CN101443661B - Active constituent, nanostructure composition containing the active constituent and preparation method thereof - Google Patents

Active constituent, nanostructure composition containing the active constituent and preparation method thereof Download PDF

Info

Publication number
CN101443661B
CN101443661B CN2006800516076A CN200680051607A CN101443661B CN 101443661 B CN101443661 B CN 101443661B CN 2006800516076 A CN2006800516076 A CN 2006800516076A CN 200680051607 A CN200680051607 A CN 200680051607A CN 101443661 B CN101443661 B CN 101443661B
Authority
CN
China
Prior art keywords
activation
nanostructured
nano
carrier
group
Prior art date
Legal status (The legal status 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 status listed.)
Expired - Fee Related
Application number
CN2006800516076A
Other languages
Chinese (zh)
Other versions
CN101443661A (en
Inventor
邹方霖
陈春生
王建霞
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Chengdu Kuachang Science and Technology Co Ltd
Original Assignee
Chengdu Kuachang Medical Industrial 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
Application filed by Chengdu Kuachang Medical Industrial Ltd filed Critical Chengdu Kuachang Medical Industrial Ltd
Priority to CN2006800516076A priority Critical patent/CN101443661B/en
Priority claimed from PCT/CN2006/002659 external-priority patent/WO2007085156A1/en
Publication of CN101443661A publication Critical patent/CN101443661A/en
Application granted granted Critical
Publication of CN101443661B publication Critical patent/CN101443661B/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Abstract

A nanostructured composition is provided, which includes at least one or more than one sets of such functionalized nanostructures comprising active components and functionalized components: A. functionalized directional nanostructured zone; B. functionalized nondirectional nanostructured zone 1; C. functionalized nondirectional nanostructured zone 2; D. functionalized nanoparticles. In the functionalized nanostructured zone, functionalized nanoparticles or nanostructured protuberant particles for immobilizing functionalized reagents have the average distribution density of more than 10/[mu]m<2>. In said active components, active structures include one or more than one such groups: immobilizing coating or/and one or more than one covalent bonding active groups: amino acid groups, amino hydrazine groups, amino hydrazine derivative groups. Due to the active components, the nanostructured compositions of the present invention have higher reaction efficiency.

Description

Active constituent, the nanostructured that contains active constituent are formed and preparation method thereof
Technical field
The present invention relates to a kind of active constituent, the nanostructured that particularly contains active constituent is formed, with and preparation method thereof.
Background technology
The present invention is the continuation of International Patent Application PCT/CN2004/000437.
Exploitation and application thereof that the separation of nanostructure-containing or analysis are formed; Increasing scheme is disclosed; For example: U.S. Patent application 20030207296, United States Patent (USP) 6025202, International Patent Application WO 0183825, International Patent Application WO 00/72018A1, U.S. Patent application 20030211488, U.S. Patent application 20030232388, U.S. Patent application 20030166297, U.S. Patent application 20020142480, United States Patent (USP) 6986989; And following document: Brust et al.; Adv.Mater., 7,795-797 (1995); Bain&Whitesides, Angew.Chem.Int.Ed.Engl., 28,506-512 (1989); Dubois&Nuzzo, Annu.Rev.Phys.Chem., 43,437-464 (1992).
Yet present nanostructured chemistry still is confined within several kinds of limited activation structures, and made function nano structure aspect reaction efficiency (for example sensitivity) and stability (especially biological stability), all waits improvements.And, more alternative activation structures are provided, also be the multifarious requirement that nanostructured is formed thing.
Summary of the invention
Fundamental purpose of the present invention is: provide a kind of new nanostructured with higher reaction efficiency to form.The objective of the invention is to realize through the continuation of International Patent Application PCT/CN2004/000437.This continues to comprise: 1). the selection that nanostructured is formed: the functionalized nano structural area must have a minimum distribution density of nano structured unit, and its nanometer response effect makes the nanostructured composition reach the object of the invention; 2). this choice criteria, not only be suitable for functionalization non-directional nanostructured district, also be suitable for functionalized directional nanostructured zone; 3). this choice criteria, not only be suitable for known activation structure, also be suitable for new activation structure.In fact; The present invention also comes from the more following of the embodiment of the invention beat all result relevant with the activation structure: 1). usually as the amino acid of passivator during, but the activated group of reactive group, particularly immobilization of biological substances can be provided unexpectedly as the derivating agent of carrier; 2). usually as the coating of reactor separant, also can be used as the activation structure of carrier, come effective immobilization of biological substances; 3). amino hydrazine not only can be used as the active agent of conventional carrier, and can be used as the active agent of nano-carrier, and the character that exists an amino diazanyl distribution density that the activation nano-carrier is had clear improvement; 4). above-mentioned activation structure can and be used.
So; First aspect of the present invention; Provide a kind of nanostructured to form; It is characterized in that: comprise one of the following group that formed by active constituent combined function component or multiple function nanostructured: A, functionalized directional nanostructured zone at least, its contained function component comprises function reagent or/and the functionalized nano particle, and its contained active constituent comprises activation oriented nano structure carrier; And said activation oriented nano structure carrier comprises the activation structure and contains the directional nano carrier that aligns the nanometer convex body, and wherein said activation structure comprises coating material solidified at least or/and the following activated group group of one or more of covalent bonding: comprise amino acid based group, amino diazanyl and amino hydrazine derivate base; B, functionalization non-directional nanostructured district 1; Its contained function component comprises function reagent or/and the functionalized nano particle; Its contained active constituent comprises activation non-directional nanostructured carrier; And said activation non-directional nanostructured carrier comprises activation structure and the non-directional nano-carrier that contains non-directional arrangement nanometer convex body; Wherein said activation structure comprises coating material solidified at least or/and the following activated group group of one or more of covalent bonding: comprise amino acid based group, amino diazanyl and amino hydrazine derivate base, and said amino diazanyl is at 1m 2The lip-deep density of said nanometer convex body is greater than 1 μ mol; C, functionalization non-directional nanostructured district 2; Its contained function component comprises the functionalized nano particle; Contained active constituent comprises activation non-nano structure carrier; And said activation non-nano structure carrier comprises non-nano carrier and activation structure, and wherein said activation structure comprises coating material solidified at least or/and the following activated group group of one or more of covalent bonding: comprise amino acid based group and amino hydrazine derivate base; D, functionalized nano particle, it can participate in forming functionalized nano structural area or other nanostructured, and its contained function component comprises function reagent, and contained active constituent is the activation nano particle; Wherein said activation nano particle comprises nano particle and activation structure; Said activation structure comprises coating material solidified at least or/and the following activated group group of one or more of covalent bonding: comprise amino acid based group, amino diazanyl and amino hydrazine derivate base, and said amino diazanyl is at 1m 2Density on the said nanoparticle surface is greater than 1 μ mol activation nano particle; And in above-mentioned A, B, C, described each the functionalized nano structural area of D, said functionalized nano particle or be fixed with function reagent said nanometer convex body be evenly distributed density greater than 10/μ m 2The example of each functionalized nano structural area, as: said functionalized directional nanostructured zone, said functionalization non-directional nanostructured district 1, said functionalization non-directional nanostructured district 2 or said functionalized nano structural area.
Second aspect of the present invention; Provide and can be used for preparing a kind of active constituent that nanostructured of the present invention is formed; Said active constituent comprises one of following group or many groups: A. activation oriented nano structure carrier; Said activation oriented nano structure carrier comprises the activation structure and contains the directional nano carrier that aligns the nanometer convex body, and wherein said activation structure comprises coating material solidified at least or/and the following activated group group of one or more of covalent bonding: comprise amino acid based group, amino diazanyl and amino hydrazine derivate base; B. activation non-directional nanostructured carrier; Said activation non-directional nanostructured carrier comprises activation structure and the non-directional nano-carrier that contains non-directional arrangement nanometer convex body; Wherein said activation structure comprises coating material solidified at least or/and the following activated group group of one or more of covalent bonding: comprise amino acid based group, amino diazanyl and amino hydrazine derivate base, and said amino diazanyl is at 1m 2The lip-deep density of said nanometer convex body is greater than 1 μ mol; C. activation non-nano structure carrier; Said activation non-nano structure carrier comprises non-nano carrier and activation structure, and wherein said activation structure comprises coating material solidified at least or/and the following activated group group of one or more of covalent bonding: comprise amino acid based group and amino hydrazine derivate base; D. activation nano particle; Wherein said activation nano particle comprises nano particle and activation structure; Wherein said activation structure comprises coating material solidified at least or/and the following activated group group of one or more of covalent bonding: comprise amino acid based group, amino diazanyl and amino hydrazine derivate base, and said amino diazanyl is at 1m 2Density on the said nanoparticle surface is greater than 1 μ mol activation nano particle; And on said activation oriented nano structure carrier or said activation oriented nano structure carrier, have an activation directional nanostructured zone or an activation non-directional nanostructured district at least with following characteristics: said nanometer convex body therein be evenly distributed density greater than 10/μ m 2
The 3rd aspect of the present invention, the preparation method who provides nanostructured of the present invention to form comprises following a kind of or plurality of step: (1). use synthetic peptide method that said activated group is fixed on said nanometer convex body or/and on the said nano particle; (2). said coating is encapsulated to said oriented nano structure carrier, non-directional nanostructured carrier or non-nano carrier; (3). said coating is encapsulated to said oriented nano structure carrier, non-directional nanostructured carrier or non-nano carrier, introduce activated group encapsulating on the coating successively to introduce coupling group and activated group or to encapsulate on the coating then.
The detailed description of summary of the invention
Term definition
Among the present invention, term " nanostructured " is meant such structure, in its structural unit that comprises, comprises the object (for example nano wire, nanotube, nanocone, nano particle, or the like) of nano-scale; Term " nano particle " is meant that particle diameter is the particle of nano-scale (preferred 3nm-300nm); Term " nanometer convex body " is meant the nanometer ridge on the solid surface; In the said nanometer ridge three-dimensional structure at least one dimension (for example particle diameter, caliber, line footpath, or the like) be nano-scale, for example: immobilization nano particle, immobilization nanotube, immobilization nanofiber, and the nanostructured of the self assembly formation of nano particle on carrier, or the like.
Among the present invention: comprise (only comprise or not only comprise) oriented nano structure unit and nano structured unit distribution density and reach 1/μ m 2Above nanostructured carrier is called for short the oriented nano structure carrier; Do not comprise the oriented nano structure unit but non-directional nano structured unit distribution density reaches 1/μ m 2Above nanostructured carrier is called for short non-directional nanostructured carrier; Nano structured unit surface distributed density is less than 1/μ m 2Carrier, be called for short the non-nano structure carrier.
Among the present invention, term " activation structure " is meant a kind of like this composition, its be artificially introduce on the carrier surface and the effective structure of combined function reagent.The chemical activation structure contains activated group at least, also can contain coupling group.Term " activated group " is meant in order to the group (for example amino acid) or the compound group (for example amino acid derivativges, synthetic peptidyl, synthetic peptide derivant base) of the group that combines with function reagent (for example amino, carboxyl, or the like) to be provided; Term " activation nanostructured " is meant a kind of like this composition, its nanostructure-containing and its activation structure of go up fixing, for example activation nano particle, activation nanometer convex body, activation nano structured unit, or the like.Among the present invention, term " coupling group " is meant that combination (the for example covalent bonding of hydroxyl reaction on silane coupling agent and the nanostructured surface) is on nanostructured surface, in order to the fixing group of activated group; Term " coupling nanostructured " is meant the structure of the coupling group that nanostructured and its go up to be fixed, for example coupling nano particle.
Among the present invention, term " active constituent " is meant that part that contains the activation structure in the nanostructured composition thing.Term " activation nanostructured carrier " is meant the nanostructure-containing carrier and the effective active constituent of the activation structure of fixed function reagent.Term " chemical activation component " is meant that the activation structure comprises the active constituent through the activated group of direct or indirect (for example the passing through coupling group) bonding of covalent bond.Term " coating active constituent " is meant that the activation structure comprises coating material solidified active constituent.Term " many active constituents " is meant the active constituent that comprises multiple activation structure (for example coating and activated group).Term " many region of activations component " is meant the active constituent that comprises multiple component structure (for example directional nanostructured zone and non-nano structural area).
Among the present invention, term " functionalized nano structure " is meant a kind of like this composition, its nanostructure-containing and its function reagent of go up fixing, for example functionalized nano particle, functionalized nano convex body, or the like.Among the present invention, term " function reagent " is meant to giving the reagent of said nanostructured with reactivity (for example with the object reactive activity).Function reagent is caught object through interact (comprise affinity interaction, ion-exchange, oleophilic function, or the like), it comprise aglucon (being equivalent to the Ligand in the English), ion exchanger, or the like.Aglucon is for example: the aptamer molecule of antigen, antibody, part, part index enhanced system evolution technology screening, polypeptide, polysaccharide, enzyme, co-factor, microbiotic, steroids, virus, cell etc. altogether.
Among the present invention, term " functionalized nano structure carrier " is meant a kind of like this material, and it contains on carrier and the carrier fixing functionalized nano structure (for example functionalized nano particle, functionalized nano convex body, or the like) at least; Term " sheet base " be meant its have fixed function, one side has the conventional carrier on macroscopical plane, for example: analysis chip sheet base, ELISA Plate sheet base, electrophoresis film, planar chromatography carrier etc.
Among the present invention, term " nanostructured composition " is meant the composition that contains the functionalized nano structure that is useful on reaction (for example analyze or separate and form), for example the device of nanostructure-containing probe or kit, nanometer label, or the like; " device " is meant the articles for use that contain the reactor with specific function, for example, analysis chip, ELISA Plate, affinity electrophoresis bar, affinity column, planar chromatography reagent strip, or the like.
Among the present invention; Term " analysis chip " abbreviates " chip " as; Include but not limited to Biochip, Microarray, Bioarray in the English; Be meant a kind of pick-up unit in qualitative and/or the quantitative test, micro-function reagent can be discerned with addressable mode with the result that the target molecule in the sample reacts in its reactor; Term " nanometer analysis chip " (abbreviation nano chips) is meant a kind of like this analysis chip, and wherein at least one reactor is a nano-reactor, has a nano-probe point in the said nano-reactor at least.The nano-probe point comprises the functionalized nano structural area, has functionalized nano structure (for example function reagent/activation nanometer convex body).In nano-reactor, can be that whole probe points all are the nano-probe points, also can be the part probe points is the nano-probe point.On the nanometer analysis chip; Can be that only the nano-probe point has nanostructured and other zone does not have nanostructured (for example fixed function nano particle former on the conventional sheet base of activation), also can be nano-probe point and other zone at least partly all has nanostructured (for example fixed function nano particle former on activation nanometer convex body sheet base).The present invention is suitable for various chips certainly, for example single reaction chamber chip, many reaction tanks chip, the chip that flows, non-current chip, or the like.
Among the present invention, term " chromatography " is equivalent to English " Chromatography ", comprise affinity chromatography, reversed phase chromatography, hydrophobic chromatography, ion-exchange chromatography, or the like, it is divided into planar chromatography (for example fast check reagent bar and fast check reagent box) and column chromatography etc.
Among the present invention; Term " polypeptide " is equivalent to " polypeptide " in the English; Comprise natural or synthetic protein, protein fragments, synthetic peptide, or the like, in the immune detection common object with detect in general aglucon, for example antigen, antibody, or the like all belong to polypeptide; Term " molecular labeling material " is meant in order to the material that forms or participate in forming detecting signal and have molecular conformation when the mark, the for example rhodamine in the chip detection label commonly used, CY3, CY5 etc.
First aspect of the present invention provides like " summary of the invention " described nanostructured and forms, and it comprises following nanostructured composition.
First kind of nanostructured of the present invention formed, and it comprises at least one functionalized directional nanostructured zone at least, and its contained function component comprises function reagent or/and the functionalized nano particle, and its contained active constituent comprises activation oriented nano structure carrier; And said activation oriented nano structure carrier comprises the activation structure and contains the directional nano carrier that aligns the nanometer convex body; Wherein said activation structure comprises coating material solidified at least or/and the following activated group group of one or more of covalent bonding: comprise amino acid based group, amino diazanyl and amino hydrazine derivate base; And wherein at least one said functionalized directional nanostructured zone, said functionalized nano particle or be fixed with function reagent said nanometer convex body be evenly distributed density greater than 10/μ m 2Amino diazanyl in we previous International Patent Application PCT/CN2004/000437, is used as the activated group of activation non-directional nanostructured carrier, and we are with its activated group as activation oriented nano structure carrier here.A kind of first kind of nanostructured of the present invention formed, and wherein said nanometer convex body comprises one of following group: nano wire, nanotube, nanocone.
Second kind of nanostructured of the present invention formed; It comprises at least one functionalization non-directional nanostructured district 1 at least; Its contained function component comprises function reagent or/and the functionalized nano particle; Its contained active constituent comprises activation non-directional nanostructured carrier; And said activation non-directional nanostructured carrier comprises activation structure and the non-directional nano-carrier that contains non-directional arrangement nanometer convex body, and wherein said activation structure comprises coating material solidified at least or/and the following activated group group of one or more of covalent bonding: comprise amino acid based group, amino diazanyl and amino hydrazine derivate base, and said amino diazanyl is at 1m 2The lip-deep density of said nanometer convex body is greater than 1 μ mol, and wherein at least one said functionalization non-directional nanostructured district, said functionalized nano particle or be fixed with function reagent said nanometer convex body be evenly distributed density greater than 10/μ m 2Although in we previous International Patent Application PCT/CN2004/000437; Amino diazanyl has been used as the activated group of activation non-directional nanostructured carrier; Following examples will explain that amino diazanyl is a principal character of decision reaction efficiency in the lip-deep density of said nanometer convex body.
The third nanostructured of the present invention is formed; It comprises at least one functionalization non-directional nanostructured district 2 at least; Wherein contained function component comprises the functionalized nano particle; Contained active constituent comprises activation non-nano structure carrier; And said activation non-nano structure carrier comprises non-nano carrier and activation structure; Wherein said activation structure comprises coating material solidified at least or/and the following activated group group of one or more of covalent bonding: comprise amino acid based group and amino hydrazine derivate base, and wherein at least one said functionalization non-directional nanostructured district, said functionalized nano particle or be fixed with function reagent said nanometer convex body be evenly distributed density greater than 10/μ m 2
The 4th kind of nanostructured of the present invention formed; It comprises the functionalized nano particle at least; Said functionalized nano particle can participate in forming functionalized nano structural area or other nanostructured, and its contained function component comprises function reagent, and contained active constituent is the activation nano particle; Wherein said activation nano particle comprises nano particle and activation structure; Said activation structure comprises coating material solidified at least or/and the following activated group group of one or more of covalent bonding: comprise amino acid based group, amino diazanyl and amino hydrazine derivate base, and said amino diazanyl is at 1m 2Density on the said nanoparticle surface is greater than 1 μ mol activation nano particle, and in above-mentioned functions nanostructured district, said functionalized nano particle be evenly distributed density greater than 10/μ m 2The functionalized nano particle.
The 5th kind of nanostructured of the present invention formed, and it comprises the different functions district more than two or two, and wherein: at least one said functional zone is the functionalized nano structural area; With at least one said functionalized nano structural area be one of said each functionalized nano structural area (for example: said functionalized directional nanostructured zone, said functionalization non-directional nanostructured district 1, said functionalization non-directional nanostructured district 2 or said functionalized nano structural area).
Different functions nanostructured district; Said different functions nanostructured district comprises different activated district and function reagent or/and the functionalized nano particle; Wherein said different region of activation is provided by many region of activations nanostructured carrier, comprises an activation nanostructured district on the nanostructured carrier of said many region of activations at least and comprises following group one or more multiple: (1).; (2). said activation non-directional nanostructured district; (3). said activation non-nano structural area.
The 6th kind of nanostructured of the present invention formed, and is a kind of kit that contains nanodevice and nanometer Mk system.A kind of the 6th kind of nanostructured of the present invention formed, and wherein nanodevice is that the present invention first is to one of the 5th kind of nanostructured composition; The nanometer Mk system is formed for the 4th kind of nanostructured of the present invention.
A kind of nanostructured of the present invention is formed, and wherein saidly comprises amino acid based group and comprises amino acid based or/and the amino acid derivativges base.During nanostructured of the present invention is formed, saidly comprise amino acid based group and do not comprise and be fixed to the anti-propylhomoserin that gathers on the component through covalent bonding.Gather anti-propylhomoserin and encapsulate slide come to light serious heterogeneity and instability.A kind of nanostructured of the present invention is formed, and wherein saidly comprises that amino acid based group comprises synthetic peptide group or/and synthetic peptide derivant base.A kind of nanostructured of the present invention is formed, and wherein said synthetic peptide comprises and contains 2-10 amino acid whose synthetic peptide.Amino acid kind wherein identical (for example arginine, N, glutaminase, or the like the monamino acid peptidyl that forms) or different (for example arginine and N, N and glycocoll, glutaminase and lysine, or the like the amino acids peptidyl of formation).A kind of nanostructured of the present invention is formed, and wherein said amino acid comprises one of following group: arginine, N, glutaminase, glycocoll, lysine, glutamine.
In the embodiment of the invention, said activation structure also contains the coupling group that connects said carrier and activated group, and said coupling group comprises silane group.Used silane coupling agent comprises: 3-aminopropyl trimethoxysilane, aminopropyl triethoxysilane, 3-isocyanates propyl-triethoxysilicane.
A kind of nanostructured of the present invention is formed, and wherein said coating comprises water resistant coating.A kind of nanostructured of the present invention is formed, and wherein said water resistant coating is selected from elementary organic paint.A kind of nanostructured of the present invention is formed, and wherein said elementary organic paint is selected from and contains organosilyl high-molecular coating.A kind of nanostructured of the present invention is formed, and wherein said organosilicon comprises the organosilicon based on following organic silicon monomer: dimethyl siloxane, methacrylic acid 3-(methyl silicane), the dimethyl siloxane that contains (methacrylic acid acyl-oxygen) propyl group terminal and 3-aminopropyl trimethoxysilane, aminopropyl triethoxysilane, 3-isocyanates propyl-triethoxysilicane.
A kind of nanostructured of the present invention is formed, and wherein said activation structure comprises activated group fixing on said coating and the said coating.A kind of nanostructured of the present invention is formed, and wherein said activated group comprises one of following group at least: aldehyde radical, epoxy radicals, amino diazanyl, said amino diazanyl derivant and said comprises amino acid based group.
A kind of nanostructured of the present invention is formed, and on wherein said activation oriented nano structure carrier, activation non-directional nanostructured carrier or the activation non-nano structure carrier, also there is the zone that contains other activation structure in the existing zone that contains said activation structure.For example, a kind of chip slapper base of the present invention, existing chemical activation district on it (for example amino region of activation), again coating of the present invention region of activation arranged.
A kind of first, second or the third nanostructured of the present invention is formed; Wherein said functionalized nano particle; Comprise function reagent fixing on activation nano particle and the activation nano particle; Wherein said activation nano particle comprises activation structure fixing on nano particle and the nano particle, and said activation structure can be, also can not be the activation structure described in the functionalized nano particle of the present invention.
A kind of nanostructured of the present invention is formed, and wherein said nanometer convex body comprises one of following group: immobilized nano wire, nanotube, nanocone, nano particle, or the like.A kind of nanostructured of the present invention is formed, and wherein said nanometer convex body or nano particle contain inorganics.A kind of nanostructured of the present invention is formed, and wherein said inorganics comprises metal, slaine.A kind of nanostructured of the present invention is formed, and wherein said slaine comprises one of following group: monox, titanium dioxide, aluminium oxide.
A kind of nanostructured of the present invention is formed, and wherein said function reagent comprises biological substance.A kind of nanostructured of the present invention is formed, and wherein said biological substance comprises polypeptide and nucleic acid.A kind of nanostructured of the present invention is formed, and wherein said polypeptide comprises antigen and the antibody that is fixed in the same reactor.
A kind of first kind of nanostructured of the present invention formed; Of following examples, wherein said activation oriented nano structure carrier comprises one of following group: said activated group/coupling group/oriented nano structure carrier, the activation nano particle/coupling group/oriented nano structure carrier that comprises said activated group, said activated group/coupling nano particle/oriented nano structure carrier, the activation nano particle/coupling nano particle/oriented nano structure carrier that comprises said activated group, said activated group/nano particle/oriented nano structure carrier, the activation nano particle/nano particle/oriented nano structure carrier that comprises said activated group, the activation nano particle/oriented nano structure carrier that comprises said activated group, the activation oriented nano structure carrier, coating activation oriented nano structure carrier, the coating/chemical activation oriented nano structure carrier that comprise the said activated group of the activation nano particle of said activated group/comprise, the activation nano particle/coating that comprises said activated group encapsulate the oriented nano structure carrier.A kind of first kind of nanostructured of the present invention formed, and it comprises one of following nanodevice group that contains said activation oriented nano structure carrier: analysis chip, ELISA Plate, biology sensor.
A kind of second kind of nanostructured of the present invention formed; Of following examples, wherein said activation non-directional nanostructured carrier comprises one of following group: main activated group/coupling group/non-directional nanostructured carrier, the activation nano particle/coupling group/non-directional nanostructured carrier that comprises said activated group, main activated group/coupling nano particle/non-directional nanostructured carrier, the activation nano particle/coupling nano particle/non-directional nanostructured carrier that comprises said activated group, the activation nano particle/non-directional nanostructured carrier that comprises said activated group, the activation non-directional nanostructured carrier, coating activation non-directional nanostructured carrier, the coating/chemical activation non-directional nanostructured carrier that comprise the said activated group of the activation nano particle of said activated group/comprise, the activation nano particle/coating that comprises said activated group encapsulate non-directional nanostructured carrier.A kind of second kind of nanostructured of the present invention formed, and it comprises one of following nanodevice group that contains said activation non-directional nanostructured carrier: analysis chip, ELISA Plate, biology sensor.
A kind of the third nanostructured of the present invention is formed; Of following examples, wherein said activation non-nano structure carrier comprises one of following group: said activated group/coupling group/non-nano structure carrier, coating activation non-nano structure carrier, coating/chemical activation non-nano structure carrier.A kind of the third nanostructured of the present invention is formed, and said non-nano structure comprises by one of following group or organizes in that the material or derivatives thereof is processed, the three-dimensional at least two-dimensional greater than the carrier of 1000nm more: glass, silicon chip, silica gel, pottery, metal oxide, metal, polymeric material and their compound.They comprise one of following group: bead-type substrate (for example chromatography gel, particularly micro particles chromatography gel), planar carrier (the sheet bases of biological example chip, ELISA Plate etc.), and membrane carrier (for example planar chromatography bar).A kind of the third nanostructured of the present invention is formed, and it comprises one of following nanodevice group that contains said activation non-nano structure carrier and functionalized nano particle: analysis chip, ELISA Plate, biology sensor, pick up reagent strip, affinity chromatography glue soon.
A kind of the 4th kind of nanostructured of the present invention formed, and wherein said functionalized nano structure carrier comprises and contains one of said following nanodevice group that contains said functionalized nano particle: analysis chip, ELISA Plate, biology sensor, pick up reagent strip, affinity chromatography glue soon.A kind of the 4th kind of nanostructured of the present invention formed, and it comprises the nanometer Mk system, and said nanometer Mk system comprises said function reagent and labelled reagent fixing on said activation nano particle, the activation nano particle at least.Said Mk system comprises one of following group: analysis chip Mk system, ELISA Plate Mk system, planar chromatography reagent strip Mk system.
A kind of the 5th kind of nanostructured of the present invention formed, and it comprises following group two kinds at least: the functionalization non-directional nanostructured district described in functionalization non-directional nanostructured district described in the functionalization non-directional nanostructured district described in the functionalized directional nanostructured zone described in above-mentioned first kind of nanostructured formed, above-mentioned second kind of nanostructured are formed, above-mentioned the third nanostructured are formed and above-mentioned the 4th kind of nanostructured are formed.A kind of the 5th kind of nanostructured of the present invention formed, and can be a kind of nanostructured device, for example both contained functionalized directional nanostructured zone, contains the biochip in functionalization non-directional nanostructured district again.
A kind of nanostructured of the present invention is formed, and it comprises one of following nanodevice group: nanometer analysis chip, nano enzyme target, nano biological sensor, nanometer are picked up reagent strip, nanometer affinity chromatography glue soon.A kind of the 4th kind of nanostructured of the present invention formed, and it comprises the nanometer Mk system, and said nanometer Mk system comprises said function reagent and labelled reagent fixing on said activation nano particle, the activation nano particle at least.A kind of nanostructured of the present invention is formed, and it comprises and contains said nanodevice or/and the kit of said nanometer Mk system.A kind of kit of the present invention, it comprises nanometer Mk system and said nanodevice.A kind of kit of the present invention, it comprises nanodevice and said nanometer Mk system.
Second aspect of the present invention provides like " summary of the invention " described active constituent.A kind of active constituent of the present invention, it is the said active constituent in the above-mentioned nanostructured composition of the present invention.
First kind of active constituent of the present invention; Be activation oriented nano structure carrier; Said activation oriented nano structure carrier comprises the activation structure and contains the directional nano carrier that aligns the nanometer convex body; Wherein said activation structure comprises coating material solidified at least or/and the following activated group group of one or more of covalent bonding: comprise amino acid based group, amino diazanyl and amino hydrazine derivate base; And on said activation oriented nano structure carrier, have an activation directional nanostructured zone at least with following characteristics: said nanometer convex body therein be evenly distributed density greater than 10/μ m 2A kind of first kind of active constituent of the present invention, it is the said activation oriented nano structure carrier during first kind of nanostructured of the present invention formed.It comprises one of following base group: analysis chip sheet base, ELISA Plate sheet base, biology sensor sheet base.
Second kind of active constituent of the present invention; Be activation non-directional nanostructured carrier; Said activation non-directional nanostructured carrier comprises activation structure and the non-directional nano-carrier that contains non-directional arrangement nanometer convex body; Wherein said activation structure comprises coating material solidified at least or/and the following activated group group of one or more of covalent bonding: comprise amino acid based group, amino diazanyl and amino hydrazine derivate base, and said amino diazanyl is at 1m 2The lip-deep density of said nanometer convex body is greater than 1 μ mol, and on said activation non-directional nanostructured carrier, has an activation non-directional nanostructured district with following characteristics at least: said nanometer convex body therein be evenly distributed density greater than 10/μ m 2A kind of second kind of active constituent of the present invention is the said activation non-directional nanostructured carrier during second kind of nanostructured formed.It comprises one of following base group: analysis chip sheet base, ELISA Plate sheet base, biology sensor sheet base, pick up reagent silver base soon.
The third active constituent of the present invention; It is an activation non-nano structure carrier; Said activation non-nano structure carrier comprises non-nano carrier and activation structure, and wherein said activation structure comprises coating material solidified at least or/and the following activated group group of one or more of covalent bonding: comprise amino acid based group and amino hydrazine derivate base.A kind of the third active constituent of the present invention, it is the said activation non-nano structure carrier during the third nanostructured of the present invention is formed.It comprises one of following base group: analysis chip sheet base, ELISA Plate sheet base, biology sensor sheet base, pick up reagent silver base soon.
The 4th kind of active constituent of the present invention; It is the activation nano particle; Wherein said activation nano particle comprises nano particle and activation structure; Wherein said activation structure comprises coating material solidified at least or/and the following activated group group of one or more of covalent bonding: comprise amino acid based group, amino diazanyl and amino hydrazine derivate base, and said amino diazanyl is at 1m 2Density on the said nanoparticle surface is greater than 1 μ mol.A kind of the 4th kind of active constituent of the present invention, it is the said activation nano particle during the 4th kind of nanostructured of the present invention formed.
The 5th kind of active constituent of the present invention, it comprises the different activated district more than two or two, and wherein: at least one said region of activation is activation nanostructured district; With at least one said activation nanostructured district for to provide by said activation oriented nano structure carrier or activation non-directional nanostructured district.A kind of the 5th kind of active constituent of the present invention, it is the said many activation nanostructured carrier during the 5th kind of nanostructured of the present invention formed.
A kind of active constituent of the present invention, it is said activation oriented nano structure carrier, activation non-directional nanostructured carrier, activation non-nano structure carrier, activation nano particle or many region of activations nanostructured carrier during nanostructured of the present invention is formed.For example:
A kind of active constituent of the present invention wherein saidly comprises amino acid based group and comprises amino acid based or/and the amino acid derivativges base.Of the present invention a kind of active constituent wherein saidly comprises that amino acid based group comprises synthetic peptide group or/and synthetic peptide derivant base.A kind of active constituent of the present invention, wherein said synthetic peptide comprise and contain 2-10 amino acid whose synthetic peptide.Amino acid kind wherein identical (for example arginine, N, glutaminase, or the like the monamino acid peptidyl that forms) or different (for example arginine and N, N and glycocoll, glutaminase and lysine, or the like the amino acids peptidyl of formation).A kind of active constituent of the present invention, wherein said amino acid comprise one of following group: arginine, N, glutaminase, glycocoll, lysine, glutamine.In the embodiment of the invention, said activation structure also contains the coupling group that connects said carrier and activated group, and said coupling group comprises silane group.Used silane coupling agent comprises: 3-aminopropyl trimethoxysilane, aminopropyl triethoxysilane, 3-isocyanates propyl-triethoxysilicane.
A kind of active constituent of the present invention, wherein said coating comprises water resistant coating.A kind of active constituent of the present invention, wherein said water resistant coating is selected from elementary organic paint.A kind of active constituent of the present invention, wherein said elementary organic paint are selected from and contain organosilyl high-molecular coating.A kind of active constituent of the present invention, wherein said organosilicon comprises the organosilicon based on following organic silicon monomer: dimethyl siloxane, methacrylic acid 3-(methyl silicane), the dimethyl siloxane that contains (methacrylic acid acyl-oxygen) propyl group terminal and 3-aminopropyl trimethoxysilane, aminopropyl triethoxysilane, 3-isocyanates propyl-triethoxysilicane.
The 3rd aspect of the present invention provides the preparation method who forms like " summary of the invention " described nanostructured of the present invention.
First kind of preparation method of the present invention, said method comprise that the synthetic peptide method of use is fixed on said nanometer convex body or/and on the said nano particle with said activated group.A kind of preparation method of the present invention, wherein said synthetic peptide method comprises following a kind of or plurality of step: the reactant that contains blocking group is provided and in step thereafter, at least partly sloughs said blocking group;-NH 2Base and-reaction between the COOH base; The amino group chain growth.
Second kind of preparation method of the present invention; Said method comprises said coating encapsulated to said oriented nano structure carrier, non-directional nanostructured carrier or non-nano carrier, and wherein said encapsulating comprises said carrier is immersed in the solution of the said coating between the w/v concentration 1/100 to 1/500000.A kind of preparation method of the present invention, said method also comprise the said curing that encapsulates coating, and condition of cure comprises: temperature is between 40-80 ℃; Time is between 3-10 hour.
The third preparation method of the present invention; Said method comprises said coating encapsulated to said oriented nano structure carrier, non-directional nanostructured carrier or non-nano carrier, introduces activated group encapsulating successively to introduce coupling group and activated group or encapsulating on the coating on the coating then.
Of the present invention prepare groups of nanostructures or method, except that being used to separate or the nanostructured analyzed forms, also can be used to generate nanostructured, for example be used for the nanostructured of devices such as computing machine, mobile phone, chip card in order to preparation.
Embodiment
In following examples, relevant chip preparation method, application process, with reference to Schena, M., Microarray Analysis, 2003, John Wiley&Sons, Inc., New York.
The preparation of first's active constituent
In following examples, used following reagent can be buied on market:
1). coating: said activation structure comprises the paint coatings of curing.Used coating is selected from water resistant coating.Water resistant coating is selected from elementary organic paint, preferably clear or translucent elementary organic paint.Elementary organic paint is selected from and contains organosilyl polymkeric substance, for example contains organosilyl condensation copolymers or/and the high molecular hydrophobic coating of graft copolymer.More specifically, used coating is selected from the following organosilyl polymkeric substance group that contains: organic siliconresin A, organic siliconresin B, organic siliconresin GRT-350 (providing by organosilicon center, morning twilight chemical design institute).Three kinds contain organosilyl polymkeric substance and are silicone resin coating, and the mean molecular weight of organic siliconresin is all greater than 10000 even greater than 30000.Explanation by the supplier: organic siliconresin A is the silicone grafted copolymer coating based on dimethyl siloxane and methacrylic acid 3-(methyl silicane base) multipolymer; Organic siliconresin B is based on the dimethyl siloxane that contains (methacrylic acid acyl-oxygen) propyl group terminal and the silicone grafted copolymer coating of methacrylic monomer; Organic siliconresin GRT-350 is for being the organic siliconresin that primary raw material makes through hydrolytic condensation with organic silicon monomer (siloxane alcohols).Wherein, comprising of organic silicon monomer: dimethyl siloxane, methacrylic acid 3-(methyl silicane), contain the dimethyl siloxane at (methacrylic acid acyl-oxygen) propyl group terminal.3-aminopropyl trimethoxysilane, aminopropyl triethoxysilane, 3-isocyanates propyl-triethoxysilicane, or the like, also can be in a suitable manner as synthesis material.In fact, in following examples, contain suitable organosilyl coating,, also can be used as the coupling material, carry out chemical activation above that except that as the activated material.Just with coating coupling material, than the coupling agent of usefulness routine, make things convenient for, considerably cheaper.
2). coupling agent: used coupling agent comprises organo-silicon coupling agent; For example silane coupling agent comprises: 3-aminopropyl trimethoxysilane (Cathay China flourish new chemical materials company), aminopropyl triethoxysilane (Cathay China flourish new chemical materials company), 3-isocyanates propyl-triethoxysilicane (chemical company limited is contained by China).
3). active agent: used active agent comprises the basic active agent that the part activated group that combines with coupling group can be provided at least and the active agent of deriving (or second active agent) of the deriveding group on the basic activated group is provided.Used basic active agent selects white peptide synthetic agent (reagent for peptide synthesis), especially many nitride with contain amino (NH 2) and carboxyl (they all contain-NH for multi-functional group reagent COOH), for example peptide coupling agent (peptide coupling reagent) and amino acid 2Or-the COOH group.In the embodiment of the invention, used peptide coupling agent comprises amino hydrazine (NH 3NH 3).In the embodiment of the invention, used amino acid comprises: arginine, N, glutaminase, glycocoll, lysine, glutamine.Among the embodiment, the preferred multi-functional group reagent of said active agent, more preferably contain amino (NH 2) or/and carboxyl (multi-functional group reagent COOH).In addition, also preferably contain the peptide coupling agent of the synthetic blocking group (for example Fmoc) of peptide and contain amino acid based peptide synthetic agent, for example amino hydrazine of Fmoc-and Fmoc-amino acid.As everyone knows, blocking group has important effect to blocking group in building-up process (for example amino or carboxyl) activity.The amino hydrazine of Fmoc is provided by the triumphant safe new technology in Chengdu Ltd, and amino acid or Fmoc-amino acid are provided by Chengdu Tag chemical institute.The peptide synthetic agent that contains other blocking group (for example Boc-, CBZ-, or the like) also can be used for the method for following corresponding embodiment.The used active agent of deriving comprises and does not contain amino multi-functional group reagent (for example glutaraldehyde, 1,4-butanediol diglycidyl ether).
In following examples, used nano structured unit can be any nano structured unit of fixing said activation structure, preparing the activation nano structured unit, for example contains the nano structured unit of organism or inorganics.Said inorganics comprise inorganic oxide, metal (for example gold, silver), or the like.Said inorganic oxide comprises metal oxide.Said nano structured unit can have different shapes, for example nano wire, nanocone, nanotube, or the like.The nano structured unit of used containing metal oxide comprises the on-chip nano wire of the nano wire of silicon oxide-containing.(line footpath 80nm, line length 400nm-3 μ m, nano wire are evenly distributed the about 50-100 nano wire of density/μ m to the nano wire substrate that aligns on carrier available from American I mmuna company 2).For practicing thrift consumption, be cut to reduced size according to experiment condition.Contain the nanostructured carrier of the nano structured unit of other inorganic material of aligning (for example other inorganic oxide, metal, or the like), the method that also can be used for following examples prepares activation nanostructured carrier.The organic material nanostructured also can be selected to down the method for embodiment, directly or indirectly (for example encapsulating inanimate matter) preparation activation nanostructured and functionalized nano structure.
The various coupling carriers of following examples preparation, its coupling group is selected from: 3-aminopropyl trimethoxysilane base, aminopropyl triethoxysilane base, 3-isocyanates propyl-triethoxysilicane base.The various chemical activation components of following examples preparations, its activated group comprise amino diazanyl, amino hydrazine derivate base, amino acid based, amino acid derivativges base, synthetic peptide activated group, synthetic peptide derivant base.Wherein: said amino hydrazine derivate base comprises: glutaraldehyde-amino diazanyl, epoxy alkyl-amino diazanyl, glutaraldehyde-amino diazanyl, or the like; Said amino acid based comprising: arginine base, N base, glutamy amino, glycocoll base, lysine base, glutamy amido, or the like; Said amino acid derivativges base comprises: glutaraldehyde-arginine base, epoxy alkyl-arginine base, glutaraldehyde-N base, or the like; Said synthetic peptide activated group comprises the synthetic peptidyl of wherein amino acid based number n more than or equal to 2 (for example 2-10), the amino acid kind identical (for example, (arginine) in the synthetic peptidyl n, (N) n, (glutaminase) n, or the like), or different (for example, (arginine-N) N/2, (N-glycocoll) N/2, (glutaminase-lysine) N/2, or the like); Said synthetic peptide derivant base comprises: glutaraldehyde-(arginine) nBase, epoxy alkyl-(arginine) n, or the like, wherein n is more than or equal to 2 (for example 2-7).
The various activation nanostructured carriers of following examples preparation; The height of activation nano structured unit on it (for example the activation nano wire is or/and immobilization activation nano particle), the minimum dimension of half eminence and be evenly distributed the mensuration of density utilize SPA-300HV type scanning probe microscopy (SPM) and analysis software to carry out.Prepare the activation nanostructured carrier that various nanostructureds are formed as following examples, have a region of activation on it at least, wherein the density of activation nano structured unit is preferably greater than 10/μ m 2, more preferably greater than 50/μ m 2
Embodiment 1: the activation nanometer particle process method
In following examples, used nano particle can be any nano particle of fixing said activation structure in order to fixed function reagent, for example contains the nano particle of organism or inorganics.Said inorganics comprise inorganic oxide, metal (for example gold, silver), or the like.Said inorganic oxide comprises metal oxide.The nano particle of used containing metal oxide comprises: Si oxide nano particle (silicon oxide nanoparticle LUDOX AS-40, particle average-size 25nm, the about 135m of specific surface area 2/ g, Sigma-Aldrich company), aluminum oxide nano particle (MC2R γ-phase nano aluminium oxide, particle average-size 60nm, specific surface area 140m 2/ g, the great bright material in Zhejiang Science and Technology Co., Ltd.), titanium oxide nano particle (TiOx nano particle, particle average-size＜80nm, specific surface area 120m 2/ g, Zhejiang Zhoushan Tomorrow Nanomaterials Co., Ltd).
In the embodiment of the invention, be example with the nano particle of used containing metal oxide, the activation nanometer particle process method comprises:
1). couplingization: nano particle is mixed with coupling agent solution, and carry out coupling reaction.Its reaction conditions is following: nano particle concentration (w/v) 1 ‰-2%; Coupling agent concentration (v/v) 1-3%; Reaction medium is moisture alcohol; Temperature of reaction is below room temperature to the reaction medium boiling point between 5 ℃; Reaction time 0.5-5 hour.The professional and technical personnel can obtain required optimal conditions through regulating these parameters.Earlier nano particle is carried out surface treatment (for example deionization processing), can improve coupling reaction efficient.
2). activation: above-mentioned coupling nano particle is mixed with activator solution, and carry out priming reaction.Reaction conditions is following: the concentration of nano particle (w/v) 1 ‰-2%; Activator concentration (v/v) is at 0.5-5%; Temperature of reaction is below room temperature to the reaction medium boiling point between 5 ℃; Reaction time 0.5-15 hour; Reaction medium is DMF.If be necessary, can before adding coupling agent, carry out purifying (for example ion-exchange passivation) to nano particle.The professional and technical personnel can obtain required optimal conditions through regulating these parameters.If active agent contains blocking group (for example Fmoc), also to slough these blocking groups.Deprotection method is selected from the deprotection method in the known peptide synthetic method.
Wherein, use supercentrifuge to isolate nano particle in system.
Other method for example prepares activated group-coupling group compound (for example 3-isocyanates propyl-triethoxysilicane-amino hydrazine) earlier, secures it on the nano particle again, also can prepare activation nanostructured carrier.If active agent contains blocking group (for example Fmoc), also to slough these blocking groups.
Among the present invention, represent the coupling nano particle with coupling group/nano particle.The coupling nano particle of embodiment of the invention preparation comprises silane coupled group/oxide nano-particles.Wherein, silane coupled group and oxide nano-particles are all as previously mentioned.Among the present invention, represent the activation nano particle with main activated group/coupling nano particle.In the activation nano particle of embodiment of the invention preparation: the coupling nano particle is selected from above-mentioned coupling group/nano particle; Main activated group is foregoing activated group.
The coupling nano particle and the activation nano particle of embodiment of the invention preparation; Can analyze through ultimate analysis (for example C, H, N ultimate analysis), NMR, or the like, calculate unit area is fixed on the nanoparticle surface the coupling group and the density of activated group.Response parameter is different, and the variation of above-mentioned groups density is bigger.For example, coupling nano particle nitrogen content (ultimate analysis) is equivalent to coupling group fixing on the 1g nano particle and between 179-464 μ mol, changes between 0.25-0.65N%, or 1m 2Coupling group fixing on the nanoparticle surface changes between 1.3-3.4 μ mol.Again for example, 1m 2Activated group fixing on the activation nanoparticle surface changes between 0.1-2.85 μ mol.The coupling nano particle that preferably has following composition characteristic is in order to carry out the preparation of activation nanostructured in following examples or functionalized nano structure: 1m 2Coupling group fixing on the nanoparticle surface greater than 1.85 μ mol, be preferably greater than 2.0 μ mol, more preferably greater than 2.5 μ mol.The activation nano particle that preferably has following composition characteristic is in order to carry out the preparation of functionalized nano structure in following examples: 1m 2Activated group fixing on the nanoparticle surface greater than 0.5 μ mol, be preferably greater than 1 μ mol, more preferably greater than 1.5 μ mol.Need to prove that the group distribution density among the present invention is based on that ultimate analysis calculates.The group distribution density of utilizing other method to measure out, data maybe be quite variant, needs to convert by the relation of the difference between the method.
The prepared activation nano particle of the embodiment of the invention can be used as the activation nano particle for preparing functionalized nano particle probe, functionalized nano separate particles medium or nanometer label at least.Below to the activation step among the above-mentioned preparation method, do further to replenish.
1. amino diazanyl/coupling nanometer particle process method
For example, the coupling group on the coupling nano particle of method for preparing (for example 3-isocyanates propyl-triethoxysilicane) is carried out carbonylation handle, carry out above-mentioned activation with the amino hydrazine of Fmoc-as active agent again, take off Fmoc then.
2. amino hydrazine derivate base/coupling nanometer particle process method
For example; Earlier obtain amino diazanyl/coupling nano particle with said method; Again with second active agent (glutaraldehyde, 1 for example; 4-butanediol diglycidyl ether, amino acid or synthetic peptide) carry out the activation second time, then isolate for the second time activation products, or the like, the condition of each priming reaction is similar with above-mentioned reaction conditions.
3. amino acid based/the coupling nanometer particle process method
For example, prepare the coupling nano particle as stated above earlier, carry out above-mentioned activation with amino acid or Fmoc-amino acid as said active agent again.One of preferred reaction is on the amino acid-COOH base and coupling agent or coupling group on-NH 2Radical reaction.
4. amino acid derivativges base/coupling nanometer particle process method
For example; Obtain amino acid based/coupling nano particle with said method earlier; Again with second active agent (glutaraldehyde, 1 for example; The 4-butanediol diglycidyl ether) carry out the activation second time, then isolate for the second time activation products, or the like, the condition of each priming reaction is similar with above-mentioned reaction conditions.Also can be with amino acid and the reaction of second active agent, preparation contains the active agent (for example aldehyde radical amino acid) of the basic activated group/activated group compound of deriving, again with the active agent of this active agent as above-mentioned priming reaction.
5. synthetic peptide activated group/coupling nanometer particle process method
For example; Obtain amino acid based/coupling nano particle with said method earlier, re-use the polypeptide solid phase synthesis process of standard, on the immobilization amino acid group; Select suitable Fmoc-amino acid for use; Carry out condensation-wash-go to protect-neutralize and wash-mode of next round condensation, amino acid is connected up successively, meet the demands up to amino acid based number.Another kind method is: carry out peptide by known peptide synthetic method and synthesize, up to obtaining the synthetic peptide of amino acid needed radix purpose, carry out above-mentioned priming reaction as said active agent and above-mentioned coupling nanostructured carrier then.
6. synthetic peptide derivant group/coupling nanometer particle process method
For example; Obtain synthetic peptide activated group/coupling nano particle with said method earlier; Again with second active agent (glutaraldehyde, 1 for example; The 4-butanediol diglycidyl ether) carry out the activation second time, then isolate for the second time activation products, or the like, the condition of each priming reaction is similar with above-mentioned reaction conditions.Also can be with synthesizing the reaction of the peptide and second active agent, preparation contains the active agent (for example aldehyde radical is combined to peptide) of the synthetic peptide activated group/activated group compound of deriving, again with the active agent of this active agent as above-mentioned priming reaction.
Embodiment 2: the preparation method of activation non-nano structure carrier
In the embodiment of the invention, although used non-nano structure carrier is slide or chromatographic silica gel, it can be any non-nano structure carrier of fixing said activation structure in order to fixed function reagent, for example contains the non-nano structure carrier of organism or inorganics.The used solid phase carrier of present embodiment is the solid phase carrier that is suitable for as chip substrate, for example slide.The activation non-nano structure carrier that the embodiment of the invention is prepared can be used as the chip slapper base.Through selecting suitable solid phase carrier for use, prepared product also can be used as nano enzyme mark porous plate base, biology sensor sheet base, or the like.Following examples are done further to replenish.
The preparation of embodiment 2.1 chemical activation non-nano structure carriers
In the present embodiment, the preparation method comprises at least:
1). couplingization: non-nano structure carrier (for example slide) is mixed with coupling agent solution, and carry out coupling reaction.Its reaction conditions is following: coupling agent concentration (v/v) 1-3%; Reaction medium is moisture alcohol; Temperature of reaction is below room temperature to the reaction medium boiling point between 5 ℃; Reaction time 0.5-5 hour.Can obtain required optimal conditions through regulating these parameters.Earlier carrier is carried out surface treatment (for example etching), can improve coupling reaction efficient.
2). activation: above-mentioned coupling carrier and active agent (for example, the active agent of basic active agent or above-mentioned basic activated group-second activated group compound) solution are mixed, and carry out priming reaction.Reaction conditions is following: activator concentration (v/v) is at 0.5-5%; Temperature of reaction is below room temperature to the reaction medium boiling point between 5 ℃; Reaction time 0.5-15 hour; Reaction medium is DMF.Can obtain required optimal conditions through regulating these parameters.If active agent contains blocking group (for example Fmoc), also to slough these blocking groups.Deprotection method is selected from the deprotection method in the known peptide synthetic method.
Use other method, for example prepare activated group-coupling group compound (for example 3-isocyanates propyl-triethoxysilicane-amino hydrazine) earlier, secure it to again on the carrier, also can prepare chemical activation non-nano structure carrier.If active agent contains blocking group (for example Fmoc), also to slough these blocking groups.
Among the present invention, represent chemical activation non-nano structure carrier with main activated group/coupling group/non-nano structure carrier.In the chemical activation non-nano structure carrier of embodiment of the invention preparation: coupling group, main activated group are respectively foregoing coupling group, main activated group.
In the present embodiment; The preparation method of amino acid based/coupling non-nano structure carrier, amino acid derivativges base/coupling non-nano structure carrier, synthetic peptidyl/coupling non-nano structure carrier, synthetic peptide derivant group/coupling non-nano structure carrier is identical with the activation nanometer particle process method that contains identical activated group among the embodiment 1.
The preparation of embodiment 2.2 coating activation non-nano structure carriers
A kind of preparation method of present embodiment comprises that coating encapsulates.For example, the non-nano structure carrier of cleaning is contacted and encapsulates with the high molecular hydrophobic coating solution, then encrusting substance is cured.Wherein, the condition of encapsulating comprises: high molecular hydrophobic coating w/v concentration is between 1/1000 to 1/100000, and temperature is between 10-30 ℃, and the time is between 0.5-10 hour; Condition of cure comprises: temperature is between 40-80 ℃, and the time is between 3-10 hour.Through regulating these Reaction conditions range, can obtain required optimal conditions.Among the present invention,, represent coating activation non-nano structure carrier with coating/non-nano structure carrier.In the coating/non-nano structure carrier of present embodiment preparation, coating as previously mentioned, the non-nano structure carrier is a slide.
The preparation of embodiment activation more than 2.3 non-nano structure carrier
Among the present invention, many active constituents are active constituents that different activation structures are arranged on zones of different, for example contain the active constituent of said coating and at least a kind of other activation structure.A kind of preparation method of present embodiment, it comprises at least: 1). preparation chemical activation non-nano structure carrier; 2). with suitable coating solution, be applied in the subregion on the chemical activation non-nano structure carrier, and carry out coating and encapsulate and solidify.Encapsulate condition and condition of cure, identical with embodiment 2.1.In the present embodiment,, represent many activation non-nano structure carrier with coating/chemical activation non-nano structure carrier.Wherein: coating comprises aforementioned coating; Chemical activation non-nano structure carrier is selected from the prepared product of embodiment 2.1.Other chemical activation non-nano structure carrier (for example amination slide, aldehyde radical slide, epoxy radicals slide, or the like) also can be used for many activation of method for preparing non-nano structure carrier.
The coating of embodiment 2.4 chemical activations encapsulates the preparation of non-nano structure carrier
For example, first method by embodiment 2.2 prepares coating and encapsulates the non-nano structure carrier, presses the method for embodiment 2.1 then respectively, activation again after the couplingization, or direct activation.In the present embodiment, encapsulate the non-nano structure carrier, represent that the coating of chemical activation encapsulates the non-nano structure carrier with main activated group/coating.Wherein: coating comprises aforementioned coating; Main activated group as previously mentioned.Other activated group, for example aldehyde radical, epoxy radicals, or the like, the coating that the method that also can be used for present embodiment is prepared chemical activation encapsulates the non-nano structure carrier.
Embodiment 3: the preparation method of activation oriented nano structure carrier
In the embodiment of the invention, although used oriented nano structure carrier (for example nano wire substrate) as previously mentioned, it can be any oriented nano structure carrier of fixing said activation structure.The activation oriented nano structure carrier that the embodiment of the invention is prepared, can be used as chip activation nanostructured sheet base, nano enzyme mark porous plate activation nanostructured sheet base, biology sensor activation nanostructured sheet base, or the like.Following examples are done further to replenish.
The preparation of embodiment 3.1 chemical activation oriented nano structure carriers
The method of the embodiment of the invention has prepared following activation oriented nano structure carrier:
1). first kind of activation oriented nano structure carrier, its composition comprises: activated group or the activation nano particle fixed on coupling group of fixing on the nano structured unit that aligns on solid phase carrier, the solid phase carrier, the nano structured unit and the coupling group.It is represented with main activated group/coupling group/oriented nano structure carrier or activation nano particle/coupling group/oriented nano structure carrier.Its preparation method comprises: earlier the oriented nano structure carrier carry out couplingization, again coupling oriented nano structure carrier is introduced fixedly activated group or/and the activation of activation nano particle.
2). second kind of activation oriented nano structure carrier, its composition comprises: activated group fixing on coupling nano particle of fixing on the nano structured unit that aligns on solid phase carrier, the solid phase carrier, the nano structured unit and the coupling nano particle is or/and the activation nano particle.It is represented with main activated group/coupling nano particle/oriented nano structure carrier or activation nano particle/coupling nano particle/oriented nano structure carrier.Its preparation method comprises: the oriented nano structure carrier through encapsulating fixedly coupling group of coupling nano particle, is introduced fixedly activated group or/and the activation of activation nano particle to coupling oriented nano structure carrier again.
3). the third activation oriented nano structure carrier, its composition comprises: activated group or the activation nano particle fixed on coupling group of fixing on the nano particle of fixing on the nano structured unit that aligns on solid phase carrier, the solid phase carrier, the nano structured unit, the nano particle and the coupling group.It is represented with main activated group/coupling group/nano particle/oriented nano structure carrier or activation nano particle/coupling group/nano particle/oriented nano structure carrier.Its preparation method comprises: the oriented nano structure carrier is encapsulated nano particle, carry out couplingization then, again coupling oriented nano structure carrier is introduced fixedly activated group or/and the activation of activation nano particle.
4). the 4th kind of activation oriented nano structure carrier, its composition comprises: the activation nano particle of fixing on the nano structured unit that aligns on solid phase carrier, the solid phase carrier, the nano structured unit.It is represented with activation nano particle/oriented nano structure carrier.Its preparation method comprises: the oriented nano structure carrier is encapsulated the activation nano particle.
5). the 5th kind of activation oriented nano structure carrier, its composition comprises: the activation nano particle of fixing on the activation nano structured unit that aligns on solid phase carrier, the solid phase carrier, the activation nano structured unit.It is represented with activation nano particle/activation oriented nano structure carrier.Its preparation method comprises: activation oriented nano structure carrier (for example first kind of activation oriented nano structure carrier) is encapsulated the activation nano particle.
Wherein: the example of solid phase carrier comprises: the slide of slide, containing metal or semiconductor film, sheet metal, semiconductor chip, or the like; The example of nano structured unit comprises: nano wire, nanocone, nanotube, or the like.Among above-mentioned each preparation method, the reaction conditions of committed step is following:
(1). couplingization: coupling agent concentration (v/v) 1-3%; Reaction medium is moisture alcohol; Temperature of reaction is below room temperature to the reaction medium boiling point between 5 ℃; Reaction time 0.5-5 hour.Can obtain required optimal conditions through regulating these parameters.Earlier carrier is carried out surface treatment (for example etching), can improve coupling reaction efficient.
(2). activation: activator concentration (v/v) is at 0.5-5%; Temperature of reaction is below room temperature to the reaction medium boiling point between 5 ℃; Reaction time 0.5-15 hour; Reaction medium is DMF.Can obtain required optimal conditions through regulating these parameters.If active agent contains blocking group (for example Fmoc), also to slough these blocking groups.Deprotection method is selected from the deprotection method in the known peptide synthetic method.Wherein, introduce the activation of amino diazanyl, amino acid based, amino acid derivativges base, synthetic peptidyl or synthetic peptide derivant group, identical with the activation in the activation nanometer particle process method that contains identical activated group among the embodiment 1.
(3). nano particle, coupling nano particle or activation nano particle encapsulate: the nano particle concentration in the nanoparticle suspension (w/v concentration) is between 1/1000 to 1/50000; Encapsulate between temperature 10-30 ℃; The time that encapsulates is more than 5 hours.
(4). the encapsulating of activation nano particle on the activation nano structured unit, tube is stated as follows: above-mentioned nano wire substrate is carried out activation (for example above-mentioned activation), make it have reactive group 1 (for example contain-COOH amino acid based); Selection has and can (for example contain-NH with reactive group 1 reactive activity group 2 2Amino acid based) the activation nano particle; Make (for example activation nano particle w/v concentration is between 1/100 to the 1/10000) reaction under optimized conditions of reactive group 1 and reactive group 2, thereby the activation nano particle is encapsulated to the nanostructured carrier.
In addition, use other method, for example prepare activated group-coupling group compound (for example 3-isocyanates propyl-triethoxysilicane-amino hydrazine) earlier, secure it to again on the carrier, also can prepare chemical activation oriented nano structure carrier.If active agent contains blocking group (for example Fmoc), also to slough these blocking groups.
Wherein: used coupling agent and active agent are as previously mentioned; Used coupling nano particle and activation nano particle are the coupling nano particle and the activation nano particle of embodiment 1 preparation; Coupling group in the prepared product is aforementioned coupling group; Activated group in the prepared product is aforementioned activated group, for example amino diazanyl, various amino hydrazine derivate base, various amino acid based, various amino acid derivativges base, various synthetic peptidyl, various synthetic peptide derivant base.Used coupling nano particle has following characteristics: 1m 2Coupling group fixing on the nanoparticle surface greater than 1.85 μ mol, be preferably greater than 2.0 μ mol, more preferably greater than 2.50 μ mol.Used activation particle has following characteristics: 1m 2Activated group fixing on the nanoparticle surface greater than 0.5 μ mol, be preferably greater than 1 μ mol, more preferably greater than 1.5 μ mol.
The preparation of embodiment 3.2 coating activation oriented nano structure carriers
The 6th kind of activation oriented nano structure carrier of the present invention is coating activation oriented nano structure carrier, and wherein said activation structure contains coating, with coating/oriented nano structure carrier.A kind of preparation method in the present embodiment, it comprises at least: the oriented nano structure carrier of cleaning is contacted and encapsulates with the high molecular hydrophobic coating solution, then encrusting substance is cured.Wherein, the condition of encapsulating comprises: high molecular hydrophobic coating w/v concentration is between 1/1000 to 1/100000, and temperature is between 10-30 ℃, and the time is between 0.5-10 hour; Condition of cure comprises: temperature is between 40-80 ℃, and the time is between 3-10 hour.Through regulating these Reaction conditions range, can obtain required optimal conditions.Wherein said coating comprises above-mentioned coating.
The preparation of embodiment activation more than 3.3 oriented nano structure carrier
The 7th kind of activation oriented nano structure carrier of the present invention is many activation oriented nano structure carrier, and wherein said activation structure contains at least 2 kinds of activated materials.Among the present invention,, represent so a kind of many activation oriented nano structure carrier: have one or more coating activating areas and chemical activation zone on its surface respectively with coating/chemical activation oriented nano structure carrier.Wherein: said coating comprises above-mentioned coating; Said chemical activation oriented nano structure carrier is selected from the prepared product of embodiment 3.1.Contain the chemical activation oriented nano structure carrier of other activated group (for example amino, aldehyde radical, epoxy radicals, or the like), also can be used for many activation of method for preparing nanostructured carrier.
A kind of preparation method in the present embodiment, it comprises at least: 1). preparation chemical activation oriented nano structure carrier; 2). suitable coating solution is applied in the subregion on the oriented nano structure carrier and encapsulates, then encrusting substance is cured.Encapsulate condition and condition of cure, identical with embodiment 3.1.Embodiment 3.4 activation nano particle/coating encapsulate the preparation method of oriented nano structure carrier
The 8th kind of activation oriented nano structure carrier of the present invention encapsulates the active constituent that forms on the oriented nano structure carrier for the activation nano particle is fixed on coating, and note is made activation nano particle/coating and encapsulated the oriented nano structure carrier.A kind of preparation method of present embodiment is included on the coating activation oriented nano structure carrier of embodiment 3.2 preparations at least, encapsulates the activation nano particle.The condition of encapsulating comprises: activation nano particle w/v concentration is between 1/1000 to 1/100000; Temperature is between 10-30 ℃; Time is between 10-48 hour; Encapsulating medium is the WS.Through regulating these Reaction conditions range, can obtain required optimal conditions.Wherein, the activation nano particle is selected from the activation nano particle of embodiment 1 preparation, but also can use other activation nano particle that can be fixed on the dope layer.
The coating of embodiment 3.5 chemical activations encapsulates the preparation of oriented nano structure carrier
The 9th kind of activation oriented nano structure carrier of the present invention is for the coating of chemical activation encapsulates the oriented nano structure carrier.Its preparation method for example earlier prepares coating by the method for embodiment 3.2 and encapsulates the oriented nano structure carrier, presses the method for embodiment 3.1 then respectively, activation again after the couplingization, or direct activation.In the present embodiment, encapsulate the oriented nano structure carrier, represent that the coating of chemical activation encapsulates the oriented nano structure carrier with main activated group/coating.Wherein: coating comprises aforementioned coating; Main activated group as previously mentioned.Other activated group, for example aldehyde radical, epoxy radicals, or the like, the coating that the method that also can be used for present embodiment is prepared chemical activation encapsulates the oriented nano structure carrier.
Embodiment 4: the preparation method of activation non-directional nanostructured carrier
In the embodiment of the invention, encapsulate solid phase carrier (for example encapsulating slide) although used non-directional nanostructured carrier is a nano particle, it can be any non-directional nanostructured carrier of fixing said activation structure in order to fixed function reagent.The activation non-directional nanostructured carrier that the embodiment of the invention is prepared, can be used as chip activation nanostructured sheet base, nano enzyme mark porous plate activation nanostructured sheet base, biology sensor activation nanostructured sheet base, or the like.Following examples are done further to replenish.
The preparation of embodiment 4.1 chemical activation non-directional nanostructured carriers
The embodiment of the invention has prepared following activation non-directional nanostructured carrier:
1). first kind of activation non-directional nanostructured carrier, composition comprises: fixing activated group or activation nano particle on fixing coupling group and the coupling group on the nano structured unit that non-directional is arranged on solid phase carrier (for example the slide of slide, containing metal or semiconductor film, sheet metal, semiconductor chip, or the like), the solid phase carrier (for example nano particle, other nanometer convex body that forms through nano particle self assembly phenomenon, or the like), the nano structured unit.It is represented with main activated group/coupling group/non-directional nanostructured carrier or activation nano particle/coupling group/non-directional nanostructured carrier.Its preparation method comprises: non-directional nanostructured carrier carry out couplingization, then coupling non-directional nanostructured carrier is carried out activation.
2). second kind of activation non-directional nanostructured carrier, composition comprises: fixing activated group or activation nano particle on fixing coupling nano particle and the coupling nano particle on the nano structured unit that non-directional is arranged on solid phase carrier (for example the slide of slide, containing metal or semiconductor film, sheet metal, semiconductor chip, or the like), the solid phase carrier (for example nano particle, other nanometer convex body that forms through nano particle self assembly phenomenon, or the like), the nano structured unit.It is represented with main activated group/coupling nano particle/non-directional nanostructured carrier or activation nano particle/coupling nano particle/non-directional nanostructured carrier.Its preparation method comprises: non-directional nanostructured carrier through encapsulating fixedly coupling group of coupling nano particle, is carried out activation to coupling non-directional nanostructured carrier then.
3). the third activation non-directional nanostructured carrier, composition comprises: fixing activated group or activation nano particle on fixing coupling group and the coupling group on fixing nano particle, the nano particle on the nano structured unit that non-directional is arranged on solid phase carrier (for example the slide of slide, containing metal or semiconductor film, sheet metal, semiconductor chip, or the like), the solid phase carrier (for example nano particle, other nanometer convex body that forms through nano particle self assembly phenomenon, or the like), the nano structured unit.It is represented with main activated group/coupling group/nano particle/non-directional nanostructured carrier or activation nano particle/coupling group/nano particle/non-directional nanostructured carrier.Its preparation method comprises: non-directional nanostructured carrier is encapsulated nano particle, carry out couplingization again, then coupling non-directional nanostructured carrier is carried out activation.
4). the 4th kind of activation non-directional nanostructured carrier, composition comprises: fixing activation nano particle on the nano structured unit that non-directional is arranged on solid phase carrier (for example the slide of slide, containing metal or semiconductor film, sheet metal, semiconductor chip, or the like), the solid phase carrier (for example nano particle, other nanometer convex body that forms through nano particle self assembly phenomenon, or the like), the nano structured unit.It is represented with activation nano particle/non-directional nanostructured carrier.Its preparation method comprises: non-directional nanostructured carrier is encapsulated the activation nano particle.
5). the 5th kind of activation non-directional nanostructured carrier, composition comprises: fixing activation nano particle on the nano structured unit that non-directional is arranged on solid phase carrier (for example the slide of slide, containing metal or semiconductor film, sheet metal, semiconductor chip, or the like), the solid phase carrier (for example nano particle, other nanometer convex body that forms through nano particle self assembly phenomenon, or the like), the activation nano structured unit.It is represented with activation nano particle/activation non-directional nanostructured carrier.Its preparation method comprises: activation non-directional nanostructured carrier (the first kind of activation non-directional nanostructured carrier that for example prepares in the present embodiment) is encapsulated the activation nano particle.
Among above-mentioned each preparation method, the reaction conditions of each committed step, for example couplingization; Activation; Nano particle, coupling nano particle or activation nano particle encapsulate, and the encapsulating of activation nano particle on the activation nano structured unit, respectively the reaction conditions of corresponding steps in the reference implementation example 3.1.In the present embodiment, used coupling agent, active agent, coupling nano particle, activation nano particle, and prepared product in contained coupling group and activated group, also respectively with embodiment 3.1 in respective substance identical.Main activated group also comprise aforementioned it: amino diazanyl, amino acid based, amino acid derivativges base, synthetic peptidyl, synthetic peptide derivant group.
Embodiment 4.2 comprises the preparation of the activation non-directional nanostructured carrier of coating
The 6th kind of activation non-directional nanostructured carrier of the present invention, wherein said activation structure contains coating, is coating activation non-directional nanostructured carrier.The 7th kind of activation non-directional nanostructured carrier of the present invention, wherein said activation structure contains at least 2 kinds of activated materials, is many activation non-directional nanostructured carrier.Among the present invention,, represent so a kind of many activation non-directional nanostructured carrier: have one or more coating activating areas and chemical activation zone on its surface respectively with coating/chemical activation non-directional nanostructured carrier.The 8th kind of activation non-directional nanostructured carrier of the present invention encapsulates the active constituent that forms on the non-directional nanostructured carrier for the activation nano particle is fixed on coating, and note is made activation nano particle/coating and encapsulated non-directional nanostructured carrier.The 9th kind of activation non-directional nanostructured carrier of the present invention, for the coating of chemical activation encapsulates non-directional nanostructured carrier, note is made main activated group/coating and is encapsulated non-directional nanostructured carrier.
In the present embodiment, the preparation method of the above-mentioned the 6th to the 9th kind of activation non-directional nanostructured carrier is identical with the preparation method of the 6th to the 9th kind of activation oriented nano structure carrier among the embodiment 3.2-3.5 respectively.Wherein: coating, activated group, activation nano particle, also respectively with embodiment 3.2-3.5 in coating, activated group, activation nano particle identical.
Embodiment 5: the preparation method of many region of activations nanostructured carrier
In the embodiment of the invention; Many region of activations nanostructured carrier to contain activation non-nano structural area and activation directional nanostructured zone is an example; A kind of preparation method is: will comprise the zone that is fixed with nano wire and not be fixed with the nano wire substrate in the zone of nano wire; Select the preparation method of suitable activation nanostructured carrier for use, make the identical activation structure of introducing on 2 zones.For example, the preparation method of reference implementation example 3.2-3.5 activation oriented nano structure carrier can introduce the identical multiple activation structure that comprises coating.
The many region of activations nanostructured carrier that contains activation non-nano structural area and activation non-directional nanostructured district uses the substrate that comprises the zone that is fixed with nano particle and be not fixed with the zone of nano particle, also can prepare as stated above.
The preparation that the second portion nanostructured is formed
In following examples, said function reagent comprises any material that is fixed on the said activated group and don't loses its function, and for example nucleic acid is or/and polypeptide.Used function reagent comprises in following examples: polypeptide, antigen, antibody, and other function reagent.Wherein: used synthetic polypeptide comprise EBV-VCA-P18 antigen (self-control, the preparation method is with reference to Tranchand-Bunel, D.; Auriault, C., Diesis; E.; Gras-Masse, H. (1998) Detection of human antibodies using " convergent " combinatorial peptide libraries or " mixotopes " designed form anonvariable antigen:Application to the EBV viral capsid antigen p18, J.PeptideRes.52; 1998,495-508); Used antigen comprises: hepatitis C virus antigen (HCV Ag), hiv antigen (HIV Ag), syphilis antigen (being hepatopathy research institute of The People's Hospital of Peking University provides); Used antibody comprises anti-hepatitis virus surface antibody (HBs Ab, hepatopathy research institute of The People's Hospital of Peking University provides) and monoclonal antibody or polyclone goat-anti people two anti-(Beijing Biological Product Inst.); Used other function reagent comprises a-protein (Shanghai Vaccine and Serum Institute).The method of present embodiment also is suitable for other function reagent, for example: medicine, polysaccharide, vitamin, microbiotic, biotin, Avidin, function organism, strand or multichain DNA, RNA and virus, cell or their composition.
In the embodiment of the invention nanostructured form, the preparation method of nanostructured device (for example nano chips, nano enzyme mark reactor, the fast check reagent bar of nanometer, nanostructured chromatography glue, biology sensor, or the like) especially; Do not have the different of essence with the preparation method of corresponding non-nano constructional device in principle; All be through point sample or encapsulate, probe (function reagent is or/and the functionalized nano particle) is fixed on the sheet base.For example, in the preparation of nano chips, comprise with suitable existing craft or mechanical deposition method, with 3 appearance of every kind of probe points on the sheet base.Wherein, the immobilized reactant condition is following: functionalized nano particle concentration (w/v) 0.01-3%; PH of buffer 5.0-9.5; Temperature of reaction 20-37 ℃; Reaction time 0.5-72 hour.Can obtain required optimal conditions through regulating these parameters.Also comprise the passivation of nanostructured device in case of necessity.Passivator commonly used comprises protein and amino acid.Must be noted that; Below the nanostructured of each embodiment preparation form, especially in the nano chips; And the unnecessary all functions district (for example probe points) that asks is functionalized nano structural area (for example nano-probe point), also can contain functionalization non-nano structural area (for example non-nano probe points).
The nanostructured device (biological example chip) of following examples preparations, its nanostructure probe point go up the functionalized nano particle or/and the functionalized nano convex body be evenly distributed density, utilize SPA-300HV type scanning probe microscopy (SPM) and analysis software mensuration.The nanostructured device of following examples preparation preferably is evenly distributed density greater than 10/μ m 2, more preferably be evenly distributed density greater than 50/μ m 2
Nanostructured of the present invention is formed, and a weight or multi-functional reagent (for example aglucon/part) can be arranged or/and a heavy or multiple nanostructured is arranged or/and a heavy or multi-functional reagent is arranged between said at least one heavy nanostructured and another the heavy nanostructured between one or more function reagent and the carrier between one or more nanostructureds in the wherein said functionalized nano structural area and the carrier.For example; The nano-structure activity media that multiple aglucon is arranged between one or more nano particles of following method preparation and the carrier: respectively carrier is encapsulated reprovision body formation part base and encapsulate carrier; And nano particle is encapsulated a reprovision base (between aglucon and the part compatible reaction being arranged) form aglucon/nano-particle compound; Again aglucon/nano-particle compound is encapsulated or point sample to part encapsulates on the carrier, form the compound of aglucon-nano particle-aglucon-part-carrier format.When the aglucon number of plies greater than 1 the time by that analogy.
Following examples are further described above-mentioned preparation method.
The preparation that 6: the first kinds of nanostructureds of embodiment are formed
Characterization functionalized nano structure carrier during first kind of nanostructured of the present invention formed is represented with function reagent/activation oriented nano structure carrier, functionalized nano particle/activation oriented nano structure carrier and function reagent and functionalized nano particle/activation oriented nano structure carrier respectively.In the present embodiment, activation oriented nano structure carrier is selected from the activation oriented nano structure carrier of embodiment 3 preparations; Function reagent is selected from above-mentioned difference in functionality combination of agents; The functionalized nano particle is selected from the combination of the difference in functionality nano particle of embodiment 9 preparations.Other functionalized nano particle also first kind of nanostructured of method preparation of available present embodiment is formed.
In the present embodiment; The preparation of nano chips; Comprise with one or more function reagent solutions (the function reagent concentration is between the 0.1-2mg/ml) or/and the functionalized nano particle (function reagent concentration between the 0.1-2mg/ml, nano particle concentration is between 0.01-10mg/ml), manual point sample forms dot matrix on the activation oriented nano structure carrier of the foregoing description 3 preparations, carries out immobilized reactant; Use passivator (for example bovine serum albumin(BSA)) passivation then, form nano chips.The immobilized reactant condition as previously mentioned.Same reaction conditions, also can in order to preparation contain functionalization oriented nano structure carrier nano enzyme mark reactor, biology sensor, or the like.In fact, all activation oriented nano structure carriers of embodiment 3 preparations all can be through the fixing various function reagent of said method or/and the functionalized nano particle be prepared different nanodevices.Table 1 is listed the part nano chips example of present embodiment preparation.
Table 1
Figure GPA00000523127800271
*: A1-A8: see the following form 4; *: wherein a-protein is fixed on organic siliconresin GRT-350 and encapsulates in the zone, and other probe stationary is in arginine base activating area; C9: activation nanostructured carrier is that activation nano particle/coating encapsulates the oriented nano structure carrier; C11: activation nanostructured carrier is that main activated group/coating encapsulates the oriented nano structure carrier
The preparation that 7. second kinds of nanostructureds of embodiment are formed
The characteristic functionalized nano structure carrier that second kind of nanostructured of the present invention formed is represented with functionalized nano particle/activation non-directional nanostructured carrier.Its preparation method comprises the preparation of functionalization non-directional nanostructured carrier, is about to the functionalized nano particle and is fixed on the activation non-directional nanostructured carrier.The immobilized reactant condition as previously mentioned.Wherein: activation non-directional nanostructured carrier is selected from the activation non-directional nanostructured carrier of preparation among the embodiment 4; The functionalized nano particle is selected from the functionalized nano particle (also available other functionalized nano particle) of preparation among the embodiment 9.In fact, all non-directional nanostructured carriers of embodiment 4 preparations all can be prepared different nano chips through the fixing various functionalized nano particles of said method.Table 2 is listed the part nano chips example of present embodiment preparation.
Table 2
*: A1-A8: see the following form 4; B4: wherein a-protein is fixed on organic siliconresin GRT-350 and encapsulates in the zone, and other probe stationary is in arginine base activating area; B8: activation nanostructured carrier is that activation nano particle/coating encapsulates non-directional nanostructured carrier; B9: activation nanostructured carrier is that main activated group/coating encapsulates non-directional nanostructured carrier
The preparation that embodiment 8. the third nanostructureds are formed
Characterization functionalized nano structure carrier during the third nanostructured of the present invention is formed is represented with function reagent/activation non-nano structure carrier, functionalized nano particle/activation non-nano structure carrier and function reagent and functionalized nano particle/activation non-nano structure carrier respectively.In the present embodiment, activation non-nano structure carrier is selected from the prepared product of embodiment 2; Function reagent is selected from above-mentioned functions reagent; The functionalized nano particle is selected from the functionalized nano particle of embodiment 9 preparations.Other functionalized nano particle also the third nanostructured of method preparation of available present embodiment is formed.The nanodevice of present embodiment preparation comprises nano chips, nanostructured chromatography glue, nano enzyme target.
The preparation method of present embodiment nanodevice; Comprise one or more function reagent solutions (the function reagent concentration is between the 0.1-2mg/ml) or/and the functionalized nano particle (function reagent concentration between the 0.1-2mg/ml, nano particle concentration is between 0.01-10mg/ml); Contact with the activation non-nano structure carrier of the foregoing description 2 preparations, carry out immobilized reactant.The immobilized reactant condition as previously mentioned.In fact, the activation non-nano structure carrier of embodiment 2 preparations all can be through the fixing various function reagent of said method or/and the functionalized nano particle be prepared different nanodevices.Table 3 is listed the part nano chips example of present embodiment preparation.
Table 3
Figure GPA00000523127800291
*: A1-A8: see the following form 4; D7: wherein a-protein is fixed on organic siliconresin GRT-350 and encapsulates in the zone, and other probe stationary is in arginine base activating area; D8: activation non-nano structure carrier is that main activated group/coating encapsulates the non-nano structure carrier
The preparation that the 4th kind of nanostructured of embodiment 9. formed
The 4th kind of nanostructured of the present invention formed and comprised: 1). functionalized nano particle probe: for example, can be fixed on the solid phase carrier in order to catch the probe (for example functionalized nano particle) of separation or evaluating objects thing; 2). functionalized nano separate particles medium: for example, be used in the nanofiltration system, separate or the nanometer separating medium of evaluating objects thing to catch; With 3). the nanometer label.
Embodiment 9.1: the preparation of functionalized nano particle
The preparation of functionalized nano particle is about to the function immobilization of reagents on the activation nano particle.In the present embodiment, the immobilized reactant condition is following: activation nano particle concentration (w/v) 0.01-3%; Function reagent concentration (w/v) 0.1-3.0mg/ml; PH of buffer 5.0-9.5; Temperature of reaction 20-37 ℃; Reaction time 0.5-72 hour.Can obtain required optimal conditions through regulating these parameters.The purifying that also comprises the functionalized nano particle in case of necessity is or/and passivation.Passivator commonly used comprises protein and amino acid.Used activation nano particle is selected from the prepared activation nano particle of the foregoing description 1.The activation nano particle of the preferred following characteristic of tool: 1m 2Coupling group fixing on the nanoparticle surface greater than 1.85 μ mol, be preferably greater than 2.0 μ mol, more preferably greater than 2.50 μ mol, or/and 1m 2Activated group fixing on the nanoparticle surface greater than 0.5 μ mol, be preferably greater than 1 μ mol, more preferably greater than 1.5 μ mol.
Among the present invention, come the presentation function nano particle with function reagent/activation nano particle.In the functionalized nano particle of present embodiment preparation: function reagent is selected from above-mentioned functions reagent, and the activation nano particle is selected from the activation nano particle of embodiment 1 preparation.It comprises: antigen/activation nano particle (for example HCVAg/ activation nano particle, HIV Ag/ activation nano particle, syphilis antigen/activation nano particle, or the like), antibody/activation nano particle (for example HBs Ab/ activation nano particle), other function reagent/activation nano particle (for example Protein A/ activation nano particle).In fact, all preferred activation nano particles of embodiment 1 preparation all can be prepared the difference in functionality nano particle through the fixing various function reagent of said method.Table 4 is listed the partial function nano particle example of present embodiment preparation.
Embodiment 9.2: the preparation of nanodevice
In the present embodiment, used base can be any sheet base that can be used for effective fixed function nano particle, comprising: flat carrier, bead-type substrate, membrane carrier.Flat carrier comprises activation microslide and ELISA porous plate; Bead-type substrate comprises activation chromatography glue; Membrane carrier comprises the tunica fibrosa bar.The activation microslide comprise according to the amino slide of own disclosed method preparation (with reference to Schena, M., Microarrayanalysis; John Wiley&Sons, INC., New York), aldehyde slide is (with reference to Schena; M., Microarray analysis, John Wiley&Sons; INC., New York).The ELISA porous plate comprises polystyrene porous plate (the bright firelight or sunlight Ltd of Shenzhen gold).Activation chromatography glue comprises: the activation chromatography glue (AH-Sepharose CL, Pharmacia company) that contains Agarose.The tunica fibrosa bar comprises nitrocellulose film bar and nylon fiber film bar (Fujian Changli Biochem. Co., Ltd., Quanzhou City).The preparation method of present embodiment is suitable for the sheet base processed by following material or derivatives thereof equally: silicon chip, silica gel, pottery, metal oxide, metal, other polymeric material and their compound.
In the present embodiment, the functionalized nano particle is selected from the aforementioned functional nano particle of present embodiment preparation.In fact, all functions nano particle of present embodiment preparation is separately fixed on the respective flap base by the said apparatus preparation method, just prepares the fast check reagent bar of various nano chips, nano enzyme target and nanometer respectively.Table 4 is listed the part nanodevice example of present embodiment preparation.Wherein: A18 and A19 are the example of nano chips, and A20 is the example of nano enzyme target, and A21 and A22 are the example of the fast check reagent bar of nanometer, the example of A23 nanostructured chromatography glue.
Embodiment 9.3: the preparation of nanometer label
Among the present invention, be that mark is represented the nanometer label with mark substance/activation nano particle/function reagent.In the present embodiment, used activation nano particle is selected from the activation nano particle of embodiment 1 preparation; Used function reagent comprises two anti-and used antigens, the corresponding antigen of antibody, antibody (married put supplies dual-antigen sandwich method, double antibody sandwich method to use); Used mark substance comprises: fluorescent material (for example rhodamine, CY3, CY4), marker enzyme (for example horseradish peroxidase), coloring agent (for example crystal violet).The method of present embodiment also is suitable for other mark substance, for example chemiluminescent substance, chemiluminescence catalyzer, non-ferrous metal salt, dyestuff and pigment.
In the present embodiment, the preparation of nanometer label comprises two kinds of methods: A). mark substance is fixed on carries out purifying on the functionalized nano particle again; B). mark substance is fixed on the function reagent, and the mark substance/function reagent complex with purifying is fixed on the activation nano particle again.Used labeling method and purification process adopt the labeling method and the purification process of the conventional label of known preparation basically.Purification condition can be with reference to the purification process of label among the known conventional label preparation method (for example filtration method, chromatography, or the like), but comprise centrifuge method in the preferable methods.The preferred condition of labeling method comprises the much longer reaction time (for example greater than 12 hours).
The part nanometer label of present embodiment preparation is following: 1). fluorescent material/activation nano particle/pairing antigen, fluorescent material/activation nano particle/pairing antibody, fluorescent material/activation nano particle/antiantibody, fluorescent material/activation nano particle/a-protein; 2). horseradish peroxidase/activation nano particle/pairing antibody, horseradish peroxidase/activation nano particle/antiantibody; 3). coloring agent/activation nano particle/antiantibody.In fact, all preferred activation nano particles of embodiment 4 preparations all can be prepared different nanometer labels through the fixing various function reagent of said method.Table 4 is listed the part nanometer label example of present embodiment preparation.
Table 4
Figure GPA00000523127800311
Figure GPA00000523127800321
The preparation that the 5th kind of nanostructured of embodiment 10. formed
In the present embodiment, the preparation example that the 5th kind of nanostructured formed (for example core jin) is: use many activation nanostructured carrier, again by functionalized reagent among the embodiment 6 or/and the process for fixation of functionalized nano particle carries out point sample and immobilized reactant.Wherein, An example does; A1 in the table 4 and A2 (or HIV Ag and HCV Ag) are separately fixed in directed (or non-directional) nanostructured of activation district of many region of activations nanostructured carrier and form directed (or non-directional) nanostructured of functionalization district, and A4 is fixed on and forms functionalization non-nano structural area on the activation non-nano structural area.Used many region of activations nanostructured carrier is that embodiment 5 is prepared in the present embodiment.
The preparation that the 6th kind of nanostructured of embodiment 11. formed
The 6th kind of nanostructured of present embodiment preparation formed, and is the kit that contains nanodevice and nanometer Mk system, and for example: wherein nanodevice is the nanodevice of the present invention first to one of the 5th kind of nanostructured composition; The nanometer Mk system that the nanometer Mk system is formed for the 4th kind of nanostructured of the present invention.Nanodevice and nanometer Mk system are selected from the prepared product of above-mentioned each related embodiment respectively.Certainly, also available other nanodevice or nanometer Mk system, but contain a kind of nanodevice of the present invention or nanometer Mk system in the kit at least.
The nanostructured of embodiment 6-10 preparation is formed, and can carry out appropriate combination, prepares different Donna rice structures and forms kit.Table 5 is listed the part Donna rice structure of present embodiment preparation and is formed the kit example.Various numbering reference table 1-tables 4.
Table 5
Figure GPA00000523127800331
The application that the third part nanostructured is formed
In following examples, sample is respectively: HCV antibody positive serum, HIV 1+2The antibody positive human serum, HBS Ag positive serum, EBV antibody positive serum, syphilis antibody positive serum, and negative serum (HCV antibody, HIV 1+2Antibody, HBs Ag and all negative serum of syphilis antibody).All samples are all through using classical ELISA method under 10 times of diluting reaction conditions of serum, to detect in advance.
Contain device of the present invention or kit, can go by the application process of known related device or kit to use.Its analysis test method, can with reference to and select the suitable analysis test method of related device or kit to carry out.For example; Chip detecting method is following: (1). and the test of non-current chip: the specimen 5 μ l that will suitably dilute add respectively in the reaction tank of respective chip; 37 ℃ of reactions were washed with cleansing solution after 30 minutes; The label that adds 5 μ l debita spissitudos again washes with cleansing solution after 30 minutes 37 ℃ of reactions, and drying scans again then.Scanner is confocal laser scanner (GMS of Afymetrix company 418 chip scanners); Scanning excitation wavelength 532nm, wavelength of transmitted light 570nm, laser intensity 35/50-55/70; The treated software of the signal that reads (JAGUARII) is handled, and obtains the result after averaging then.(2) test of mobile chip: the specimen that will suitably dilute is heated to 37 ℃; Add the chip reactor with flow velocity 10-50 μ l/min; 60 minutes application of sample time added the washing lotion washing then, and the label that adds 5-10 μ l debita spissitudo again carries out mark; Washing, drying scan by the method identical with the test of non-current chip more at last.
In following examples, the ELISA method of testing is identical with classical way, and for example: the specimen 100 μ l that will suitably dilute add respectively in the corresponding above-mentioned 96 hole ELISA Plates; 37 ℃ were reacted 0.5-1 hour, added 3 times (each 300 μ l) of cleansing solution washing again, added 100 μ l labels then; 37 ℃ were reacted 30 minutes; Add substrate again, utilize ELIASA (Thermo Labsystems, Shanghai Lei Bo Analytical Instrument Co., Ltd) to carry out colorimetric analysis after the reaction.In following examples, fast check reagent bar detection method and known method of testing.For example, the sample that suitably dilutes is added the above-mentioned paper slip of inspecting soon respectively, add cleansing solution again, make test strips slowly be drawn to nature controlling line and occur.
In following examples, the affinity chromatography detection method is identical with known method of testing.For example, present embodiment has detected the dynamics adsorption capacity of the nanometer affinity chromatography system (for example containing the post that function reagent/activation nano particle/micron silica gel particle and functionalized nano particle/activation gather the polysaccharide particle respectively) of the foregoing description preparation.Dynamics adsorption capacity testing conditions is following: be used to fill the pillar internal diameter 0.5cm and the length 2cm of above-mentioned medium, damping fluid is 0.01M Tris-HCl/pH 7.40, and flow velocity is 1ml/min, and used chromatograph is HP 1090.Is example with the affinity reagent during for a-protein, specimen in use behaviour antibody.Affinity chromatography dynamics adsorption capacity assay method is a known method.
In following examples, provide some comparative studies and come further to explain.
Embodiment 12: the application that first kind of nanostructured of the present invention formed
In the present embodiment, the nano chips (for example C1-C3 in the table 1 and C6-C7) of chip used being respectively: embodiment 6 preparations and contrast nano chips 1 and 2.In the contrast nano chips 1, used base be the activation non-nano structure carrier of preparation among the embodiment 2, identical in fixing above activation structure and the nano chips that embodiment 6 prepares.But the chip probe point contains distribution density less than 10/μ m 2The functionalized nano particle, identical in the nano chips of the function reagent in the functionalized nano particle and embodiment 6 preparations.In the contrast nano chips 2; Used base is the coupling oriented nano structure carrier of preparation among the embodiment 3; The activation structure is above-mentioned coupling group; Amino (amino oriented nano structure carrier) is arranged on it, and chip probe puts contained function reagent or/and identical in the nano chips of functionalized nano particle and embodiment 6 preparations.In the present embodiment, the label that uses during chip testing is conventional label, and for example: rhodamine mark two is anti-, the corresponding antigen of rhodamine mark, the corresponding antibody of rhodamine mark, the corresponding antigen of rhodamine mark and corresponding antibody.
In the present embodiment; Nanostructured chip of the present invention and contrast nano chips are relatively: 1). and under preferred function reagent concentration (for example function reagent concentration 0.1-1.0mg/ml during point sample), the average signal reading when using identical positive under the same scan condition is high more than 300%.2). under preferred function reagent concentration (for example function reagent concentration 0.1-1.0mg/ml during point sample), the positive least concentration that can detect will hang down more than 5 times respectively.Thereby nanostructured chip of the present invention (more more specifically, functionalized nano structural unit density and activation structure), the effect with obvious raising reaction sensitivity.
Other nanostructured of embodiment 5 preparations is formed the comparison of (for example C4, C8-C10 in the table 1); More similar (for example the contrasting with coating activation oriented nano structure carrier and activation oriented nano structure carrier) of method and said chip, the result who is obtained is also consistent.It is to be noted that especially the activation oriented nano structure carrier that contains coating activation structure of the present invention has than higher nanometer convex body stability, in preparation and application process, does not see to come off.And this situation happens occasionally on some chemical activation oriented nano structure carrier.
Embodiment 13: the application that second kind of nanostructured of the present invention formed
In the present embodiment, the nano chips (the for example D1-C3 in the table 2, D5 and D6) of chip used being respectively: embodiment 7 preparations and contrast nano chips 1 and 2.In the contrast nano chips 1, used base be the activation non-nano structure carrier of preparation among the embodiment 2, identical in fixing above activation structure and the nano chips that embodiment 7 prepares.But the chip probe point contains distribution density less than 10/μ m 2The functionalized nano particle, identical in the nano chips of the function reagent in the functionalized nano particle and embodiment 7 preparations.In the contrast nano chips 2, used base is the coupling non-directional nanostructured carrier of preparation among the embodiment 4, and the activation structure is above-mentioned coupling group, and amino (amino non-directional nanostructured carrier) is arranged on it.Chip probe is put identical in the nano chips of contained function reagent and functionalized nano structural unit density and embodiment 7 preparations.In the present embodiment, the label that uses during chip testing is conventional label, and for example: rhodamine mark two is anti-, the corresponding antigen of rhodamine mark, the corresponding antibody of rhodamine mark, the corresponding antigen of rhodamine mark and corresponding antibody.
In the present embodiment; Nanostructured chip of the present invention and contrast nano chips are relatively: 1). and under preferred function reagent concentration (for example function reagent concentration 0.1-1.0mg/ml during point sample), the average signal reading when using identical positive under the same scan condition is high more than 100%.2). under preferred function reagent concentration (for example function reagent concentration 0.1-1.0mg/ml during point sample), the positive least concentration that can detect will hang down more than 2 times respectively.Thereby nanostructured chip of the present invention (more more specifically, functionalized nano structural unit density and activation structure), the effect with obvious raising reaction sensitivity.
Other nanostructured of embodiment 7 preparation is formed the comparison of (for example D4 in the table 2), more similar (for example the contrasting) of method and said chip with coating activation oriented nano structure carrier and activation oriented nano structure carrier, and the result who is obtained is unanimity also.
Embodiment 14: the application that the third nanostructured of the present invention is formed
In the present embodiment, the nano chips (the for example B5 in the table 3, B6) of chip used being respectively: embodiment 8 preparations and contrast nano chips 1 and 2.In the contrast nano chips 1, used base be the activation non-nano structure carrier of preparation among the embodiment 2, identical in fixing above activation structure and the nano chips that embodiment 7 prepares.But the chip probe point contains distribution density less than 10/μ m 2The functionalized nano particle, identical in the nano chips of the function reagent in the functionalized nano particle and embodiment 7 preparations.In the contrast nano chips 2, used base is the coupling slide of preparation among the embodiment 2, and the activation structure is above-mentioned coupling group, and amino (amino non-directional nanostructured carrier) is arranged on it, is fixed with the identical function nano particle.In the present embodiment, the label that uses during chip testing is conventional label, and for example: rhodamine mark two is anti-, the corresponding antigen of rhodamine mark, the corresponding antibody of rhodamine mark, the corresponding antigen of rhodamine mark and corresponding antibody.
In the present embodiment; Nanostructured chip of the present invention and contrast nano chips are relatively: 1). and under preferred function reagent concentration (for example function reagent concentration 0.1-1.0mg/ml during point sample), the average signal reading when using identical positive under the same scan condition is high more than 250%.2). under preferred function reagent concentration (for example function reagent concentration 0.1-1.0mg/ml during point sample), the positive least concentration that can detect will hang down more than 4 times respectively.Thereby nanostructured chip of the present invention (more more specifically, functionalized nano structural unit density and activation structure), the effect with obvious raising reaction sensitivity.
Other nanostructured of embodiment 8 preparations is formed the comparison of (for example B1-B3, B4, B6 in the table 3); More similar (for example do contrast, contrast with chemical sheet base and amino sheet base with paint chips base and substrate) of method and said chip, the result who is obtained is unanimity also.
Embodiment 15: the application that the 4th kind of nanostructured of the present invention formed
Embodiment 15.1: the application of functionalized nano
Functionalized nano of embodiment 9 preparations can be widely used.In following examples, provided the example of certain applications.
Embodiment 15.2: the application of nanodevice
With nano chips relatively is example.In the present embodiment, the nano chips (for example A18 in the table 4 and A19) of chip used being respectively: embodiment 9 preparations, contrast nano chips 1,2 and 3.In all experiment chips, all contain the functionalized nano particle, and identical in used base and the nano chips that embodiment 9 prepares.In the contrast nano chips 1, identical in the nano chips that functionalized nano particle and embodiment 9 prepares, but the chip probe point contains distribution density less than 10/μ m 2The functionalized nano particle.In the contrast nano chips 2, the activation nano particle in the used functionalized nano particle is the coupling nano particle of preparation among the embodiment 1, and the activation structure is above-mentioned coupling group, and amino is arranged on it.In the contrast nano chips 3; Activation nano particle in the used functionalized nano particle is the low activation nano particle of preparation among the embodiment 1; The activated group of the activation nano particle on the nano chips of activated group and embodiment 9 preparations in the used functionalized nano particle is identical, but 1m 2Coupling group fixing on the nanoparticle surface is less than 1.85 μ mol, or/and 1m 2Activated group fixing on the nanoparticle surface is less than 0.5 μ mol.In the contrast nano chips 2 and 3, the chip probe point contains distribution density greater than 10/μ m 2The functionalized nano particle.In the present embodiment, the label that uses during chip testing is conventional label, and for example: rhodamine mark two is anti-, the corresponding antigen of rhodamine mark, the corresponding antibody of rhodamine mark, the corresponding antigen of rhodamine mark and corresponding antibody.
In the present embodiment; Nanostructured chip of the present invention and each contrast nano chips are relatively: 1). and under preferred function reagent concentration (for example function reagent concentration 0.1-1.0mg/ml during point sample), the average signal reading when using identical positive under the same scan condition is high more than 200%.2). under preferred function reagent concentration (for example function reagent concentration 0.1-1.0mg/ml during point sample), the positive least concentration that can detect will hang down more than 3 times respectively.In the embodiment of the invention, the bottom line surveyed of positive is diluted to the critical concentration of judging positive and negative through positive and representes.Thereby nanostructured chip of the present invention (more more specifically, functionalized nano structural unit density and activation structure), the effect with obvious raising reaction sensitivity.
The comparison of other nanodevice (for example the nano enzyme target A20 in the watch 4, the fast check reagent A21 of nanometer and A22, bar and nanostructured chromatography glue A23) of embodiment 9 preparations, method is more similar with above-mentioned nano chips, and the result who is obtained is also consistent.The fast check reagent bar of nanostructured of embodiment 9 preparations compares with the fast check reagent bar of contrast nanostructured, and the positive least concentration that can detect will hang down more than 1 times, and 37 ℃ of signal values of placing identical positive after 71 hours do not reduce.The nanostructured affinity chromatography glue of embodiment 9 preparations is compared with nanostructured affinity chromatography glue, and it is high more than 30% that the dynamics adsorption capacity is wanted.
Embodiment 15.3: the application of nanometer Mk system
With the nanometer Mk system of chip agent box relatively is example.In the present embodiment, used chip agent box of the present invention is: 1) go up stationary probe group HCV Ag and the chip of HIV Ag and the nanometer label A15 of table 4 respectively at epoxy radicals slide (Super-Epoxy); 2) go up stationary probe group HCVAg and the chip of HBs Ab and the nanometer label A12 and the A14 of table 4 respectively at epoxy radicals slide (Super-Epoxy).Used contrast agents box, wherein: chip is identical with chip in the chip agent box of the present invention; Used function reagent, mark substance and the nano particle of nanometer label is identical, the preparation method is identical, but the activation structure on the activation nano particle is different.In first kind of contrast nanometer label, used activation nano particle is the coupling nano particle of preparation among the embodiment 1, and the activation structure is above-mentioned coupling group, and amino is arranged on it.In second kind of contrast nanometer label, used activation nano particle be the low activation nano particle of preparation among the embodiment 1, and the activated group of used activation nano particle is identical on activated group and the nano chips that embodiment 9 prepares, but 1m 2Coupling group fixing on the nanoparticle surface is less than 1.85 μ mol, or/and 1m 2Activated group fixing on the nanoparticle surface is less than 0.5 μ mol.
In the present embodiment, chip agent box of the present invention and each contrast chip agent box relatively, under preferred function reagent concentration (for example function reagent concentration 0.1-1.0mg/ml during point sample), the positive least concentration that can detect will hang down more than 1 times respectively.Thereby nanometer label of the present invention has higher sensitivity.This also explains, preferred activation nano particle of the present invention (more more specifically, the activation structure), the effect with obvious raising reaction sensitivity.
Other of embodiment 9 preparation contains the comparison of the kit (the enzyme marking reagent box that for example contains A16 in the table 4 or A17) of nanometer label, and method is more similar with the said chip kit, and the result who is obtained is unanimity also.
Embodiment 16: the application that the 5th kind of nanostructured of the present invention formed
In the present embodiment, the nano chips of chip used being respectively: embodiment 10 preparations and contrast nano chips.The nano chips of embodiment 10 preparation for example contain the chip of functionalized directional nanostructured zone (nano-probe point) and functionalization non-nano structural area (non-nano probe points).The contrast nano chips are the nano chips that contain functionalized directional nanostructured zone of embodiment 6 preparations.In two kinds of nano chips: used function reagent is or/and the functionalized nano particle is identical.In the present embodiment, the label that uses during chip testing is conventional label, and for example: rhodamine mark two is anti-, the corresponding antigen of rhodamine mark, the corresponding antibody of rhodamine mark, the corresponding antigen of rhodamine mark and corresponding antibody.
In the present embodiment, the nano chips of embodiment 10 preparations compare with the contrast nano chips, and when using indirect method to measure, the functionalization non-nano structural area that functionalized directional nanostructured zone compares has stronger ground unrest.Thereby the 5th kind of nanostructured of the present invention formed has greater flexibility, helps improving the sensitivity that particular probe is used particular detection method.This also explains, preferred sheet base of the present invention (more more specifically, many region of activations) has the effect of obvious raising reaction efficiency.
The comparison that other nanostructured of embodiment 10 preparations is formed, method is more similar with said chip, and the result who is obtained is also consistent.It is to be noted that especially the activation oriented nano structure carrier that contains coating activation structure of the present invention has than higher nanometer convex body stability, in preparation and application process, does not see to come off.And this situation happens occasionally on some chemical activation oriented nano structure carrier.
Embodiment 17: the application that the 6th kind of nanostructured of the present invention formed
In the present embodiment, chip used kit is respectively: nano chips kit (the for example E1-E7 in the table 5) and contrast agents box 1 and 2 of embodiment 11 preparations.Contrast agents box 1 contains nano chips and foregoing conventional label, and contrast agents box 2 contains non-nano chip and nanometer label.Conventional chip is used and nano chips identical functions reagent, through identical chip preparation method, processes at the activation non-nano structure carrier with identical activation structure.Activation non-nano structure carrier is selected from embodiment 2.In three kinds of kits, used nano chips nanometer label is identical.
In the present embodiment, the contrast of kit of the present invention and contrast agents box, under preferred function reagent concentration (for example function reagent concentration 0.1-1.0mg/ml during point sample), the positive least concentration that can detect will hang down more than 1 times respectively.Thereby kit of the present invention has higher sensitivity.
The comparison of other kit (for example E8 in the table 5) of embodiment 11 preparations, method is more similar with said chip, and the result who is obtained is also consistent.

Claims (38)

1. a nanostructured is formed, and it is characterized in that: comprise one of following group or the multiple function nanostructured that are formed by active constituent combined function component at least:
A, functionalized directional nanostructured zone, its contained function component comprises function reagent or/and the functionalized nano particle, and its contained active constituent comprises activation oriented nano structure carrier; And said activation oriented nano structure carrier comprises the activation structure and contains the directional nano carrier that aligns the nanometer convex body, and wherein said activation structure comprises coating material solidified at least or/and the following activated group group of one or more of covalent bonding: comprise amino acid based group, amino diazanyl and amino hydrazine derivate base;
B, functionalization non-directional nanostructured district 1; Its contained function component comprises function reagent or/and the functionalized nano particle; Its contained active constituent comprises activation non-directional nanostructured carrier; And said activation non-directional nanostructured carrier comprises activation structure and the non-directional nano-carrier that contains non-directional arrangement nanometer convex body; Wherein said activation structure comprises coating material solidified at least or/and the following activated group group of one or more of covalent bonding: comprise amino acid based group, amino diazanyl and amino hydrazine derivate base, and said amino diazanyl is at 1m 2The lip-deep density of said nanometer convex body is greater than 1 μ mol;
C, functionalization non-directional nanostructured district 2; Its contained function component comprises the functionalized nano particle; Contained active constituent comprises activation non-nano structure carrier; And said activation non-nano structure carrier comprises non-nano carrier and activation structure, and wherein said activation structure comprises coating material solidified at least or/and the following activated group group of one or more of covalent bonding: comprise amino acid based group and amino hydrazine derivate base;
D, functionalized nano particle, it can participate in forming functionalized nano structural area or other nanostructured, and its contained function component comprises function reagent, and contained active constituent is the activation nano particle; Wherein said activation nano particle comprises nano particle and activation structure; Said activation structure comprises coating material solidified at least or/and the following activated group group of one or more of covalent bonding: comprise amino acid based group, amino diazanyl and amino hydrazine derivate base, and said amino diazanyl is at 1m 2Density on the said nanoparticle surface is greater than 1 μ mol activation nano particle;
And in above-mentioned A, B, C, described each the functionalized nano structural area of D, said functionalized nano particle or be fixed with function reagent said nanometer convex body be evenly distributed density greater than 10/μ m 2
Said coating comprises water resistant coating.
2. form according to the described nanostructured of claim 1, wherein saidly comprise amino acid based group, comprise amino acid based or/and the amino acid derivativges base.
3. form according to the described nanostructured of claim 1, wherein saidly comprise amino acid based group, comprise synthetic peptide group or/and synthetic peptide derivant base.
4. form according to the described nanostructured of claim 3, wherein said synthetic peptide comprises and contains 2-10 amino acid whose synthetic peptide.
5. form according to the described nanostructured of one of claim 2-4, wherein said amino acid comprises following one or more: arginine, N, glutaminase, glycocoll, lysine, glutamine.
6. form according to the described nanostructured of claim 1, wherein said water resistant coating is selected from elementary organic paint.
7. form according to the described nanostructured of claim 6, its said elementary organic paint is selected from and contains organosilyl high-molecular coating.
8. form according to the described nanostructured of claim 7, wherein said organosilicon comprises following one or more: dimethyl siloxane, methacrylic acid 3-(methyl silicane), contain the dimethyl siloxane at (methacrylic acid acyl-oxygen) propyl group terminal.
9. form according to the described nanostructured of one of claim 1-8, wherein said activation structure comprises activated group fixing on said coating and the said coating.
10. form according to the described nanostructured of claim 9, wherein said activated group comprises following one or more at least: aldehyde radical, epoxy radicals, amino diazanyl, said amino diazanyl derivant and said amino acid based group.
11. form according to the described nanostructured of claim 1; The zone of other activation structure is also contained in the zone of both having contained said activation structure on wherein said activation oriented nano structure carrier, activation non-directional nanostructured carrier or the activation non-nano structure carrier.
12. form according to the described nanostructured of claim 1, wherein said nanometer convex body comprises following one or more: nano wire, nanotube, nanocone, immobilization nano particle.
13. form according to claim 1 or 12 described nanostructureds, wherein said nanometer convex body or nano particle contain inorganics.
14. form according to the described nanostructured of claim 13, wherein said inorganics comprises metal, slaine.
15. form according to the described nanostructured of claim 14, wherein said slaine comprises following one or more: monox, titanium dioxide, aluminium oxide.
16. form according to the described nanostructured of claim 1, wherein said function reagent comprises biological substance.
17. form according to the described nanostructured of claim 16, wherein said biological substance comprises polypeptide and nucleic acid.
18. form according to the described nanostructured of claim 17, wherein said polypeptide comprises antigen and the antibody that is fixed in the same reactor.
19. form according to the described nanostructured of one of claim 1-18; Wherein said activation oriented nano structure carrier comprises one of following group: the activation nano particle/coupling group of said activated group/coupling group/oriented nano structure carrier, said activated group/oriented nano structure carrier, said activated group/coupling nano particle/oriented nano structure carrier, the activation nano particle/coupling nano particle/oriented nano structure carrier that comprises said activated group, said activated group/nano particle/oriented nano structure carrier, the activation nano particle/nano particle/oriented nano structure carrier that comprises said activated group, the activation nano particle/oriented nano structure carrier that comprises said activated group, the activation oriented nano structure carrier, coating activation oriented nano structure carrier, the coating/chemical activation oriented nano structure carrier that comprise the said activated group of the activation nano particle of said activated group/comprise, activation nano particle/coating of comprising said activated group encapsulate the oriented nano structure carrier, main activated group/coating encapsulates the oriented nano structure carrier.
20. form according to the described nanostructured of one of claim 1-18, wherein said activation non-directional nanostructured carrier comprises one of following group: main activated group/coupling group/non-directional nanostructured carrier, the activation nano particle/coupling group/non-directional nanostructured carrier that comprises said activated group, main activated group/coupling nano particle/non-directional nanostructured carrier, the activation nano particle/coupling nano particle/non-directional nanostructured carrier that comprises said activated group, the activation nano particle/non-directional nanostructured carrier that comprises said activated group, the activation non-directional nanostructured carrier, coating activation non-directional nanostructured carrier, the coating/chemical activation non-directional nanostructured carrier that comprise the said activated group of the activation nano particle of said activated group/comprise, activation nano particle/coating of comprising said activated group encapsulate non-directional nanostructured carrier, main activated group/coating encapsulates non-directional nanostructured carrier.
21. form according to the described nanostructured of one of claim 1-18, wherein said activation non-nano structure carrier comprises one of following group; Said activated group/coupling group/non-nano structure carrier, coating activation non-nano structure carrier, coating/chemical activation non-nano structure carrier, main activated group/coating encapsulate the non-nano structure carrier.
22. form according to the described nanostructured of one of claim 1-18, it comprises the different functions district more than two or two, wherein: at least one said functional zone is the functionalized nano structural area; With at least one said functionalized nano structural area be one of said each functionalized nano structural area.
23. form according to the described nanostructured of one of claim 19-22, it comprises one of following nanodevice group: nanometer analysis chip, nano enzyme target, nano biological sensor, nanometer are picked up reagent strip, nanometer affinity chromatography glue soon.
24. form according to the described nanostructured of one of claim 1-18, it comprises the nanometer Mk system, said nanometer Mk system comprises said function reagent and labelled reagent fixing on said activation nano particle, the activation nano particle at least.
25. form according to claim 23 or 24 described nanostructureds, it comprises and contains said nanodevice or/and the kit of said nanometer Mk system.
26. form according to the described nanostructured of claim 25, said kit comprises nanometer Mk system and said nanodevice.
27. form according to the described nanostructured of claim 25, said kit comprises nanodevice and said nanometer Mk system.
28. an active constituent, said active constituent comprise one of following group or many groups:
A .Activation oriented nano structure carrier; Said activation oriented nano structure carrier comprises the activation structure and contains the directional nano carrier that aligns the nanometer convex body, and wherein said activation structure comprises coating material solidified at least or/and the following activated group group of one or more of covalent bonding: amino diazanyl and amino hydrazine derivate base;
B .Activation non-directional nanostructured carrier; Said activation non-directional nanostructured carrier comprises activation structure and the non-directional nano-carrier that contains non-directional arrangement nanometer convex body; Wherein said activation structure comprises coating material solidified at least or/and the following activated group group of one or more of covalent bonding: comprise amino acid based group, amino diazanyl and amino hydrazine derivate base, and said amino diazanyl is at 1m 2The lip-deep density of said nanometer convex body is greater than 1 μ mol;
C .Activation non-nano structure carrier; Said activation non-nano structure carrier comprises non-nano carrier and activation structure, and wherein said activation structure comprises coating material solidified at least or/and the following activated group group of one or more of covalent bonding: comprise amino acid based group and amino hydrazine derivate base;
D .The activation nano particle; Wherein said activation nano particle comprises nano particle and activation structure; Wherein said activation structure comprises coating material solidified at least or/and the following activated group group of one or more of covalent bonding: comprise amino acid based group, amino diazanyl and amino hydrazine derivate base, and said amino diazanyl is at 1m 2Density on the said nanoparticle surface is greater than 1 μ mol activation nano particle,
And on said activation oriented nano structure carrier or said activation oriented nano structure carrier, have an activation directional nanostructured zone or an activation non-directional nanostructured district at least with following characteristics: said nanometer convex body therein be evenly distributed density greater than 10/μ m 2
29., comprise the said active constituent in the described nanostructured composition of one of claim 1-27 according to the described active constituent of claim 28.
30., be said activation oriented nano structure carrier according to the described active constituent of claim 29.
31., be said activation non-directional nanostructured carrier according to the described active constituent of claim 29.
32., be said activation non-nano structure carrier according to the described active constituent of claim 29.
33., be said activation nano particle according to the described active constituent of claim 29.
34. according to the described active constituent of claim 29, comprise the different activated district more than two or two, wherein: at least one region of activation is activation nanostructured district; With at least one activation nanostructured district, for providing by said activation oriented nano structure carrier or activation non-directional nanostructured district.
35. the preparation method according to the described nanostructured of one of claim 1-27 is formed comprises following a kind of or plurality of step:
(1) .Use synthetic peptide method that said activated group is fixed on said nanometer convex body or/and on the said nano particle;
(2) .Said coating is encapsulated to said oriented nano structure carrier, non-directional nanostructured carrier or non-nano carrier;
(3) .Said coating is encapsulated to said oriented nano structure carrier, non-directional nanostructured carrier or non-nano carrier, introduce activated group encapsulating on the coating successively to introduce coupling group and activated group or to encapsulate on the coating then.
36. according to the preparation method that the described nanostructured of claim 35 is formed, wherein said synthetic peptide method comprises following a kind of or plurality of step: the reactant that contains blocking group is provided and in step thereafter, at least partly sloughs said blocking group;-NH 2Base and-reaction between the COOH base; The amino group chain growth.
37. according to the preparation method that the described nanostructured of claim 35 is formed, wherein said encapsulating comprises said carrier is immersed in the solution of the said coating between the w/v concentration 1/100 to 1/500000.
38. the preparation method according to the described nanostructured of claim 35 is formed comprises the said curing that encapsulates coating, condition of cure comprises: temperature is between 40-80 ° of C; Time is between 3-10 hour.
CN2006800516076A 2006-01-25 2006-10-11 Active constituent, nanostructure composition containing the active constituent and preparation method thereof Expired - Fee Related CN101443661B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN2006800516076A CN101443661B (en) 2006-01-25 2006-10-11 Active constituent, nanostructure composition containing the active constituent and preparation method thereof

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN200610020226.3 2006-01-25
CN 200610020226 CN1811427A (en) 2006-01-25 2006-01-25 Active carrier for biological chip, producing method and application thereof
PCT/CN2006/002659 WO2007085156A1 (en) 2006-01-25 2006-10-11 Active component, nanostructured composition comprising active components and its preparation
CN2006800516076A CN101443661B (en) 2006-01-25 2006-10-11 Active constituent, nanostructure composition containing the active constituent and preparation method thereof

Publications (2)

Publication Number Publication Date
CN101443661A CN101443661A (en) 2009-05-27
CN101443661B true CN101443661B (en) 2012-11-14

Family

ID=36844469

Family Applications (2)

Application Number Title Priority Date Filing Date
CN 200610020226 Pending CN1811427A (en) 2006-01-25 2006-01-25 Active carrier for biological chip, producing method and application thereof
CN2006800516076A Expired - Fee Related CN101443661B (en) 2006-01-25 2006-10-11 Active constituent, nanostructure composition containing the active constituent and preparation method thereof

Family Applications Before (1)

Application Number Title Priority Date Filing Date
CN 200610020226 Pending CN1811427A (en) 2006-01-25 2006-01-25 Active carrier for biological chip, producing method and application thereof

Country Status (1)

Country Link
CN (2) CN1811427A (en)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6221602B1 (en) * 1998-11-10 2001-04-24 Bio-Pixels Ltd. Functionalized nanocrystals and their use in labeling for strand synthesis or sequence determination
US6365362B1 (en) * 1998-02-12 2002-04-02 Immunivest Corporation Methods and reagents for the rapid and efficient isolation of circulating cancer cells
WO2003006676A2 (en) * 2001-07-13 2003-01-23 Nanosphere, Inc. Method for immobilizing molecules onto surfaces
CN1434295A (en) * 2003-03-13 2003-08-06 成都夸常科技有限公司 Test device and method for making qualitative and/or quantitative analysis to object
WO2004089819A1 (en) * 2003-04-14 2004-10-21 Centre National De La Recherche Scientifique Functionalized carbon nanotubes, a process for preparing the same and their use in medicinal chemistry
WO2004102196A1 (en) * 2003-04-30 2004-11-25 Chengdu Kuachang Medical Industrial Limited Apparatus including nanostructures used for separation or analysis, and the preparation and application thereof
WO2005108625A2 (en) * 2001-07-13 2005-11-17 Nanosphere, Inc. Method for preparing substrates having immobilized molecules and substrates

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6365362B1 (en) * 1998-02-12 2002-04-02 Immunivest Corporation Methods and reagents for the rapid and efficient isolation of circulating cancer cells
US6221602B1 (en) * 1998-11-10 2001-04-24 Bio-Pixels Ltd. Functionalized nanocrystals and their use in labeling for strand synthesis or sequence determination
WO2003006676A2 (en) * 2001-07-13 2003-01-23 Nanosphere, Inc. Method for immobilizing molecules onto surfaces
WO2005108625A2 (en) * 2001-07-13 2005-11-17 Nanosphere, Inc. Method for preparing substrates having immobilized molecules and substrates
CN1434295A (en) * 2003-03-13 2003-08-06 成都夸常科技有限公司 Test device and method for making qualitative and/or quantitative analysis to object
WO2004089819A1 (en) * 2003-04-14 2004-10-21 Centre National De La Recherche Scientifique Functionalized carbon nanotubes, a process for preparing the same and their use in medicinal chemistry
WO2004102196A1 (en) * 2003-04-30 2004-11-25 Chengdu Kuachang Medical Industrial Limited Apparatus including nanostructures used for separation or analysis, and the preparation and application thereof

Also Published As

Publication number Publication date
CN101443661A (en) 2009-05-27
CN1811427A (en) 2006-08-02

Similar Documents

Publication Publication Date Title
CN100410664C (en) Device of containing Nano structure for analysis or separation, preparation method and application
US9260656B2 (en) Fluorescent silica nano-particle, fluorescent nano-material, and biochip and assay using the same
US5846724A (en) Highly specific surface for biological reactions having an exposed ethylenic double bond, process of using the surface, and method for assaying for a molecule using the surface
CN100412203C (en) Bio-chip prepared by gelation on a chip substrate
US10191045B2 (en) Sol composition for sol-gel biochip to immobilize probe on substrate without surface treatment and method and screening thereof
JP2007502998A5 (en)
WO2006125124A2 (en) Substrate functionalization method for high sensitivity applications
CN101166693A (en) Structure, porous body, sensor, and method for manufacturing structure, and method for detecting specimen
JPH07260790A (en) Biotin silane compound and bond matrix containing compound thereof
CN1207810A (en) Storage-stable particle, in particular carrier for carrier-bound reactions, and methods of producing said particle
CN101443661B (en) Active constituent, nanostructure composition containing the active constituent and preparation method thereof
JP2005037331A (en) Substrate for detecting substance derived from organism and its manufacturing method
WO2007115444A1 (en) Seperation or analysis composition comprising active nanostructure and seperation or analysis method
CN101013128A (en) Analysis or separation facility containing nano structure
CN1987454B (en) Nano structure component containing oriented nano structure functional zone and its preparing and applying method
CN107486270B (en) Preparation method of microarray chip based on ball-brush double-layer nanostructure substrate
CN1250969C (en) Test device and method for making qualitative and/or quantitative analysis to object
ES2298738T3 (en) IMMOBILIZATION METHOD AND KIT FOR IT.
CN101195473A (en) Immobilization nanowire, containing device and production method thereof
WO2004102196A1 (en) Apparatus including nanostructures used for separation or analysis, and the preparation and application thereof
DE60308176T2 (en) Device for the presentation of polypeptides, process for their preparation and their uses
Apriyani et al. Preliminary Study of Modified Fluorescent Silica Nanoparticles for the Detection of IgY Antibody
AU739219B2 (en) Highly specific surfaces for biological reactions, method of preparation and utilization
WO2007085156A1 (en) Active component, nanostructured composition comprising active components and its preparation
WO2004081571A1 (en) The detecting method and device of polypeptide, and the ligand compound comprising nanoparticles

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C14 Grant of patent or utility model
GR01 Patent grant
ASS Succession or assignment of patent right

Owner name: CHENGDU KUACHANG SCI-TECH CO., LTD.

Free format text: FORMER OWNER: CHENGDU KUACHANG MEDICAL IND. LTD.

Effective date: 20131211

C41 Transfer of patent application or patent right or utility model
COR Change of bibliographic data

Free format text: CORRECT: ADDRESS; FROM: CHENGDU, SICHUAN PROVINCE TO: 610041 CHENGDU, SICHUAN PROVINCE

TR01 Transfer of patent right

Effective date of registration: 20131211

Address after: High tech Zone Gaopeng road in Chengdu city of Sichuan province 610041 No. 5 Chengdu Overseas Students Pioneer Park

Patentee after: Chengdu Kuachang Sci-Tech Co., Ltd.

Address before: Dr. Park No. 5 in Sichuan province Chinese Chengdu high tech Zone Gaopeng Avenue

Patentee before: Chengdu Kuachang Medical Ind. Ltd.

CF01 Termination of patent right due to non-payment of annual fee
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20121114

Termination date: 20191011