bovine serum albumin solution and then used.
  • control chip in this example is based on an aldehyde group, and according to a well-known spotting method, HCV antigen (1 mg / ml) without magnetic nanoparticles and HIV 1 + 2 antigen are spotted (1 mg / ml). ) The prepared chip.
  • the magnetic nanomarker in this embodiment is a ligand / magnetic nanoparticle / molecular labeling substance complex.
  • the ligand was goat anti-human secondary antibody (Jackson ImmnoResearch Laboratories).
  • the magnetic chip kit in this embodiment may contain one, two, three, or four magnetic particle-containing components.
  • 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 the magnetic nanoparticle ( Has been blocked with calf serum albumin). See Table 11 for different kits formed by their different combinations.
  • the magnetic chip kit is used to detect the yin (one) and yang (+) 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 The dynamic magnetic field is conducive to the internal flow of the liquid sample added to the chip reactor 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 separately [the magnetic nanoparticle concentration after mixing is 1/2000 (w / v), affinity magnetic Nanoparticle concentration is 1/4000 (w / v)]; 15 ⁇ of serum samples were added to the chip reaction cell and reacted at 37 ° C for 10 minutes under a pulsating magnetic field; washing; 15 ⁇ of magnetic nano-markers were added and It was reacted at 37 ° C for 10 minutes under a pulsating magnetic field; 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 .12. Table 12
  • the kit of this embodiment is an enzyme-labeled kit containing a nanostructured active carrier.
  • the ligand used in this example is a synthetic peptide, which is prepared according to the methods disclosed in the following documents
  • EBV VCA-P18 antigen Tranchand-Bunel, D., Auriault, C., Diesis, E., Gras-Masse, H. (1998) Detection of human antibodies using "convergent” combinatorial peptide libraries or 'mixotopes "designed form a nonvariable antigen: Application to the EBV viral capsid antigen pi 8, J. Peptide Res. 52, 1998, 495-508.
  • the nanostructured microtiter plate used in the examples is obtained by immobilizing a ligand on the bottom of a microwell of the nanostructured microwell plate.
  • Example 3 For the preparation and identification of nanostructured microwell plates, see Example 3.
  • the synthetic peptides with a concentration of 0.3 g / ml were coated onto the 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 is coated with 8 wells. After coating, block with bovine serum albumin solution before use.
  • the prepared nanostructured microplate is shown in Table 6.
  • the control microplate in this example is a microplate with a 96-well plate as the base and coated with the EBV VCA-P18 antigen according to the coating method described above.
  • the nanoparticle concentration in the active nanoparticle with an EBV VCA-P18 antigen concentration of 0.1 g ml was 1: 4000, and the coating was carried out in the microwells of a 96-well plate in Table 2 according to a well-known enzyme plate coating method. Eight wells were sealed with bovine serum albumin solution after coating and used.
  • the samples used are EBV IgA positive serum and EBV IgA negative serum. All samples were pre-tested using the classic ELISA method under serum 20-fold dilution reaction conditions.
  • the sample was appropriately diluted during sample loading, the sample volume was 100 ⁇ l, and the reaction temperature was 37 ° C.
  • the washing solution was added 300 ⁇ l each time and washed 3 times.
  • the marker was enzyme-labeled goat anti-human IgA (Beijing Tiantan Biological Products Co., Ltd.), the addition amount was 100 ⁇ l, the reaction temperature was 37 degrees, and the reaction time was 30 minutes.
  • the reaction conditions for adding the substrate were the same as those of the classical ELISA method.
  • the nano-structured microtiter plate has at least higher sensitivity and a faster reaction speed than the control microtiter 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 method.
  • the detection result using the control enzyme plate was negative, while the detection result using the ligand / nanoparticle / plate-based complex enzyme plate prepared in this example was still Positive.
  • Example 15 Preparation and application of a peptide quantitative or / and qualitative detection kit (2)
  • the kit of this embodiment is a quick pick-up kit containing a nanostructured active carrier.
  • 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 method for preparing a nanoparticle-coated planar chromatography test strip used in this embodiment is: contacting a nanoparticle liquid having a concentration of 1/12500 to 1/25000 (w / v) with a substrate and reacting at room temperature for 5 minutes. Hours, reuse Wash repeatedly with distilled water.
  • the prepared nanostructured planar chromatographic bands are listed in Table 6.
  • the photographs are based on nitrocellulose membrane strips and nylon fiber membrane strips not treated with nanoparticle-containing buffer.
  • the ligand used in this example is HCV antigen (Institute of Liver Diseases, Beijing People's Hospital, China).
  • HCV antigen at a concentration of 0.5 mg / ml was spotted on the four kinds of nanostructured planar chromatography bands prepared above and two kinds of photographs (detection lines), and then the rabbit anti-goat IgG quality control was spotted by conventional methods. After the line, it was closed with bovine serum albumin solution, and then assembled with a sample addition area, a colloidal gold-labeled sheep anti-human marker area, and a water absorption area.
  • the control quick test reagent strip is a quick test reagent strip prepared based on the photo.
  • samples 1 and 2 are HCV antibody-positive serum and HCV antibody-negative serum, respectively, and all samples were tested for yin and positive with a classic ELISA method in advance.
  • Control reagent strips Nitrocellulose membrane strips None + ⁇ 2 minutes. Nanoparticle coated nitrocellulose membrane strips Silicon oxide + ⁇ 1 minute Quick test reagent strips (LUDOX AS-40)
  • Nanoparticle coated nitrocellulose membrane strip Silica nanoparticles + ⁇ 1 minute Quick Test Reagent Strip (STN-3)
  • Nanoparticle-coated nylon fiber membrane strips Silicon oxide nanoparticles + ⁇ 1 minute quick test reagent strip (STN-3) From the results in Table 14 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 16 Preparation and application of a nanostructured active carrier separation medium
  • the separation medium prepared in this embodiment is a chromatographic stationary phase.
  • the ligand used in this example is DEAE-dextran (Pharmacia), the nanoparticles used are silica nanoparticles (STN-3) in Table 1, and the carrier used is an average particle size of 60 ⁇ m for chromatography.
  • Silica particles Institute of Chemistry, Chinese Academy of Sciences).
  • a DEAE-dextran solution at a concentration of 1 / 10,000 (w / v) was mixed with silicon oxide nanoparticles (STN-3) at a concentration of 1/6250 (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 particle composites.
  • it can also apply the classic method of dextran derivation A variety of chromatographic stationary phases are derived.
  • the kinetic adsorption capacity of the above silica gel particles, DEAE-dextran / silica gel particle complex, and DEAE-dextran / nanoparticles / silica gel particle complex were tested.
  • the inner diameter of the column used to fill the above medium is 0.5 cm and the length is 2 cm
  • the buffer solution is 0.01M Tris-HCl / pH 7.40
  • the flow rate is 1 ml / min
  • the chromatography used is HP 1090.
  • the sample was human albumin.

Abstract

The invention relates to an active nanostructured carrier with high sensitivity for separation and/or analysis, its preparation method, and a nano-label and its labeling method. The invention also relates to a biochip and a polypeptide detection device, all of which contain the high-sensitive active nanostructured carrier and /or the nano-label, especially analysis chips, enzyme labeling plates and chromatography test strips, and their applications.

Description

包含纳米结构活性载体的分析或分离用装置、 制备方法及其应用  Device for analysis or separation containing nanostructure active carrier, preparation method and application thereof
技术领域 Technical field
本发明涉及用于分离或 /和分析的高灵敏度纳米结构活性载体及其制备方 法、 纳米标记物及其标记方法。 本发明还涉及含高灵敏度纳米结构活性载体和 / 或纳米标记物的生物芯片及其应用， 含高灵敏度纳米结构活性载体和 /或纳米标 记物的多肽检测装置、 特别是分析芯片、 酶标板、 平面层析条及其应用。 背景技术  The invention relates to a high-sensitivity nanostructure active carrier for separation or / and analysis, a preparation method thereof, a nanolabel and a label method thereof. The invention also relates to a biochip containing a high-sensitivity nanostructure active carrier and / or a nano-label and its application, a polypeptide detection device containing a high-sensitivity nano-structure active carrier and / or a nano-label, especially an analysis chip, and an enzyme labeling plate. , Plane chromatography strip and its application. Background technique
具有选择性反应活性的活性载体， 在很多方面、 尤其是在对样品中的目标 物进行定性和 /或定量分析或 /和分离方面获得了广泛的应用。分离方面应用的活 性载体， 例如有亲和层析凝胶。 检测方面应用的活性载体， 例如有抗原或 /和抗 体分析芯片。 在生物检测分析中， 根据样品目标物不同可以分为核酸检测、 多 肽检测等。 下面以生物芯片检测为例来简单地说明现有检测方法及装置尚待解 决的问题。  Active carriers with selective reactivity have been widely used in many aspects, especially in the qualitative and / or quantitative analysis or / and separation of target substances in samples. Examples of active carriers for separation are affinity chromatography gels. Examples of active carriers for detection include antigen or / and antibody analysis chips. In biological detection analysis, it can be divided into nucleic acid detection and peptide detection according to the different target objects of the sample. In the following, biochip detection is used as an example to briefly explain the problems that need to be solved with existing detection methods and devices.
生物芯片由于其高通量和微型化特点， 有着广泛的应用范围， 包括基因表 达检测、 基因筛选、 药物筛选、 疾病诊断治疗、 环境监测和治理、 司法鉴定等 领域。 生物芯片检测的主要质量指标之一是灵敏度。 而活性载体是决定灵敏度 的关键之一。 目前的芯片活性载体， 主要是将活性试剂 （例如配基）直接固定 在固相载体表面上形成的。 所用固相载体主要是玻璃、 金属、 塑料等材料及其 衍生物制作的平面片基。 其中玻片衍生物， 例如胺基玻片、 醛基玻片、 环氧基 玻片和聚氨基酸包被玻片等， 是目前使用的主要片基。 目前的第二种芯片活性 载体是将配基固定在膜等具有较高比表面积的片基上 （例如膜一玻璃片基） 。  Because of its high throughput and miniaturization, biochips have a wide range of applications, including gene expression testing, gene screening, drug screening, disease diagnosis and treatment, environmental monitoring and governance, and judicial identification. One of the main quality indicators of biochip detection is sensitivity. The active carrier is one of the keys to determining sensitivity. Current chip active carriers are mainly formed by immobilizing active reagents (such as ligands) directly on the surface of solid-phase carriers. The solid phase carriers used are mainly flat substrates made of glass, metal, plastic and other materials and their derivatives. Among them, slide derivatives, such as amine-based slides, aldehyde-based slides, epoxy-based slides, and polyamino acid-coated slides, etc., are the main slide substrates currently used. At present, the second kind of chip active carrier is to fix the ligand on a film substrate (such as a film-glass substrate) with a high specific surface area, such as a film.
活性试剂在片基上的固定， 是影响生物芯片检测灵敏度的关健之一。 固定 化方法主要有： 共价键固定法、 物理化学吸附法、 包埋法和交联法。 物理化学 吸附法包括特异性吸附法和非特异性吸附法。非特异性吸附的例子有离子吸附。 特异性吸附的例子有亲和吸附。 抗原一抗体反应、 核苷酸配对反应、 亲油反应 等， 都属于亲和吸附。 目前的第一种芯片活性载体， 由于活性载体比表面较小 等原因， 其反应动力学条件尚待优化， 表现为灵敏度尚待提高、 或 /和反应时间 尚待缩短。 第二种芯片活性载体， 理论上讲较高的比表面可以改善灵敏度， 然 而实际情况却并非总是如此。 例如， 至少部分以膜为片基的反应器由于其背景 噪音高而降低了灵敏度。 而且， 以膜、 微颗粒等为片基的反应器还有其它实际 问题 （例如膜的清洗、 微颗粒的粘结等） ， 结果是目前其应用甚至不如第一种 方法制备的反应器。 决定灵敏度和检测时间的另一关键因素是标记系统。 目前 芯片检测中使用的标记系统主要是分子分散标记系统， 其灵敏度尚待提高。 由 于灵敏度尚待提高， 对芯片片基的选择自由度亦有待提高， 可用的芯片片基的 种类不多。 目前， 其它含片基的检测装置， 例如酶标板等等， 也都有与生物芯 片同样的问题。 The fixation of the active reagent on the substrate is one of the key factors that affect the detection sensitivity of the biochip. The immobilization methods mainly include: covalent bond immobilization method, physicochemical adsorption method, embedding method and cross-linking method. Physicochemical adsorption methods include specific adsorption methods and non-specific adsorption methods. Examples of non-specific adsorption are ion adsorption. An example of specific adsorption is affinity adsorption. Antigen-antibody reaction, nucleotide pairing reaction, lipophilic reaction, etc. are all affinity adsorption. At present, the first kind of chip active carrier, due to the smaller surface area of the active carrier, etc., its reaction kinetics conditions need to be optimized, and the performance is still to be improved, or the reaction time To be shortened. The second kind of chip active carrier, in theory, 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 chip detection 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.
仅管目前有不少努力用微粒或纳米微粒来改变活性载体， 但奇怪的是， 并 未见报道改变的活性载体具有明显高得多的灵敏度。 例如， 在国际专利申请公 幵 WO 0183825中， 将胶体、特别是尺寸 100 nm— 500 nm的有机粒子用作配基载 体， 其目的以及所要达到的效果仅为减小芯片的加工劳动量例如加工步骤以加 工出探针点均匀的芯片。 此外， 在美国专利申请号 20030207296中， 纳米微粒仅 被用作用于核酸检测的配基载体。 此外， 在分析芯片检测中， 核酸检测多用基 因芯片， 多肽检测多用多肽芯片。 尽管使用基因芯片的核酸检测已经取得 Γ公 认的进步 （例如人基因组测序工程中的核酸检测） ， 芯片多肽检测则尚在发展 中。  Notwithstanding the current efforts to modify active carriers with microparticles or nanoparticles, weirdly, no significantly higher sensitivity has been reported for modified active carriers. For example, in International Patent Application Publication WO 0183825, colloids, especially organic particles with 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 nucleic acid detection multipurpose gene chip is used, and a polypeptide detection multipurpose polypeptide chip is used. Although the use of gene chips for nucleic acid detection has made recognized progress (such as nucleic acid detection in the human genome sequencing project), chip peptide detection is still in development.
此外， 在分离装置例如层析装置中也大量使用活性载体， 例如亲和层析固 定相。 尽管用作层析固定相基质的微粒比之基片有更大的比表面积， 然而在层 析时间和层析收率等方面， 仍有待改进。  In addition, active carriers 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 chromatographic stationary phase have a larger specific surface area than the substrate, there is still room for improvement in terms of analysis time and chromatographic yield.
此外， 在定性和 /或定量检测分析中， 含有配基的标记系统是最广泛使用的 标记系统。 此外， 尽管目前生物芯片的标记系统中巳引入某些纳米微粒来提高 检测灵敏度 （如 WO 00/72018 A1 , 第 20030211488号美国专利申请， 第 02133538.9、 02137418.X 01133527.0号中国专利申请， 它们使用胶体金作为标 记物， 加上银增强系统） ， 但它们或是标记物质本身 （例如第 02141718.0、 01109551.2号中国专利申请， 其使用荧光稀土络合物纳米颗粒标记物） 、 或是 标记物质增强剂（例如第 20030232388、20030166297、20020142480、20030211488 号美国专利申请， 使用具有拉曼增强效应的金属） 。 发明内容 In addition, in qualitative and / or quantitative detection analysis, a labeling system containing a ligand is the most widely used labeling system. In addition, despite the introduction of certain nanoparticles in the current biochip labeling systems to improve detection sensitivity (such as WO 00/72018 A1, US Patent Application No. 20030211488, Chinese Patent Application Nos. 02333538.9, 02137418.X 01133527.0, they use colloids Gold is used as a marker, plus a silver enhancement system), but they are either the marking substance itself (for example, Chinese patent applications No. 02141718.0, 01109551.2, which uses fluorescent rare earth complex nanoparticle markers), or a marking substance enhancer ( For example, U.S. Patent Nos. 20030232388, 20030166297, 20020142480, 20030211488, using metals with Raman enhancement effect). Summary of the invention
本发明的主要目的是提高分析、 分离中活性载体（或固定化活性试剂） 的 反应效率，从而提高检测灵敏度或 /和降低检测时间或 /和提供更多种类的具有足 够高灵敏度的片基。 本发明的目标通过对载体、 活性载体和标记系统的开发来 实现。  The main purpose of the present invention is to improve the reaction efficiency of the active carrier (or the immobilized active reagent) in the analysis and separation, thereby improving the detection sensitivity or / and reducing the detection time or / and providing more kinds of substrates with sufficient high sensitivity. The object of the present invention is achieved by the development of vectors, active vectors and labeling systems.
由于对载体和活性载体的研究， 本发明的另一个目的是提高分离介质的分 离效率。  As a result of studies on carriers and active carriers, another object of the present invention is to improve the separation efficiency of the separation medium.
根据本发明的一个方面， 其提供一种用于多肽分析的装置， 该装置包括含 有活性纳米微粒的活性载体， 所述活性载体包含固相载体及固定在所述固相载 体局部或全部表面上的活性纳米微粒， 所述活性纳米微粒包含纳米微粒及固定 于所述纳米微粒上的活性试剂， 所述活性试剂选自于以下组中： 离子交换剂、 药物、 多肽、 多糖、 维生素、 抗生素、 功能有机物、 抗原、 以及病毒、 细胞或 它们的组成。 所述活性载体可以通过多种介质获得， 但本发明活性载体的制备 为本发明的活性载体优选的制备方法。 特别要强调的是， 本发明的活性载体在 检测灵敏度方面比不引入纳米微粒、 在常规片基上仅固定有相同配基的活性载 体高 25 %、 甚至高 50%、 更甚至高 50 %。 而用某些高浓度纳米微粒 （例如 w/v 浓度大于 1 % ) 的纳米微粒 /配基混合液点样在芯片片基上形成的活性载体， 其 灵敏度的提高非常有限。  According to an aspect of the present invention, it provides a device for polypeptide analysis, the device includes an active carrier containing active nanoparticles, the active carrier comprises a solid phase carrier and is fixed on a part or the entire surface of the solid phase carrier. Active nanoparticles comprising nanoparticles and an active agent fixed on the nanoparticles, the active agent is selected from the group consisting of an ion exchanger, a drug, a polypeptide, a polysaccharide, a vitamin, an antibiotic, Functional organisms, antigens, and viruses, cells or their composition. The active carrier can be obtained through a variety of media, but the preparation of the active carrier of the present invention is a preferred method of preparation of the active carrier of the present invention. It should be particularly emphasized that the active carrier of the present invention has a detection sensitivity 25%, even 50%, or even 50% higher than that of an active carrier that does not introduce nanoparticles and has only the same ligand immobilized on a conventional substrate. However, with some high concentration nanoparticles (such as w / v concentration greater than 1%), a nanoparticle / ligand mixed solution spotted on an active carrier formed on a chip substrate has a very limited increase in sensitivity.
所述活性载体中一种或多种纳米微粒与固相载体之间有一重或多重配基、 或 /和一种或多种配基与固相载体之间有一重或多重纳米微粒、 或 /和所述至少 一重纳米微粒与另一重纳米微粒之间有一重或多重配基。  There is a heavy or multiple ligand between one or more nanoparticles in the active support and the solid support, or / and a heavy or multiple nano particles between the one or more ligands and the solid support, or / There is a heavy or multiple ligand between the at least one heavy nanoparticle and another heavy nanoparticle.
在根据本发明的装置中， 所述活性载体包括单活性载体和多活性载体， 其 中所述单活性载体在同一个固相载体上只固定有包含一种活性试剂的活性纳米 微粒， 而所述多活性载体在同一个固相载体上固定有包含二种以上活性试剂的 活性纳米微粒。 在第 20030207296号美国专利申请中， 只有单活性载体。  In the device according to the present invention, the active carrier includes a single active carrier and a multi-active carrier, wherein the single active carrier has only active nanoparticles containing one active agent immobilized on the same solid-phase carrier, and the Multi-active carriers have active nanoparticles containing two or more active agents fixed on the same solid-phase carrier. In US patent application 20030207296, there is only a single active carrier.
在根据本发明的装置中， 包含多活性载体的装置例如是物芯片和平面层析 试剂条， 而包含单活性载体的装置例如是酶标板。  In the device according to the present invention, a device containing a multi-active carrier is, for example, a biochip and a planar chromatography reagent strip, and a device containing a single-active carrier is, for example, an enzyme plate.
在根据本发明的装置中， 所述活性纳米微粒与所述固相载体之间不通过核 苷酸配对结合， 但通过下述一种或多种方式结合： 共价键结合、 非特异性物理 化学吸附、 抗原一抗体吸附、 及亲和吸附， 而且所述结合是通过固相载体或 /和 纳米粒子表面的连接剂实现的， 所述连接剂包括表面基团或 /和包被有机物或 / 和所述活性试剂。在 20030207296号美国专利申请中， 纳米粒子与片基之间的结 合必须通过核酸的配对作用来进行，. 不适合处核酸以外的物质例如多肽的检测 和分离。 In the device according to the present invention, the active nano-particles and the solid-phase carrier are not bound through nucleotide pairing, but are bound by one or more of the following methods: covalent bonding, non-specific physical chemistry Adsorption, antigen-antibody adsorption, and affinity adsorption, and the binding is achieved through a solid phase support or / and a linker on the surface of the nanoparticle, the linker including surface groups or / and coating organic matter or / And the active agent. In US Patent Application No. 20030207296, the binding between the nanoparticles and the substrate must be performed by the pairing of nucleic acids. It is not suitable for the detection and separation of substances other than nucleic acids such as polypeptides.
在根据本发明的装置中， 所述纳米微粒的平均粒径为 1一 100 nm、 优选 1一 50 nm的微粒。  In the device according to the present invention, the nanoparticles have an average particle diameter of 1 to 100 nm, preferably 1 to 50 nm.
在根据本发明的装置中， 所述纳米微粒包括无机纳米微粒或 /和有机纳米微 粒及它们的衍生物， 所述无机纳米微粒包括非磁性无机纳米微粒和磁性无机纳 米微粒， 所述非磁性无机纳米微粒包括非磁性金属纳米微粒和非磁性非金属纳 米微粒， 所述衍生物包括结合有表面基团或 /和包被有机物的衍生物。  In the device according to the present invention, the nanoparticles include inorganic nanoparticles or / and organic nanoparticles and their derivatives, the inorganic nanoparticles include non-magnetic inorganic nanoparticles and magnetic inorganic nanoparticles, and the non-magnetic inorganic The nanoparticles include non-magnetic metal nanoparticles and non-magnetic non-metal nanoparticles, and the derivative includes a derivative having a surface group bound thereto and / or a coated organic substance.
在根据本发明的装置中， 所述无机非磁性非金属纳米微粒包括氧化硅、 氧 化钛、 氧化铝纳米微粒， 所述非磁性金属纳米微粒包括金、 钒、 铅、 银、 铁及 其氧化物纳米微粒， 所述有机纳米微粒包括塑料、 多糖、 乳胶、树脂纳米微粒。 虽然纳米微粒金等金属粒子已被用于快检试剂条， 但它是作为微粒分子标记物 质， 而不是本发明方法的固相载体使用的。  In the device according to the present invention, the inorganic non-magnetic non-metal nanoparticles include silicon oxide, titanium oxide, and alumina nanoparticles, and the non-magnetic metal nanoparticles include gold, vanadium, lead, silver, iron, and oxides thereof. Nano particles, the organic nano particles include plastic, polysaccharide, latex, resin nano particles. Although metal particles such as nanoparticle gold have been used for the rapid detection reagent strip, it is used as a microparticle molecular marker substance, not as a solid-phase carrier in the method of the present invention.
在根据本发明的装置中， 所述表面基团包括下述一种或多种基团： 氨基、 醛基、环氧基、 氨基肼、 二乙氨基乙基、 二乙基一 （2—羟丙基）氨乙基、 羧甲 基、 磺酸丙基、 巯乙基吡啶基、硅氧烷基、 硫醇基、 垸基;所述包被有机物包括 下述一种或多种有机物： 包括聚乙烯吡咯烷酮、 吐温类表面活性剂在内的表面 活性剂， 包括聚氨基酸在内的聚电解质， 包括聚硅氧烷在内的亲油有机物， 包 括葡聚糖衍生物、 琼脂糖衍生物、 纤维素衍生物、 聚丙烯酰胺在内的离子交换 聚合物， 和包括肝素钠、 生物素、 活性素、 抗原、 抗体在内的亲和物质。 所述 表面基团还可以是长臂衍生基团 R— (CH2) x—， 其中 R是衍生基团， X等于或 大于 2、优选大于 4、更优选大于 6。在本发明中，特别是由于包被衍生物的制备， 使得纳米微粒表面可以引入的活性有机基团的范围很大。 例如在本发明实施例 中， 纳米微粒衍生物如超疏水氧化硅 （CS7)_上有烷基， 包被衍生物表面有包 被有机物的基团，例如聚氨基酸具有氨基、氨基肼-聚氨基酸具有氨基和氨基肼 基、 DEAE-Dextran具有二乙氨乙基、 等等。 另一方面， 表面活性剂如聚乙烯吡 咯垸酮、 聚电解质如聚氨基酸、离子交换聚合物如 DEAE-Dextran、 亲和物质如 蛋白质 A等均用在本发明方法中制备配基 /纳米微粒 /片基复合物和配基 /纳米微 粒 /分子标记物质复合物。 实际上， 微粒衍生物的重要方面是包被衍生物。本发 明方法中的包被衍生物可以是一重或多重包被衍生物。例如， 包被有分散剂或 / 和分散稳定剂的氧化硅粒子上 （一重包被） ， 还可以包被 PVP (二重包被） ， 继续还可 、―■以包—被一■蛋白质 A ― (三―■―重 »_包— 被） ..等.— »等一。 因此， 可以选择的包被有机物的 范围是非常的大。 In the device according to the present invention, the surface group includes one or more of the following groups: amino, aldehyde, epoxy, aminohydrazine, diethylaminoethyl, diethylmono (2-hydroxy Propyl) aminoethyl, carboxymethyl, sulfonic acid propyl, mercaptoethylpyridyl, siloxane, thiol, fluorenyl; the coated organics include one or more of the following: Surfactants including polyvinylpyrrolidone and Tween-based surfactants, polyelectrolytes including polyamino acids, lipophilic organics including polysiloxanes, including dextran derivatives, agarose derivatives, Ion exchange polymers including cellulose derivatives, polyacrylamide, and affinity substances including heparin sodium, biotin, actives, antigens, and antibodies. The surface group may also be a long-arm derived group R— (CH 2 ) x— , where R is a derived group, and X is equal to or greater than 2, preferably greater than 4, and more preferably greater than 6. In the present invention, in particular, 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 an alkyl group, and the surface of the coated derivative has an organic-coated group, for example, a polyamino acid has an amino group, and an aminohydrazine-polyamino acid Has amino and aminohydrazine, DEAE-Dextran has diethylaminoethyl, and the like. 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 / Tablet 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 invention may be a single or multiple coated derivative. For example, coated with a dispersant or / And dispersion stabilizer silicon oxide particles (one-layer coating), can also be coated with PVP (two-layer coating), continue to be, ― ■ with coating—coated with a ■ protein A ― (three-■-heavy »_ Package — be) .. etc. — »wait one. Therefore, the range of organic coatings that can be selected is very large.
在根据本发明的装置中， 所述活性试剂选自于以下组中： 离子交换剂、 药 物、 多肽、 多糖、 维生素、 抗生素、 功能有机物、 抗原、 以及病毒、 细胞或它 们的组成。 这些物质均是公知的可与目标多肽作用的物质。  In the device according to the present invention, the active agent is selected from the group consisting of an ion exchanger, a drug, a polypeptide, a polysaccharide, a vitamin, an antibiotic, a functional organic substance, an antigen, and a virus, a cell, or a composition thereof. These substances are all known substances which can interact with the target polypeptide.
在根据本发明的装置中， 所述固相载体包括常规载体和纳米结构载体， 其 中所述常规载体包括由以下材料或其衍生物制成的固相物质： 玻璃、 硅片、 硅 胶、 陶瓷、 金属氧化物、 金属、 聚合物材料及它们的复合物， 所述纳米结构载 体包括在所述常规载体表面形成纳米尺寸的结构的载体。  In the device according to the present invention, the solid-phase carrier includes a conventional carrier and a nanostructured carrier, wherein the conventional carrier includes a solid-phase substance made of the following materials or derivatives thereof: glass, silicon wafer, silica gel, ceramic, Metal oxides, metals, polymer materials and their composites, and the nanostructured carrier includes a carrier that forms a nano-sized structure on the surface of the conventional carrier.
根据本发明的另一个方面，其提供一种用于分离或 /和分析的纳米结构活性 载体， 该载体包括固相载体及所述固相载体表面上的活性纳米结构， 所述活性 纳米结构^"纳米结构及所述纳米结构上的活性试剂。 在所述纳米结构活性载体 上根据其表面有无活性试剂分为活性区及非活性区，非活性区是任选存在的。 在所述活性区中,根据其表面是否主要分布着活性纳米结构分为纳米结构活性 区和非纳米结构活性区， 非纳米结构活性区任选存在的 (当所述活性区中不存 在基本上无活性纳米结构的分布区域时,其不存在)， 而且所述活性区至少具有 下述一种或多种特征： （a)相同投影面积的所述活性区与所述固相载体的表面 积之比大于 1.5; (b)所述活性纳米结构包括凸出高度大于 3 nm、 且凸出半高 处至少一维尺寸在 1一 500 nm之间的纳米凸体，且在所述纳米结构活性区中所述 纳米凸体在所述固相载体的垂直投影面上的分布密度大于 1个 / μ ηι2; (c) 所 述纳米结构活性区与所述非纳米结构活性区在固相载体上的垂直投影面积之比 大于 1。在本发明中，所述活性区是指所述纳米结构活性载体表面上分布有所述 活性试剂的区域， 所述非活性区是指其表面上除所述活性区以外的区域； 所述 纳米结构活性区是指其表面上主要分布有所述活性纳米结构的区域， 所述非纳 米结构活性区是指除所述活性区内纳米结构活性区以外的区域。 特别要强调的 是， 固相载体上构成的活性纳米结构是本发明纳米结构活性载体的关健,本发明 中的纳米结构活性载体不同于一般意义上含有纳米微粒的活性载体。 尽管某些 文件（例如第 20030207296号美国专利申请）中的活性载体是包括固相载体和固 相载体上的纳米微粒的活性载体， 而我们在大量的试验中惊奇地发现： 不同表 面形貌特征的活性载体， 其活性 （例如检测灵敏度） 可以是非常不同的。 本发 明的纳米结构活性载体具有确定的表面形貌特征， 因而具有高活性 （例如检测 灵敏度） 。 而不具有这些形貌特征的含纳米微粒活性载体表面上可能没有足够 多的活性纳米结构来令其获得高活性。 在本发明的一个实施例中， 我们将讨论 研究它们的形貌差别。 实际上， 载体表面附着的纳米微粒不一定形成具有纳米 尺寸的结构区。 在电子探针显微镜（DFM)和电子扫描显微镜帮助下， 可以观 察到表面一些凸起但凸出尺寸远大于纳米尺寸的凸体， 很可能是纳米微粒的聚 集体。而且，大尺寸的纳米微粒聚集体可能失去了、至少部分失去了部分纳米结 构特性。例如，在实施例中活性氧化硅浓度大于 1 %时形成的活性载体，其中这 类凸体 （非纳米结构活性区）在活性区中的比例就比较大， 因而以其为载体固 定活性试剂制作的芯片的检测灵敏度就较低。 总之， 在没有严格的结构设计的 条件下形成的含活性纳米微粒的活性载体是与本发明的纳米结构活性载体不相 同的。 只有拥有上述纳米结构参数的活性载体， 才是本发明的纳米结构活性载 体。 According to another aspect of the present invention, there is provided a nanostructure active support for separation or / and analysis, the carrier comprising a solid phase support and an active nanostructure on a surface of the solid phase support, the active nanostructure ^ "Nanostructures and the active agents on said nanostructures. On the nanostructured active support, active areas and inactive areas are divided according to the presence or absence of active agents on the surface. Inactive areas are optional. In the region, active nanostructures are mainly divided into nanostructure active regions and non-nanostructure active regions according to whether the surface is mainly distributed with active nanostructures. Non-nanostructure active regions are optionally present (when substantially no active nanostructures exist in the active region Distribution region, it does not exist), and the active region has at least one or more of the following characteristics: (a) the ratio of the surface area of the active region to the surface area of the solid phase support of greater than 1.5; (b) said active nanostructures comprise projecting height greater than 3 nm, and the half-height projecting at least one dimension between 1 nanometer convex body of a 500 nm, and the active region in the nanostructure Said distribution density of nano-convex body in a projection plane perpendicular to the solid support is greater than 1 / μ ηι 2; perpendicular (c) the nanostructured active region and the non-active nano-structured region on a solid phase support The ratio of the projected areas is greater than 1. In the present invention, the active region refers to a region where the active agent is distributed on the surface of the nanostructured active support, and the inactive region refers to the surface except the active region. Other regions; the nanostructured active region refers to a region where the active nanostructure is mainly distributed on the surface, and the non-nanostructured active region refers to a region other than the nanostructured active region in the active region. It should be emphasized that the active nanostructure formed on the solid support is the key to the nanostructure active support of the present invention. The nanostructure active support in the present invention is different from the active support containing nanoparticles in general. Although some documents ( For example, the active carrier in US Patent Application No. 20030207296) is an active carrier including a solid carrier and nanoparticles on the solid carrier. Surprisingly found that: different active carrier surface topography, its activity (e.g., sensitivity) can be very different from the present invention. The lucid nanostructured active support has a defined surface morphology and is therefore highly active (eg, detection sensitivity). There may not be enough active nanostructures on the surface of a nanoparticle-containing active support that does not have these morphological features to achieve high activity. In one embodiment of the present invention, we will discuss studying their topographical differences. In fact, the nanoparticles attached to the surface of the carrier do not necessarily form structural regions with nanometer size. With the help of the electron probe microscope (DFM) and the electron scanning microscope, it is possible to observe that some protrusions on the surface, but the protrusions are much larger than the nanometer, are likely to be aggregates of nanoparticles. Moreover, large-sized nanoparticle aggregates may be lost, at least partially, of nanostructure characteristics. For example, in the embodiment, the active carrier formed when the active silica concentration is greater than 1%, in which the proportion of such convex bodies (non-nanostructured active regions) in the active region is relatively large, so it is made by using the carrier as the carrier to fix the active reagent. The detection sensitivity of the chip is lower. In short, the active nanoparticle-containing active support formed without strict structural design is different from the nanostructure active support of the present invention. Only the active carrier having the above nanostructure parameters is the nanostructure active carrier of the present invention.
在根据本发明的纳米结构活性载体中， 所述活性区与所述固相载体的表面 积之比大于 3， 优选大于 6。  In the nanostructured active support according to the present invention, the ratio of the surface area of the active region to the solid-phase support is greater than 3, preferably greater than 6.
在根据本发明的纳米结构活性载体中， 所述凸体分布密度大于 5个 /μ m2， 优选大于 10个 / m2。 In the nanostructured active support according to the present invention, the distribution density of the convex body is more than 5 pieces / μm 2 , preferably more than 10 pieces / m 2 .
在根据本发明的纳米结构活性载体中， 所述纳米结构活性区与所述非纳米 结构活性区在固相载体上的投影面积之比大于 2， 优选大于 4。  In the nanostructured active support according to the present invention, a ratio of a projected area of the nanostructured active area to the non-nanostructured active area on a solid support is greater than 2, preferably greater than 4.
在根据本发明的纳米结构活性载体中，所述分离或 /和分析的目标物包括多 肽、 核酸、 及与它们相互作用的药物。 所述纳米结构活性载体包括： 生物芯片 或其活性载体、 酶标板、 平面层析试剂条、 层析活性凝胶。  In the nanostructure active carrier according to the present invention, the target of the separation or / and analysis includes a peptide, a nucleic acid, and a drug interacting with them. The nanostructured active carrier includes: a biochip or an active carrier thereof, an enzyme-labeled plate, a planar chromatography reagent strip, and a chromatography active gel.
在根据本发明的纳米结构活性载体中， 所述活性试剂选自于以下组中： 离 子交换剂、 药物、 多肽、 多糖、 维生素、 抗生素、 功能有机物、 抗原、 单链或 多链 DNA、 RNA、 核苷酸、 以及病毒、 细胞或它们的组成。  In the nanostructure active carrier according to the present invention, the active agent is selected from the group consisting of an ion exchanger, a drug, a polypeptide, a polysaccharide, a vitamin, an antibiotic, a functional organic substance, an antigen, a single- or multi-stranded DNA, RNA, Nucleotides, as well as viruses, cells or their composition.
在根据本发明的纳米结构活性载体中， 所述活性纳米结构中含有活性纳米 微粒， 所述活性纳米微粒包括纳米微粒和固定于其上的一种或多种所述活性试 剂或任选的连接剂， 其中所述纳米微粒为在三维空间中至少有一维为大于 1 nm 且小于 100 nm、 优选大于 1 nm且小于 50 nm的微粒。  In the nanostructure active carrier according to the present invention, the active nanostructure contains an active nanoparticle, and the active nanoparticle includes a nanoparticle and one or more of the active agents or optional links fixed thereto. An agent, wherein the nanoparticle is a particle 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 50 nm.
在根据本发明的纳米结构活性载体中， 所述活性纳米微粒与所述固相载体 之间通过下述一种或多种方式结合： 共价键结合、 非特异性物理化学吸附、 抗 原 -抗体吸附、 亲和吸附、及核苷酸配对结合。优选的是， 所述结合是通过固相 载体或 /和纳米粒子表面的连接剂实现的， 所述连接剂包括表面基团或 /和包被 有机物。 In the nanostructured active support according to the present invention, the active nanoparticle and the solid-phase support are bound by one or more of the following methods: covalent bonding, non-specific physicochemical adsorption, anti- Proto-antibody adsorption, affinity adsorption, and nucleotide pairing. Preferably, the binding is achieved by a solid-phase support or / and a linker on the surface of the nanoparticle, the linker comprising surface groups or / and coating organic matter.
在根据本发明的纳米结构活性载体中， 所述纳米微粒包括无机纳米微粒或 / 和有机纳米微粒及它们的衍生物， 所述无机纳米微粒包括非磁性无机纳米微粒 和磁性无机纳米微粒， 所述非磁性无机纳米微粒包括非磁性金属纳米微粒和非 磁性非金属纳米微粒， 所述衍生物包括结合有表面基团或 /和包被有机物的衍生 物。优选的是， 所述无机非磁性非金属纳米微粒包括氧化硅微粒、氧化钛微粒、 氧化铝微粒、 氧化铁微粒在内的氧化物微粒， 所述非磁性金属纳米微粒包括金 微粒、 钒微粒、 铅微粒， 所述非金属纳米微粒包括包括塑料、 多糖、 乳胶、 树 脂微粒的有机微粒。  In the nanostructure active support according to the present invention, the nanoparticles include inorganic nanoparticles or / and organic nanoparticles and their derivatives, the inorganic nanoparticles include non-magnetic inorganic nanoparticles and magnetic inorganic nanoparticles, and Non-magnetic inorganic nanoparticles include non-magnetic metal nanoparticles and non-magnetic non-metal nanoparticles, and the derivative includes a derivative having a surface group bound thereto and / or a coated organic substance. Preferably, the inorganic non-magnetic non-metallic nanoparticles include silicon oxide particles, titanium oxide particles, alumina particles, iron oxide particles, and the non-magnetic metal nanoparticles include gold particles, vanadium particles, Lead particles, the non-metal nanoparticles include organic particles including plastic, polysaccharide, latex, and resin particles.
在根据本发明的纳米结构活性载体中， 所述表面基团包括下述一种或多种 基团： 氨基、 醛基、环氧基、氨基肼、 二乙氨基乙基、 二乙基一 （2—羟丙基） 氨乙基、 羧甲基、 磺酸丙基、巯乙基吡啶基、 硅氧烷基、 硫醇基、 烷基;所述包 被有机物包括下述一种或多种有机物： 包括聚乙烯吡咯烷酮、 吐温类表面活性 剂在内的表面活性剂， 包括聚氨基酸在内的聚电解质， 包括聚硅氧烷在内的亲 油有机物， 包括葡聚糖衍生物、 琼脂糖衍生物、 纤维素衍生物、 聚丙烯酰胺在 内的离子交换聚合物， 和包括肝素钠、 生物素、 活性素、 抗原、 抗体在内的亲 和物质。 在此所述的衍生基团和功能有机物可参考前述活性载体中的描述。  In the nanostructure active support according to the present invention, the surface group includes one or more of the following groups: amino, aldehyde, epoxy, aminohydrazine, diethylaminoethyl, diethyl- ( 2-hydroxypropyl) aminoethyl, carboxymethyl, sulfopropyl, mercaptoethylpyridyl, siloxane, thiol, alkyl; the coated organics include one or more of the following Organics: Surfactants including polyvinylpyrrolidone, Tween-based surfactants, polyelectrolytes including polyamino acids, lipophilic organics including polysiloxanes, including dextran derivatives, agarose Derivatives, cellulose derivatives, polyacrylamide, ion exchange polymers, and affinity substances including sodium heparin, biotin, actives, antigens, and antibodies. The derivatizing groups and functional organics described herein can be referred to the description in the aforementioned active support.
在根据本发明的纳米结构活性载体中， 所述固相载体包括常规载体和纳米 结构载体， 其中： 所述常规载体包由以下材料或其衍生物制成的固相物质： 玻 璃、 硅片、 硅胶、 陶瓷、 金属氧化物、 金属、 聚合物材料及它们的复合物；所述 纳米结构载体为表面分布纳米尺寸的结构的载体。  In the nanostructure active support according to the present invention, the solid phase support includes a conventional support and a nanostructure support, wherein: the conventional support includes a solid phase substance made of the following materials or derivatives thereof: glass, silicon wafer, Silica gel, ceramics, metal oxides, metals, polymer materials and their composites; the nanostructured carrier is a carrier with a nanometer-sized structure distributed on the surface.
根据本发明的又一个方面，其提供一种含有上述活性载体的分析或 /和分离 装置。 所述装置的例子为： 包含多活性载体的装置如生物芯片和平面层析试剂 条， 包含单活性载体的装置如酶标板。  According to still another aspect of the present invention, there is provided an analysis or / and separation device containing the above-mentioned active carrier. Examples of such devices are: devices containing multiple active carriers such as biochips and planar chromatography reagent strips, devices containing single active carriers such as microplates.
根据本发明的另一个方面， 其提供一种制备根据本发明的装置中所包括的 包含活性纳米微粒的活性载体以及根据本发明的活性载体的方法， 其包括以下 步骤： （a)准备所述活性试剂、 载体、 纳米微粒、 及任选的连接剂； （b)将 所述活性试剂固定在所述纳米微粒上制备活性纳米微粒， 如有必要预先用所述 连接剂对所述纳米微粒进行有利于固定所述活性试剂的活化； （c) 将步骤（b) 中制备的所述活性纳米微粒的悬浮液与所述载体接触和进行固定化反应， 如有 必要预先用所述连接剂对载体进行有利于所述固定的活化， 固定化反应时所述 活性纳米微粒悬浮液中所述纳米微粒的浓度为：纳米微粒重量 /体积浓度二百分 之一至六万分之一、 或纳米微粒摩尔浓度 0.12— 37.4 nM。 所述活性纳米微粒可 以是纳米微粒和配基以与其它物质的混合物的方式被准备，例如含有着色剂或 / 和粘结剂的纳米微粒悬浮液、 含有稳定剂的配基溶液等。 本发明方法的一个例 子是： 纳米微粒悬浮液与配基溶液混合反应后形成亲和纳米微粒、 通过点样将 混合液点至芯片基片片基上进行结合反应等。 本发明所述的载体也可以是以与 其它结构共存的方式被准备， 例如多片基池基片中的片基等。 所述亲和纳米微 粒悬浮液包括混合物（例如纳米微粒与配基混合反应后的未纯化物） 和纯化物 (例如纳米微粒与配基混合反应后结离心分离去除自由配基的纯化物） ， 而且 其纯化物中一种纳米微粒上固定一种或多种配基。 需要特别强调的是， 当纳米 微粒不够稀释或过分稀释时， 利用某些纳米微粒制备的所述复合物观察不到灵 敏度的提高； According to another aspect of the present invention, it provides a method for preparing an active carrier including active nanoparticles and an active carrier according to the present invention, which are included in a device according to the present invention, including the following steps: (a) preparing the An active reagent, a carrier, a nanoparticle, and an optional linking agent; (b) fixing the active reagent to the nanoparticle to prepare an active nanoparticle, and if necessary, performing a pre-treatment on the nanoparticle with the linker; Facilitates the activation of the active agent; (c) Step (b) The active nanoparticle suspension prepared in contact with the carrier and undergoes an immobilization reaction, if necessary, the carrier is activated in advance with the linker to facilitate the immobilization, and the active nanoparticle is immobilized during the immobilization reaction. The concentration of the nano-particles in the micro-particle suspension is: a nano-particle weight / volume concentration of two-hundredths to sixty thousandths, or a nano-particle molar concentration of 0.12 to 37.4 nM. The active nanoparticle may be a nanoparticle and a ligand prepared as a mixture with other substances, such as a nanoparticle suspension containing a colorant or / and a binder, a ligand solution containing a stabilizer, and the like. An example of the method of the present invention is: after the nanoparticle suspension is mixed and reacted with the ligand solution to form affinity nanoparticles, the mixed solution is spotted on the chip substrate to perform a binding reaction by spotting. The carrier according to the present invention may also be prepared in a manner coexisting with other structures, such as a substrate in a multi-chip base substrate. The affinity nanoparticle suspension 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 centrifugation to remove a purified substance that is free of the ligand), Furthermore, one or more kinds of ligands are immobilized on a nanoparticle in the purified product. It needs to be particularly emphasized that when the nanoparticles are not sufficiently diluted or over-diluted, no increase in sensitivity is observed for the composites prepared using certain nanoparticles;
一种或多种纳米微粒与载体之间有多重配基的纳米结构活性载体例如是如 下制备的： 分别将载体包被一重配基 1形成配基 1包被载体， 并将纳米微粒包被 一重另一种配基 2 (配基 1和 2之间可发生配对反应）形成配基 2/纳米微粒复合物， 再将配基 2/纳米微粒复合物包被或点样至配基 1包被载体上， 形成配基 2—纳米 微粒一配基 2—配基 1一载体形式的复合物。 当配基层数大于 2时以此类推。  A nanostructured active carrier having multiple ligands between one or more nanoparticles and a carrier is prepared, for example, as follows: the carrier is coated with a reguiding group 1 to form a ligand 1 coated carrier, and the nanoparticles are coated with a Another ligand 2 (pairing reaction between ligands 1 and 2 can take place) to form a ligand 2 / nanoparticle complex, and then coat or spot the ligand 2 / nanoparticle complex to ligand 1 On the carrier, a complex in the form of ligand 2-nanoparticle-ligand 2-ligand 1-carrier is formed. When the number of ligand layers is greater than 2, and so on.
同样的方法还可制备至少一重纳米微粒与另一层纳米微粒之间有多重配基 的纳米结构活性载体，例如配基 3—纳米微粒一配基 3—配基 2—纳米微粒一配基 2—配基 1一载体或配基 2—纳米微粒一配基 2—配基 1一纳米微粒一配基 1一载体 等。  The same method can also be used to prepare at least one heavy nanoparticle and another layer of nanostructured nanostructure active carrier, such as ligand 3—nanoparticles—ligand 3—ligand 2—nanoparticles—ligand 2 -Ligand 1-carrier or ligand 2-nanoparticle-ligand 2-ligand 1-nanoparticle-ligand 1-carrier, etc.
一种或多种配基与载体之间有多重纳米微粒的纳米结构活性载体可如下制 备： 先将一种或一种以上所述纳米微粒与多种所述 ψ華结合在一起形成多种亲 和纳米微粒（例如配基 2—纳米微粒—配基 2、配基 3—纳米微粒一配基 2、配基 1 一纳米微粒一配基 1等），再将这些亲和纳米微粒先后或同时结合在所述载体上， 形成诸如配基 2—纳米微粒一配基 2—配基 1一纳米微粒一配基 1一载体、 配基 3 一纳米微粒—配基 2—配基 1一纳米微粒一配基 1一载体等形式的纳米结构活性 载体。 ―  A nanostructured active 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 ψs to form a plurality of affinity groups. And nanoparticles (for example, ligand 2—nanoparticles—ligand 2, ligand 3—nanoparticles—ligand 2, ligand 1—nanoparticles—ligand 1, etc.), and then these affinity nanoparticles are successively or simultaneously Binding 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-Nanoparticles A nanostructured active support in the form of a ligand, a support, and the like. ―
在根据本发明的方法中，所述纳米微粒的重量 /体积浓度为二千分之一至六 万分之一、 优选为一万分之一至四万分之一， 或者纳米微粒的摩尔浓度为 0.12 — 3.74 nM、 优选为 0.19— 0.75 nM。 In the method according to the present invention, the weight / volume concentration of the nanoparticles is from one thousandth to six thousandths. One ten-thousandth, preferably one ten-thousandth to one forty-thousandth, or the molar concentration of the nanoparticles is 0.12-3.74 nM, preferably 0.19-0.75 nM.
根据本发明的再一个方面， 其提供另一种制备根据本发明的装置中所包括 的包含活性纳米微粒的活性载体以及根据本发明的活性载体的方法， 其包括以 下步骤： （a) 准备所述活性试剂、 载体、 纳米微粒、 及任选的连接剂； （b) 将所述纳米微粒的悬浮液与所述载体接触和进行固定化反应形成纳米结构载 体，如有必要预先用所述连接剂对所述纳米微粒或 /和载体进行有利于所述固定 的活化， 固定化反应时所述悬浮液中的所述纳米微粒的浓度为： 纳米微粒重量 / 体积浓度二百分之一至六万分之一、 或纳米微粒摩尔浓度 0.12— 37.4 nM; (c) 将所述活性试剂固定在所述纳米结构载体上。 According to yet another aspect of the present invention, it provides another method for preparing an active carrier including an active nanoparticle included in a device according to the present invention and an active carrier according to the present invention, which includes the following steps: (a) preparing an The active reagent, the carrier, the nanoparticle, and an optional linker; (b) contacting the suspension of the nanoparticle with the carrier and performing an immobilization reaction to form a nanostructured carrier, and if necessary, use the linker in advance The agent performs activation of the nanoparticles or / and the carrier in favor of the immobilization. The concentration of the nanoparticles in the suspension during the immobilization reaction is: nanoparticle weight / volume concentration of two to six percent 1 / 10,000, or a molar concentration of nanoparticles of 0.12 to 37.4 nM ; (c) immobilizing the active agent on the nanostructure carrier.
在根据本发明的方法中，所述纳米微粒的重量 /体积浓度为二千分之一至六 万分之一、 优选为一万分之一至四万分之一， 或者纳米微粒的摩尔浓度为 0.12 — 3.74 nM。 优选为 0.19— 0.75 nM。  In the method according to the present invention, the nanoparticle has a weight / volume concentration of one thousandth to sixty thousandth, preferably one ten thousandth to one forty thousandth, or the molar concentration of the nanoparticle. 0.12 to 3.74 nM. It is preferably 0.19-0.75 nM.
根据本发明的又一个方面， 其提供一种用于分离或 /和分析的纳米结构载 体， 其包括固相载体及所述固相载体上的纳米结构， 所述纳米结构载体包括纳 米结构区和任选的非纳米结构区， 而且所述纳米结构区至少具有下述一种或多 种特征： （a)相同投影面积的所述纳米结构区与所述固相载体的表面积之比大 于 1.5; (b)所述纳米结构包括凸出高度大于 3 nm、 且凸出半高处至少一维尺 寸在 1一 500 nm之间的纳米凸体,且在所述纳米结构区中所述纳米载体在所述固 相载体的垂直投影面上的分布密度大于 1个 /ΙΟΟ μ ηι2; (c) 所述纳米结构区与 所述非纳米结构区在固相载体上的垂直投影面积之比大于 1。所述纳米结构区是 指所述纳米结构载体表面上主要分布有所述纳米结构的区域， 所述非纳米结构 区是指所述纳米结构区以外的区域。 如同上述对本发明的纳米结构活性载体的 讨论， 本发明的纳米结构载体与目前的含纳米微粒的载体是有非常不同的确切 含义。 According to yet another aspect of the present invention, it provides a nanostructure support for separation or / and analysis, which includes a solid phase support and nanostructures on the solid phase support. The nanostructure support includes a nanostructure region and An optional non-nano-structured region, and the nano-structured region has at least one or more of the following characteristics: (a) the ratio of the surface area of the nano-structured region to the surface area of the solid phase support of greater than 1.5; (b) The nanostructure includes a nanoconvex having a protrusion height greater than 3 nm and a half-height at least one-dimensional dimension between 1 and 500 nm, and the nanocarrier in the nanostructure region is The distribution density on the vertical projection surface of the solid-phase support is greater than 1 / ΙΟΟ μ ηι 2 ; (c) the ratio of the vertical projection area of the nanostructured region to the non-nanostructured region on the solid-phase support is greater than 1 . The nanostructured region refers to a region where the nanostructures are mainly distributed on the surface of the nanostructured carrier, and the non-nanostructured region refers to a region other than the nanostructured region. As discussed above with respect to the nanostructured active support of the present invention, the nanostructured support of the present invention and the current nanoparticle-containing support have very different exact meanings.
在根据本发明的纳米结构载体中， 所述纳米结构区与所述固相载体的所述 表面积之比大于 2、 或 /和所述纳米结构分布密度大于 1个 /10 m2、 或 /和纳米结 构区与所述非纳米结构区在固相载体上的投影面积之比大于 2。 In the nanostructure carrier according to the present invention, a ratio of the nanostructure region to the surface area of the solid-phase carrier is greater than 2, or / and the nanostructure distribution density is greater than 1/10 m 2 , or / and The ratio of the projected area of the nanostructured region to the non-nanostructured region on the solid support is greater than two.
在根据本发明的纳米结构载体中， 所述纳米结构中如上所述的纳米微粒， 而且该纳米微粒与所述固相载体之间的连接也可通过以上所述的方式进行。  In the nanostructure carrier according to the present invention, the nanoparticle in the nanostructure is as described above, and the connection between the nanoparticle and the solid phase carrier can also be performed in the manner described above.
根据本发明的纳米结构载体可以作为分析芯片片基、 分析芯片通道、 酶标 板片基、 平面层析试剂条片基、 及层析凝胶的载体。 The nanostructure carrier according to the present invention can be used as an analysis chip substrate, an analysis chip channel, and an enzyme label. Plate base, planar chromatography reagent strip base, and carrier for chromatography gel.
根据本发明的又一个方面， .其提供 种含有上述纳米结抅载体的分析或 /.一. 和分离装置。所述装置的例子为:含纳米结构凝胶的层析柱、含纳米结构通道的 芯片 (例如层片上试验室)、 等等。  According to still another aspect of the present invention, there is provided an analysis or / and a separation device containing the above-mentioned nanocrusted carrier. Examples of such devices are: chromatographic columns containing nanostructured gels, chips containing nanostructured channels (e.g., a laboratory on a slice), and the like.
根据本发明的再一个方面，其提供一种制备如上所述纳米结构载体的方法， 其包括以下步骤： （a) 准备所述载体、 纳米微粒、 及任选的连接剂； （b)如 有必要， 用所述连接剂对载体、 或 /和纳米微粒进行活化； （c)将所述纳米微粒 的悬浮液与所述载体接触和进行固定化反应， 固定化反应时所述悬浮液中的所 述纳米微粒的重量 /体积浓度为二百分之一至六万分之一、优选为二千分之一至 六万分之一、 更优选为一万分之一至四万分之一， 或者纳米微粒的摩尔浓度为 According to yet another aspect of the present invention, it provides a method for preparing a nanostructured carrier as described above, which comprises the following steps: (a) preparing the carrier, nanoparticles, and optional linker; (b) if If necessary, use the linker to activate the carrier, or nanoparticles, and (c) contact the suspension of the nanoparticles with the carrier and perform an immobilization reaction. The nanoparticle has a weight / volume concentration of one to two hundredths to sixty thousandths, preferably one to two thousandth to sixty thousandths, and more preferably one to ten thousandth to one forty thousandths. , Or the molar concentration of nanoparticles is
0.12— 37.4 nM、优选为 0.12— 3.74 nM、更优选为 0.19— 0.75 nM。如同上述本发 明的活性载体的制备方法一样， 本发明的纳米结构载体制备方法中， 确定的纳 米粒子浓度是关键所在。 0.12 to 37.4 nM, preferably 0.12 to 3.74 nM, and more preferably 0.19 to 0.75 nM. As with the above-mentioned method for preparing the active carrier of the present invention, in the method for preparing the nanostructured carrier of the present invention, the determined nanoparticle concentration is the key.
根据本发明的再一个方面，其提供一种标记系统，其含活性试剂 /纳米结构 / 分子标记物质复合物， 所述活性试剂 /纳米结构 /分子标记物质复合物为含有活 性试剂、 分子标记物质、 纳米微粒、 及任选的封闭剂的混合物或纯化物， 其中 所述活性试剂为赋于所述复合物反应活性的物质， 所述纳米微粒为粒径 1一 100 nm且本身不是标记物质增强剂的非磁性无机非金属微粒。 所述配基 /纳米微粒 / 分子标记物质复合物含有一种或多种分子标记物质、 一种或多种纳米微粒、 一 种或多种配基以及任选的封闭剂， 所述纳米微粒 /配基 /分子标记物质为混合物 或纯化物。 在此方面中， 本发明的配基 /纳米微粒 /分子标记物质复合物不同于 配基一纳米微粒复合物， 例如配基―微粒配基（第 02137418.X、 02115771.5和 01133527.0号中国专利申请）、配基一纳米磁性微粒（第 02103867.8号中国专利 申请） 、 分子标记物质一微粒配基（第 0211411903号中国专利申请） 等。 本发 明将分子标记物质与配基和纳米微粒结合形成配基 /纳米微粒 /分子标记物质复 合物，也不同于配基一微球一荧光微粒复合物（第 02121391.7号中国专利申请）， 因为本发明复合物中的标记物质是分子标记物质 （例如罗丹明）而不是微粒， 本发明的复合物中的载体是纳米微粒（例如纳米氧化硅）而不是尺寸较大的微 球。  According to still another aspect of the present invention, it provides a labeling system containing an active agent / nanostructure / molecular labeling substance complex, wherein the active agent / nanostructure / molecular labeling substance complex contains an active agent and a molecular labeling substance , Nano-particles, and a mixture or purification of optional blocking agents, wherein the active agent is a substance that imparts reactivity to the complex, and the nano-particles have a particle size of 1-100 nm and are not themselves labeled substances. Agent of non-magnetic inorganic non-metal particles. The ligand / nanoparticle / molecular labeling substance complex contains one or more molecular labeling substances, one or more nanoparticles, one or more ligands, and an optional blocking agent. The ligand / molecularly labeled substance is a mixture or a purified product. In this aspect, the ligand / nanoparticle / molecular labeling substance complex of the present invention is different from the ligand-nanoparticle complex, for example, ligand-microparticle ligand (No. 02137418.X, 02115771.5, and 01133527.0 Chinese patent applications) Ligand-nano magnetic particles (Chinese Patent Application No. 02103867.8), molecular tagging substance-microparticle ligands (Chinese Patent Application No. 0211411903), etc. 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 particle complex (Chinese Patent Application No. 02121391.7), because the present invention The labeling substance in the composite of the invention is a molecular labeling substance (such as rhodamine) rather than a microparticle, and the carrier in the composite of the present invention is a nanoparticle (such as nano-silica) instead of a microsphere with a larger size.
根据本发明的标记系统中， 所述复合物中包含的非磁性无机非金属纳米微 粒包括尺寸 1一 lOO nm的氧化物微粒及其衍生物， 所述氧化物微粒包括氧化硅、 氧化钛、氧化铝， 所述衍生物包括表面含衍生基团或 /和包被功能有机物的衍生 物。 所述衍生基团包括下述: 种或多种基团:. 氨基、 醛基、 环氧基、 氨基肼、 二乙氨基乙基、 二乙基一（2—羟丙基）氨乙基、羧甲基、磺酸丙基、巯乙基吡 啶基、 硅氧烷基、 硫醇基、 烷基;所述功能有机物包括下述一种或多种有机物： 包括聚乙烯吡咯烷酮、 吐温类表面活性剂在内的表面活性剂， 包括聚氨基酸在 内的聚电解质， 包括聚硅氧烷在内的亲油有机物， 包括葡聚糖衍生物、 琼脂糖 衍生物、 纤维素衍生物、 聚丙烯酰胺在内的离子交换聚合物， 和包括肝素钠、 生物素、 活性素在内的活性物质；其中所述微载体包被衍生物中的微载体包括 所述纳米微粒。 According to the marking system of the present invention, the non-magnetic inorganic non-metallic nanoparticles contained in the composite include oxide particles having a size of 110 nm and derivatives thereof, and the oxide particles include silicon oxide, Titanium oxide and aluminum oxide, and the derivatives include derivatives containing derivatizing groups on the surface or / and coating functional organic matter. The derived groups include the following: one or more types of groups: amino, aldehyde, epoxy, aminohydrazine, diethylaminoethyl, diethylmono (2-hydroxypropyl) aminoethyl, Carboxymethyl, sulfopropyl, mercaptoethylpyridyl, siloxane, thiol, alkyl; the functional organics include one or more of the following organics: including polyvinylpyrrolidone, Tween-like surfaces Surfactants including surfactants, polyelectrolytes including polyamino acids, lipophilic organics including polysiloxanes, including dextran derivatives, agarose derivatives, cellulose derivatives, polyacrylamides Ion exchange polymers, and active substances including heparin sodium, biotin, and active substance; wherein the microcarriers in the microcarrier-coated derivative include the nanoparticles.
根据本发明的标记系统中，所述复合物中包含的活性试剂包括抗原、抗体、 亲和素、 生物素、 单链或多链 DNA、 RNA、 核苷酸、 以及病毒、 细胞或它们的 组成。  In the labeling system according to the present invention, the active agents contained in the complex include antigens, antibodies, avidin, biotin, single- or multi-stranded DNA, RNA, nucleotides, and viruses, cells, or their components .
根据本发明的标记系统中， 所述复合物中包含的所述分子标记物质包括下 述一种或多种物质： 荧光物质、化学发光物质、化学发光催化剂、有色金属盐、 染料和颜料。 具体而言， 这些分子标记物质包括下述物质之一种或多种： 荧光 素、 罗丹明、 海藻蛋白、 银盐、 酶、 碱性黑、 碱性紫、 胺基黑、 考马斯亮蓝、 结晶紫。  In the labeling system according to the present invention, the molecular labeling substance contained in the composite includes one or more of the following substances: 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.
在上述标记系统中， 活性试剂 /纳米结构 /分子标记物质复合物的制备方法 为- 将所述一种或多种配基、 一种或多种纳米微粒、 以及一种或多种所述分子 标记物质按下列之一种方式结合： 将所述配基与所述纳米微粒结合再与所述分 子标记物质结合、将所述纳米微粒与所述分子标记物质结合再与所述配基结合、 将所述配基与所述分子标记物质结合再与所述纳米微粒结合、 将所述配基与所 述分子标记物质和所述纳米微粒同时结合、 及基于这些方式的组合。  In the above-mentioned labeling system, the preparation method of the active reagent / nanostructure / molecular labeling substance complex is-combining the one or more ligands, one or more nanoparticles, and one or more of the molecules The labeling substance is combined in one of the following ways: combining the ligand with the nanoparticle and then with the molecular labeling substance, combining the nanoparticle with the molecular labeling substance and then with the ligand, The ligand is combined with the molecular labeling substance and then with the nanoparticle, the ligand is simultaneously bonded with the molecular labeling substance and the nanoparticle, and combinations based on these methods.
根据本发明的又一个方面， 其提供一种标记方法， 其至少包括一些步骤： (a)淮备上述含活性试剂 /纳米结构 /分子标记物质复合物的标记系统； (b) 用 所述活性试剂 /纳米结构 /分子标记物质复合物进行标记， 标记时所述活性试剂 / 纳米结构 /分子标记物质复合物中所述纳米微粒的重量 /体积浓度大于四万分之 一， 或纳米微粒的摩尔浓度大于 0.19 nM。本发明的复合物的制备方法简单、 制 备物水溶性好且灵敏度高。 与上述制备纳米结构活性载体方法中观察的纳米微 粒的影响完全不同的是， 本发明的制备所述复合物的方法中， 复合物检测灵敏 度是随纳米微粒浓度升高而升高的。 在根据本发明的方法中， 所述纳米微粒的重量 /体积浓度大于千分之一、优 选大于百分之一， 纳米微粒的摩尔浓度大于 7.48 nM、 优选大于 74.8 nM。 According to still another aspect of the present invention, it provides a labeling method, which includes at least some steps: (a) preparing the above-mentioned labeling system containing an active reagent / nanostructure / molecular labeling substance complex; (b) using said activity The reagent / nanostructure / molecular labeling substance complex is used for labeling, and the weight / volume concentration of the nanoparticle in the active reagent / nanostructure / molecular labeling substance complex is greater than 1 / 40,000 or the mole of the nanoparticle during labeling. The concentration is greater than 0.19 nM. The preparation method of the complex of the invention is simple, the preparation has good water solubility and high sensitivity. Different from the effect of the nanoparticles observed in the method for preparing a nanostructured active carrier described above, in the method for preparing the composite according to the present invention, the detection sensitivity of the composite increases as the concentration of the nanoparticles increases. In the method according to the invention, the weight / volume concentration of the nanoparticles is greater than one thousandth, preferably greater than one hundredth, and the molar concentration of the nanoparticles is greater than 7.48 nM, preferably greater than 74.8 nM.
根据本发明的另一个方面， 其提供一种分析芯片检测方法， 其包括（a)提 供根据本发明之包含活性纳米微粒的活性载体或分析芯片并将待检测样品与它 们接触和反应， （b)利用根据本发明的标记方法进行标记， 其中所述活性载体 为多活性载体。  According to another aspect of the present invention, it provides a method for detecting an analysis chip, which comprises (a) providing an active carrier or an analysis chip containing active nanoparticles according to the present invention and contacting and reacting a sample to be detected with them, (b) ) Labeling is performed using the labeling method according to the present invention, wherein the active carrier is a multi-active carrier.
根据本发明的另一个方面， 其提供一种分析芯片试剂盒， 其包括包含活性 纳米微粒的活性载体或包含纳米结构活性载体的分析芯片、 和 /或活性试剂 /纳 米结构 /分子标记物质复合物。  According to another aspect of the present invention, it provides an analysis chip kit comprising an active carrier containing active nanoparticles or an analysis chip containing a nanostructured active carrier, and / or an active reagent / nanostructure / molecular labeling substance complex .
根据本发明的另一个方面， 其提供一种分析芯片检测方法， 其包括（a)提 供根据本发明之包含活性纳米微粒的活性载体或包含纳米结构活性载体的分析 芯片， （b)将待检测样品与它们接触和反应，其中所述活性载体为多活性载体。  According to another aspect of the present invention, it provides a method for detecting an analysis chip, which includes (a) providing an active carrier containing active nanoparticles or an analysis chip containing a nanostructure active carrier according to the present invention, (b) detecting The samples are contacted and reacted with them, wherein the active carrier is a multi-active carrier.
根据本发明的又一个方面， 其提供一种分析芯片检测方法， 其包括利用根 据本发明的标记方法进行标记。  According to yet another aspect of the present invention, it provides a method for detecting an analysis chip, which includes using a labeling method according to the present invention for labeling.
根据本发明的再一个方面， 其提供一种分析芯片试剂盒， 其包含如上所述 的活性试剂 /纳米结构 /分子标记物质复合物。  According to yet another aspect of the present invention, it provides an analysis chip kit comprising the active reagent / nanostructure / molecular labeling substance complex as described above.
根据本发明的另一个方面， 其提供一种分析芯片检测方法， 其至少包括以 下步骤： （a)提供纳米结构微流路芯片， 所述纳米结构微流路芯片含纳米结构 微通道或 /和纳米结构分离介质，所述纳米结构微通道含有根据本发明的纳米结 构载体， 所述纳米结构分离介质为根据本发明的纳米结构载体或包含活性纳米 微粒的活性载体或纳米结构活性载体； （b)将样品加入所述微流路芯片并进行 分离、 分析。在该方法中， 所述纳米结构微通道或 /和纳米结构分离介质包括下 述一种或多种： 纳米结构分子筛、 含纳米粒子的微流路涂布物、 含纳米粒子的 固定相、 含纳米粒子的流动相。  According to another aspect of the present invention, it provides a detection method for an analysis chip, which includes at least the following steps: (a) providing a nanostructured microfluidic chip, wherein the nanostructured microfluidic chip includes a nanostructured microchannel or / and A nanostructure separation medium containing a nanostructure carrier according to the present invention, and the nanostructure separation medium is a nanostructure carrier according to the present invention or an active carrier or a nanostructure active carrier comprising active nanoparticles; (b ) The sample is added to the microfluidic chip and separated and analyzed. In this method, the nanostructured microchannel or / and nanostructured separation medium includes one or more of the following: a nanostructured molecular sieve, a nanochannel-containing microchannel coating, a nanoparticle-containing stationary phase, Nanoparticles mobile phase.
根据本发明的又一个方面， 其提供一种分析芯片试剂盒， 其中含有如上所 述的纳米结构微通道或 /和'纳米结构分离介质。  According to another aspect of the present invention, it provides an analysis chip kit, which contains the nanostructured microchannel or / and the nanostructured separation medium as described above.
根据本发明的另一个方面， 其提供一种多肽分析的分析芯片检测方法， 其 至少包括下述一个、 二个、 三个或四个步骤：  According to another aspect of the present invention, it provides an analysis chip detection method for polypeptide analysis, which includes at least one, two, three or four steps as follows:
(a)将样品与磁微粒或 /和磁微片混合；  (a) mixing the sample with magnetic particles or / and magnetic flakes;
(b)将样品与活性试剂 /磁纳米微粒复合物混合；  (b) mixing the sample with the active reagent / magnetic nanoparticle complex;
(c)将样品与芯片接触并反应， 反应时可任选存在外加磁场， 所述芯片含 有如上所述的纳米结构活性载体， 而且所述纳米微粒包括磁纳米微粒；(c) contacting and reacting the sample with the chip, and an external magnetic field may optionally be present during the reaction, and the chip contains There is a nanostructure active carrier as described above, and the nanoparticle includes a magnetic nanoparticle;
( d)将活性试剂 /磁纳米微粒 /分子标记物质复合物用于标记反应， 在标记 时可任选存在外加磁场， 所述活性试剂 /磁纳米微粒 /分子标记物质复合物含为 有一种或多种分子标记物质、 一种或多种磁纳米微粒、 一种或多种活性试剂以 及任选的封闭剂的混合物或纯化物； (d) the active reagent / magnetic nanoparticle / molecular labeling substance complex is used for the labeling reaction, and an external magnetic field may optionally be present during labeling; the active reagent / magnetic nanoparticle / molecular labeling substance complex contains one or A mixture or purification of multiple molecularly labeled substances, one or more magnetic nanoparticles, one or more active agents, and optionally a blocking agent;
其中所述磁纳米微粒 三维空间中至少有一维为 1—200 nm、 优选 1一 100 ran,更优选 1一 50 nm，且其本身不是分子标记物质增强剂的磁微粒及其衍生物; 所述活性试剂选自以下组中能与多肽作用的物质： 多肽、 多糖、 维生素、 抗生 素、 病毒、 细胞、 及功能有机物， 所述活性试剂 /磁纳米微粒 /分子标记物质复 合物在标记时的磁纳米微粒的重量 /体积浓度大于三万分之一、优选大于三千分 之一、更优选大于五百分之一， 或纳米微粒的摩尔浓度大于 0.24 nM、优选大于 2.4 nM、 更优选大于 14.4 nM。  Wherein the magnetic nanoparticle has at least one dimension in the three-dimensional space of 1-200 nm, preferably 1-100 ran, more preferably 1-50 nm, and is not itself a magnetic particle and a derivative of a molecular marker substance enhancer; The active agent is selected from the group of substances that can interact with polypeptides: peptides, polysaccharides, vitamins, antibiotics, viruses, cells, and functional organic matter, the active agent / magnetic nanoparticle / molecular labeling substance complex at the time of labeling magnetic nanometer The weight / volume concentration of the particles is greater than 1 / 30,000, preferably greater than 1 / 3,000, more preferably greater than 5%, or the molar concentration of the nanoparticles is greater than 0.24 nM, preferably greater than 2.4 nM, more preferably greater than 14.4 nM .
在上述方法中， 所述磁微粒选自于包括四氧化三铁、 三氧化二铁在内的铁 氧体及其衍生物，所述衍生物包括表面含衍生基团的表面修饰或 /和功能有机物 包被衍生物。  In the above method, the magnetic particles are selected from the group consisting of ferrite and ferric oxide, and derivatives thereof, and the derivatives include a surface modification or / and a function containing a derivative group on the surface. Organic-coated derivatives.
在上述方法中， 所述外加磁场是脉冲式的。  In the above method, the applied magnetic field is pulsed.
在上述方法中， 所述其中所述分子标记物质包括下述一种或多种物质： 荧 光物质、 化学发光物质、 化学发光催化剂、 有色金属盐、 染料和颜料。  In the above method, the molecularly labeled substance includes one or more of the following substances: a fluorescent substance, a chemiluminescent substance, a chemiluminescent catalyst, a non-ferrous metal salt, a dye, and a pigment.
根据本发明的另一个方面， 其提供一种分析芯片试剂盒， 其包括以下组成 中的至少一种： 磁微粒或 /和磁微片、 活性试剂 /磁纳米微粒复合物、 含磁纳米 结构活性载体的芯片、 活性试剂 /磁纳米微粒 /分子标记物质复合物， 这些物质 的详细描述都可参考以上段落。在该试剂盒， 所述磁微粒或 /和微片用以产生反 应介质混合， 配基 /磁纳米微粒用以浓缩样品目标物， 配基 /磁纳米微粒 /分子标 记物质复合物用以标记反应。  According to another aspect of the present invention, it provides an analysis chip kit, which includes at least one of the following components: magnetic particles or / and magnetic microchips, active reagent / magnetic nanoparticle complex, magnetic nanostructure-containing activity The carrier chip, active reagent / magnetic nanoparticle / molecular labeling substance complex can be described in detail in the above paragraphs. In this kit, the magnetic particles or / and microchips are used to generate a reaction medium mixture, the ligand / magnetic nanoparticle is used to concentrate the sample target, and the ligand / magnetic nanoparticle / molecular labeling substance complex is used to label the reaction. .
根据本发明的再一个方面， 其提供一种多肽定量或 /和定性检测方法， 其至 少包括以下步骤： （a)提供根据本发明之包含活性纳米微粒的活性载体或活性 试剂 /纳米结构 /分子标记物质复合物， （b)将待检测样品与所述活性载体接触 并反应； 和 /或 （c)用所述标记系统进行标记反应， 其中所述活性试剂选自以 下组中能与目标多肽作用的物质： 多肽、 多糖、维生素、抗生素、功能有机物、 病毒及细胞及它们的天然组成或合成组成。该方法可用于芯片检测、酶标检测、 和平面层析检测。 根据本发明的另一个方面， 其提供一种多肽定量或 /和定性检测试剂盒， 其 包括根据本发明之包含活性纳米微粒的活性载体、和 /或权利要求 47所述的活性 试剂 /纳米结构 /分子标记物质复合物。 该试剂盒可以式分析芯片试剂盒酶标试 剂盒和平面层析试剂盒。 According to yet another aspect of the present invention, it provides a method for quantitative or / and qualitative detection of polypeptides, which comprises at least the following steps: (a) providing an active carrier or active agent / nanostructure / molecule comprising active nanoparticles according to the present invention A labeling substance complex, (b) contacting a sample to be detected with the active carrier and reacting; and / or (c) performing a labeling reaction with the labeling system, wherein the active agent is selected from the group consisting of a reagent capable of interacting with a target polypeptide Substances that act: Peptides, polysaccharides, vitamins, antibiotics, functional organics, viruses and cells and their natural or synthetic composition. The method can be used for chip detection, enzyme label detection, and planar chromatography detection. According to another aspect of the present invention, it provides a polypeptide quantitative or / and qualitative detection kit, which comprises the active carrier comprising active nanoparticles according to the present invention, and / or the active reagent / nanostructure according to claim 47. / Molecularly labeled substance complex. The kit can be used as an analytical chip kit, an enzyme labeling kit, and a planar chromatography kit.
根据本发明的另一个方面， 其提供一种分离方法， 其中使用根据本发明的 包含活性纳米微粒的活性载体、 纳米结构载体和纳米结构活性载体中的一种或 者多种作为分离介质。  According to another aspect of the present invention, it provides a separation method in which one or more of an active carrier, a nanostructure carrier, and a nanostructure active carrier including active nanoparticles according to the present invention are used as a separation medium.
本发明的定量或 /和定性检测方法，特别是多肽定量或 /和定性检测方法的优 点是既利用纳米结构活性载体捕捉样品中的目标多肽， 又利用配基 /纳米微粒 / 分子标记物质复合物进行标记，大大提高了检测灵敏度或 /和大大提高了检测速 度。  The advantage of the quantitative or / and qualitative detection method of the present invention, especially the quantitative or / and qualitative detection method of polypeptide, is that both a nanostructured active carrier is used to capture a target polypeptide in a sample, and a ligand / nanoparticle / molecular labeling substance complex is used. Marking greatly improves detection sensitivity or / and greatly improves detection speed.
本发明的检测装置， 包括多肽检测装置的优点是既含有纳米结构活性载体 捕捉样品中的目标多肽， 又含有配基 /纳米微粒 /分子标记物质复合物进行标记， 大大提高了检测灵敏度、 或 /和大大提高了检测速度。  The detection device of the present invention includes a polypeptide detection device, which has the advantage of containing both a nanostructured active carrier to capture a target polypeptide in a sample and a ligand / nanoparticle / molecular labeling substance complex for labeling, which greatly improves detection sensitivity, or And greatly improved the detection speed.
本发明之制备用于多肽检测或组分分离的纳米结构包被载体的方法的优点 是不需加热的包被使宏观固相载体表面受到的变化更小。  An advantage of the method for preparing a nanostructure-coated carrier for polypeptide detection or component separation of the present invention is that coatings that do not require heating cause less change in the surface of the macroscopic solid-phase carrier.
本发明的纳米结构包被载体的优点是更容易制备成本低廉、 灵敏度足够高 的各种活性载体。  The nanostructure coating carrier of the invention has the advantage that it is easier to prepare various active carriers with low cost and sufficient sensitivity.
本发明的制备纳米结构活性载体的方法的优点是简便有效。  The method for preparing a nanostructure active carrier of the present invention has the advantage of being simple and effective.
本发明之用于多肽检测或组分分离的纳米结构活性载体的优点是与样品目 标物反应效率更高， 可检出目标物的浓度下限更低， 而且反应速度更高。  The advantages of the nanostructured active carrier used for polypeptide detection or component separation of the present invention are higher reaction efficiency with the target substance of the sample, lower concentration limit of detectable target substance, and higher reaction speed.
本发明之制备配基 /纳米微粒 /分子标记物质复合物的方法的优点是简便有 效。  The method for preparing the ligand / nanoparticle / molecular labeling substance complex of the present invention has the advantage of being simple and effective.
本发明之用于多肽检测的配基 /纳米微粒 /分子标记物质复合物的优点是与 被标记物的反应效率更高。  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.
本发明之含纳米结构活性载体的检测装置或分离装置的优点是与样品目标 物反应效率更高、 可检出目标物的浓度下限更低、 反应速度更高。  The detection device or separation device of the nanostructure-containing active support of the present invention has the advantages of higher reaction efficiency with the target substance of the sample, lower concentration limit of detectable target substance, and higher reaction speed.
本发明之含配基 /纳米微粒 /分子标记物质复合物的检测装置的优点是含有 配基 /纳米微粒 /分子标记物质复合物进行标记， 提高了检测灵敏度或 /和提高了 检测速度。  The detection device of the ligand / 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.
本发明之多肽定量或 /定性检测方法的优点是至少利用纳米结构活性载体 捕捉样品中的目标多肽， 提高了检测灵敏度或 /和提高了检测速度。 本发明之多肽定量或 /定性检测方法的优点是至少利用配基 /纳米微粒 /分子 标记物质复合物进行标记， 提高了检测灵敏度或 /和提高了检测速度。 An advantage of the method for quantitative or qualitative detection of a polypeptide of the present invention is that it uses at least a nanostructured active carrier Capturing a target polypeptide in a sample increases detection sensitivity or / and speeds up detection. 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.
本发明之多肽定量或 /和定性的芯片检测方法的优点是快速和高灵敏度。 本发明的多肽检测芯片和试剂盒的优点是快速和高灵敏度。  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.
本发明的芯片检测仪的优点是快速和高灵敏度。  The advantages of the chip detector of the present invention are fast and high sensitivity.
以下将通过实施例更为详细地说明本发明。 附图说明  Hereinafter, the present invention will be described in more detail through examples. BRIEF DESCRIPTION OF THE DRAWINGS
图 1是根据本发明之纳米结构载体的 DFM平面图。  FIG. 1 is a DFM plan view of a nanostructured carrier according to the present invention.
图 2A是根据本发明之纳米结构活性载体的 DFM平面图。  Figure 2A is a DFM plan view of a nanostructured active support according to the present invention.
图 2B是图 2A所示 DFM的立体图。  FIG. 2B is a perspective view of the DFM shown in FIG. 2A.
需要说明的是， 该图只给出本发明的纳米结构活性载体的不同 DFM平面图 中的一种， 不可视为代表本发明的其它纳米结构活性载体。 具体实施方式  It should be noted that this figure only shows one of the different DFM plan views of the nanostructured active carrier of the present invention, and it cannot be regarded as representing other nanostructured active carriers of the present invention. detailed description
术语定义 Definition of Terms
本发明术语 "检测装置"是指定量或 /和定性检测过程中所用的包含有用以 同样品目标物作用的配基的用品， 例如含有捕获配基的仪器、 耗材和含有捕获 配基和标记配基的标记试剂盒。 例子有分析芯片、 酶标板、 亲和电泳条、 亲和 层析柱、平面层析试剂条、分析芯片试剂盒、酶标板试剂盒、亲和电泳试剂盒、 等等。 定量或 /和定性检测过程可以在体外进行、 也可以在体内进行。  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 are analysis chips, microplates, affinity electrophoresis strips, affinity chromatography columns, planar chromatography reagent strips, analysis chip kits, microplate readers, affinity electrophoresis kits, and the like. Quantitative or / and qualitative detection can be performed in vitro or in vivo.
本发明术语 "分离装置"是指分离过程中所用的包含有具有分离功能的物 质的分离用品。 分离过程是通过分离方法获得样品的全部或部分组分的过程， 分离装置的例子可举层析装置， 具有分离功能的物质的例子有层析载体等。  The term "separation device" as 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.
本发明术语 "配基 /磁纳米微粒 /固相载体"是指一种至少包含有配基、磁纳 米微粒、 和载体的复合物， 其中配基、 磁纳米微粒、 载体间的结合可以有不同 的形式， 包括直接结合和间接结合。  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, and the binding between the ligand, the magnetic nanoparticle, and the carrier may be different. The forms include direct binding and indirect binding.
本发明术语 "活性试剂"是指用以通过相互作用 （包括亲和作用、 离子交 换、 亲油作用、 等等） 捕获目标物的物质。 可用作活性试剂的物质很多， 例如 下述之一种或多种物质： 二乙氨乙基（DEAE) 、 二乙基一 （2—羟丙基）氨乙 基 （QAE) 、 羧甲基 （CM) 、 磺酸丙基 （SP) 、 巯乙基吡啶基 （MEP) 、 一 NR3+、 一 RCOOE 硅氧烷基、 硫醇基、 垸基、 抗原、 抗体、 配体、 配体指数增 强系统进化技术筛选的适配分子、 配基、 多肽、 多糖、 共酶、 辅因子、 抗生素、 类固醇、 病毒、 细胞、 生物素、 亲和素等。 活性试剂包括配基（相当于英语中 的 Ligand) 。 The term "active agent" in the present invention refers to a substance for capturing a target substance through interaction (including affinity, ion exchange, lipophilicity, etc.). There are many substances that can be used as active reagents, 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 RCOOE siloxane group, thiol group, fluorenyl group, antigen, Antibodies, ligands, ligand index enhancement phylogenetic techniques, adaptation molecules, ligands, peptides, polysaccharides, coenzymes, cofactors, antibiotics, steroids, viruses, cells, biotin, avidin, etc. Active reagents include ligands (equivalent to Ligand in English).
本发明术语 "多肽"相当于英语中的 "polypeptide"， 包括天然或合成蛋白 质、 蛋白质片断、 合成肽、 等等， 免疫检测中通常的目标物和检测中通用的配 基、 例如抗原、 抗体、 等等都属于多肽。  The term "polypeptide" in the present invention is equivalent to "polypeptide" in English, and includes natural or synthetic proteins, protein fragments, synthetic peptides, etc., common targets in immunoassays and commonly used ligands in tests, such as antigens, antibodies, And so on are all peptides.
本发明术语 "纳米微粒"是指在三维空间中至少有一维小于 100 nm、 优选 小于 50 nm的固体微粒。  The term "nanoparticles" in the present invention refers to solid particles having a dimension of less than 100 nm, preferably less than 50 nm in at least one dimension in a three-dimensional space.
本发明术语 "固相载体" ， 简称载体， 是指具有固相形态的、 尺寸远大于 上述纳米微粒的载体， 例如： 分析芯片片基、 酶标板片基、 酶标小球载体、 层 析载体、 电泳胶、 层析凝胶等。  The term “solid phase carrier” in the present invention, referred to as carrier for short, refers to a carrier having a solid phase morphology and a size much larger than the above-mentioned nanoparticles, for example, an analysis chip substrate, an enzyme plate substrate, an enzyme-labeled bead carrier, and chromatography. Carrier, electrophoresis gel, chromatography gel, etc.
本发明术语"片基"是指其具有固定功能的一面具有宏观平面的固相载体， 例如： 分析芯片片基、 酶标板片基、 电泳胶片、 平面层析载体等。  The term “film substrate” in the present invention refers to a solid-phase carrier having a fixed function and a macroscopic plane on one side, such as an analysis chip substrate, an enzyme-labeled plate substrate, an electrophoresis film, and a planar chromatography carrier.
本发明术语 "配基 /纳米微粒 /片基复合物"是指包含有配基、纳米微粒、片 基的一种复合组成， 其中配基、 纳米微粒、 片基间的结合可以有不同的形式， 例如： 配基一纳米微粒一片基、 配基一纳米微粒一配基一片基、 配基一纳米微 粒一配基一纳米微粒一片基、 配基一纳米微粒一配基一纳米微粒一配基一片基 等。  The term “ligand / nanoparticle / tablet complex” in the present invention refers to a composite composition containing 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, ligand-nanoparticles-ligands-nanoparticles, ligand-nanoparticles-ligands-nanoparticles-ligands A piece of base and so on.
本发明术语 "活性纳米微粒"， 是指活性试剂与纳米微粒通过共价或 /和非 共价结合形成的复合物。  The term "active nanoparticle" in the present invention refers to a complex formed by the active agent and the nanoparticle by covalent or / and non-covalent bonding.
本发明术语 "反应器"是指其中固定有活性载体与目标分子发生特异性反 应的场所及与其连通的其它相关结构， 例如开放式多反应器生物芯片中的反应 池和相关的隔离结构和进出液结构等、 96孔酶标板中的孔、 快速检测试剂盒的 试剂条等。  The term "reactor" in the present invention refers to a place in which an active carrier is immobilized to specifically react with a target molecule and other related structures communicating with it, such as a reaction cell and related isolation structures in and out of an open multi-reactor biochip Liquid structure, wells in 96-well microtiter plates, reagent strips for rapid detection kits, etc.
本发明术语 "基片"是指以片基为基础的、 结合有无其它结构 （例如隔离 结构）的用以在固定配基后形成芯片的产品。基片上可以有一个或多个片基池。 单片基池基片上通常没有隔离结构，此时基片既是片基 (例如市售的氨基玻片）。 多片基池基片上有隔离结构， 此时基片包括片基和隔离结构。 片基池在固定上 配基后形成反应器， 多片基池片基形成多反应器芯片。 片基是用以固定配基及 其它助剂 （假如有的话） 的片基， 其表面化学性质和光学性质是影响芯片性能 及成本的重要因素。 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 the substrate is fixed. There can be one or more substrate pools on the substrate. Monolithic substrates usually do not have an 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 base is used to fix the ligand and For the substrate of other additives (if any), the surface chemical and optical properties are important factors affecting chip performance and cost.
本发明术语 "片基池"是指片基与其隔离结构形成的结构。  The term “film base pool” in the present invention refers to a structure formed by a film base and its isolation structure.
本发明术语 "分析芯片"简称为 "芯片"， 包括但不限于英语中的 Bioc ip、 Microarray> Bioarray, 是指定性和 /或定量分析中的一种检测装置， 其反应器中 微量配基同样品中的目标分子发生特异反应的结果可以以可寻址的方式进行识 别。 芯片的核心是其中的反应器， 反应器的核心是其中的芯片片基和固定在芯 片片基上的配基。芯片包括微通道芯片（相当于英语中的 MicroChannel Biochip) 和微阵列芯片 （相当于英语中的 Bioc ip、 Microarray、 Bioarray) ， 但众所周知 不包括现有的快检试剂条。 本发明的芯片含有单反应器或多反应器且有无标记 系统， 反应器中配基在片基上的分布密度大于 10点 /cm2、 优选方案大于 20点 /cm2, 且每个配基点的面积不大于 1 mm2。 The term "analysis chip" in the present invention is referred to as "chip" for short, and includes, but is not limited to, Bioc ip, Microarray> Bioarray in English, which is a detection device in designated and / or quantitative analysis. The result of the specific reaction of the target molecule in the 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 MicroChannel Biochip in English) and a microarray chip (equivalent to Bioc ip, 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 a labeling system. The distribution density of the ligand on the substrate in the reactor is greater than 10 points / cm 2 , and the preferred solution is greater than 20 points / cm 2 . The area of the base point is not more than 1 mm 2 .
本发明术语 "层析"相当于英语" Chromatography" ， 包括亲和层析、 反相 层析、 疏水层析、 离子交换层析、 等等， 其分为平面层析 （例如快检试剂条和 快检试剂盒）和柱层析等。  The term "chromatography" in the present invention is equivalent to "Chromatography" 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 Kit) and column chromatography.
本发明术语 "分子标记物质"是指用以形成或参与形成检出信号、 并在标 记时具有分子形态的物质， 例如芯片检测常用标记物中的罗丹明、 CY3、 CY5 等。  The term "molecularly labeled substance" in the present invention refers to a substance used to form or participate in the formation of a detection signal and has a molecular form at the time of labeling.
本发明术语 "纳米结构"是指具有纳米尺寸的结构，例如具有纳米尺寸的 无规则的树枝状、 深丘状、 网状、 孔隙状、 有规则的圆球、 等等。 又例如， 所 述树枝状可以有向上的或平行于表面的走向。 纳米结构通常反应出部分或全部 纳米效应 （例如表面效应、 尺寸效应等） 。  The term "nanostructure" in the present invention refers to a structure having a nanometer size, such as a random dendrimer, a deep mound, a network, a pore, a regular sphere, and the like having a nanometer size. As another example, the dendrimer may have an upward or parallel direction to the surface. Nanostructures usually reflect some or all of the nano-effects (such as surface effects, size effects, etc.).
本发明术语 "凸体"是指有凸起于表面的立体几何形状，其仅有一个顶部, 例如上述树枝状结构的一条无分支的 "枝"、深丘状结构的一座 "峰"、等等。  The term "convex" in the present invention refers to a three-dimensional geometric shape with a protrusion on the surface, which has only one top, such as a branchless "branch" of the above dendritic structure, a "peak" of a deep mound-like structure, etc. Wait.
本发明术语 "凸出距离"是指上述凸体 其顶部至其底部的距离。  The term "projecting distance" in the present invention refers to the distance from the top of the convex body to the bottom of the convex body.
本发明术语 "凸出半距处横断面"是指上述凸体在其凸出距离一半处垂直 于这一距离的面。  In the present invention, the term "cross-section at a half-distance" refers to the surface of the above-mentioned convex body which is perpendicular to this distance at half of the projection distance.
本发明术语 "单活性载体"是指只固定有一种上述活性试剂的活性载体。 本发明术语 "多活性载体"是指固定有一种以上的多种上述活性试剂的活 性载体。  The term "single active carrier" in the present invention means an active carrier to which only one of the above active agents is immobilized. The term "multi-active carrier" in the present invention means an active carrier to which one or more of the above-mentioned active agents are immobilized.
本发明术语 "纳米微粒摩尔浓度" 是指将纳米微粒作为分子的摩尔浓度， 反映的是单位体积液体中纳米微粒的数目。 本发明中的纳米微粒的分子量是以 一种碳原子的质量的 1/12作为标准， 纳米微粒跟它比较所得的数值， 这种碳原 子指的是核内有 6个质子和 6个中子的碳原子。 具体计算如下： p V 2.65 X 4/3 X π x(20xl0_7)3xl0"3 kg The term “molar concentration of nanoparticles” in the present invention refers to the molar concentration of nanoparticles as molecules, and reflects the number of nanoparticles in a unit volume of liquid. The molecular weight of the nanoparticles in the present invention is A carbon atom has a mass of 1/12 as a standard. The value obtained by comparing a nanoparticle with it. This carbon atom refers to a carbon atom with 6 protons and 6 neutrons in the nucleus. The specific calculation is as follows: p V 2.65 X 4/3 X π x (20xl0 _7 ) 3 xl0 " 3 kg
= = 5.35xl07 = = 5.35xl0 7
1/12碳原子的质量 1.66 x 10'27 kg Mass of 1/12 carbon atom 1.66 x 10 '27 kg
P为密度， Si02为 2.65/cm3， V为纳米微粒的体积， 碳原子的质量的 1/12的 重量为 1.66 x l0_27 kg。 所用 nM的含义为纳摩尔。 实施例 P is the density, Si0 2 is 2.65 / cm 3 , V is the volume of the nanoparticle, and the weight of 1/12 of the mass of the carbon atom is 1.66 x l0_ 27 kg. The meaning of nM used is nanomolar. Examples
在以下实施例使用的纳米微粒及其衍生物如以下表 1所示。 表 1  The nanoparticles and their derivatives used in the following examples are shown in Table 1 below. Table 1
Figure imgf000020_0001
在以下实施例中所用的片基如下表 2所示。 表 2
Figure imgf000020_0001
The bases used in the following examples are shown in Table 2 below. Table 2
Figure imgf000021_0001
Figure imgf000021_0001
*： 制作方法参考 Melnyk 0等， Peptide arrays for highly sensitive and special antibody-binding fluorescence arrays， Bioconjug Chem.13： 713-20.2002。  *: For the preparation method, refer to Melnyk 0, etc., Peptide arrays for highly sensitive and special antibody-binding fluorescence arrays, Bioconjug Chem. 13: 713-20.2002.
**： 制作方法参考第 03135618.4号中国专利申请 在以下实施例中使用的活性试剂如下表 3所示。 表 3  **: For the preparation method, please refer to Chinese Patent Application No. 03135618.4. The active reagents used in the following examples are shown in Table 3 below. table 3
Figure imgf000021_0002
Figure imgf000021_0002
* 制作方法参考： Tranchand-Bunel, D.， Auriault, C.， Diesis, E.， Gras-Masse, H. ( 1998) Detection of human antibodies using "convergent" combinatorial peptide libraries or "mixotopes" designed form a nonvariable antigen: Application to the EBV viral capsid antigen pi 8, J. Peptide Res. 52, 1998， 495-508。 实施例 1: 纳米微粒衍生物的制备 * Production method reference: Tranchand-Bunel, D., Auriault, C., Diesis, E., Gras-Masse, H. (1998) Detection of human antibodies using "convergent" combinatorial peptide libraries or "mixotopes" designed form a nonvariable antigen: Application to the EBV viral capsid antigen pi 8, J. Peptide Res. 52, 1998, 495-508. Example 1: Preparation of nanoparticle derivatives
本实施例所制备的纳米微粒衍生物为用功能高分子包被的衍生物，所用纳米 微粒包括在表 1中的纳米微粒， 所用的功能高分子列于下表 3中， 包括聚离子 型有机物（聚赖氨酸） 、 离子型衍生高聚物（DEAE-葡聚糖、 QAE-纤维素、 氨 基肼-聚赖氨酸)、 高分子表面活性剂（聚乙烯吡咯垸酮） 。  The nanoparticle derivatives prepared in this embodiment are derivatives coated with functional polymers. The nanoparticles used are included in Table 1. The functional polymers used are listed in Table 3 below, including polyionic organic compounds. (Polylysine), ionic derived polymers (DEAE-dextran, QAE-cellulose, aminohydrazine-polylysine), polymer surfactants (polyvinylpyrrolidone).
有机物包被纳米微粒的制备方法为：将纳米微粒在超声振荡下分散配制成浓 度 1/5000— 1/10000 (w/v) 的纳米微粒液， 再与等体积的浓度 1/5000 (w/v) 的 有机物溶液混合， 在超声振荡下在 37°C反应 1小时。 反应产物滴入装有凝胶的 旋转管，在 4000 rpm/min条件下离心，取收集管液体备用（在所有条件优化后， 也可以略去离心分离步骤）。 The method for preparing organic-coated nanoparticles is: dispersing the nanoparticles under ultrasonic oscillation to prepare a nanoparticle solution with a concentration of 1 / 5000-1 / 10000 (w / v), and then equalizing the volume with a concentration of 1/5000 (w / v) The organic solution was mixed and reacted at 37 ° C for 1 hour under ultrasonic vibration. The reaction product was added dropwise to a rotary tube with gel, centrifugation, standby liquid collection tube at 4000 rpm / mi n conditions (when all conditions are optimized, the separation step may be omitted centrifugation).
本发明制备的纳米微粒包被衍生物列于下表 4中。
Figure imgf000022_0001
The nanoparticle-coated derivatives prepared by the present invention are listed in Table 4 below.
Figure imgf000022_0001
纳米微粒衍生物 纳米微粒 包被物  Nanoparticle derivative nanoparticle coating
功能高分子 厂商  Functional polymer manufacturers
DEAE-葡聚糖包被 氧化硅 DEAE-葡聚糖 Pharmacia公司  DEAE-dextran coated silica DEAE-dextran Pharmacia
纳米微粒 (LUDOX  Nanoparticles (LUDOX
AS-40)  AS-40)
QAE-纤维素包被 氧化硅纳米微 QAE-纤维素 晨光化工研究院  QAE-cellulose coated silica nano-micro QAE-cellulose Chenguang Chemical Research Institute
纳米微粒 粒 （STN-3)  Nanoparticles (STN-3)
聚乙烯吡咯垸酮包被纳 氧化硅 聚乙烯吡咯烷酮 天津市津宇精细化工 Polyvinylpyrrolidone coated with silica Silicon polyvinylpyrrolidone Tianjin Jinyu Fine Chemical Industry
米微粒 (LUDOX 有限公司  Rice particles (LUDOX Co., Ltd.
AS-40)  AS-40)
聚赖氨酸包被纳米微粒 氧化硅纳米微 聚赖氨酸 Sigma公司 Polylysine-coated Nanoparticles Silicon Nanoparticles Polylysine Sigma
粒 （STN-3)  Grain (STN-3)
氨基肼-聚赖氨酸包被纳 氧化硅纳米微 氨基肼-聚赖氨酸 成都凯泰新技术有限 Aminohydrazine-polylysine coating nanometer silica nano-aminohydrazine-polylysine Chengdu Kaitai New Technology Co., Ltd.
米微粒 粒（STN-3) 责任公司 实施例 2: 活性纳米微粒的制备 Rice grains (STN-3) Example 2: Preparation of active nanoparticles
本实施例所制备的活性纳米微粒，所用纳米微粒包括选自于表 1中的纳米微 粒及实施例 1制备的纳米微粒衍生物， 所用配基选自于表 3中的配基。  The active nanoparticles prepared in this example include nanoparticles selected from Table 1 and the nanoparticle derivatives prepared in Example 1. The ligand used is selected from the ligands listed in Table 3.
本实施例活性纳米微粒制备方法为：将纳米微粒或纳米微粒多聚物在超声振 荡下分散配制成浓度为 1/5000 (w/v) 的纳米微粒液， 再将浓度为 2 mg/ml的配 基溶液分别与其作 1 : 1混合， 在室温下反应 1小时。 如需纯化， 纯化方法为： 将产物滴入装有凝胶的旋转管， 在 4000 rpm/min条件下离心， 取收集管中的液 体。 所获活性纳米微粒见表 5。 表 5 The method for preparing active nanoparticles in this embodiment is: dispersing nano particles or nano particle polymers under ultrasonic oscillation to prepare a nano particle liquid with a concentration of 1/5000 (w / v), and then adding 2 mg / ml The ligand solution was mixed with each other 1: 1, and reacted at room temperature for 1 hour. For purification, the purification method as follows: The product was added dropwise to a gel with the rotating tube, centrifuged at 4000 rpm / mi n conditions for liquid collection tube. See Table 5 for the obtained active nanoparticles. table 5
活性纳米微粒 纳米微粒 活性试剂 （配基）Active Nanoparticles Nanoparticles Active Reagents (Ligands)
EBV抗原 -STN-3 氧化硅纳米微粒 （ST -3) EBV-VCA-P18抗原 HCV抗原 -STN-3 氧化硅纳米微粒 （STN-3) HCV抗原 EBV antigen-STN-3 silicon oxide nanoparticles (ST -3) EBV-VCA-P18 antigen HCV antigen -STN-3 silicon oxide nanoparticles (STN-3) HCV antigen
HIV抗原 -STN-3 氧化硅纳米微粒 （STN-3) HIV抗原 HIV Antigen-STN-3 Silica Nanoparticles (STN-3) HIV Antigen
HBs抗原 -STN-3 氧化硅纳米微粒 （STN-3) HBs抗原 HBs Antigen-STN-3 Silica Nanoparticles (STN-3) HBs Antigen
EBV抗原 -LUDOX 氧化硅 （LuDOX As-40) EBV-VCA-P18抗原 HCV抗原 -LuDOX 氧化硅 （LuDOX As-40) HCV抗原 EBV Antigen-LUDOX Silicon Oxide (LuDOX As-40) EBV-VCA-P18 Antigen HCV Antigen-LuDOX Silicon Oxide (LuDOX As-40) HCV Antigen
HIV抗原 -LuDOX 氧化硅（LuDOX As-40) HIV抗原 HIV Antigen-LuDOX Silicon Oxide (LuDOX As-40) HIV Antigen
HBs抗原 -LuDOX 氧化硅（LuDOX As-40) HBs抗原 HBs Antigen-LuDOX Silica (LuDOX As-40) HBs Antigen
HBs抗体 -LuDOX 氧化硅 （LuDOX As-40) HBs抗体 HBs Antibody-LuDOX Silica (LuDOX As-40) HBs Antibody
HCV抗原-氧化钛 氧化钛纳米微粒 HCV抗原 HCV Antigen-Titanium Oxide Nanoparticles HCV Antigen
HIV抗原-氧化钛 氧化钛纳米微粒 HIV抗原 HIV antigen-titanium oxide titanium oxide nanoparticles
HCV抗原 -CDS7 疏水硅纳米微粒 （CDS7) HCV抗原 HCV Antigen-CDS7 Hydrophobic Silicon Nanoparticles (CDS7) HCV Antigen
HIV抗原 -CDS7 疏水纳米微粒 （CDS7) HIV抗原 HIV Antigen-CDS7 Hydrophobic Nanoparticles (CDS7) HIV Antigen
EBV抗原-胶体金 胶体金 EBV VCA-P18抗原 EBV原 -胺基胶体金 胺基胶体金 EBV VCA-P18抗原 HCV抗原 -DEAE粒子 DEAE-葡聚糖包被纳米微粒 HCV抗原 EBV antigen-colloidal gold colloidal gold EBV VCA-P18 antigen EBV pro-amine-based colloidal goldamine-based colloidal gold EBV VCA-P18 antigen HCV antigen-DEAE particles DEAE-dextran coated nanoparticles HCV antigen
HIV抗原 -DEAE粒子 DEAE-葡聚糖包被纳米微粒 HIV抗原 HIV antigen-DEAE particles DEAE-dextran coated nanoparticles HIV antigen
HCV抗原 -QAE粒子 QAE-葡聚糖包被纳米微粒 HCV抗原  HCV antigen-QAE particles QAE-dextran coated nanoparticles HCV antigen
HIV抗原 -QAE粒子 QAE-葡聚糖包被纳米微粒 HIV抗原  HIV antigen-QAE particles QAE-dextran coated nanoparticles HIV antigen
HCV抗原-聚乙烯 聚乙烯吡咯垸酮 HCV抗原  HCV Antigen-Polyvinylpyrrolidone HCV Antigen
吡咯垸酮粒子 包被纳米微粒 Pyrrolidinone particles coated nanoparticles
HIV抗原-聚乙烯 聚乙烯吡咯烷酮 HIV抗原  HIV Antigen-Polyvinylpyrrolidone HIV Antigen
吡咯烷酮粒子 包被纳米微粒 Pyrrolidone particles coated nanoparticles
HCV抗原-聚赖氨酸粒子 聚赖氨酸包被纳米微粒 HCV抗原 HCV antigen-polylysine particles Polylysine-coated nanoparticles HCV antigen
HIV抗原-聚赖氨酸粒子 聚赖氨酸包被纳米微粒 HIV抗原  HIV antigen-polylysine particles Polylysine-coated nanoparticles HIV antigen
HCV抗原 -氨基肼赖氨酸粒子 氨基肼聚赖氨酸包被纳米微粒 HCV抗原 HCV antigen-aminohydrazine lysine particles aminohydrazine polylysine coated nanoparticles HCV antigen
HIV抗原 -氨基肼赖氨酸粒子 氨基肼聚赖氨酸包被纳米微粒 HIV抗原 实施例 3: 纳米结构载体的制备  HIV antigen-aminohydrazine lysine particles Aminohydrazine polylysine coated nanoparticles HIV antigen Example 3: Preparation of nanostructured carrier
1 ) 纳米结构芯片片基的制备  1) Preparation of nano-structured chip substrate
本实施例所用纳米微粒包括表 1中的氧化硅（LuDOX AS-40)和实施例 1制 备的纳米微粒衍生物， 所用片基为表 2中的载玻片。  The nanoparticles used in this embodiment include the silicon oxide (LuDOX AS-40) in Table 1 and the nanoparticle derivatives prepared in Example 1. The substrate used is the glass slide in Table 2.
将载玻片先用浓度为 10%的 NaOH及 HN03处理并洗净， 干燥后放入纳米微 粒浓度 1/25000 (w/v) 中的悬浮液中浸泡 15小时， 然后洗涤烘干， 所制得的纳 米微粒包被芯片片基列于表 6中。 The slides were first treated with 10% NaOH and HN0 3 and washed. After drying, the slides were immersed in a suspension with a nanoparticle concentration of 1/25000 (w / v) for 15 hours, then washed and dried. The resulting nanoparticle-coated chip substrates are listed in Table 6.
2) 纳米微粒包被微孔板的制备 2) Preparation of nanoparticle-coated microplates
本实施例所用片基为 96孔聚苯乙烯板（深圳金灿华实业有限公司），所用纳 米微粒分别为胶体金、 氧化硅纳米微粒（STN-3)和氧化硅（LUDOX AS-40) (表 1 ) 。  The substrate used in this example 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). ).
本实施例纳米微粒包被微孔板的制备方法为： 将纳米微粒浓度为 1/25000 (w/v)的纳米微粒液与聚苯乙烯板微孔底部接触， 在室温下反应 15小时， 再用 蒸熘水反复清洗。 所制备的纳米微粒包被片基列于表 6中。 3)纳米结构微通道的制备 In this embodiment, a method for preparing a nanoparticle-coated microwell plate is as follows: a nanoparticle liquid with a nanoparticle concentration of 1/25000 (w / v) is brought into contact with the bottom of a microwell of a polystyrene plate, reacted at room temperature for 15 hours, and then Wash repeatedly with distilled water. The prepared nanoparticle-coated tablets are listed in Table 6. 3) Preparation of nanostructured microchannels
本实施例所用的基片为表 2中的载玻片 （国际申请号： PCT/CN03/01091 )。 将浓度为 1/25000 (w/v) 的氧化硅纳米微粒 （STN-3 )悬浮液涂到片基上形成 宽度为 0.3— l.O nrni, 长度 3n皿的线条， 然后在室温放置 15小时， 然后洗尽、 干燥。 在玻片片基上制备的纳米结构为通道， 经检定均具有类似毛细现象的输 水性能。 所用纳米微粒分别为： DEAE—葡聚糖包被纳米微粒、 QAE-纤维素包 被纳米微粒和聚乙烯吡咯烷酮包被纳米微粒。 尽管不拟进入理论讨论， 但有可 能本发明的微通道中， 由于其上的纳米结构的作用 （例如更高的表面积， 更多 微小凹体）， 是形成此一类 "毛细现象"的原因或部分原因。 尽管本发明的微 通道很简单， 但专业人士都知道， 结合本发明的微通道， 利用公知的微通道芯 片制备的其它技术， 是容易制成各种不同类型的微通道芯片的。  The substrate used in this embodiment is the slide glass in Table 2 (International Application No .: PCT / CN03 / 01091). Apply a suspension of silicon oxide nanoparticles (STN-3) at a concentration of 1/25000 (w / v) to the substrate to form a line with a width of 0.3-lO nrni and a length of 3n, and then leave it at room temperature for 15 hours. Rinse and dry. The nanostructures prepared on the glass substrate are channels, and they have been verified to have capillary-like water transport performance. The nanoparticles used were: DEAE-dextran-coated nanoparticles, QAE-cellulose-coated nanoparticles, and polyvinylpyrrolidone-coated nanoparticles. Although it is not intended to enter the theoretical discussion, it is possible that the microchannels of the present invention, due to the effect of the nanostructures on them (for example, higher surface area, more micro-concavities), are the reasons for this type of "capillary phenomenon" Or part of the reason. Although the microchannels of the present invention are very simple, professionals know that in combination with the microchannels of the present invention, it is easy to make various types of microchannel chips by using other known microchannel chip preparation techniques.
4) 纳米结构载体的鉴定 4) Identification of nanostructured carriers
利用电子探针显微镜（DFM) 和扫描电子显微镜， 可以区分纳米结构区和 非纳米结构区并可以检测载体上纳米凸体的高度、 其半高处横截面最小维尺寸 及纳米凸体结构的分布密度。所述形貌特征可参考图 1。利用全自动氦吸附比表 面仪可以分别检测固相载体和纳米结构载体的比表面积并计算它们的表面积 比。 DFM检测在成都电子科技大学分析测试中心进行。 扫描电子显微镜检测在 四川大学分析测试中心进行。 比表面的检测在中国科学有机化学研究所分析试 验室进行。 尽管利用不同的检测仪器和计算方法可能会有数据差别， 但原则上 一些数据明显大于另一些数据的时候， 这些数据还是有意义的。 所得结果列于 表 6中， 且所述凸体的分布密度均大于 1个 / μ ηι2。 Using electron probe microscopy (DFM) and scanning electron microscopy, it is possible to distinguish between nanostructured regions and non-nanostructured regions and to detect the height of the nanoconvex on the carrier, the minimum dimension of its cross-section at half height and the distribution of the nanoconvex structure. density. The morphological features can be referred to FIG. 1. With a fully automatic helium adsorption specific surface analyzer, the specific surface area of the solid phase support and the nanostructured support can be detected and their surface area ratios calculated. DFM testing was performed at the Analysis and Testing Center of Chengdu University of Electronic Science and Technology. Scanning electron microscopy was performed at the Analysis and Testing Center of Sichuan University. The specific surface area was tested in the analytical laboratory of the Chinese Academy of Organic Chemistry. Although there may be differences in data using different detection instruments and calculation methods, in principle, when some data are significantly larger than others, these data are still meaningful. The obtained results are listed in Table 6, and the distribution densities of the convex bodies are all greater than 1 / μηι 2 .
表 6 Table 6
Figure imgf000026_0001
实施例 4: 含纳米结构活性载体的芯片的制备 (1)
Figure imgf000026_0001
Example 4: Preparation of a chip containing a nanostructured active carrier (1)
本试施所用基片为实施例 3中制备的纳米结构芯片片基， 所用活性试剂为 选自于表 3中的配基。  The substrate used in this test is the nanostructured chip substrate prepared in Example 3, and the active agent used is a ligand selected from Table 3.
本实施例所制备的纳米结构活性载体为一个点上只含一种配基的纳米结构 活性载体， 包括配基一纳米微粒包被片基和配基一纳米结构一配基一纳米微粒 包被片基。 本实施例中所述配基既作为试剂又作为连接剂使用。  The nanostructured active carrier prepared in this embodiment is a nanostructured active carrier containing only one kind of ligand at one point, including a ligand-nanoparticle-coated tablet base and a ligand-nanostructure-ligand-nanoparticle coating Film base. The ligands described in this example are used as both reagents and linkers.
1 )含纳米结构的多片基池基片的制备  1) Preparation of nano-structure-containing multi-cell substrates
本实施例中的多片基池基片是如下制备的： 用高疏水有机硅涂料（成都晨 光化工研究院）涂在片基上隔离结构处， 然后干燥成膜（膜厚小于 0.05 mm) ， 由此在片基上形成片基池的隔离结构 （参考中国专利申请号 03117397.7) 。 每 1 个基片上有 8个片基池，每个片基池尺寸为 4.5 nunX4.5 mm，片基池间隔离结构 宽度为 4.5 mm。 The multi-basin substrate in this embodiment is prepared as follows: a highly hydrophobic organic silicon coating (Chengdu Chenguang Chemical Research Institute) is applied to the isolation structure on the substrate, and then dried to form a film (film thickness less than 0.05 mm), Thus, the isolation structure of the substrate pool is formed on the substrate (refer to Chinese Patent Application No. 03117397.7). Every 1 There are 8 substrate pools on each substrate, and the size of each substrate pool is 4.5 nunX4.5 mm, and the width of the isolation structure between the substrate pools is 4.5 mm.
2) 多反应池芯片的制备 2) Preparation of multi-reaction cell chip
( 1 ) 配基一纳米微粒包被片基型  (1) Ligand-nanoparticle coated tablet type
本实施例所用配基为 HCV抗原（中国北京人民医院肝病研究所）和 HIV1+2 抗原 （中国北京人民医院肝病研究所） 。 在上述 1 ) 中制备的基片的一个片基 池中， 分别将 HCV抗原 （l.O mg/ml) 和 HIV1+2抗原溶液（l.O mg/ml) 按照通 用的点样方法点样在片基池中，两种抗原各点 3个直径为 200μπι的点， 间点距 为 600— 700μιη， 形成 2 X 3阵列。反应池片基上的配基密度大于 30点 /cm2。所 制备的芯片列于表 7中的芯片 101-104。 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 film base pool of the substrate prepared in 1) above, HCV antigen (10 mg / ml) and HIV 1 + 2 antigen solution (10 mg / ml) were spotted on the film base according to a general spotting method. In the cell, each of the two antigens has three dots with a diameter of 200 μm, and the distance between the dots is 600-700 μm, forming a 2 × 3 array. The density of the ligand on the substrate of the reaction cell is greater than 30 dots / cm 2 . The prepared chips are listed in chips 101-104 in Table 7.
(2)配基一纳米结构一配基一纳米微粒包被片基型  (2) Ligand-nanostructure-ligand-nanoparticle-coated tablet type
将纳米微粒浓度为 1/25000 (W/V)的上述活性纳米微粒悬浮液分别按通常 的点样方法点样至上述制备的多片基池基片片基池中， 每种活性纳米微粒点 3 个点， 形成 3 X2配基一纳米微粒阵列， 然后用牛血清白蛋白溶液封闭后备用。 所得芯片为表 7中芯片 105-108。 表 7  The above-mentioned active nanoparticle suspensions with a nanoparticle concentration of 1/25000 (W / V) were respectively spotted into the above-prepared multi-chip base substrate base pool according to the usual spotting method, and each active nanoparticle spot Three spots were formed to form a 3 X2 ligand-nanoparticle array, and then blocked with a bovine serum albumin solution before use. The chips obtained are chips 105-108 in Table 7. Table 7
Figure imgf000027_0001
Figure imgf000027_0001
※见表 6， ※※见表 5 实施例 7: 含纳米结构活性载体的芯片的制备（2)  ※ See Table 6, ※※ See Table 5 Example 7: Preparation of chip containing nanostructured active carrier (2)
本实施例所用片基选自于表 2中的芯片片基， 所用活性试剂表 3中的配基。 本实施例所制备的纳米结构活性载体为一个点上只含一种配基的纳米结构 活性载体： 配基一纳米微粒一.配基一片基。. .本实施例中所述配基既作为试剂使 用又作为连接剂使用。 The base used in this embodiment is selected from the chip base in Table 2, and the ligands in Table 3 of the active reagent used. The nanostructured active support prepared in this embodiment is a nanostructured active support that contains only one kind of ligand at one point: a ligand, a nanoparticle, and a ligand. .. The ligands described in this example are used both as reagents and as linkers.
1 )含纳米结构活性载体的芯片的制备 1) Preparation of a chip containing a nanostructured active carrier
本实施例所制备的芯片为多反应池芯片。  The chip prepared in this embodiment is a multi-reaction cell chip.
本实施例的配基为中的 HCV抗原和 HIV1+2抗原。本实施例的纳米微粒包括金 属纳米微粒、 氧化物纳米微粒、 纳米微粒疏水衍生物、 纳米微粒表面修饰衍生 物、 聚离子型有机物包被纳米微粒、 离子型衍生高聚物包被纳米微粒、 高分子 表面活性剂包被纳米微粒 （见表 1、 3) 。 本实施例的片基包括表面修饰玻片、 表面包被有机物玻片 （见表 2)及纳米结构片基（见表 6) 。 The ligands of this example are HCV antigen and HIV 1 + 2 antigen. 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 substrate in this embodiment includes a surface-modified glass substrate, a surface-coated organic glass substrate (see Table 2), and a nanostructured substrate (see Table 6).
( 1 ) 多片基池基片及活性纳米微粒的准备  (1) Preparation of multi-chip substrates and active nanoparticles
本实施例所用多片基池基片其制备方法如同实施例 3中的多片基池基片制备 方法。 本实施例所用活性纳米微粒为实施例 2中制备的活性纳米微粒。  The method for preparing the multi-basin-cell substrate used in this embodiment is the same as the method for preparing the multi-basin-cell substrate in Example 3. The active nanoparticles used in this example are the active nanoparticles prepared in Example 2.
(2)纳米结构活性载体的制备  (2) Preparation of nanostructured active support
本实施例的制备方法为： 将纳米微粒浓度为 1/25000 (W/V) 的上述活性 纳米微粒分别按通常的点样方法点样至上述制备的多片基池基片片基池中， 每 种活性纳米微粒点 3个点， 形成 3 X2配基一纳米微粒阵列， 然后用牛血清白蛋 白溶液封闭后备用。 所得芯片为表 8中芯片 201至芯片 222。  The preparation method of this embodiment is as follows: the above active nanoparticles with a nanoparticle concentration of 1/25000 (W / V) are respectively spotted into the above-mentioned prepared multi-substrate base substrate base pool according to a general spotting method, Three dots of each active nanoparticle were formed to form a 3 X2 ligand-nanoparticle array, and then blocked with a bovine serum albumin solution for later use. The chips obtained are chips 201 to 222 in Table 8.
2) 纳米结构活性载体的鉴定 2) Identification of nanostructured active carriers
利用电子探针显微镜 （DFM)和扫描电子显微镜， 可以区分纳米结构活性 区和非纳米结构活性区， 并可以检测载体上活性纳米凸体高度、 其半高处最小 维尺寸及活性纳米凸体的分布密度。图 2A和 3B给出所制备的纳米结构活性载体 的平面图和立体图。 SPA-300HU型扫描探针显微镜（DFM)检测在成都电子科 技大学分析测试中心进行。扫描电子显微镜检测在四 ) 11大学分析测试中心进行。 利用全自动氦吸附比表面仪可以分别检测固相载体和纳米结构载体的比表面积 并计算它们的表面积比。 比表面的检测在中国科学院成都有机化学研究所分析 试验室进行。 尽管利用不同的检测仪器和计算方法可能会有数据差别， 但原则 上一些数据明显大于另一些数据的时候， 这些数据还是有意义的。表 8给出按照 上述 1 ) 中纳米结构活性载体的制备分法， 但所述纳米微粒浓度分别为 1/200、 1/25000 1/125000 (w/v) 制备的三种活性载体的检测结果。 表 8 Using electron probe microscopy (DFM) and scanning electron microscopy, it is possible to distinguish between nanostructured active regions and non-nanostructured active regions, and to detect the height of the active nanoconvex on the carrier, the minimum dimension at its half-height, and the Distribution density. 2A and 3B show plan and perspective views of the prepared nanostructured active support. The SPA-300HU scanning probe microscope (DFM) test was performed at the Analysis and Testing Center of Chengdu University of Electronic Technology. Scanning electron microscopy was performed at the University of Analytical Testing Center of D.11. With a fully automatic helium adsorption specific surface analyzer, the specific surface area of the solid phase support and the nanostructured support can be detected and their surface area ratios calculated. Specific surface area was tested in the Analysis Laboratory of Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences. Although there may be differences in data using different detection instruments and calculation methods, in principle, when some data are significantly larger than others, these data are still meaningful. Table 8 shows the preparation method of the nanostructured active carrier according to the above 1), but the concentration of the nanoparticles is 1/200, 1/25000 1/125000 (w / v) Test results of three active carriers. Table 8
Figure imgf000029_0001
Figure imgf000029_0001
※见表 2， ※※见表 5 本实施例中制备的片基， 具有同表 8中纳米浓度为 1/25000 (w/v) 时获得的 芯片类似的形貌特征。 实施例 8: 含纳米结构活性载体的芯片的制备（3)  ※ See Table 2 and ※※ See Table 5 The substrates prepared in this example have similar morphological characteristics to those obtained in Table 8 when the nanoconcentration is 1/25000 (w / v). Example 8: Preparation of a chip containing a nanostructured active carrier (3)
. 本实施例所制备的纳米结构活性载体为一个点上含一种以上配基的纳米结 构活性载体， 包括两种纳米结构活性载体： 配基 2—纳米微粒一配基 2—配基 1 —纳米微粒一配基 1一片基和配基 2—纳米微粒—配基 2—配基 1一片基。 其中配 基 1可以与配基 2进行配对反应且配基 1比配基 2更易与片基结合。 本实施例中一 种配基（配基 2)既作为试剂使用又作为连接剂使用， 另一种配基仅作为连接剂 使用（配基 1 )。 本实施例中配基 1的例子为 HBsAg (中国北京人民医院肝病研究 所） ， 配基 2的例子为 HBsAb (中国北京人民医院肝病研究所） 。 本实施例中的 片基为表 2中的氨基玻片。  The nanostructured active support prepared in this embodiment is a nanostructured active support containing more than one ligand at one point, and includes two types of nanostructured active support: Ligand 2—Nanoparticles—Ligand 2—Ligand 1 — The nanoparticle has one ligand and one ligand and two ligands—nanoparticles—ligand 2—ligand 1. Among them, Ligand 1 can be paired with Ligand 2 and Ligand 1 is easier to bind to the base than Ligand 2. In this embodiment, one kind of ligand (ligand 2) is used as both a reagent and a linker, and the other kind of ligand is used only as a linker (ligand 1). 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 substrate in this example is the amino slide in Table 2.
1 )含纳米结构活性载体的芯片的制备  1) Preparation of a chip containing a nanostructured active carrier
( 1 ) 多片基池基片、 活性纳米微粒、 活性纳米微粒复合物的淮备  (1) Multi-chip base substrate, active nanoparticle, active nanoparticle composite
本实施例所用多片基池基片其制备方法如同实施例 2中的多片基池基片制备 方法。本实施例所用活性纳米微粒和活性纳米微粒复合物为实施例 2中制备的活 性纳米微粒和活性纳米微粒复合物。 (2) 纳米结构活性载体的制备 The method for preparing the multi-basin-cell substrate used in this embodiment is the same as the method for preparing the multi-basin-cell substrate in Embodiment 2. The active nanoparticles and the active nanoparticle composite used in this embodiment are the active nanoparticle and the active nanoparticle composite prepared in Example 2. (2) Preparation of nanostructured active support
配基 2—纳米微粒一配基 2—配基 1一纳米微粒一配基 1一片基的制备方法 为： 将纳米微粒浓度为 1/10000 (w/v) 的配基 2—纳米微粒一配基 2和配基 1 一纳米微粒一配基 1悬浮液作 1 : 1混合后， 按通常的点样方法点样至片基上。 每种活性纳米微粒点 2个点， 形成 3 X2阵列，然后用牛血清白蛋白溶液封闭后 备用。 所得芯片记作芯片 223。 点样时 HBsAb浓度为 3 mg/ml。  Ligand 2—nanoparticles—ligand 2—ligand 1—nanoparticles—ligand 1—one base is prepared as follows: Ligand 2—nanoparticles—compounds with a nanoparticle concentration of 1/10000 (w / v) After mixing base 2 and ligand 1, a nanoparticle and a ligand 1 suspension as 1: 1, spot the sample onto the substrate according to the usual spotting method. Each active nanoparticle was spotted at 2 spots to form a 3 X2 array, and then blocked with bovine serum albumin solution before use. The obtained chip is referred to as a chip 223. The HBsAb concentration was 3 mg / ml when spotted.
配基 2—纳米微粒—配基 2—配基 1一片基复合物的制备方法为：将纳米微粒 浓度为 1/5000 (w/v) 的配基 2—纳米微粒一配基 2和配基 1作 1 : 1混合后， 按通 常的点样方法点样至片基上。每种活性纳米微粒点 2个点， 形成 3 X2阵列， 然后 用牛血清白蛋白溶液封闭后备用。所得芯片记作芯片 224。 点样时 HBsAb浓度为 3 mg/mL 实施例 9: 配基 /纳米微粒 /分子标记物质复合物的制备  Ligand 2—Nanoparticles—Legand 2—Legand 1 One-piece complex preparation method is as follows: Ligand 2—Nanoparticles—Ligand 2 and Ligand—with a nanoparticle concentration of 1/5000 (w / v) After 1 to 1 mixing, apply the spot to the substrate according to the usual spotting method. Each active nanoparticle was spotted at 2 spots to form a 3 × 2 array, and then blocked with bovine serum albumin solution before use. The obtained chip is referred to as a chip 224. HBsAb concentration was 3 mg / mL when spotting Example 9: Preparation of ligand / nanoparticle / molecular labeling substance complex
本实施例所制备的配基 /纳米微粒 /分子标记物质复合物为用于芯片的纳米微 粒标记物。  The ligand / nanoparticle / molecular labeling substance complex prepared in this embodiment is a nanoparticle labeling substance for a chip.
本实施例的纳米微粒包括氧化物纳米微粒及其衍生物 （表 9) ， 所用分子标 记物质为罗丹明（Molecular probes公司）， 所用配基为羊抗人二抗（北京天坛生 物制品股份有限公司）。  The nanoparticles in this embodiment include oxide nanoparticles and their derivatives (Table 9). The molecular marker used is rhodamine (Molecular probes), and the ligand used is goat anti-human secondary antibody (Beijing Tiantan Biological Products Co., Ltd.). ).
本实施例对照标记物为未用含纳米微粒液体处理的常规标记物（罗丹明标记 羊抗人二抗， 美国 Jackson ImmunoRresearch Laboratories公司) 。  The control label in this example is a conventional label (rhodamine-labeled goat anti-human secondary antibody, Jackson ImmunoRresearch Laboratories, USA) that has not been treated with a nanoparticle-containing liquid.
1 )活性纳米微粒的制备  1) Preparation of active nanoparticles
本实施例中所用的活性纳米微粒制备方法为：将纳米微粒在超声振荡下分散 配制成浓度为 1/1000 (w/v) 的纳米微粒液， 再将浓度为 2 mg/ml的配基溶液分 别与其作 1 : 1混合， 在室温下反应 2小时。 混合物的纯化方法为， 混合产物滴 入装有凝胶的旋转管， 在 4000 r/min条件下离心， 取收集管液体。  The preparation method of the active nano-particles used in this embodiment is: dispersing the nano-particles under ultrasonic oscillation to prepare a nano-particle liquid with a concentration of 1/1000 (w / v), and then preparing a ligand solution with a concentration of 2 mg / ml They were mixed 1: 1 with each other and reacted at room temperature for 2 hours. The purification method of the mixture is that the mixed product is dropped into a gel-filled rotating tube, centrifuged at 4000 r / min, and the collection tube liquid is taken.
2) 纳米微粒一分子标记物质的制备 2) Preparation of nanoparticle-molecular labeling substance
本实施例纳米微粒一分子标记物质复合物制备方法为：将纳米微粒在超声振 荡下分散配制成浓度为 1/1000 (w/v)的纳米微粒液， 再将浓度为 2 mg/ml的分 子标记物质溶液分别与其作 1 : 1混合， 在室温下反应 1小时。 如有必要纯化， 混合物的纯化方法为： 混合产物滴入装有凝胶的旋转管， 在 4000 r/min条件下 离心， 取收集管液体。 In this embodiment, a nanoparticle-molecular labeling substance complex is prepared by dispersing the nanoparticle under ultrasonic oscillation to prepare a nanoparticle liquid with a concentration of 1/1000 (w / v), and then dispersing 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, the purification method of the mixture is as follows: The mixed product is dropped into a gel-filled rotating tube at 4000 r / min. Centrifuge and take the liquid from the collection tube.
3) 配基一分子标记物质的制备 3) Preparation of a ligand-labeled substance
本实施例配基一分子标记物质复合物制备方法为公知的罗丹明标记抗抗体 的制备方法。  The preparation method of the ligand-molecular labeling substance complex in this embodiment is a well-known preparation method of rhodamine-labeled anti-antibody.
4) 配基 /纳米微粒 /分子标记物质复合物的制备 4) Preparation of ligand / nanoparticle / molecular labeling substance complex
本实施例制备方法， 其中所述配基、 分子标记物质和纳米微粒的结合， 包 括下列一种或多种方式： 将上述活性纳米微粒与分子标记物质结合（A)、将上 述纳米微粒一分子标记物质与配基结合（B)、将按公知方法制备的分子标记物 质标记配基与纳米微粒结合（C)、将配基与分子标记物质和纳米微粒同时结合 (D) ， 其中所述结合其产物为混合物和纯化物均可。  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 active nanoparticle with the molecularly labeled substance (A), and combining the above nanoparticle with a molecule Binding of a labeling substance to a ligand (B), binding of a molecular labeling substance-labeled ligand prepared according to a known method with a nanoparticle (C), simultaneous binding of a ligand with a molecular-labeling substance and a nanoparticle (D), wherein said binding The product can be a mixture or a purified product.
5) 鉴定 5) Identification
本实施例所用芯片为按公知芯片制备方法制备的 （片基为环氧基玻片， 配 基分别为 HCV抗原和 HIV抗原） 。  The chip used in this embodiment is prepared according to a known chip preparation method (the film base is an epoxy-based glass slide, and the ligands are HCV antigen and HIV antigen, respectively).
鉴定方法为： 可参考实施例 10中芯片的应用方法。 鉴定结果列于表 9中。 表 9  The identification method is as follows: Refer to the application method of the chip in Example 10. The identification results are listed in Table 9. Table 9
标记物 纳米微粒 制备 检测结果  Marker nanoparticle preparation test result
方法 稀释倍 样品 1 样品 2 样品 3 数  Method Dilution Sample 1 Sample 2 Sample 3 Number
对照标记物 无 200 + + ― 对照标记物 无 400 ― ― ― 标记物 1 纳米氧化硅 (STN-3) (A) 400 + + ― 标记物 2 纳米氧化硅（STN-3) (B) 400 + + 一 标记物 3 纳米氧化硅 (STN-3) (C) 400 + + ― 标记物 4 纳米氧化硅 (STN-3) (D) 400 + + ― 标记物 5 聚乙烯吡咯烷酮包被纳米微粒 (C) 400 + + 一 标记物 6 氨基肼-聚赖氨酸包被纳米微 (C) 400 + + 一  Control mark without 200 + + ― Control mark without 400 ― ― ― Label 1 Nanometer silicon oxide (STN-3) (A) 400 + + ― Labeler 2 Nanometer silicon oxide (STN-3) (B) 400 + + One label 3 nanometer silicon oxide (STN-3) (C) 400 + + ― Label 4 Nanometer silicon oxide (STN-3) (D) 400 + + ― label 5 Polyvinylpyrrolidone coated nanoparticles (C ) 400 + + one label 6 aminohydrazine-polylysine coated nanometer (C) 400 + + one
粒 实施例 10: 芯片试剂盒的制备及应用 （1) . Grain Example 10: Preparation and application of chip kit (1).
1 )试剂盒的制备  1) Preparation of the kit
本实施例试剂盒为含本发明活性载体的分析芯片试剂盒。 本实施例含本发 明活性载体的分析芯片 （表 10)为实施例 7和实施例 8中制备的分析芯片。  The kit of this embodiment is an analysis chip kit containing the active carrier of the present invention. The analysis chip (Table 10) containing the active carrier of the present invention in this example is the analysis chip prepared in Examples 7 and 8.
2)试剂盒的应用 2) Application of the kit
在本实施例中， 1号样为 HCV抗体阳性血清， 2号样为 HIV1+2抗体阳性人 血清， 3号样为阴性对照物（HCV抗体和 HIV1+2抗体都为阴性的血清对照物）。 所有的样品均经使用经典的 ELISA方法在血清 20倍稀释反应条件下预先检测。 本实施例的标记物为罗丹明标记羊抗人二抗 （美国 Jackson ImmunoRresearch Laboratories公司)。 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).
实验时前述 3种样品分别加入表 10中所述芯片的反应池中。 其中对照片为 以表 1 中的环氧基玻片按常规的点样方法固定有相同配基制备的芯片。 加样量 为 15 μ 1， 反应 30分钟后洗涤 5次， 洗涤液每次加入量为 25 μ 1。 标记物加入量 为 15 μ 1， 反应后洗涤 5次， 洗涤液每次加入量为 25 μ ΐ, 干燥后在 35/50下进行 扫描。 扫描仪为共聚焦激光扫描仪（Afymetrix公司 GMS 418芯片扫描仪） ， 扫描激发光波长 532 nm,发射光波长 570 nni,读取的信号经处理软件（JAGUAR II )处理， 然后取平均值后根据 Cut-off值判定阴 （一） 阳 （+)性得到结果如 表 10。 实施例 8中制备的其它芯片 （芯片 222-223) ， 具有灵敏度类似的检测 结果。实施例 7中制备的其它芯片（芯片 101-108)也表现出灵敏度提高的结果。 During the experiment, the aforementioned three samples were respectively added to the reaction cell of the chip described in Table 10. The photo is a chip prepared by using the epoxy glass slide in Table 1 with the same ligand fixed by a conventional spotting method. The sample volume was 15 μ1. After 30 minutes of reaction, the sample was washed 5 times. The washing solution was added 25 μ1 each time. The labeling amount was 15 μ1, and the reaction was washed 5 times. The washing solution was added 25 μΐ each time. After drying, scanning was performed at 35/50. The scanner is a confocal laser scanner (Afymetrix's GMS 418 chip scanner). It scans the excitation light wavelength of 532 nm and the emission light wavelength of 570 nni. The read signal is processed by the processing software (JAGUAR II). The cut-off value judges the yin (one) and yang (+) sex. The results are shown in Table 10. The other chips (chips 222-223) prepared in Example 8 have detection results with similar sensitivity. Other chips (chips 101-108) prepared in Example 7 also showed results of improved sensitivity.
表 10 Table 10
芯片号 . 配基 /纳米微粒 /片基复合物 . 检测结果 Chip No.. Ligand / Nanoparticles / Tablet Complex. Test Results
配基或活性纳米微粒 样品 1 样品 2 样品 3 样品 稀释 对照片 醛基玻片 配基 + + ― 100倍 对照片 醛基玻片 配基 ― ― 一 500倍 配基一纳米微粒一配基一片基复合物  Ligand or Active Nanoparticles Sample 1 Sample 2 Sample 3 Sample Dilution Photo Aldehyde Ligand + 100 times Photo Aldehyde Ligand-500 times Ligand One Nanoparticle One Ligand One Base Complex
201 醛基玻片 配基胶体金 + + ― 500倍 201 Aldehyde glass with colloidal gold + + ― 500 times
202 醛基玻片 配基胺基胶体金（2020) + + 一 500倍202 Aldehyde slides Ligand amine based colloidal gold (2020) + + 500 times
203 醛基玻片 配基 DEAE—葡聚糖 + + ― 500倍 包被纳米微粒 203 Aldehyde slide Ligand DEAE-dextran + 500 times coated nanoparticles
204 醛基玻片 配基聚乙烯吡咯烷酮 + + ― 500倍 包被纳米微粒  204 Aldehyde slide Ligand polyvinylpyrrolidone + ― 500 times coated nanoparticles
205 麵 片 配基氨基肼一聚赖氨酸包被 + + ― 500倍 纳米微粒  205 Facial film Ligidine aminohydrazine-polylysine coating ++ 500 times Nanoparticles
206 醛基玻片 配基疏水氧化硅 + + ― 500倍 纳米微粒（CDS7)  206 Aldehyde glass slide Ligand hydrophobic silica + + ― 500 times Nanoparticles (CDS7)
207 配基氧化硅纳米微粒 + + ― 500倍 玻片 (STO-3)  207 Lithium Oxide Nanoparticles + + ― 500 times Slide (STO-3)
208 配基氧化硅 + + ― 500倍 玻片 (LUDOX AS-40)  208 Lithium Oxide + +-500 times slide (LUDOX AS-40)
209 环氧基 配基多孔氧化硅（AEROSIL + + ― 500倍 玻片 200)  209 Epoxy-based ligand porous silica (AEROSIL + + ― 500 times slide 200)
210 配基氧化钛纳米微粒 + + ― 500倍 玻片  210 Ligand Titanium Nanoparticles ++ 500 times slide
211 配基 DEAE—葡聚糖 + + ― 500倍 玻片 包被纳米微粒  211 Ligand DEAE-Dextran + +-500 times glass slide coated with nanoparticles
212 配基聚乙烯吡咯烧酮 + + ― 500倍 玻片 ' 包被纳米微粒  212 Ligand polyvinylpyrrolidone + +-500 times glass slide '' coated nanoparticles
213 配基氨基肼一聚赖氨酸包被 + + ― 500倍 玻片 纳米微粒 214 环氧基 配基疏水氧化硅 + + ― 500倍 玻片 纳米微粒 （CDS7) 213 Ligidine aminohydrazine-polylysine coated ++ 500-fold glass slide nanoparticles 214 Epoxy Ligand Hydrophobic Silica + +-500 times Glass Nanoparticles (CDS7)
215 氨基肼 配基疏水氧化硅 + + ― 500倍 玻片 纳米微粒（CDS7)  215 Aminohydrazine Ligand hydrophobic silica + + ― 500 times glass slide Nanoparticles (CDS7)
216 氨基肼 配基氧化硅纳米微粒 + + ― 500倍 玻片 (STN-3)  216 Aminohydrazine Ligand Silica Nanoparticles ++ 500 times slide (STN-3)
217 氨基肼 . 配基氧化硅 + + ― 500倍 玻片 (LUDOX AS-40)  217 Amino Hydrazine. Ligand Silica + + ― 500 times Slide (LUDOX AS-40)
218 氨基肼 配基氨基肼一聚赖氨酸包被 + + ― 500倍 玻片 纳米微粒  218 Aminohydrazine Ligidine aminohydrazine-polylysine coating ++ 500 times slide glass nanoparticle
219 PVP包被玻 配基疏水氧化硅 + + ― 500倍 片 纳米微粒 （CDS7)  219 PVP Coated Glass Ligand Hydrophobic Silica + +-500-fold Nanoparticles (CDS7)
220 PVP包被玻 配基氧化硅纳米微粒 + + 一 500倍 片 (STN-3 )  220 PVP-coated vitreous silica nanoparticles + 500 times (STN-3)
221 PVP包被玻 配基氧化硅 + + 一 500倍 片 (LUDOX AS-40)  221 PVP Coated Glass Lithium Silica + + 500 times (LUDOX AS-40)
222 PVP包被玻 配基氨基肼一聚赖氨酸包被 + + ― 500倍 片 纳米微粒  222 PVP-coated glass ligand aminohydrazine-polylysine-coated ++ 500-fold tablets Nanoparticles
配基一纳米微粒一配基一纳米微粒一片基复合物  Ligand-nanoparticles-ligand-nanoparticles
223 片基 2 配基氧化硅 + + ― 500倍  223 substrate 2 Ligand silicon oxide + +-500 times
(LUDOX AS-40)  (LUDOX AS-40)
224 片基 2 配基氨基肼一聚赖氨酸包被 + + ― 500倍 纳米微粒 实施例 11: 芯片试剂盒的制备及应用 （2)  224 Film base 2 Ligidine aminohydrazine-polylysine coating ++ 500 times Nanoparticles Example 11: Preparation and application of chip kit (2)
1 ) 试剂盒的制备  1) Preparation of the kit
本实施例试剂盒为含配基 /纳米微粒 /分子标记物质复合物的分析芯片试剂盒。本 实施例中使用的配基 /纳米微粒 /分子标记物质复合物为实施例 8制备（表 9) 。本 实施例试剂盒中所含芯片为实施例 10中所述的对照片。 因此， 本实施例可以形 成数量众多的不同试剂盒。 2)试剂盒的应用 The kit of this embodiment is an analysis chip kit containing a ligand / nanoparticle / molecular labeling substance complex. The ligand / nanoparticle / molecular labeling substance complex used in this example was prepared in Example 8 (Table 9). The chip contained in the kit of this example is the pair of photos described in Example 10. Therefore, this embodiment can form a large number of different kits. 2) Application of the kit
本实施例中， 所用样品及检测方法同实施例 8中的样品和检测方法相同， 所得 结果如表 10所示。 表 9中的其它标记物（标记物 1、 2及 4) .具有与表 10中标 记物类似的检测结果。 实施例 12: 芯片试剂盒的制备及应用 （3) 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. The other markers in Table 9 (Labels 1, 2 and 4) have similar results to those in Table 10. Example 12: Preparation and application of chip kit (3)
1 )试剂盒的制备  1) Preparation of the kit
本实施例试剂盒包括含活性纳米结构载体的分析芯片和含配基 /纳米微粒 /分 子标记物质复合物的标记系统。 本实施例含活性纳米结构载体的分析芯片同实施 例 8。本实施例配基 /纳米微粒 /分子标记物质复合物实施例 9。 因此， 本实施例可 以形成数量众多的不同试剂盒。  The kit of this embodiment includes an analysis chip containing an active nanostructure carrier and a labeling system containing a ligand / nanoparticle / molecular labeling substance complex. The analysis chip containing the active nanostructure carrier in this embodiment is the same as that in Embodiment 8. Example 9 of the ligand / nanoparticle / molecular labeling substance complex of this embodiment. Therefore, this embodiment can form a large number of different kits.
本实施例中，所用样品及检测方法同实施例 8中的样品和检测方法相同，所 得结果如表 11所示。 表 11  In this embodiment, the samples and detection methods used are the same as those in Example 8, and the results are shown in Table 11. Table 11
Figure imgf000035_0001
实施例 13: 芯片试剂盒的制备及应用 （4)
Figure imgf000035_0001
Example 13: Preparation and application of chip kit (4)
1 )试剂盒的制备  1) Preparation of the kit
本实施例试剂盒包括磁芯片。 The kit of this embodiment includes a magnetic chip.
本实施例中所用磁纳米粒子为表 1中的水基磁液（NG-21A),所用片基为表 2环氧基玻片， 所用的配基为实施例 2中所用配基 HCV抗原和 HIV1+2抗原。 1 )磁纳米微粒芯片的制备 The magnetic nanoparticles used in this example are the water-based magnetic fluid (NG-21A) in Table 1. The substrate used is the epoxy glass slide in Table 2. The ligand used is the HCV antigen and the ligand used in Example 2. HIV 1 + 2 antigen. 1) Preparation of magnetic nanoparticle chip
本实施例中的磁纳米微粒芯片为一种配基 /磁纳米微粒 /片基复合物。  The magnetic nanoparticle chip in this embodiment is a ligand / magnetic nanoparticle / sheet-based composite.
本实施例中的磁纳米芯片为多反应器芯片， 其中多片基池基片的制备与实 施例题中多片基池基片的制备相同。 本实施例的亲和磁纳米微粒的制备与实施 例如中活性纳米微粒的制备。  The magnetic nanochip in this embodiment is a multi-reactor chip, in which 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 example are the preparation of active nanoparticles.
将制备好的分别含 HCV抗原 （l mg/ml)和含 HIV1+2抗原 （1 mg/ml) 的亲 和磁纳米微粒点样至多片基池基片的片基池中，每种亲和磁纳米微粒点 2个点， 形成 2X 2阵列。 然后在外磁场作用下于 37Ό反应一小时。 外磁场的作用在于 有利亲和磁纳米微粒向片基的移动及固定。 包被完成后， 用牛血清白蛋白溶液 封闭后备用。 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 substrate substrate, and And magnetic nanoparticle dots to form a 2 × 2 array. Then reacted at 37 ° F 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 coating was completed, it was blocked with bovine serum albumin solution and then used.
本实施例中的对照芯片为以醛基为片基，按公知的点样方法点有不含磁纳米 微粒的 HCV抗原 （l mg/ml)和含 HIV1+2抗原.（1 mg/ml) 制备的芯片。 The control chip in this example is based on an aldehyde group, and according to a well-known spotting method, HCV antigen (1 mg / ml) without magnetic nanoparticles and HIV 1 + 2 antigen are spotted (1 mg / ml). ) The prepared chip.
2)磁纳米标记物的制备 2) Preparation of magnetic nano-markers
本实施例中的磁纳米标记物为一种配基 /磁纳米微粒 /分子标记物质复合物。 其中配基为羊抗人二抗 ( Jackson ImmnoResearch Laboratories公司)。  The magnetic nanomarker in this embodiment is a ligand / magnetic nanoparticle / molecular labeling substance complex. The ligand was goat anti-human secondary antibody (Jackson ImmnoResearch Laboratories).
3 )磁芯片试剂盒的制备 3) Preparation of magnetic chip kit
本实施例中的磁芯片试剂盒， 其中可含一种、 二种、 三种或四种含磁粒子 的组分。 所述含磁粒子组分分别为： 上述制备的磁纳米微粒芯片， 上述制备的 磁纳米微粒标记物， 上述制备的亲和磁纳米微粒 （已用小牛血清白蛋白封闭） 和磁纳米微粒（已用小牛血清白蛋白封闭）。因其不同组合而形成的不同试剂盒 见表 11。  The magnetic chip kit in this embodiment may contain one, two, three, or four magnetic particle-containing components. 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 the magnetic nanoparticle ( Has been blocked with calf serum albumin). See Table 11 for different kits formed by their different combinations.
4)磁芯片试剂盒的应用  4) Application of magnetic chip kit
本实施例中磁芯片试剂盒用于检测样品血清中的 HIV1+2抗体和 HCV抗体 的阴 （一）、 阳 （+) 性。 所用血清样品与实施例 2中的血清样品同。 检测方法 与公知的芯片检测方法不同之处在于， 当试剂盒中除磁芯片外尚含有含磁粒子 组分 （例如磁纳米粒子） 时， 检测反应是在芯片底部有外界磁场的条件下进行 的。 特别是定脉冲磁场。 在外磁场作用下， 有利于磁纳米微粒标记物及亲和磁 纳米微粒捕捉的目标物向反应器底部的运动。 而加入样品中的磁纳米微粒在脉 动磁场的作用下有利于加入芯片反应器中的液相样品内部产生流动以有利于样 品的混合。 由于试剂盒种类多， 下面取一个试剂盒为例来说明其使用方法， 其 它试剂盒的使用方法可根据此例类推。 In this embodiment, the magnetic chip kit is used to detect the yin (one) and yang (+) 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 The dynamic magnetic field is conducive to the internal flow of the liquid sample added to the chip reactor 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.
本例中使用试剂盒的组成为： 对照芯片，磁纳米粒子， 亲和磁纳米粒子与 四种血清样品分别混合 [混合后的磁纳米粒子浓度为 1/2000 (w/v), 亲和磁纳米 粒子浓度为 1/4000 (w/v) ]； 将 15 μ ΐ血清样品加入芯片反应池中并在脉动磁场 作用下于 37°C反应 10分钟； 洗涤； 加入 15 μ ΐ磁纳米标记物并在脉动磁场作用 下于 37°C反应 10分钟； 洗涤并干燥； 然后按与实施例 2中相同的扫描及分析 方法进行扫描及分析。 所得结果见表 .12。 表 12  The composition of the kit used in this example is: control chip, magnetic nanoparticles, affinity magnetic nanoparticles and four serum samples were mixed separately [the magnetic nanoparticle concentration after mixing is 1/2000 (w / v), affinity magnetic Nanoparticle concentration is 1/4000 (w / v)]; 15 μΐ of serum samples were added to the chip reaction cell and reacted at 37 ° C for 10 minutes under a pulsating magnetic field; washing; 15 μΐ of magnetic nano-markers were added and It was reacted at 37 ° C for 10 minutes under a pulsating magnetic field; 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 .12. Table 12
Figure imgf000037_0001
Figure imgf000037_0001
I一对照芯片， II一磁纳米微粒芯片， III一磁纳米微粒， IV—亲和磁纳米微粒， V—磁纳米微粒标记物， VI—对照标记物 实施例 14: 多肽定量或 /和定性检测试剂盒的制备及应用  I—control chip, II—magnetic nanoparticle chip, III—magnetic nanoparticle, IV—affinity magnetic nanoparticle, V—magnetic nanoparticle label, VI—control label Example 14: Quantitative or / and qualitative detection of peptides Preparation and application of kit
1 )试剂盒的制备  1) Preparation of the kit
本实施例试剂盒为含纳米结构活性载体的酶标试剂盒  The kit of this embodiment is an enzyme-labeled kit containing a nanostructured active carrier.
本实施例所用配基为合成肽，其为根据以下文献中公开的方法方法制得的 The ligand used in this example is a synthetic peptide, which is prepared according to the methods disclosed in the following documents
EBV VCA-P18抗原： Tranchand-Bunel, D.， Auriault, C.， Diesis, E.， Gras-Masse, H. ( 1998) Detection of human antibodies using "convergent" combinatorial peptide libraries or 'mixotopes" designed form a nonvariable antigen: Application to the EBV viral capsid antigen pi 8, J. Peptide Res. 52, 1998, 495-508。 本实施 例中所用的纳米结构酶标板是通过在上述纳米结构微孔板的微孔底部固定配基 而成。 EBV VCA-P18 antigen: Tranchand-Bunel, D., Auriault, C., Diesis, E., Gras-Masse, H. (1998) Detection of human antibodies using "convergent" combinatorial peptide libraries or 'mixotopes "designed form a nonvariable antigen: Application to the EBV viral capsid antigen pi 8, J. Peptide Res. 52, 1998, 495-508. The nanostructured microtiter plate used in the examples is obtained by immobilizing a ligand on the bottom of a microwell of the nanostructured microwell plate.
( 1 )从纳米微粒包被载体制备纳米结构活性载体：  (1) Preparation of a nanostructured active carrier from a nanoparticle-coated carrier:
纳米结构微孔板的制备及鉴定见实施例 3。 将浓度为 0.3 g/ml的合成肽按照 通常的酶标板制作方法分别包被到上述纳米微粒包被微孔板 （胶体金包被微孔 板、 氧化硅包被微孔板和氧化硅包被微孔板）和对照微孔板上 （96孔聚苯乙烯 板）， 每一种微孔板上包被 8个孔。 包被后用牛血清白蛋白溶液封闭后备用。 所 制备的纳米结构酶标板列于表 6中，本实施例中的对照酶标板为以 96孔板为片基 按照上述包被方法包被有 EBV VCA-P18抗原的酶标板。  For the preparation and identification of nanostructured microwell plates, see Example 3. The synthetic peptides with a concentration of 0.3 g / ml were coated onto the 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 is coated with 8 wells. After coating, block with bovine serum albumin solution before use. The prepared nanostructured microplate is shown in Table 6. The control microplate in this example is a microplate with a 96-well plate as the base and coated with the EBV VCA-P18 antigen according to the coating method described above.
(2) 从活性纳米微粒制备纳米结构活性载体- 含有 EBV VCA-P18抗原和氧化硅纳米微粒 （STN-3) 的活性纳米微粒的制 备方法同例 2中活性纳米微粒的制备方法。  (2) Preparation of nanostructured active carrier from active nanoparticles-The preparation method of active nanoparticles containing EBV VCA-P18 antigen and silicon oxide nanoparticles (STN-3) is the same as that in Example 2.
将 EBV VCA-P18抗原浓度为 0.1 g ml的活性纳米微粒中的纳米微粒浓度为 1： 4000， 按公知的酶标板包被方法包被到表 2中的 96孔板微孔内， 包被 8个孔， 包被后用牛血清白蛋白溶液封闭后备用。  The nanoparticle concentration in the active nanoparticle with an EBV VCA-P18 antigen concentration of 0.1 g ml was 1: 4000, and the coating was carried out in the microwells of a 96-well plate in Table 2 according to a well-known enzyme plate coating method. Eight wells were sealed with bovine serum albumin solution after coating and used.
2) 应用 2) Application
在本实施例中， 所用样品为 EBV IgA阳性血清、 EBV IgA阴性血清。 所有 的样品均经使用经典的 ELISA方法在血清 20倍稀释反应条件下预先检测。  In this embodiment, the samples used are EBV IgA positive serum and EBV IgA negative serum. All samples were pre-tested using the classic ELISA method under serum 20-fold dilution reaction conditions.
实验时 2种样品分别加入上述对照酶标板及 3个纳米结构酶标板中， 每种 样品加 4个反应池。加样时样品作适当稀释， 加样量为 100μ 1， 反应温度 37°C。 洗涤液每次加入量为 300 μ 1，洗涤 3次。标记物为酶标记的羊抗人 IgA (北京天 坛生物制品股分有限公司）， 加入量为 ΙΟΟμ Ι, 反应温度 37度， 反应时间 30分 钟。 加入底物的反应条件和与经典的 ELISA法相同。 利用酶标仪 （Thermo Labsystems, 上海雷勃分析仪器有限公司）进行比色分析， 8 孔平均结果根据 Cut-off值判定阴 ( _ ) 阳 (+)性， 结果见表 13。 表 13 During the experiment, two kinds of samples were added to the control microplate and three nano-structured microplates respectively, and each sample was added with four reaction cells. The sample was appropriately diluted during sample loading, the sample volume was 100 μl, and the reaction temperature was 37 ° C. The washing solution was added 300 μl each time and washed 3 times. The marker was enzyme-labeled goat anti-human IgA (Beijing Tiantan Biological Products Co., Ltd.), the addition amount was 100 μl, the reaction temperature was 37 degrees, and the reaction time was 30 minutes. The reaction conditions for adding the substrate were the same as those of the classical ELISA method. Colorimetric analysis was performed using a microplate reader (Thermo Labsystems, Shanghai Leibo Analytical Instrument Co., Ltd.). The average results of the 8 wells were used to determine the yin (_) positivity (+) based on the Cut-off value. The results are shown in Table 13. Table 13
Figure imgf000039_0001
由表 13中的结果可见， 纳米结构酶标板比对照酶标板至少具有较高的灵敏 度， 还有更快的反应速度。
Figure imgf000039_0001
It can be seen from the results in Table 13 that the nano-structured microtiter plate has at least higher sensitivity and a faster reaction speed than the control microtiter plate.
本实施例中所用的血清样品、 检测方法均同于实施例 3， 所用对照酶标板为 用相同浓度、相同方法包被的 EBV VCA-P18抗原酶标板。结果是，在抗 EBV IgA 阳性血清 1000倍稀释时，利用对照酶标板的检测结果为阴性， 而利用本实施例 制备的配基 /纳米微粒 /片基复合物酶标板其检测结果仍为阳性。 实施例 15: 多肽定量或 /和定性检测试剂盒的制备及应用 （2)  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 method. As a result, when the anti-EBV IgA-positive serum was diluted 1000-fold, the detection result using the control enzyme plate was negative, while the detection result using the ligand / nanoparticle / plate-based complex enzyme plate prepared in this example was still Positive. Example 15: Preparation and application of a peptide quantitative or / and qualitative detection kit (2)
本实施例试剂盒为含纳米结构活性载体的快捡试剂盒。 The kit of this embodiment is a quick pick-up kit containing a nanostructured active carrier.
1 )试剂盒的制备 1) Preparation of the kit
( 1 ) 纳米微粒包被平面层析试纸条的制备  (1) Preparation of nanoparticle-coated planar chromatography test strips
本实施例所用的片基为硝酸纤维膜条（福建泉州长立生化有限公司）和尼龙 纤维膜条 （福建泉州长立生化有限公司） ， 所用的纳米微粒分别为氧化硅纳米 微粒 （STN-3) (浙江舟山明日纳米材料有限公司）和氧化硅 （LUDOX AS-40) ( Sigma- Aldrich公司) 。  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).
本实施例中所用的纳米微粒包被平面层析试纸条的制备方法为： 将浓度为 1/12500至 1/25000 (w/v)的纳米微粒液与片基接触， 在室温下反应 5小时， 再用 蒸馏水反复清洗。 所制备的纳米结构平面层析带列于表 6中。 The method for preparing a nanoparticle-coated planar chromatography test strip used in this embodiment is: contacting a nanoparticle liquid having a concentration of 1/12500 to 1/25000 (w / v) with a substrate and reacting at room temperature for 5 minutes. Hours, reuse Wash repeatedly with distilled water. The prepared nanostructured planar chromatographic bands are listed in Table 6.
对照片基为未用含纳米微粒缓冲液处理的硝酸纤维膜条和尼龙纤维膜条。  The photographs are based on nitrocellulose membrane strips and nylon fiber membrane strips not treated with nanoparticle-containing buffer.
(2)纳米结构活性载体的快捡试剂条的制备  (2) Preparation of quick picking reagent strip for nanostructured active carrier
本实施例所用配基为 HCV抗原 （中国北京人民医院肝病研究所）。  The ligand used in this example is HCV antigen (Institute of Liver Diseases, Beijing People's Hospital, China).
将浓度 0.5 mg/ml的 HCV抗原分别在上述制备的 4种纳米结构平面层析带 和 2种对照片基上进行点样（检测线)， 再按常规方法分别点上兔抗羊 IgG质控 线后用牛血清白蛋白溶液封闭， 再组装上加样区、胶体金标记羊抗人标记物区、 吸水区组合而成。  HCV antigen at a concentration of 0.5 mg / ml was spotted on the four kinds of nanostructured planar chromatography bands prepared above and two kinds of photographs (detection lines), and then the rabbit anti-goat IgG quality control was spotted by conventional methods. After the line, it was closed with bovine serum albumin solution, and then assembled with a sample addition area, a colloidal gold-labeled sheep anti-human marker area, and a water absorption area.
对照快检试剂条为其中以对照片基制备的快检试剂条。  The control quick test reagent strip is a quick test reagent strip prepared based on the photo.
2)应用 2) Application
在本实施例中，样品 1和 2分别为 HCV抗体阳性血清和 HCV抗体阴性血 清， 所有的样品均预先用经典的 ELISA方法检测确定其阴、 阳性。  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 with a classic ELISA method in advance.
实验时， 2种样品分别加入上述制备的 6种快检试纸条。加样时样品作适当 稀释， 加样量为 50 μ1， 加样后接着加入洗涤液试纸条缓慢吸至质控线出现。 结 果见表 14。 表 14  During the experiment, two kinds of samples were added to the six kinds of quick test strips prepared above. The sample was diluted appropriately during the sample loading, and the sample volume was 50 μ1. After the sample was added, the washing liquid test strip was slowly sucked until the quality control line appeared. The results are shown in Table 14. Table 14
快检试剂条 . 纳米结构平面层析带 检 测  Quick Test Reagent Strips
常规片基 包被纳米微粒 样 样品 2 检测所需时间 品 1  Conventional film-coated nanoparticles Sample 2 Time required for detection Product 1
对照试剂条 硝酸纤维膜条 无 + ― 2分钟 . 纳米微粒包被 硝酸纤维膜条 氧化硅 + ― 1分钟 快检试剂条 (LUDOX AS-40) Control reagent strips Nitrocellulose membrane strips None + ― 2 minutes. Nanoparticle coated nitrocellulose membrane strips Silicon oxide + ― 1 minute Quick test reagent strips (LUDOX AS-40)
纳米微粒包被 硝酸纤维膜条 氧化硅纳米微粒 + ― 1分钟 快检试剂条 (STN-3) Nanoparticle coated nitrocellulose membrane strip Silica nanoparticles + ― 1 minute Quick Test Reagent Strip (STN-3)
对照试剂条 尼龙纤维膜条 无 + ― 2分钟 纳米微粒包被 尼龙纤维膜条 氧化硅 + ― 1分钟 快检试剂条 (LUDOX AS-40) Control reagent strip Nylon fiber membrane strip None + ― 2 minutes Nanoparticle coating Nylon fiber membrane strip Silicon oxide + ― 1 minute Quick detection reagent strip (LUDOX AS-40)
纳米微粒包被 尼龙纤维膜条 氧化硅纳米微粒 + ― 1分钟 快检试剂条 (STN-3) 由以上表 14的结果可以看出， 纳米微粒包被快检试剂条比对照试剂条仅需 要短一倍的时间。 实施例 16: 纳米结构活性载体分离介质的制备及应用 Nanoparticle-coated nylon fiber membrane strips Silicon oxide nanoparticles + ― 1 minute quick test reagent strip (STN-3) From the results in Table 14 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 16: Preparation and application of a nanostructured active carrier separation medium
本实施例制备的分离介质为层析固定相。  The separation medium prepared in this embodiment is a chromatographic stationary phase.
1 )制备  1) Preparation
本实施例中所用配基为 DEAE-葡聚糖（Pharmacia公司） 、 所用纳米微粒为 表 1中的氧化硅纳米微粒（STN-3)、所用载体为平均粒径 60 μ πι的用于层析的硅 胶粒子（中国科学院化学研究所） 。  The ligand used in this example is DEAE-dextran (Pharmacia), the nanoparticles used are silica 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).
将浓度为 1/万 (w/v) 的 DEAE-葡聚糖溶液与浓度 1/6250 (w/v) 的氧化硅 纳米微粒 （STN-3)混合并在室温下搅拌 4小时， 然后将预干燥的硅胶粒子浸泡 于其中， 再按照本发明之一发明人发表的方法（参考本发明发明人之一的文章 ( COATED SILICA SUPPORTS FOR HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY OF PROTEIN, Journal of Chromatography, 476， ( 1989) 195-203)进行葡聚糖交联并得 DEAE-葡聚糖 /纳米微粒 /硅胶粒子复合物。实际 上， 由于在纳米葡聚糖的引入， 其还可以应用经典的葡聚糖衍生方法衍生出多 种层析固定相出来。  A DEAE-dextran solution at a concentration of 1 / 10,000 (w / v) was mixed with silicon oxide nanoparticles (STN-3) at a concentration of 1/6250 (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 particle composites. In fact, due to the introduction of nanodextran, it can also apply the classic method of dextran derivation A variety of chromatographic stationary phases are derived.
作为对比的 DEAE-葡聚糖 /硅胶粒子复合物的制备方法参考本发明之一发 明人发表的方法 （同上） 。  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)配基 /纳米微粒 /载体复合物分离介质的应用 2) Application of ligand / nanoparticle / carrier complex separation media
本实施例检测了上述硅胶粒子、 DEAE-葡聚糖 /硅胶粒子复合物和 DEAE-葡 聚糖 /纳米微粒 /硅胶粒子复合物的动力学吸附容量。动力学吸附容量检测中， 用 于填充上述介质的柱子内径 0.5 cm和长度 2 cm, 缓冲液为 0.01M Tris-HCl/pH 7.40, 流速为 1 ml/min， 所用层析仪为 HP 1090， 所用样品为人白蛋白。 按照公 知的人白蛋白动力学吸附容量分别为 1.2 mg/ml、 7.8 mg/ml和 13.5 mg/ml， 其中 DEAE-葡聚糖 /纳米微粒 /硅胶粒子复合物具有最高的动力学吸附容量。  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 inner diameter of the column used to fill the above medium is 0.5 cm and the length is 2 cm, the buffer solution is 0.01M Tris-HCl / pH 7.40, the flow rate is 1 ml / min, and the chromatography used is HP 1090. The sample was human albumin. According to the known kinetic adsorption capacities of human albumin, 1.2 mg / ml, 7.8 mg / ml, and 13.5 mg / ml, respectively, of which the DEAE-dextran / nanoparticle / silica gel particle complex has the highest kinetic adsorption capacity.

Claims

权利要求  Rights request
- 1、一种用于多肽分析的装置，其包括含有活性纳米微粒的活性载体， 所述 活性载体包含固相载体及固定在所述固相载体局部或全部表面上的活性纳米微 粒， 所述活性纳米微粒包含纳米微粒及固定于所述纳米微粒上的活性试剂， 所 述活性试剂选自于以下组中： 离子交换剂、 药物、 多肽、 多糖、 维生素、 抗生 素、 功能有机物、 抗原、 以及病毒、 细胞或它们的组成。 -1. A device for polypeptide analysis, comprising an active carrier containing active nanoparticles, the active carrier comprising a solid-phase carrier and an active nano-particle immobilized on a part or the entire surface of the solid-phase carrier, said The active nanoparticle includes nanoparticle and an active agent fixed on the nanoparticle, and the active agent is selected from the group consisting of an ion exchanger, a drug, a polypeptide, a polysaccharide, a vitamin, an antibiotic, a functional organic substance, an antigen, and a virus. , Cells or their composition.
2、根据权利要求 1或 2所述的装置，其中所述活性载体包括单活性载体和多 活性载体。  The device according to claim 1 or 2, wherein the active carrier comprises a single active carrier and a multiple active carrier.
3、 根据权利要求 2所述的装置， 其中包含多活性载体的装置包括生物芯片 和平面层析试剂条， 而包含单活性载体的装置包括酶标板。  3. The device according to claim 2, wherein the device containing a multi-active carrier includes a biochip and a planar chromatography reagent strip, and the device containing a single-active carrier includes a microplate.
4、 根据权利要求 1一 3之一所述的装置， 其中所述活性纳米微粒与所述固相 载体之间不通过核苷酸配对结合， 但通过下述一种或多种方式结合： 共价键结 合、 非特异性物理化学吸附、 抗原一抗体吸附、 及亲和吸附， 而且所述结合是 通过固相载体或 /和纳米粒子表面的连接剂实现的， 所述连接剂包括表面基团或 / 和包被有机物或 /和所述活性试剂。  4. The device according to any one of claims 1 to 3, wherein the active nanoparticles and the solid-phase carrier are not bound through nucleotide pairing, but are bound by one or more of the following methods: Valence bonding, non-specific physico-chemical adsorption, antigen-antibody adsorption, and affinity adsorption, and the binding is achieved through a solid-phase support or / and a linker on the surface of the nanoparticle, the linker comprising a surface group or / And coated organics or / and the active agent.
5、 根据权利要求 1一 4之一所述的装置， 其中所述纳米微粒的平均粒径为 1 一 100 nm、 优选 1—50 nm的微粒  5. The device according to any one of claims 1 to 4, wherein the average particle diameter of the nanoparticles is 1 to 100 nm, preferably 1 to 50 nm.
6、 根据权利要求 1一 5之一所述的装置， 其中所述纳米微粒包括无机纳米微 粒或 /和有机纳米微粒及它们的衍生物， 所述无机纳米微粒包括非磁性无机纳米 微粒和磁性无机纳米微粒， 所述非磁性无机纳米微粒包括非磁性金属纳米微粒 和非磁性非金属纳米微粒， 所述衍生物包括结合有表面基团或 /和包被有机物的 衍生物。  6. The device according to any one of claims 1 to 5, wherein the nanoparticles include inorganic nanoparticles or / and organic nanoparticles and their derivatives, and the inorganic nanoparticles include non-magnetic inorganic nanoparticles and magnetic inorganics. Nano-particles, the non-magnetic inorganic nanoparticles include non-magnetic metal nanoparticles and non-magnetic non-metal nanoparticles, and the derivative includes a derivative bound to a surface group or / and an organic substance.
7、 根据权利要求 6所述的装置， 其中所述无机非磁性非金属纳米微粒包括 氧化硅、 氧化钛、 氧化铝微粒， 所述非磁性金属纳米微粒包括金、 钒、 铅、 银、 铁及其氧化物微粒， 所述有机纳米微粒包括塑料、 多糖、 乳胶、 树脂微粒。  7. The device according to claim 6, wherein the inorganic non-magnetic non-metal nanoparticles include silicon oxide, titanium oxide, and alumina particles, and the non-magnetic metal nanoparticles include gold, vanadium, lead, silver, iron, and Its oxide particles, the organic nanoparticles include plastic, polysaccharide, latex, and resin particles.
8、 根据权利要求 4一 7之一所述的装置， 其中所述表面基团包括下述一种或 多种基团：氨基、 醛基、 环氧基、 氨基肼、 二乙氨基乙基、 二乙基一 （2—羟丙 基）氨乙基、 羧甲基、 磺酸丙基、 巯乙基吡啶基、 硅氧垸基、 硫醇基、 烷基;所 述包被有机物包括下述一种或多种有机物： 包括聚乙烯吡咯垸酮、吐温类表面活 性剂在内的表面活性剂， 包括聚氨基酸在内的聚电解质， 包括聚硅氧垸在内的 亲油有机物， 包括葡聚糖衍生物、 琼脂糖衍生物、 纤维素衍生物、 聚丙烯酰胺 在内的离子交换聚合物， 和包括肝素钠、 生物素、 活性素、 抗原、 抗体在内的 亲和物质。  8. The device according to any one of claims 4 to 7, wherein the surface group includes one or more of the following groups: amino, aldehyde, epoxy, aminohydrazine, diethylaminoethyl, Diethylmono (2-hydroxypropyl) aminoethyl, carboxymethyl, sulfopropyl, mercaptoethylpyridyl, siloxanyl, thiol, alkyl; the coated organics include the following One or more organics: Surfactants including polyvinylpyrrolidone, Tween-based surfactants, polyelectrolytes including polyamino acids, lipophilic organics including polysiloxane, including glucose Ion exchange polymers including glycan derivatives, agarose derivatives, cellulose derivatives, polyacrylamides, and affinity substances including heparin sodium, biotin, actives, antigens, and antibodies.
9、根据权利要求 1一 8之一所述的装置，其中所述固相载体包括固相载体和 纳米结构载体， 所述固相载体包括由以下材料或其衍生物制成的固相物质：玻 璃、 硅片、„—硅一胶、. 瓷:^ 属 ft _、 金 J§、 聚合物材料及它们的复合物， 而 所述纳米结构载体 分布形成纳米尺寸的结构的载体。 9. The device according to any one of claims 1 to 8, wherein the solid phase support comprises a solid phase support and A nano-structured support, the solid-phase support includes a solid-phase substance made of the following materials or derivatives thereof: glass, silicon wafer, „silicone glue, ceramics: ^ genus ft_, gold J§, polymer materials And their complexes, and the nano-structured carriers are distributed to form a nano-sized structured carrier.
10、 一种用于分离或 /和分析的纳米结构活性载体， 其包括固相载体及所述 固相载体表面上的活性纳米结构， 所述活性纳米结构包含纳米结构及固定在所 述纳米结构上的活性试剂， 所述纳米结构活性载体包括活性区及任选存在的非 活性区， 所述活性区包括纳米结构活性区和任选存在的非纳米结构活性区， 而 且所述活性区至少具有下述一种或多种特征： （a)相同投影面积的所述活性区 与所述固相载体的表面积之比大于 1.5; (b)所述活性纳米结构包括凸出高度 大于 3 nm、 且凸出半高处至少一维尺寸在 1一 500 nm之间的纳米凸体， 且在所 述纳米结构活性区中所述纳米凸体在所述固相载体的垂直投影面上的分布密度 大于 1个 /μ ηι2; (c)所述纳米结构活性区与所述非纳米结构活性区在固相载体 上的垂直投影面积之比大于 1。 10. A nanostructure active support for separation or / and analysis, comprising a solid phase support and an active nanostructure on a surface of the solid phase support, the active nanostructure comprising a nanostructure and fixed on the nanostructure The nanostructured active carrier includes an active region and optionally an inactive region, the active region includes a nanostructured active region and an optional non-nanostructured active region, and the active region has at least One or more of the following features: (a) the ratio of the surface area of the active area to the solid phase support of the same projected area is greater than 1.5; (b) the active nanostructure includes a protrusion height greater than 3 nm, and Protrudes at a half-height at least one-dimensional nano-convex with a dimension between 1 and 500 nm, and the distribution density of the nano-convex on the vertical projection plane of the solid support in the nano-structure active region is greater than 1 / μηι 2 ; (c) The ratio of the vertical projected area of the nanostructured active region to the non-nanostructured active region on a solid support is greater than 1.
11、 根据权利要求 10所述的纳米结构活性载体， 其中所述活性区与所述固 相载体的表面积之比大于 3。  11. The nanostructured active support according to claim 10, wherein a ratio of a surface area of the active region to the solid phase support is greater than 3.
12、 根据权利要求 11所述的纳米结构活性载体， 其中所述活性区与所述固 相载体的表面积之比大于 5。  12. The nanostructured active support according to claim 11, wherein a ratio of a surface area of the active region to the solid-phase support is greater than 5.
13、 根据权利要求 10— 12之一所述的纳米结构活性载体， 其中所述凸体分 布密度大于 5个 /μ ηι2。 13. The nanostructured active support according to any one of claims 10-12, wherein the distribution density of the convexities is greater than 5 / μηι 2 .
14、 根据权利要求 10— 12之一所述的纳米结构活性载体， 其中所述凸体分 布密度大于 10个 /μ πι2。 14. The nanostructured active support according to any one of claims 10-12, wherein the distribution density of the convex body is greater than 10 / μπι 2 .
15、 根据权利要求 10— 14之一所述的纳米结构活性载体， 其中所述纳米结 构活性区与所述非纳米结构活性区在固相载体上的投影面积之比大于 2。  15. The nanostructured active support according to any one of claims 10 to 14, wherein a ratio of a projected area of the nanostructured active area to the non-nanostructured active area on a solid support is greater than two.
16、 根据权利要求 10— 14之一所述的纳米结构活性载体， 其中所述纳米结 构活性区与所述非纳米结构活性区在固相载体上的投影面积之比大于 4。  16. The nanostructured active support according to any one of claims 10 to 14, wherein a ratio of a projected area of the nanostructured active area to the non-nanostructured active area on a solid support is greater than 4.
17、根据权利要求 10— 16之一所述的纳米结构活性载体， 其中所述分离或 / 和分析的目标物包括多肽、核酸、及与它们相互作用的药物，所述纳米结构活性 载体包括:生物芯片或其活性载体、 酶标板、 平面层析试剂条、 层析活性凝胶。  17. The nanostructured active carrier according to any one of claims 10-16, wherein the targets for separation or / and analysis include polypeptides, nucleic acids, and drugs that interact with them, and the nanostructured active carrier comprises: Biochip or its active carrier, microtiter plate, planar chromatography reagent strip, chromatography active gel.
18、 根据权利要求 10— 17之一所述的纳米结构活性载体， 其中所述活性试 剂选自于以下组中： 离子交换剂、 药物、 多肽、 多糖、 维生素、 抗生素、 功能 有机物、 抗原、 单链或多链 DNA、 RNA、 核苷酸、 以及病毒、 细胞或它们的组 成。 18. The nanostructured active carrier according to any one of claims 10-17, wherein the active agent is selected from the group consisting of an ion exchanger, a drug, a polypeptide, a polysaccharide, a vitamin, an antibiotic, a functional organic substance, an antigen, a single Stranded or multistranded DNA, RNA, nucleotides, and viruses, cells or their composition.
19、 根据权利要求 10— 18之一所述的纳米结构活性载体， 其中所述活性纳 米结构中含有活性纳米微粹，. 述活性纳米微粒包括纳米微粒和固定于其上的 一种或多种所述活性试剂或任选的连接剂， 所述纳米微粒为在三维空间中至少 有一维为大于 1 nm且小于 100 nm、 优选大于 1 nm且小于 50 nm的微粒。 19. The nanostructure active carrier according to any one of claims 10 to 18, wherein the active nanostructure contains an active nanomicrosphere, and the active nanoparticle includes a nanoparticle and one or more types fixed thereon. In the active agent or optional linking agent, the nanoparticle is a particle 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 50 nm.
20、 根据权利要求 10— 19之一所述的活性载体， 其中所述活性纳米微粒与 所述固相载体之间通过下述一种或多种方式结合： 共价键结合、 非特异性物理 化学吸附、抗原一抗体吸附、 亲和吸附、及核苷酸配对结合,且所述结合是通过 固相载体或 /和纳米粒子表面的连接剂实现的， 所述连接剂包括表面基团或 /和 包被有机物。  20. The active support according to any one of claims 10 to 19, wherein the active nanoparticles and the solid-phase support are bound by one or more of the following methods: covalent bonding, non-specific physical chemistry Adsorption, antigen-antibody adsorption, affinity adsorption, and nucleotide pairing binding, and the binding is achieved through a solid phase support or / and a linker on the surface of the nanoparticle, the linker including surface groups or / and Coated organics.
21、 根据权利要求 19或 20所述的活性载体， 其中所述纳米微粒包括无机纳 米微粒或 /和有机纳米微粒及它们的衍生物， 所述无机纳米微粒包括非磁性无机 纳米微粒和磁性无机纳米微粒， 所述非磁性无机纳米微粒包括非磁性金属纳米 微粒和非磁性非金属纳米微粒， 所述衍生物包括结合有表面基团或 /和包被有机 物的衍生物。  21. The active carrier according to claim 19 or 20, wherein the nanoparticles include inorganic nanoparticles or / and organic nanoparticles and their derivatives, and the inorganic nanoparticles include non-magnetic inorganic nanoparticles and magnetic inorganic nanoparticles Fine particles, the non-magnetic inorganic nanoparticles include non-magnetic metal nanoparticles and non-magnetic non-metal nanoparticles, and the derivative includes a derivative bound to a surface group or / and an organic substance.
22、 根据权利要求 21所述的活性载体， 其中所述无机非磁性非金属纳米微 粒包括氧化硅微粒、 氧化钛微粒、 氧化铝微粒、 氧化铁微粒在内的氧化物纳米 微粒， 所述非磁性金属纳米微粒包括金微粒、 钒微粒、 铅微粒， 所述非金属纳 米微粒包括包括塑料、 多糖、 乳胶、 树脂微粒的有机纳米微粒。  22. The active support according to claim 21, wherein the inorganic non-magnetic non-metallic nanoparticles include oxide nanoparticles including silicon oxide particles, titanium oxide particles, aluminum oxide particles, and iron oxide particles, and the non-magnetic The metal nanoparticles include gold particles, vanadium particles, and lead particles, and the non-metal nanoparticles include organic nanoparticles including plastic, polysaccharide, latex, and resin particles.
23、 根据权利要求 21— 22之一所述的活性载体， 其中所述表面基团包括下 述一种或多种基团： 氨基、 醛基、 环氧基、 氨基肼、 二乙氨基乙基、 二乙基一 (2—羟丙基）氨乙基、羧甲基、磺酸丙基、巯乙基吡啶基、硅氧烷基、硫醇基、 垸基;所述包被有机物包括下述一种或多种有机物： 包括聚乙烯吡咯垸酮、 吐温 类表面活性剂在内的表面活性剂， 包括聚氨基酸在内的聚电解质， 包括聚硅氧 垸在内的亲油有机物， 包括葡聚糖衍生物、 琼脂糖衍生物、 纤维素衍生物、 聚 丙烯酰胺在内的离子交换聚合物， 和包括肝素钠、 生物素、 活性素、 抗原、 抗 体在内的亲和物质。  23. The active support according to any one of claims 21 to 22, wherein the surface group comprises one or more of the following groups: amino, aldehyde, epoxy, aminohydrazine, diethylaminoethyl , Diethylmono (2-hydroxypropyl) aminoethyl, carboxymethyl, sulfopropyl, mercaptoethylpyridyl, siloxane, thiol, fluorenyl; the coated organics include the following Said one or more organic substances: surfactants including polyvinylpyrrolidone, Tween-type surfactants, polyelectrolytes including polyamino acids, lipophilic organic substances including polysiloxane, including Ion exchange polymers including dextran derivatives, agarose derivatives, cellulose derivatives, polyacrylamides, and affinity substances including heparin sodium, biotin, actives, antigens, and antibodies.
24、 根据权利要求 10— 23之一所述的活性载体， 其中所述固相载体包括固 相载体和纳米结构载体， 所述固相载体包由以下材料或其衍生物制成的固相物 质： 玻璃、 硅片、 硅胶、 陶瓷、 金属氧化物、 金属、 聚合物材料及它们的复合 物， 而所述纳米结构载体为表面分布纳米尺寸的结构的载体。  24. The active carrier according to any one of claims 10 to 23, wherein the solid phase carrier comprises a solid phase carrier and a nanostructured carrier, and the solid phase carrier comprises a solid phase substance made of the following materials or derivatives thereof : Glass, silicon wafer, silica gel, ceramics, metal oxides, metals, polymer materials and their composites, and the nanostructure carrier is a carrier with a nanometer-sized structure distributed on the surface.
25、 含有权利要求 10— 24之一所述活性载体的分析或 /和分离装置。  25. An analysis or / and separation device comprising the active carrier according to any one of claims 10-24.
26、 一种制备如权利要求 1一 9之一所述的包含活性纳米微粒的活性载体或 如权利要求 10— 24之一所述的活性载体的方法， 其包括以下步骤： （a)准备所 述活性试剂、 载体、 纳米微粒、 及任选的连接剂； （b)将所述活性试剂固定在所 述纳米微粒上制备活性纳米微粒， 如有必要预先用所述连接剂对所述纳米微粒 进行有利于固定所述活性试剂的活化； (c)将步骤 (b)中所制备的所述活性纳米 微粒的悬浮液与所述载体接触和进行固定化反应， 如有必要预先用所述连接剂 对载体进行有利于所述固定的活化， 固定化反应时所述活性纳米微粒悬浮液中 所述纳米微粒的浓度为:纳米微粒重量 /体积浓度二百分之一至六万分之一、 或 纳米微粒摩尔浓度 0.12— 37.4 nM。 26. A method for preparing an active carrier comprising active nanoparticles according to any one of claims 1 to 9 or an active carrier according to any one of claims 10 to 24, comprising the following steps: (a) preparing The active agent, the carrier, the nanoparticle, and an optional linker; (b) preparing the active nanoparticle by immobilizing the active agent on the nanoparticle; Performing activation that is favorable for fixing the active agent; (c) contacting the suspension of the active nanoparticle prepared in step (b) with the carrier and performing an immobilization reaction, if necessary, using the connection in advance The carrier performs an activation to facilitate the immobilization. The concentration of the nanoparticles in the active nanoparticle suspension during the immobilization reaction is: nanoparticle weight / volume concentration of two to one hundred thousand to sixty thousand, Or the molar concentration of nanoparticles is 0.12-37.4 nM.
27、根据权利要求 26所述的方法， 其中所述纳米微粒的重量 /体积浓度为二 千分之一至六万分之一， 或纳米微粒的摩尔浓度为 0.12— 3.74nM。  27. The method according to claim 26, wherein the nanoparticle has a weight / volume concentration of one thousandth to one sixty thousandth, or the nanoparticle has a molar concentration of 0.12 to 3.74 nM.
28、根据权利要求 26所述的方法， 其中所述纳米微粒的重量 /体积浓度为一 万分之一至四万分之一， 或纳米微粒的摩尔浓度为 0.19—0.75 nM。  28. The method according to claim 26, wherein the weight / volume concentration of the nanoparticles is between 1 / 10,000 and 40,000, or the molar concentration of the nanoparticles is 0.19-0.75 nM.
29、 一种制备如权利要求 1一 9之一所述的含活性纳米微粒的活性载体或如 权利要求 10— 24之一所述的活性载体的方法， 其包括以下步骤： （a)准备所述 活性试剂、 载体、 纳米微粒、 及任选的连接剂； （b)将所述纳米微粒的悬浮液 与所述载体接触和进行固定化反应形成纳米结构载体， 如有必要预先用所述连 接剂对所述纳米微粒或 /和载体进行有利于所述固定的活化， 固定化反应时所述 悬浮液中的所述纳米微粒的重量 /体积浓度为二百分之一至六万分之一，或纳米 微粒的摩尔浓度 0.12— 37.4 nM; (c)将所述活性试剂固定在所述纳米结构载体 上。  29. A method for preparing an active carrier containing active nanoparticles according to one of claims 1 to 9 or an active carrier according to one of claims 10 to 24, comprising the steps of: (a) preparing The active agent, the carrier, the nanoparticle, and an optional linker; (b) contacting the suspension of the nanoparticle with the carrier and performing an immobilization reaction to form a nanostructured carrier, and if necessary, using the linker in advance The nanoparticle or / and the carrier is beneficial to the activation of the immobilization, and the weight / volume concentration of the nanoparticle in the suspension during the immobilization reaction is from 1% to 60,000 Or, the molar concentration of the nanoparticles is 0.12 to 37.4 nM; (c) the active agent is fixed on the nanostructure carrier.
30、根据权利要求 29所述的方法， 其中所述纳米微粒的重量 /体积浓度为二 千分之一至六万分之一， 或纳米微粒的摩尔浓度为 0.12— 3.74 nM。  30. The method according to claim 29, wherein the nanoparticle has a weight / volume concentration of one thousandth to sixty thousandth, or the nanoparticle has a molar concentration of 0.12 to 3.74 nM.
31、根据权利要求 30所述的方法， 其中所述纳米微粒的重量 /体积浓度为一 万分之一至四万分之一， 或纳米微粒的摩尔浓度为 0.19— 0.75 nM。  31. The method according to claim 30, wherein the weight / volume concentration of the nanoparticles is between 1 / 10,000 and 40,000 / 10,000, or the molar concentration of the nanoparticles is from 0.19 to 0.75 nM.
32、 一种用于分离或 /和分析的纳米结构载体， 其包括固相载体及所述固相 载体上的纳米结构， 所述纳米结构载体包括纳米结构区和任选存在的非纳米结 构区， 而且所述纳米结构区至少具有下述一种或多种特征： （a)相同投影面积 的所述纳米结构区与所述固相载体的表面积之比大于 1.5; (b)所述纳米结构包 括凸出高度大于 3 nm、 且凸出半高处至少一维尺寸在 1一 500 nm之间的纳米凸 体， 且在所述纳米结构区中所述纳米载体在所述固相载体的垂直投影面上的分 布密度大于 1个 /ΙΟΟ μ ηι2; (c)所述纳米结构区与所述非纳米结构区在固相载体 上的垂直投影面积之比大于 1。 32. A nanostructure support for separation or / and analysis, comprising a solid phase support and nanostructures on the solid phase support, the nanostructure support comprising a nanostructure region and optionally a non-nanostructure region And, the nanostructure region has at least one or more of the following characteristics: (a) the ratio of the surface area of the nanostructure region to the solid phase support of the same projected area is greater than 1.5; (b) the nanostructure It includes a nano-convex body with a protrusion height greater than 3 nm, and at least a one-dimensional dimension between 1 and 500 nm at the protrusion half-height, and in the nano-structure region, the nano-carrier is perpendicular to the solid-phase carrier. The distribution density on the projection surface is greater than 1/100 μηι 2 ; (c) the ratio of the vertical projection area of the nanostructured region to the non-nanostructured region on the solid support is greater than 1.
33、 根据权利要求 32所述的纳米结构载体， 其中所述纳米结构区与所述固 相载体的所述表面积之比大于 2， 或 /和所述纳米结构分布密度大于 1个 /10 μ m2， 或 /和纳米结构区与所述非纳米结构区在固相载体上的投影面积之比大于 2。 33. The nanostructure carrier according to claim 32, wherein the nanostructure region and the solid structure The ratio of the surface area of the phase support is greater than 2, or / and the nanostructure distribution density is greater than 1/10 μm 2 , or / and the projected area of the nanostructure region and the non-nanostructure region on the solid phase support. The ratio is greater than 2.
34、 根据权利要求 32或 33所述的纳米结构载体， 其中所述纳米结构中含有 如权利要求 1一 23之一所述的纳米微粒，且其中所述纳米微粒与所述固相载体之 间通过如权利要求 20所述的连接方式进行连接。  34. The nanostructure carrier according to claim 32 or 33, wherein the nanostructure contains the nanoparticle according to any one of claims 1 to 23, and wherein the nanoparticle and the solid-phase carrier The connection is performed by the connection method according to claim 20.
35、 根据权利要求 32— 34之一所述的纳米结构载体， 其包括下述之一的载 体:分析芯片片基、 分析芯片通道、 酶标板片基、 平面层析试剂条片基、 及层析 凝胶。  35. The nanostructure carrier according to any one of claims 32 to 34, comprising one of the following: an analysis chip substrate, an analysis chip channel, an enzyme plate substrate, a planar chromatography reagent strip substrate, and Chromatography gel.
36、 含权利要求 32— 35之一所述的纳米结构载体的分析或 /和分离装置。 36. An analysis or separation device comprising the nanostructure carrier according to any one of claims 32 to 35.
37、 一种制备如权利要求 32— 36之一所述的纳米结构载体的方法， 其包括 以下步骤： （a)准备所述载体、 纳米微粒、 及任选的连接剂； （b)如有必要， 用 所述连接剂对载体、 或 /和纳米微粒进行活化； （c)将所述纳米微粒的悬浮液与 所述载体接触和进行固定化反应， 固定化反应时所述悬浮液中的所述纳米微粒 的重量 /体积浓度为二百分之一至六万分之一， 或纳米微粒的摩尔浓度为 0.12— 37. A method for preparing a nanostructured carrier according to any one of claims 32 to 36, comprising the following steps: (a) preparing the carrier, nanoparticles, and an optional linker; (b) if If necessary, use the linker to activate the carrier, or nanoparticles, and (c) contact the suspension of the nanoparticles with the carrier and perform an immobilization reaction. The weight / volume concentration of the nano-particles is between 1% and 1 / 60,000, or the molar concentration of the nano-particles is 0.12-
38、根据权利要求 37所述的方法， 其中所述纳米微粒的重量 /体积浓度为二 千分之一至六万分之一， 或纳米微粒的摩尔浓度为 0.12— 3.74 nM。 38. The method according to claim 37, wherein the nanoparticle has a weight / volume concentration of one thousandth to one sixty thousandth, or the nanoparticle has a molar concentration of 0.12 to 3.74 nM.
39、根据权利要求 37所述的方法， 其中所述纳米微粒的重量 /体积浓度为一 万分之一至四万分之一， 或纳米微粒的摩尔浓度为 0.19— 0.75 nM。  39. The method according to claim 37, wherein the weight / volume concentration of the nanoparticles is 1 / 10,000 to 40,000, or the molar concentration of the nanoparticles is 0.19-0.75 nM.
40、 一种标记系统，其含活性试剂 /纳米结构 /分子标记物质复合物，所述活 性试剂 /纳米结构 /分子标记物质复合物为含有活性试剂、 分子标记物质、 纳米 微粒、 及任选的封闭剂的混合物或纯化物， 其中所述活性试剂为赋于所述复合 物反应活性的物质，所述纳米微粒为粒径 1 - 100 nm且本身不是标记物质增强剂 的非磁性无机非金属微粒。  40. A labeling system comprising an active reagent / nanostructure / molecular labeling substance complex, wherein the active reagent / nanostructure / molecular labeling substance complex contains an active reagent, molecular labeling substance, nanoparticles, and optionally A mixture or a purified product of a blocking agent, wherein the active agent is a substance that imparts reactivity to the complex, and the nanoparticles are non-magnetic inorganic non-metal particles having a particle diameter of 1 to 100 nm and which are not themselves labeling substance enhancers. .
41、 根据权利要求 40所述的标记系统， 所述复合物中包含的非磁性无机非 金属微粒包括尺寸 1一 100 nm的氧化物微粒及其衍生物，所述氧化物微粒包括氧 化硅、氧化钛、氧化铝， 所述衍生物包括表面含衍生基团或 /和包被功能有机物 的衍生物。  41. The marking system according to claim 40, wherein the non-magnetic inorganic non-metal particles contained in the composite include oxide particles having a size of 1 to 100 nm and derivatives thereof, and the oxide particles include silicon oxide, oxide Titanium and alumina, the derivatives include derivatives containing derivatizing groups on the surface or / and coating functional organic matter.
42、 根据权利要求 41所述的标记系统， 所述衍生基团包括下述一种或多种 基团： 氨基、 醛基、 环氧基、 氨基肼、 二乙氨基乙基、 二乙基一 （2—羟丙基） 氨乙基、 羧甲基、 磺酸丙基、 巯乙基吡啶基、 硅氧烷基、 硫醇基、 垸基， 所述 功能有机物包括下述一种或多种有机物： 包括聚乙烯吡咯垸酮、 吐温类表面活 性剂在内的表面活性剂， 包括聚氨基酸在内的聚电解质， 包括聚硅氧垸在内的 亲油有机物， 包括葡聚糖衍生物、 琼脂糖衍生物、 纤维素衍生物、 聚丙烯酰胺 在内的离子交换聚合物， 和包括肝素钠、 生物素、 活性素在内的活性物质； 所 述微载体包被衍生物中的微载体包括所述纳米微粒。 42. The labeling system according to claim 41, wherein the derived group comprises one or more of the following groups: amino, aldehyde, epoxy, aminohydrazine, diethylaminoethyl, diethyl- (2-hydroxypropyl) aminoethyl, carboxymethyl, sulfopropyl, mercaptoethylpyridyl, siloxane, thiol, fluorenyl, and the functional organics include one or more of the following Organics: including polyvinylpyrrolidone, Tween-like surface actives Surfactants including surfactants, polyelectrolytes including polyamino acids, lipophilic organics including polysiloxane, including dextran derivatives, agarose derivatives, cellulose derivatives, polyacrylamide Ion exchange polymers, and active substances including heparin sodium, biotin, and active substance; the microcarriers in the microcarrier-coated derivative include the nanoparticles.
43、 根据权利要求 40— 42之一所述的标记系统， 所述复合物中包含的活性 试剂包括抗原、 抗体、 亲和素、 生物素、 单链或多链 DNA、 RNA、 核苷酸、 以 及病毒、 细胞或它们的组成。  43. The labeling system according to any one of claims 40 to 42, wherein the active agent contained in the complex includes an antigen, an antibody, avidin, biotin, single-stranded or multi-stranded DNA, RNA, nucleotides, As well as viruses, cells or their composition.
44、 根据权利要求 40— 43之一所述的标记系统， 所述复合物中包含的分子 标记物质包括下述一种或多种物质： 荧光物质、 化学发光物质、 化学发光催化 剂、 有色金属盐、 染料和颜料。  44. The labeling system according to any one of claims 40 to 43, wherein the molecular labeling substance contained in the composite includes one or more of the following substances: a fluorescent substance, a chemiluminescent substance, a chemiluminescent catalyst, and a non-ferrous metal salt , Dyes and pigments.
45、 一种标记方法， 其至少包括以下步骤： （a)淮备权利要求 40— 44之一 所述的标记系统； （b) 用所述活性试剂 /纳米结构 /分子标记物质复合物进行标 记， 标记时所述活性试剂 /纳米结构 /分子标记物质复合物中的所述纳米微粒的 重量 /体积浓度大于四万分之一， 或纳米微粒的摩尔浓度大于 0.19 nM。  45. A labeling method, comprising at least the following steps: (a) Huai Bei labeling system according to any one of claims 40 to 44; (b) using the active reagent / nanostructure / molecular labeling substance complex for labeling The weight / volume concentration of the nanoparticles in the active reagent / nanostructure / molecular labeling substance complex at the time of labeling is greater than 1 / 40,000, or the molar concentration of the nanoparticles is greater than 0.19 nM.
46、根据权利要求 45所述的方法， 其中所述纳米微粒的重量 /体积浓度大于 千分之一， 或纳米微粒的摩尔浓度大于 7.48 nM。  46. The method of claim 45, wherein the weight / volume concentration of the nanoparticles is greater than one thousandth, or the molar concentration of the nanoparticles is greater than 7.48 nM.
47、根据权利要求 46所述的方法， 其中所述纳米微粒的重量 /体积浓度大于 百分之一， 或纳米微粒的摩尔浓度大于 74.8 nM。  47. The method of claim 46, wherein the weight / volume concentration of the nanoparticles is greater than one hundredth, or the molar concentration of the nanoparticles is greater than 74.8 nM.
48、一种分析芯片检测方法， 其包括以下步骤： （a)提供分析芯片，所述芯片 为权利要求 1一 9之一所述用于多肽分析的装置、或权利要求 25所述的分析装置, 所述装置含所述含有活性纳米微粒的活性载体或所述纳米结构活性载体，且所 述活性载体为多活性载体; (b)将待检测样品与所述芯片中的所述活性载体接触 和反应；或 /和 (c)提供权利要求 40-44之一所述的标记系统，并利用如权利要求 45一 47之一所述的标记方法进行标记。  48. An analysis chip detection method, comprising the following steps: (a) providing an analysis chip, the chip being the device for polypeptide analysis according to any one of claims 1 to 9 or the analysis device according to claim 25 The device contains the active carrier containing active nanoparticles or the nanostructured active carrier, and the active carrier is a multi-active carrier; (b) contacting the sample to be detected with the active carrier in the chip And reaction; or / and (c) providing the marking system according to any one of claims 40 to 44 and using the marking method according to any one of claims 45 to 47 for marking.
49、 一种分析芯片试剂盒， 其包括：权利要求 1一 9之一所述用于多肽分析 的装置、 或权利要求 25所述的分析装置,和权利要求 40— 44之一所述的标记系 统， 其中所述装置为芯片。  49. An analysis chip kit, comprising: the device for peptide analysis according to any one of claims 1 to 9, or the analysis device according to claim 25, and the label according to one of claims 40 to 44. System, wherein the device is a chip.
50、 一种分析芯片试剂盒， 其包括权利要求 1一 9之一所述用于多肽分析的 装置、 或权利要求 25所述的分析装置,其中所述装置为芯片。  50. An analysis chip kit comprising the device for polypeptide analysis according to any one of claims 1 to 9 or the analysis device according to claim 25, wherein the device is a chip.
51、 一种分析芯片试剂盒， 其包括权利要求 40— 44之一所述的标记系统。 51. An analysis chip kit comprising the labeling system according to any one of claims 40 to 44.
52、 一种分析芯片检测方法， 其至少包括以下步骤： （a)提供纳米结构微流 路芯片， 所述纳米结构微流路芯片含纳米结构微通道或 /和纳米结构分离介质， 所述纳米结构微通道含有如权利要求 30— 35之一所述的纳米结构载体， 所述纳 米结构分离介质选自下述一种或多种材料： 如权利要求 30— 35之一所述的纳米 结构载体、 如权利要求 1一 9之一所述 _的含活性纳米微粒的活性载体、 及如权利 要求 10— 24之一所述的纳米结构活性载体； （b)将样品加入所述微流路芯片并进 行分离、 分析。 52. An analysis chip detection method, comprising at least the following steps: (a) providing a nanostructured microfluidic chip, the nanostructured microfluidic chip containing a nanostructured microchannel or / and a nanostructured separation medium, The nanostructured microchannel contains the nanostructured carrier according to any one of claims 30-35, and the nanostructured separation medium is selected from one or more of the following materials: as described in one of claims 30-35 active carrier containing the active support nanostructures nanoparticles, according to one of claim 1 to a 9 _ and nanostructures active carrier according to claim one of 10-24; (b) adding the sample to the micro The flow path chip is separated and analyzed.
53、根据权利要求 52所述的方法， 其中所述纳米结构微通道或 /和纳米结构 分离介质包括下述一种或多种： 纳米结构分子筛、含纳米粒子的微流路涂布物、 含纳米粒子的固定相、 含纳米粒子的流动相。  53. The method according to claim 52, wherein the nanostructured microchannel or / and nanostructured separation medium comprises one or more of the following: a nanostructured molecular sieve, a nanoflow-containing microfluid coating, Nanoparticle stationary phase, nanoparticle-containing mobile phase.
54、 一种分析芯片试剂盒， 其含有如权利要求 52或 53所述的纳米结构微通 道或 /和纳米结构分离介质。  54. An analysis chip kit comprising the nanostructured microchannel or the nanostructured separation medium according to claim 52 or 53.
55、 一种多肽分析的分析芯片检测方法， 其至少包括下述一个、 二个、 三 个或四个步骤：  55. An analysis chip detection method for peptide analysis, which includes at least one, two, three or four steps as follows:
(a)将样品与磁微粒或 /和磁微片混合；  (a) mixing the sample with magnetic particles or / and magnetic flakes;
(b)将样品与活性试剂 /磁纳米微粒复合物混合；  (b) mixing the sample with the active reagent / magnetic nanoparticle complex;
(c)将样品与芯片接触并反应， 反应时可任选存在外加磁场，所述芯片为 为权利要求 1一 9之一所述用于多肽分析的装置、或权利要求 25所述的分析装置， 且其中所述纳米微粒包括磁纳米微粒；  (c) contacting and reacting the sample with the chip, and an external magnetic field may optionally be present during the reaction, and the chip is the device for peptide analysis according to any one of claims 1 to 9 or the analysis device according to claim 25 And wherein the nanoparticles include magnetic nanoparticles;
(d)将活性试剂 /磁纳米微粒 /分子标记物质复合物用于标记反应， 在标记 时可任选存在外加磁场， 所述活性试剂 /磁纳米微粒 /分子标记物质复合物含为 有一种或多种分子标记物质、 一种或多种磁纳米微粒、 一种或多种活性试剂以 及任选的封闭剂的混合物或纯化物；  (d) an active reagent / magnetic nanoparticle / molecular labeling substance complex is used for the labeling reaction, and an external magnetic field may optionally be present during labeling; the active reagent / magnetic nanoparticle / molecular labeling substance complex contains one or A mixture or purification of multiple molecularly labeled substances, one or more magnetic nanoparticles, one or more active agents, and optionally a blocking agent;
其中所述磁纳米微粒在三维空间中至少有一维为 1一 200 nm、 优选 1一 100 nm、更优选 1一 50 nm，且其本身不是分子标记物质增强剂的磁微粒及其衍生物; 所述活性试剂选自以下组中能与多肽作用的物质： 多肽、 多糖、 维生素、 抗生 素、 病毒、 细胞、 及功能有机物， 所述活性试剂 /磁纳米微粒 /分子标记物质复 合物在标记时的磁纳米微粒浓度为：纳米微粒重量 /体积浓度大于三万分之一、 或纳米微粒摩尔浓度大于 0.25nM，优选为纳米微粒重量 /体积浓度大于三千分之 一、 或纳米微粒摩尔浓度大于 2.4nM， 更优选为纳米微粒重量 /体积浓度大于五 百分之一、 或纳米微粒摩尔浓度大于 15.0nM。  Wherein the magnetic nanoparticle has 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, and is not itself a magnetic particle and a derivative of a molecular marker substance enhancer; The active agent is selected from the group consisting of substances capable of interacting with polypeptides: polypeptides, polysaccharides, vitamins, antibiotics, viruses, cells, and functional organics, and the magnetic properties of the active agent / magnetic nanoparticle / molecular labeling substance complex at the time of labeling Nanoparticle concentration is: nanoparticle weight / volume concentration is greater than 1 / 30,000, or nanoparticle molar concentration is greater than 0.25nM, preferably nanoparticle weight / volume concentration is greater than 1 / 3,000th, or nanoparticle molar concentration is greater than 2.4nM More preferably, the nanoparticle weight / volume concentration is greater than one-hundredth, or the nanoparticle molar concentration is greater than 15.0 nM.
56、根据权利要求 55所述的方法，其中所述磁微粒选自于包括四氧化三铁、 三氧化二铁在内的铁氧体及其衍生物， 所述衍生物包括表面含衍生基团的表面 修饰或 /和功能有机物包被衍生物。 56. The method of claim 55, wherein the magnetic particles are selected from the group consisting of ferrite and ferric oxide, and derivatives thereof, and the derivatives include derivatizing groups on the surface Surface-modified or / and functional organic-coated derivatives.
57、 根据权利要求 55或 56所述的方法， 其中所述外加磁场是脉冲式的。57. The method according to claim 55 or 56, wherein the applied magnetic field is pulsed.
58、 根据权利要求 55—.57之一所述的方法， 其中所述其中所述分子标记物 质包括下述一种或多种物质： 荧光物质、 化学发光物质、 化学发光催化剂、 有 色金属盐、 染料和颜料。 58. The method according to any one of claims 55-57, wherein the molecularly labeled substance comprises one or more of the following: a fluorescent substance, a chemiluminescent substance, a chemiluminescent catalyst, a non-ferrous metal salt, Dyes and pigments.
59、一种分析芯片试剂盒，其包括如权利要求 55— 58之一所述的下述一种、 两种、 三种或四种组成： 所述磁微粒或 /和磁微片、 所述活性试剂 /磁纳米微粒 复合物、 所述芯片、 所述活性试剂 /磁纳米微粒 /分子标记物质复合物。  59. An analysis chip kit comprising the following one, two, three or four components according to any one of claims 55 to 58: the magnetic particles or / and magnetic microchips, the An active agent / magnetic nanoparticle complex, the chip, the active agent / magnetic nanoparticle / molecular labeling substance complex.
60、 一种多肽定量或 /和定性检测方法， 其至少包括以下步骤： （a) 提供 权利要求 1一 9之一所述用于多肽分析的装置、或权利要求 25所述的分析装置; (b) 将待检测样品与所述装置中的所述活性载体接触并反应； 或 /和 (c)提供权利要 求 40— 44之一所述的标记系统，并利用如权利要求 45— 47之一所述的标记方法 进行标记； 其中所述活性试剂选自以下组中能与目标多肽作用的物质： 多肽、 多糖、 维生素、 抗生素、 功能有机物、 病毒及细胞及它们的天然组成或合成组 成。  60. A method for quantitative or / and qualitative detection of a polypeptide, comprising at least the following steps: (a) providing a device for polypeptide analysis according to any one of claims 1 to 9, or an analysis device according to claim 25; ( b) contacting the sample to be detected with the active support in the device and reacting; or / and (c) providing a labeling system according to any one of claims 40 to 44 and using one of claims 45 to 47 The labeling method performs labeling; wherein the active agent is selected from the group of substances that can interact with the target polypeptide: polypeptides, polysaccharides, vitamins, antibiotics, functional organics, viruses, and cells, and their natural or synthetic composition.
61、 根据权利要求 60所述的方法， 其中所述检测包括分析芯片检测、 酶标 检测、 和平面层析检测。  61. The method according to claim 60, wherein the detection comprises an analysis chip detection, an enzyme label detection, and a planar chromatography detection.
62、一种多肽定量或 /和定性检测试剂盒， 其包括如权利要求 1一 9之一所述 用于多肽分析的装置、 或权利要求 25所述的分析装置，或 /和权利要求 40— 44之 一所述的标记系统。  62. A polypeptide quantitative or / and qualitative detection kit comprising the device for polypeptide analysis according to any one of claims 1 to 9, or the analysis device according to claim 25, or / and claim 40- The marking system of one of 44.
63、 根据权利要求 62所述的试剂盒， 其为分析芯片试剂盒、 酶标试剂盒或 平面层析试剂盒。  63. The kit according to claim 62, which is an analysis chip kit, an enzyme labeling kit, or a flat chromatography kit.
64、 一种分离方法， 其包括使用选自下述一种或多种材料的分离介质： 如 权利要求 32— 36之一所述的纳米结构载体、 如权利要求 1一 9之一所述的含活性 纳米微粒的活性载体、 及如权利要求 10— 24之一所述的纳米结构活性载体。  64. A separation method comprising using a separation medium selected from one or more of the following materials: a nanostructured carrier according to any one of claims 32 to 36, or a nanostructured carrier according to any one of claims 1 to 9. An active carrier containing active nanoparticles, and the nanostructured active carrier according to any one of claims 10 to 24.
65、 根据权利要求 64所述的方法， 其中所述分离介质包括层析凝胶。  65. The method of claim 64, wherein the separation medium comprises a chromatography gel.
PCT/CN2004/000203 2003-03-13 2004-03-15 Device for analysis or separation containing an active nanostructured carrier, its preparation method and applications WO2004090548A1 (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP2006529552A JP2007502998A (en) 2003-04-30 2004-04-30 Apparatus for analyzing or separating nanometer structures, and its fabrication method and application
PCT/CN2004/000437 WO2004102196A1 (en) 2003-04-30 2004-04-30 Apparatus including nanostructures used for separation or analysis, and the preparation and application thereof
EP04730459A EP1624306A4 (en) 2003-04-30 2004-04-30 Apparatus including nanostructures used for separation or analysis, and the preparation and application thereof
US11/258,996 US7842515B2 (en) 2003-04-30 2005-10-27 Nano-structured device for analysis or separation, and its preparation and application

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
CN03117446.9 2003-03-13
CN 03117446 CN1250969C (en) 2003-03-13 2003-03-13 Test device and method for making qualitative and/or quantitative analysis to object
CN03117787.5 2003-04-30
CNA031177875A CN1514243A (en) 2003-04-30 2003-04-30 Method of preceeding qualitative and lor quantitative analysis against target substance its device and marker and detecting reagent box
CNPCT/CN04/00077 2004-01-20
PCT/CN2004/000077 WO2004081571A1 (en) 2003-03-13 2004-01-20 The detecting method and device of polypeptide, and the ligand compound comprising nanoparticles

Related Child Applications (2)

Application Number Title Priority Date Filing Date
PCT/CN2004/000077 Continuation WO2004081571A1 (en) 2003-03-13 2004-01-20 The detecting method and device of polypeptide, and the ligand compound comprising nanoparticles
US11/258,996 Continuation US7842515B2 (en) 2003-04-30 2005-10-27 Nano-structured device for analysis or separation, and its preparation and application

Publications (1)

Publication Number Publication Date
WO2004090548A1 true WO2004090548A1 (en) 2004-10-21

Family

ID=33162518

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2004/000203 WO2004090548A1 (en) 2003-03-13 2004-03-15 Device for analysis or separation containing an active nanostructured carrier, its preparation method and applications

Country Status (1)

Country Link
WO (1) WO2004090548A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101216458B (en) * 2008-01-09 2011-04-20 浙江大学 Sampling volume controllable micro-fluidic chip sieving electrophoresis analytical method

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6025202A (en) * 1995-02-09 2000-02-15 The Penn State Research Foundation Self-assembled metal colloid monolayers and detection methods therewith
US6268222B1 (en) * 1998-01-22 2001-07-31 Luminex Corporation Microparticles attached to nanoparticles labeled with flourescent dye
US20010053521A1 (en) * 1999-09-27 2001-12-20 Kreimer David I. Amplification of analyte detection by substrates having particle structures with receptors
WO2002031191A2 (en) * 2000-10-12 2002-04-18 Yissum Research Development Company Of The Hebrew University Of Jerusalem Dendritically amplified detection method
CN1400467A (en) * 2002-08-30 2003-03-05 厦门大学 Fluorescence rare earth complex silicone nano granules labeling compound and its preparation method
US20030044805A1 (en) * 1996-07-29 2003-03-06 Nanosphere, Inc. Nanoparticles having oligonucleotides attached thereto and uses therefor
US20030207296A1 (en) * 1996-07-29 2003-11-06 So-Jung Park Nanoparticles having oligonucleotides attached thereto and uses therefor

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6025202A (en) * 1995-02-09 2000-02-15 The Penn State Research Foundation Self-assembled metal colloid monolayers and detection methods therewith
US20030044805A1 (en) * 1996-07-29 2003-03-06 Nanosphere, Inc. Nanoparticles having oligonucleotides attached thereto and uses therefor
US20030207296A1 (en) * 1996-07-29 2003-11-06 So-Jung Park Nanoparticles having oligonucleotides attached thereto and uses therefor
US6268222B1 (en) * 1998-01-22 2001-07-31 Luminex Corporation Microparticles attached to nanoparticles labeled with flourescent dye
US20010053521A1 (en) * 1999-09-27 2001-12-20 Kreimer David I. Amplification of analyte detection by substrates having particle structures with receptors
WO2002031191A2 (en) * 2000-10-12 2002-04-18 Yissum Research Development Company Of The Hebrew University Of Jerusalem Dendritically amplified detection method
CN1400467A (en) * 2002-08-30 2003-03-05 厦门大学 Fluorescence rare earth complex silicone nano granules labeling compound and its preparation method

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101216458B (en) * 2008-01-09 2011-04-20 浙江大学 Sampling volume controllable micro-fluidic chip sieving electrophoresis analytical method

Similar Documents

Publication Publication Date Title
US7842515B2 (en) Nano-structured device for analysis or separation, and its preparation and application
AU762294B2 (en) Force discrimination assay
JP2007502998A5 (en)
US7232691B2 (en) Bioassay and biomolecular identification, sorting, and collection methods using magnetic microspheres
EP1558750A1 (en) Microparticle-based methods and systems and applications thereof
JP5401724B2 (en) Biosensing method using coated magnetic fine particles and biosensing device used in the method
JP2004525345A (en) Immobilized microarray of biological or chemical probes immobilized on a support by magnetic force
JP2006330000A (en) Improved biochip
EP2856162B1 (en) Microplates with enhanced immobilisation capabilities controlled by magnetic field
WO2003046507A2 (en) Apparatus used in identification, sorting and collection methods using magnetic microspheres and magnetic microsphere kits
WO2007115444A1 (en) Seperation or analysis composition comprising active nanostructure and seperation or analysis method
WO2004090548A1 (en) Device for analysis or separation containing an active nanostructured carrier, its preparation method and applications
WO2004102196A1 (en) Apparatus including nanostructures used for separation or analysis, and the preparation and application thereof
WO2004081571A1 (en) The detecting method and device of polypeptide, and the ligand compound comprising nanoparticles
EP1336104B1 (en) Proteomics based drug comparison methods
US20070087382A1 (en) Molecule array and method for producing the same
CN1250969C (en) Test device and method for making qualitative and/or quantitative analysis to object
CA2100683A1 (en) Scanning probe microscopy immunoassay
CN101013128A (en) Analysis or separation facility containing nano structure
JP2004177348A (en) Method for detecting nucleic acid or protein with ultra-high sensitivity, detecting agent, and detection apparatus
Marquette et al. Beads arraying and beads used in DNA chips
US20050064446A1 (en) Detection of binding species with colloidal and non-colloidal structures
Wang et al. Microarray-Based Study of Carbohydrate–Protein Binding
Cretich et al. Peptide microarrays on coated silicon slides for highly sensitive antibody detection
CN101443661B (en) Active constituent, nanostructure composition containing the active constituent and preparation method thereof

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 11258996

Country of ref document: US

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 69(1) EPC

WWP Wipo information: published in national office

Ref document number: 11258996

Country of ref document: US

122 Ep: pct application non-entry in european phase