CN1987454B - Nano structure component containing oriented nano structure functional zone and its preparing and applying method - Google Patents
Nano structure component containing oriented nano structure functional zone and its preparing and applying method Download PDFInfo
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- CN1987454B CN1987454B CN2006100221476A CN200610022147A CN1987454B CN 1987454 B CN1987454 B CN 1987454B CN 2006100221476 A CN2006100221476 A CN 2006100221476A CN 200610022147 A CN200610022147 A CN 200610022147A CN 1987454 B CN1987454 B CN 1987454B
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Abstract
The disclosed Nano structure composition includes following one or more varieties: multiple Nano structural unitary reactor, multiple activation structural reactor, multiple space structural reactor, high activation structural reactor, multiple sortal functional reactor, and multiple Nano structural kit. The disclosed Nano structure composition provides higher applicability for ex. higher individual sensitivity, overall sensitivity, integration level, or/and application degree of freedom.
Description
Technical field
The present invention relates to a kind of nanostructured that contains oriented nano structure functional zone forms.The invention still further relates to and comprise the methods for making and using same that described nanostructured is formed.
Background technology
The present invention is the continuation of International Patent Application PCT/CN2004/000437.In International Patent Application PCT/CN2004/000437, mainly relate to comprise the nanostructured composition that non-directional is arranged the nanometer convex body.And the present invention then is it to be expanded to comprise the nanostructured composition that aligns the nanometer convex body.Comprise the nanostructured that aligns the nanometer convex body and form, disclose increasing scheme.Yet, aspect applicability (but for example indivedual sensitivity, overall sensitivity integrated level), wait improvements.And, provide more alternative nanostructureds to form, also be that it uses multifarious requirement.
Summary of the invention
Fundamental purpose of the present invention is to improve to comprise the applicability that the nanostructured that aligns the nanometer convex body is formed, but for example improves the individual sensitivity that detects, overall sensitivity integrated level or/and use degree of freedom.Purpose of the present invention is basically by the incompatible realization of various structural group.
So, first aspect of the present invention, provide a kind of nanostructured that contains oriented nano structure functional zone to form, it comprises: at least one many nanometers structural unit reactor of A., and described reactor contains at least one and comprises the oriented nano structure functional zone that aligns nanometer convex body and at least a other nano structured unit; B. at least one activates structural response devices more, and described reactor contains at least two functional zones that comprise different activation structures, and wherein at least one is an oriented nano structure functional zone; C. at least one many space structures reactor, described reactor contains at least two functional zones that comprise the different spaces structure, and wherein at least one is an oriented nano structure functional zone; D. at least one overactivity structural response device, described reactor contains at least one activated group density greater than 2 μ mol/ μ m
2Oriented nano structure functional zone; E. at least one multiple class reaction kinetic device, described multiple class reaction kinetic device contains at least two functional zones that comprise variety classes function reagent, and wherein at least one is an oriented nano structure functional zone; Or/and at least one many nanometers structure kit of F., described many nanometers structure kit contains the reactor that at least one contains oriented nano structure functional zone, with at least a other nanostructured reactant, wherein: (1). described oriented nano structure functional zone comprises at least and aligns the nanometer convex body on function reagent and the carrier; (2). in the described oriented nano structure functional zone, the described distribution density of nanometer convex body that aligns is greater than 10/μ m
2
Second aspect of the present invention provides a kind of method, and described method comprises provides and use nanostructured composition of the present invention.
The detailed description of summary of the invention
Term definition
Among the present invention, term " nanostructured " is meant the structure that includes nano structured unit; Term " nano structured unit " is meant in the three-dimensional structure structural unit of one dimension at least (for example particle diameter, caliber, line footpath, or the like) for nano-scale (preferred 3nm-300nm), for example nano particle, nanometer convex body, or the like; 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 described nanometer ridge three-dimensional structure at least one dimension (for example particle diameter, caliber, line footpath, or the like) be nano-scale, for example: the delineation protrusion of the nano-scale on immobilization nano particle, immobilization nanotube, immobilization nanofiber, the carrier surface, and the nano structured unit of the self assembly formation of nano particle on carrier, or the like.
Among the present invention, term " nanostructured composition " is meant the composition that contains nanodevice, for example analysis or tripping device or kit; Term " nanodevice " is meant the device that contains nano-reactor, for example, nano chips, nano-sensor, nano enzyme target, nanometer affinity electrophoresis bar, nanometer affinity column, nanometer planar chromatography reagent strip, or the like; Term " nano-reactor " is meant the reactor that contains at least one nano structure functional zone; Term " analysis " is meant carry out qualitative or/and quantitative test in external or body; " separation " is meant the separation of one or more components from other component of sample; Term " nano structure functional zone " is meant the functional zone that contains the functionalized nano structure, for example the probe points that contains the functionalized nano structure on the nano chips (nano-probe point), or the like.
Among the present invention, term " activation structure " is meant on the carrier surface the effectively structure of combined function reagent, for example the chemical activation structure, be coated with material package by, or the like.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, and its nanostructure-containing and its are gone up fixing activation structure, for example activate nano particle, activation nanometer convex body, activate nano structured unit, or the like; Term " coupling group " is meant in conjunction with (for example covalent bonding of hydroxyl reaction on silane coupling agent and the nanostructured surface) on nanostructured surface, in order to the fixing group of activated group; Term " coupling nanostructured " is meant that nanostructured and its go up the structure of the coupling group of fixing, for example coupling nano particle.
Among the present invention, term " activated carrier " is meant the activation structure person of containing the effectively fixed function reagent on carrier and the carrier.Term " chemical activation carrier " is meant that the activation structure comprises the activated carrier by the activated group of direct or indirect (for example the passing through coupling group) bonding of covalent bond.Term " coating activation non-nano structure carrier " is meant that the activation structure comprises coating material solidified activated carrier.Term " many activated carriers " is meant that the activation structure comprises activated carrier coating material solidified and other activation structure (organic group outside for example coating material solidified).Term " activation nanostructured carrier " is meant a kind of like this composition, and it contains activation structure fixing on nanostructured fixing on carrier, the carrier and the nanostructured at least.Among the present invention: comprise the nanostructured carrier of oriented nano structure unit, be called for short the oriented nano structure carrier; The nanostructured carrier that does not comprise the oriented nano structure unit is called for short non-directional nanostructured carrier.Term " activates the non-nano structure carrier ", is meant the activated carrier of the activation structure that contains the effectively fixed function reagent on non-nano structure carrier and the non-nano structure carrier.
Among the present invention, term " space structure " is meant the spatial relationship between function reagent and the macroscopical carrier, and it comprises following group at least: non-nano structure, oriented nano structure, non-directional nanostructured district, or the like.
Among the present invention, term " function reagent " is meant to giving the reagent of described nanostructured with reactivity (for example with the object reactive activity).Function reagent is caught object by 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.Ion exchanger is for example: diethyl amino ethyl group (DEAE), diethyl-(2-hydroxypropyl) aminoethyl (QAE), ethyloic (CM), sulfonic acid propyl group (SP), mercapto ethyl pyridinyl (MEP) ,-NH
2,-RCOOH, siloxy group, mercapto, alkyl.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 " is meant a kind of like this composition, and its nanostructure-containing and its are gone up the function reagent of fixing, for example functionalized nano particle, functionalized nano convex body, or the like.Among the present invention, term " functionalized nano structure carrier " is meant a kind of like this composition, 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 " 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 in 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 nano-reactor at least.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 all have nanostructured (for example activating fixed function nano particle former on the nanometer convex body sheet base) to other zone of small part.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 " 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 and 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, for example rhodamine, CY3, the CY5 etc. in the chip detection label commonly used.
As described in the part of front " summary of the invention ", first aspect present invention provides the nanostructured that comprises the directional nano convex body to form.
First kind of nanostructured of the present invention formed, be characterised in that it comprises at least one many nanometers structural unit reactor, described reactor contains at least one and comprises the oriented nano structure functional zone that aligns nanometer convex body and at least a other nano structured unit, wherein: (1). and described oriented nano structure functional zone comprises at least and aligns the nanometer convex body on function reagent and the carrier; (2). in the described oriented nano structure functional zone, the described distribution density of nanometer convex body that aligns is greater than 10/μ m
2
Second kind of nanostructured of the present invention formed, be characterised in that it comprises at least one many activation structural response device, described reactor contains at least two functional zones that comprise different activation structures, and wherein at least one is an oriented nano structure functional zone, wherein: (1). described oriented nano structure functional zone comprises at least and aligns the nanometer convex body on function reagent and the carrier; (2). in the described oriented nano structure functional zone, the described distribution density of nanometer convex body that aligns is greater than 10/μ m
2
The third nanostructured of the present invention is formed, be characterised in that it comprises at least one many space structures reactor, described reactor contains at least two functional zones that comprise the different spaces structure, and wherein at least one is an oriented nano structure functional zone, wherein: (1). described oriented nano structure functional zone comprises at least and aligns the nanometer convex body on function reagent and the carrier; (2). in the described oriented nano structure functional zone, the described distribution density of nanometer convex body that aligns is greater than 10/μ m
2
The 4th kind of nanostructured of the present invention formed, and is characterised in that it comprises at least one overactivity structural response device, and described reactor contains at least one activated group density greater than 2 μ mol/ μ m
2Oriented nano structure functional zone, wherein: (1). described oriented nano structure functional zone comprises at least and aligns the nanometer convex body on function reagent and the carrier; (2). in the described oriented nano structure functional zone, the described distribution density of nanometer convex body that aligns is greater than 10/μ m
2
The 5th kind of nanostructured of the present invention formed, be characterised in that it comprises at least one multiple class reaction kinetic device, described multiple class reaction kinetic device contains at least two functional zones that comprise variety classes function reagent, and wherein at least one is an oriented nano structure functional zone, wherein: (1). described oriented nano structure functional zone comprises at least and aligns the nanometer convex body on function reagent and the carrier; (2). in the described oriented nano structure functional zone, the described distribution density of nanometer convex body that aligns is greater than 10/μ m
2
The 6th kind of nanostructured of the present invention formed, be characterised in that it comprises at least one many nanometers structure kit, described many nanometers structure kit contains the reactor that at least one contains oriented nano structure functional zone, with at least a other nanostructured reactant, wherein: (1). described oriented nano structure functional zone comprises at least and aligns the nanometer convex body on function reagent and the carrier; (2). in the described oriented nano structure functional zone, the described distribution density of nanometer convex body that aligns is greater than 10/μ m
2
The 7th kind of nanostructured of the present invention formed, and is characterised in that the feature that its each nanostructured with at least two kinds of the invention described above is formed.
Of the present invention first or a scheme forming of the 7th kind of nanostructured, during described nanostructured was formed, described many nanometers structural unit reactor contains the one of the following kind or multiple nano structured unit combination: A. aligns the nanometer convex body and aligns functionalized nano particle fixing on the nanometer convex body; B. align functionalized nano particle fixing on nano particle fixing on the nanometer convex body and the nano particle; C. align functionalized reagent fixing on nano particle fixing on the nanometer convex body and the nano particle.Another scheme, the distribution density of wherein said functionalized nano particle in the many nano structure functional zones of described orientation is greater than 50/μ m
2Another scheme, the distribution density of wherein said functionalized nano particle in the many nano structure functional zones of described orientation is greater than 100/μ m
2Different with the catches kind according to nano particle size, function reagent type, the distribution density of functionalized nano particle in the many nano structure functional zones of described orientation has the different preferred upper limits.
Of the present invention second or a scheme forming of the 7th kind of nanostructured, many activation structural response devices contained following activation textural association described in described nanostructured was formed: hydrophobic coating and covalent bond activated group.Another scheme, the surface hydrophobicity angle of wherein said hydrophobic coating is greater than 60 degree.Another scheme, wherein said surface hydrophobicity angle is between the 80-100 degree.Another scheme, wherein said activated group comprise following one or more groups: amino acid based, synthetic peptidyl, amino, aldehyde radical, epoxy radicals, amino hydrazine, ethyloic, sulfonic acid propyl group, mercapto ethyl pyridinyl, mercapto.
The of the present invention the 3rd or a scheme forming of the 7th kind of nanostructured, many space structures reactor described in described nanostructured is formed contains one of the following kind or the combination of multiple functional zone: A. functionalization non-nano structural area and described functionalized directional nanostructured zone; B. functionalization non-directional nanostructured district and described functionalized directional nanostructured zone; C. functionalization non-nano structural area, functionalization non-directional nanostructured district and described functionalized directional nanostructured zone.
The of the present invention the 5th or a scheme forming of the 7th kind of nanostructured, multiple class reaction kinetic device contained the following function agent combination described in described nanostructured was formed: antigen and antibody.Another scheme, wherein said antibody is in described oriented nano structure functional zone, and antibody is in other region of activation.Another scheme, wherein said antibody comprises the hepatitis B surface antibody.Another scheme, wherein said antigen comprises hepatitis C virus antigen or/and hiv antigen.
The of the present invention the 6th or a scheme forming of the 7th kind of nanostructured, in many nanometers structure kit, described other nanostructured reactant comprised the nanometer separator or/and the nanometer label described in described nanostructured was formed.
Of the present invention first to the 7th kind of scheme that nanostructured is formed, the wherein said distribution density of nanometer convex body that aligns is greater than 50/μ m
2Different with the catches kind according to aligning nanometer convex body size, composite nanoparticle size, function reagent type, this distribution density has the different preferred upper limits.In following examples, indivedual nanostructureds are formed the described density that is evenly distributed and are reached 100/μ m
2
Of the present invention first to the 7th kind of scheme that nanostructured is formed, wherein said nanometer convex body comprises nano wire, nanotube, nanocone.
Of the present invention first to the 7th kind of scheme that nanostructured is formed, wherein said function reagent comprises biological substance.Another scheme, wherein said biological substance comprises polypeptide and nucleic acid.
The scheme that of the present invention first to the 7th kind of nanostructured formed, wherein said nanostructured are formed and are comprised one of following group: biochip, enzyme mark reactor, analyte sensors.Another scheme, wherein said nanostructured are formed and are comprised one of following group: biochip kit, enzyme mark reactor kit, analyte sensors kit.
In the method according to second aspect present invention, step that provides and use the nanostructured composition of first aspect present invention is provided for it.
Embodiment
In following examples, used function reagent comprises: polypeptide, antigen, antibody, and other function reagent.Wherein: used synthetic polypeptide comprises that EBV-VCA-P18 antigen (is called for short EBV 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.Peptide Res.52,1998,495-508); Used antigen comprises: hepatitis C virus antigen (HCVAg), 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 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.
The preparation of first's activated carrier and functionalized nano particle
In following examples, used following reagent can be buied on market:
1). coating: 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.The example of organic silicon monomer comprises: dimethyl siloxane, methacrylic acid 3-(methyl silicane), contain the dimethyl siloxane of (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.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).As seen, described activation structure comprises the paint coatings of curing.
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). activator: used activator comprises the basic activator that the part activated group that combines with coupling group can be provided at least and the activator of deriving (or second activator) of the deriveding group on the basic activated group is provided.Used basic activator is to contain amino siloxane, amino hydrazine (NH
3NH
3) and amino acid.Amino acid comprises: arginine, N, glutaminase, glycocoll, lysine, glutamine.In following examples, preferably contain the activator of the synthetic blocking group (for example Fmoc) of peptide, for example amino hydrazine of Fmoc-and Fmoc-amino acid.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 activator of deriving comprises and does not contain amino multi-functional group reagent (for example glutaraldehyde, 1,4-butanediol diglycidyl ether).
4). nano structured unit: used nano structured unit comprises the on-chip nano wire of the nano wire of silicon oxide-containing.The used nano wire substrate that aligns, (line footpath 80nm, line length 400nm-3 μ m, nano wire are evenly distributed the about 50-100 nano wire of density/μ m on carrier available from American I mmuna company
2).For saving consumption, be cut to reduced size according to experiment condition.In following examples, used nano structured unit can be any form nanostructured, in the three-dimensional own at least one dimension have the structural unit of nano-scale.The nano structured unit of other shape that aligns (for example nanometer convex body, nanocone, nanotube, or the like), and the nano structured unit of other material (for example, the nano structured unit that contains organism or inorganics), also can be selected to down the method for embodiment, prepare nanostructured of the present invention and form.Described inorganics comprise inorganic oxide, metal (for example gold, silver), or the like.Described inorganic oxide comprises metal oxide.
5). nano particle: in following examples, used nano particle 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 following examples, used nano particle can also be that any other can be fixed the nano particle of described activation structure in order to fixed function reagent, for example contains the nano particle of organism or inorganics.Wherein, the described nano particle that contains inorganics comprises non magnetic inorganic nano-particle and magnetic inorganic nano-particle, described non magnetic inorganic nano-particle comprises nonmagnetic metal nano particle and non magnetic nonmetal nano particle, and described derivant comprises and is combined with activated group or/and be coated with the derivant of machine thing.Wherein, described inorganic non magnetic nonmetal nano particle comprises metal oxide (for example, silicon oxide particle, Titanium particles, aluminium oxide particles); Described nonmagnetic metal nano particle comprises gold particle, vanadium particle, lead button.In following examples, preferably use the littler nano particle of particle diameter ratio directional nano convex body diameter of section, more preferably use the little nano particle more than 50% of particle diameter ratio directional nano convex body diameter of section.
The various coupling nano particles or the coupling oriented nano structure carrier 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 nano particles or the chemical activation oriented nano structure carrier of following examples preparations, its activated group comprise amino, amino diazanyl, amino acid based, or the like.Certainly, among the present invention, the activated group on the activated carrier also can be selected from following one or more group groups: aldehyde radical, epoxy radicals, ethyloic, sulfonic acid propyl group, mercapto ethyl pyridinyl, mercapto.
Embodiment 1: the activation nanometer particle process method
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 as follows: nano particle concentration (w/v) 1 ‰-2%; Coupling agent concentration (v/v) 1-3%; Reaction medium is moisture alcohol; Temperature of reaction in room temperature to below the reaction medium boiling point between 5 ℃; Reaction time 0.5-5 hour.The professional and technical personnel can obtain required optimal conditions by 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 as follows: the concentration of nano particle (w/v) 1 ‰-2%; Activator concentration (v/v) is at 0.5-5%; Temperature of reaction in room temperature to below 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 by regulating these parameters.If activator 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 the nano particle of making.
Among the present invention, represent the coupling nano particle, represent to activate nano particle with main activated group/coupling nano particle with coupling group/nano particle.In the prepared product of the embodiment of the invention: coupling group is silane coupled group as the aforementioned; Nano particle is included as the nano particle of containing metal oxide as the aforementioned; Main activated group is aforementioned amino diazanyl, amino acid based, synthetic peptidyl or their derivants separately.
In the present embodiment, gained activation nano particle calculate activated group by ultimate analysis or/and coupling group at 1m
2Distribution density on the nanoparticle surface.In following examples, preferably make the activation nano particle of the following feature of apparatus: 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.
Embodiment 2: the preparation of functionalized nano particle
In the present embodiment, the preparation method of functionalized nano particle comprises the function immobilization of reagents on the activation nano particle.The immobilized reactant condition is as follows: 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 by 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.
Among the present invention, come the presentation function nano particle with function reagent/activation nano particle.The functionalized nano particle of present embodiment preparation comprises: antigen/activation nano particle, antibody/activation nano particle, other function reagent/activation nano particle.Wherein: antigen, antibody, other function reagent are all as mentioned above; The activation nano particle is selected from the activation nano particle of embodiment 1 preparation.Table 1 is listed the partial function nano particle example of present embodiment preparation.
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 is for dual-antigen sandwich method, double antibody sandwich method 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).Table 1 is listed the part nanometer label of present embodiment preparation.
Table 1
| Code name | The functionalized nano particle | Other composition | |
| Function reagent | The activation nano particle | ||
| 1). the functionalized nano particle | |||
| A1 | ?HCVAg | Arginine base/coupling nano particle | Do not have |
| A2 | ?HIVAg | The same | Do not have |
| A3 | Syphilis antigen | The same | Do not have |
| A4 | ?HBs?Ab | The same | Do not have |
| A5 | Goat-anti people two is anti- | The same | Do not have |
| A6 | A-protein | The same | Do not have |
| A7 | EBV antigen | The same | Do not have |
| 2). the nanometer label | |||
| A8 | ?HCVAg | Arginine base/coupling nano particle | Rhodamine |
| A9 | ?HIVAg | The same | The same |
| A10 | ?HBs?Ab | The same | The same |
| A11 | Goat-anti people two is anti- | The same | The same |
The preparation of embodiment 3 activation oriented nano structure carriers
In the embodiment of the invention, the preparation method of activation oriented nano structure carrier, can in order to preparation chip activation nanostructured sheet base, nano enzyme mark porous plate activation nanostructured sheet base, biology sensor activation nanostructured sheet base, or the like.In following examples: used coupling agent and activator 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, amino diazanyl, various amino acid based, 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 present embodiment has prepared following first kind to the 5th kind activation oriented nano structure carrier (seeing Table 2):
1). first kind of activation oriented nano structure carrier, its composition comprises: activated group of fixing 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 or activation nano particle.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 activate the activation of nano particle.
2). second kind of activation oriented nano structure carrier, its composition comprises: fixing activated group 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.Its preparation method comprises: to the oriented nano structure carrier by bag by the fixing coupling group of coupling nano particle, again coupling oriented nano structure carrier is introduced fixedly activated group or/and activate the activation of nano particle.
3). the third activation oriented nano structure carrier, its composition comprises: activated group of fixing 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 or activation nano particle.Its preparation method comprises: oriented nano structure carrier bag by nano particle, carry out couplingization then, coupling oriented nano structure carrier is introduced fixing activated group or/and the activation of activation nano particle again.
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.Its preparation method comprises: oriented nano structure carrier bag is activated nano particle.
5). the 5th kind of activation oriented nano structure carrier, its composition comprises: fixing activation nano particle on the activation nano structured unit that aligns on solid phase carrier, the solid phase carrier, the activation nano structured unit.Its preparation method comprises: activation oriented nano structure carrier (for example first kind of activation oriented nano structure carrier) bag is activated 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 as follows:
(1). couplingization, activation: couplingization, activation method in the difference reference example 1.
(2). nano particle, coupling nano particle or activation nano particle bag quilt: between the nano particle concentration in the nanoparticle suspension (w/v concentration) 1/1000 to 1/50000; Bag is by between temperature 10-30 ℃; Bag is by time more than 5 hours.
(3). the bag quilt of activation nano particle on the activation nano structured unit, tube is stated as follows: above-mentioned nano wire substrate is activated (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 reactive group 1 and reactive group 2 under optimized conditions, (for example activate nano particle w/v concentration between 1/100 to 1/10000) and react, thereby will activate the nano particle bag by to the nanostructured carrier.
The preparation of embodiment 3.2 coating activation oriented nano structure carrier
The 6th kind of activation oriented nano structure carrier (seeing Table 2) of the present invention is coating activation oriented nano structure carrier.Its preparation method comprises: the oriented nano structure carrier of cleaning is contacted and wrap quilt with the high molecular hydrophobic coating solution, then encrusting substance is cured.Wherein, bag is comprised by condition: high molecular hydrophobic coating w/v concentration is between 1/10000 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.By regulating these Reaction conditions range, can obtain required optimal conditions.
Wherein said coating comprises above-mentioned coating.
The preparation of embodiment more than 3.3 activation oriented nano structure carriers
The 7th kind of activation oriented nano structure carrier (seeing Table 2) of the present invention is many activation oriented nano structure carriers, and wherein said activation structure contains at least 2 kinds of activated materials.The prepared product of present embodiment exists one or more coating activating areas and chemical activation zone respectively on its surface.Wherein: described coating comprises above-mentioned coating; Described 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 method for preparing and activate the nanostructured carrier more.
A kind of preparation method in the present embodiment, it comprises at least: 1). preparation chemical activation oriented nano structure carrier; 2). be applied to suitable coating solution in the subregion on the oriented nano structure carrier and wrap quilt, then encrusting substance is cured.Bag is by condition and condition of cure, and is identical with embodiment 3.1.
Embodiment 3.4 activation nano particles/the be coated with preparation method that material package is directed the nanostructured carrier
The 8th kind of activation oriented nano structure carrier of the present invention is coated with material package and is directed the activated carrier that forms on the nanostructured carrier for the activation nano particle is fixed on.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, and bag is activated nano particle.Bag is comprised by condition: activation nano particle w/v concentration is between 1/1000 to 1/100000; Temperature is between 10-30 ℃; Time is between 10-48 hour; Bag is an aqueous solution by medium.By 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.
Table 2
| Code name | Kind | The oriented nano structure carrier | The activation structure | ||
| The nanometer convex body | Other | Connect material | Activated material | ||
| B1 | 1 | Nano wire | The coupling group | Activated group | |
| B2 | 1 | Nano wire | The coupling group | The activation nano particle | |
| B3 | 2 | Nano wire | The coupling nano particle | Activated group | |
| B4 | 2 | Nano wire | The coupling nano particle | The activation nano particle | |
| B5 | 3 | Nano wire | Nano particle | The coupling group | Activated group |
| B6 | 3 | Nano wire | Nano particle | The coupling group | The activation nano particle |
| B7 | 4 | Nano wire | The activation nano particle | ||
| B8 | 5 | Nano wire | Activated group 1 | Activation nano particle 2 | |
| B9 | 5 | Nano wire | Activation nano particle 1 | Activation nano particle 2 | |
| B10 | ?6 | Nano wire | Coating |
| B11 | ?7 | Nano wire | Coating and activated group | ||
| B12 | ?7 | Nano wire | Coating and activation nano particle | ||
| B13 | ?8 | Nano wire | Coating | The activation nano particle |
The preparation that the second portion nanostructured is formed
In the embodiment of the invention, nanostructured is formed used step in the preparation of (for example nano chips, nano enzyme mark reactor, biology sensor, or the like), there is no the different of essence with the preparation process of corresponding non-nano constructional device in principle, all be by point sample or the bag quilt, with probe stationary on activated carrier.In the embodiment of the invention, probe comprises function reagent or/and the functionalized nano particle.For example, in the preparation of nano chips, comprise with suitable existing craft or mechanical deposition method, with 3 samples of every kind of probe points on activated carrier.Wherein, the immobilized reactant condition is as follows: function reagent concentration 0.1-2mg/ml; 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 by 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).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 following examples: used oriented nano structure carrier is selected from the activation oriented nano structure carrier of embodiment 3 preparations; Used functionalized nano particle is selected from the functionalized nano particle of embodiment 2 preparations.Certainly, other oriented nano structure carrier, function reagent or functionalized nano particle also can be respectively prepare nanostructured with the method for following embodiment and form.The nanostructured device (biological example chip) of following examples preparation, its nanostructure probe point are gone up the density that is evenly distributed of functionalized nano particle, utilize SPA-300HV type scanning probe microscopy and analysis software to measure.In the functionalized directional nanostructured zone that the nanostructured of following examples preparation is formed, the functionalized nano particle is evenly distributed density, is preferably greater than 50/μ m
2, more preferably greater than 100/μ m
2
Following examples are further described above-mentioned preparation method.
The preparation that 4. first kinds of nanostructureds of embodiment are formed
In the present embodiment, (for example, the many nanometers structural unit probe points on the nano chips) preparation comprises functionalized nano particle or function reagent are carried out immobilized reactant on activation oriented nano structure carrier directed many nanometers structural unit functional zone.The immobilized reactant condition as previously mentioned.Wherein, used functionalized nano particle and activation oriented nano structure carrier are selected from the prepared product of above embodiment.Table 3 is listed the part nano chips example of present embodiment preparation.
The reactor of nanometer structural unit more than first kind of present embodiment preparation, it contains and aligns the nanometer convex body and align functionalized nano particle fixing on the nanometer convex body.For example, the C1-C4 in the table 3.Wherein, directed many nanometers structural unit functional zone is for fixed function nano particle on activation oriented nano structure carrier forms.Used activation oriented nano structure carrier comprises oriented nano structure carrier and activation structure (for example B1 in the table 2 or B10).
The reactor of nanometer structural unit more than second kind of present embodiment preparation, it contains and aligns functionalized nano particle fixing on nano particle fixing on the nanometer convex body and the nano particle.For example, the C5-C8 in the table 3.Wherein, directed many nanometers structural unit functional zone forms for fixed function nano particle on activation oriented nano structure carrier.Used activation oriented nano structure carrier comprises oriented nano structure carrier, nano particle and activation structure (for example one of B2-B9 in the table 2 or B13).
The third many nanometers structural unit reactor of present embodiment preparation, it contains and for example aligns functionalized reagent fixing on nano particle fixing on the nanometer convex body and the nano particle, the C9-C12 in the table 3.Wherein, directed many nanometers structural unit functional zone forms for fixed function reagent on activation oriented nano structure carrier.Used activation oriented nano structure carrier comprises oriented nano structure carrier, nano particle and activation structure (for example one of B2-B9 in the table 2 or B13).
Table 3
| Code name | Type | Probe * | Activated carrier ** |
| C1 | 1 | A1、A2、A3 | B1 or B10 |
| C2 | 1 | A1、A2、A3、A7 | B1 |
| C3 | 1 | A1、A2、A3、A4 | B1 |
| C4 | 1 | A1、A2、A4、A6、A7 | B1 |
| C5 | 2 | A1、A2、A3 | One of B2-B9 or B13 |
| C6 | 2 | A1、A2、A3、A4 | One of B2-B9 or B13 |
| C7 | 2 | A1、A2、A4、A7 | One of B2-B9 or B13 |
| C8 | 2 | A1、A2、A4、A6、A7 | One of B2-B9 or B13 |
| ?C9 | ?3 | HCV Ag, HIV Ag, syphilis antigen | One of B2-B9 or B12 |
| ?C10 | ?3 | HCV Ag, HIV Ag, syphilis antigen, HBs Ab | One of B2-B9 or B13 |
| ?C11 | ?3 | HCV Ag, HIV Ag, syphilis antigen, HBs Ab, EBV antigen | One of B2-B9 or B13 |
| ?C12 | ?3 | HCV Ag, HIV Ag, syphilis antigen, HBs Ab, a-protein, EBV-VCA-P18 antigen | One of B2-B9 or B13 |
*: reference table 1; *: reference table 2
The preparation that 5. second kinds of nanostructureds of embodiment are formed
In the present embodiment, activate the preparation of structural response device (for example micro-array chip) more, comprise, be fixed on many activation oriented nano structure carriers two or more functionalized nano particle or function reagent.The immobilized reactant condition as previously mentioned.Wherein: used many activation oriented nano structure carriers are above-mentioned the 7th kind of activation oriented nano structure carrier, are selected from embodiment 3.3 (for example B11 in the table 2 or B12); Used functionalized nano particle is selected from the prepared product (for example A1-A7 in the table 1) of embodiment 1.In the embodiment of the invention, a preferred point sample scheme is: antibody probe is fixed on the region of activation that contains organosilicon bag quilt, and antigen probe is fixed on the region of activation that contains activated group.
The preparation that embodiment 6. the third nanostructureds are formed
In the embodiment of the invention, (for example, a nano-reactor on the nano chips) preparation comprises the functionalized nano particle or/and function reagent is fixed on many space structures of activation carrier many space structures reactor.The immobilized reactant condition as previously mentioned.Wherein: used functionalized nano particle is selected from the prepared product (for example A1-A7 in the table 1) of above embodiment; Used many space structures of activation carrier, the activation non-nano structural area that comprises the activation nanostructured district that is distributed with the directional nano convex body and be not distributed with significant nanometer convex body.For example, utilize in the previous embodiment preparation method of activation oriented nano structure carrier, a carrier that had not only contained directional nanostructured zone but also contained the non-nano structural area is coated with the material package quilt, both can obtain this and activate many space structures carrier.Utilize preferred chemical activation method, also can obtain this and activate many space structures carrier.Table 4 is listed the part nano chips example of present embodiment preparation.
The space structure reactor more than first kind of present embodiment preparation, it contains at least one functionalization non-nano structural area and at least one described functionalized directional nanostructured zone, for fixed function reagent on many space structures of activation carrier forms.For example, the D1-D4 in the table 4.One of preferred scheme of present embodiment is to be fixed for the probe of double antibody (or two antigen) sandwich method in the activation directional nanostructured zone, and is fixed for the probe of indirect method in activation non-nano structural area.
The space structure reactor more than second kind of present embodiment preparation, it contains at least one functionalization non-directional nanostructured district and at least one described functionalized directional nanostructured zone, for fixed function nano particle on many space structures of activation carrier forms.For example, the D5-D8 in the table 4.One of preferred scheme of present embodiment is to be fixed for the functionalized nano particle probe of double antibody (or two antigen) sandwich method in the activation directional nanostructured zone, and is fixed for the functionalized nano particle probe of indirect method in activation non-nano structural area.
The third many space structures reactor of present embodiment preparation, it contains at least one functionalization non-nano structural area, at least one functionalization non-directional nanostructured district and at least one described functionalized directional nanostructured zone, for fixed function reagent and functionalized nano particle on many space structures of activation carrier form.For example, the D9-D12 in the table 4.One of scheme of present embodiment choosing, be in the activation directional nanostructured zone, to be fixed for the functionalized nano particle probe of double antibody (or two antigen) sandwich method, and in activation non-nano structural area, be fixed for the functionalized nano particle probe and the function reagent probe of indirect method.
Table 4
| Code name | Type | Fixing probe in the activation directional nanostructured zone | Fixing probe in the activation non-nano structural area | |
| First kind | Second kind | |||
| D1 | 1 | HCV?Ag、HIV?Ag | HCVAg、HIVAg | - |
| D2 | 1 | HCV?Ag、HIV?Ag | Syphilis antigen | - |
| D3 | 1 | HBs?Ab | HCVAg, HIVAg, syphilis antigen | - |
| D4 | 1 | HBs?Ab | HCVAg, HIVAg, syphilis antigen, EBV antigen | - |
| D5 | 2 | A1、A2 | A1、A2 | |
| D6 | 2 | A1、A2 | A3 | |
| D7 | 2 | A4 | A1、A2、A3 | |
| D8 | 2 | A4 | A1、A2、A3、A7 | |
| D9 | 3 | A1、A2 | A1、A2 | HCVAg、HIVAg |
| D10 | 3 | A1、A2 | A3 | HCVAg、HIVAg |
| D11 | 3 | A4 | A1、A2、A3 | HCVAg、HIVAg |
| D12 | 3 | A4 | A1、A2、A3 | EBV-antigen |
*: probe code name reference table 1
The preparation that the 4th kind of nanostructured of embodiment 7. formed
In the present embodiment, in the overactivity structural response device, oriented nano structure functional zone contains density greater than 0.5 μ mol/ μ m
2Activated group, for containing on the activation oriented nano structure carrier that activates nano particle, fixed function reagent is or/and functionalized nano particle and forming.The used activation oriented nano structure carrier that activates nano particle that contains is selected from the prepared product of the foregoing description.Wherein, the activation nano 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.In fact, do not containing on the activation oriented nano structure carrier that activates nano particle, so activated group density also should arranged, during less than this density, the raising of the sensitivity of oriented nano structure functional zone, the raising when obviously being not so good as greater than this density.
The preparation that the 5th kind of nanostructured of embodiment 8. formed
In the present embodiment, the contained at least two kinds of function reagent of multiple class reaction kinetic device can be one of following combinations: nucleic acid and polypeptide, cell and polypeptide, antigen and antibody, or the like.Its preparation method comprises antigen of probe and probe antibody are fixed on the activation oriented nano structure carrier.The immobilized reactant condition as previously mentioned.Wherein used activation oriented nano structure carrier is selected from the activation oriented nano structure carrier (for example B1-B12 in the table 2) for preparing in the previous embodiment.Wherein used antigen of probe and probe antibody combination comprise: A. comprises hepatitis B surface antibody and hepatitis C virus antigen; B. comprise hepatitis B surface antibody, hepatitis C virus antigen and hiv antigen; C. comprise hepatitis B surface antibody, hepatitis C virus antigen, hiv antigen and tumor marker antigen.Antigen of probe comprises antigen or/and antigen coated nano particle.Probe antibody comprises antibody or/and the antibody sandwich nano particle.For example, some nano chips reactor in table 3 and the table 4.In addition, this forms supporting label, should non-specific antigen-antibody reaction not take place with any probe or object.Preferred label is one of following label combination: A, specific marker antibody and specific marker antigen; B, different structure specific marker antiantibody; C, species specificity mark antiantibody and specific marker antibody are or/and specific marker antigen; D, structure specific marker antiantibody and specific marker antibody are or/and specific marker antigen; E, based on the various combinations of deriving of combinations thereof
The preparation that the 6th kind of nanostructured of embodiment 9. formed
The 6th kind of nanostructured of the present invention formed, and it is characterized by, and contains at least one many nanometers structure kit.In the embodiment of the invention, the combination of prepared nanostructured comprises: first kind of nanostructured combination of A. contains at least one reactor that contains oriented nano structure functional zone and at least a nanometer label; B. second kind of nanostructured combination contains at least one reactor that contains oriented nano structure functional zone and at least a nanometer separator; C. the third nanostructured combination contains at least one reactor that contains oriented nano structure functional zone, at least a nanometer label and at least a nanometer separator.In the embodiment of the invention: used at least one reactor that contains oriented nano structure functional zone, nanometer label, the nanometer separator of containing all is selected from the prepared product of the above embodiment of the present invention.
The preparation that the 7th kind of nanostructured of embodiment 10. formed
The 7th kind of nanostructured of present embodiment preparation formed, comprise one of following group: A, at least one many nanometers structural unit/activate structural response device more, this reactor contains at least two functional zones, and wherein: at least two functional zones comprise different activation structures and at least one functional zone is directed many nanometers structural unit functional zone; B, at least one many nanometers structural unit/many space structures reactor, this reactor contains at least two functional zones, and wherein: at least two functional zones comprise the different spaces structure and at least one functional zone is directed many nanometers structural unit functional zone; C, at least one many nanometers structural unit/multiple class reaction kinetic device, this reactor contains at least two functional zones, and wherein: at least two functional zones comprise variety classes function reagent and at least one functional zone is directed many nanometers structural unit functional zone; D, at least one many nanometers structural unit reactor and at least a nanometer separator are or/and the nanometer label; E, at least one many activation structure/many space structures reactor, this reactor contains at least two functional zones, and wherein: at least two functional zones comprise the different spaces structure, at least two functional zones comprise different activation structures; F, at least one many activation structure/multiple class reaction kinetic device, this reactor contains at least two functional zones, and wherein: at least two functional zones comprise variety classes function reagent, at least two functional zones comprise different activation structures; G, at least one many activation structural response device and at least a nanometer separator are or/and the nanometer label; H, at least one many space structure/multiple class reaction kinetic device, this reactor contains at least two functional zones, and wherein: at least two functional zones comprise variety classes function reagent, at least two functional zones comprise the different spaces structure; I, at least one many space structures reactor and at least a nanometer separator are or/and the nanometer label; J, at least one multiple class reaction kinetic device and at least a nanometer separator are or/and the nanometer label; K, at least one many nanometers structural unit/activate structure/many space structures reactor more, this reactor contains at least two functional zones, and wherein: at least two functional zones comprise the different spaces structure, at least two functional zones comprise different activation structures and at least one functional zone is directed many nanometers structural unit functional zone; L, at least one many nanometers structural unit/activate structure/multiple class reaction kinetic device more, this reactor contains at least two functional zones, and wherein: at least two functional zones comprise variety classes function reagent, at least two functional zones comprise different activation structures and at least one functional zone is directed many nanometers structural unit functional zone; M, at least one many nanometers structural unit/activate structural response device and at least a nanometer separator or/and the nanometer label more, this reactor contains at least two functional zones, and wherein: at least two functional zones comprise different activation structures and at least one functional zone is directed many nanometers structural unit functional zone; N, at least one many activation structure/many space structures/multiple class reaction kinetic device, this reactor contains at least two functional zones, and wherein: at least two functional zones comprise that variety classes function reagent, at least two functional zones comprise the different spaces structure, at least two functional zones comprise different activation structures; O, at least one many activation structure/many space structures reactor and at least a nanometer separator are or/and the nanometer label, this reactor contains at least two functional zones, and wherein: at least two functional zones comprise the different spaces structure, at least two functional zones comprise different activation structures; P, at least one many space structure/multiple class reaction kinetic device and at least a nanometer separator are or/and the nanometer label, this reactor contains at least two functional zones, and wherein: at least two functional zones comprise variety classes function reagent, at least two functional zones comprise the different spaces structure; Q, at least one many nanometers structural unit/activate structure/many space structures/multiple class reaction kinetic device more, this reactor contains at least two functional zones, and wherein: at least two functional zones comprise that variety classes function reagent, at least two functional zones comprise the different spaces structure, at least two functional zones comprise different activation structures and at least one functional zone is directed many nanometers structural unit functional zone; S, at least one many nanometers structural unit/activate structure/many space structures reactor and at least a nanometer separator or/and the nanometer label more, this reactor contains at least two functional zones, and wherein: at least two functional zones comprise the different spaces structure, at least two functional zones comprise different activation structures and at least one functional zone is directed many nanometers structural unit functional zone; T, at least one many nanometers structural unit/activate structure/many space structures/multiple class reaction kinetic device and at least a nanometer separator or/and the nanometer label more, this reactor contains at least two functional zones, and wherein: at least two functional zones comprise that variety classes function reagent, at least two functional zones comprise the different spaces structure, at least two functional zones comprise different activation structures and at least one functional zone is directed many nanometers structural unit functional zone.
The reactor of present embodiment preparation all contains at least two functional zones.In the present embodiment, the preparation method of the reactor in difference in functionality district is arranged, respectively with reference to the preparation method of respective reaction device in the various embodiments described above.
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 to detect in advance under 10 times of diluting reaction conditions of serum.In following examples, used activation slide is according to the homemade activation slide of known method.
Nanostructured of the present invention is formed (device or kit), can go by the application process of known related device or kit to use.For example, chip detecting method is as follows: (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, wash with cleansing solution after 30 minutes 37 ℃ of reactions, 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 (JAGUAR II) is handled, and obtains the result after averaging then.(2). the test of the chip that flows: 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, add the washing lotion washing then, 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 the embodiment of the invention, the bottom line surveyed of positive is diluted to the critical concentration of judging positive and negative by positive and represents.In following examples, locate (for example embodiment 15 and 16) unless stated otherwise, 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 following examples, with nano chips be example relatively, provide some comparative studies and come further instruction in addition.
Embodiment 10: the application that first kind of nanostructured of the present invention formed
In the present embodiment, chip used being respectively: first kind, second kind, the third many nanometers structural unit reactor being selected from embodiment 4 (for example table 3) preparation; With contrast chip 1 that contains single nano structured unit reactor and contrast chip 2.Wherein: contrast chip 1 contains and aligns the nanometer convex body and align on the nanometer convex body fixing function reagent, for using identical activation oriented nano structure carrier, using identical function reagent prepared under identical point batten spare; Contrast chip 2 contains the functionalized nano particle that non-directional is arranged on the activation slide, for use have identical activation structure the activation slide, use the functionalized nano particle that contains identical function reagent, prepared under identical point batten spare.
In the present embodiment, nanostructured chip of the present invention and each contrast chip is relatively: 1). and under preferred function reagent concentration (for example function reagent concentration 0.1-0.2mg/ml during point sample), the average signal reading when using identical positive under the same scan condition is high more than 50%.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 1 times respectively.This explanation, preferred nano structured unit of the present invention compound, effect with obvious raising reaction sensitivity.Other first kind of nanostructured of the present invention is formed (for example nano enzyme target A20, nano-sensor), also should have and the identical result of nanostructured chip of the present invention.
Embodiment 11: the application that second kind of nanostructured of the present invention formed
In the present embodiment, chip used being respectively: the chip and the contrast chip that are selected from containing of embodiment 5 preparations of many activation structural responses device.Wherein, the contrast chip contains single activation structural response device, and its activation structure be an identical activated group in the chip with many activation structural response device, for use identical function reagent prepared under identical point batten spare.
In the present embodiment, nanostructured chip of the present invention and contrast chip be relatively: 1). under preferred function reagent concentration (for example function reagent concentration 0.1-0.2mg/ml during point sample), when using identical weak positive, the average marking signal reading of the catches of HBs Ab probe under the same scan condition, containing on the functional areas that contain amination activation directional nano convex body on the functional areas that the organosilicon bag is activated the directional nano convex body than it highly more than 50%, the average marking signal reading of the catches of HCV Ag probe then differs not obvious on two functional areas; 2). under preferred function reagent concentration (for example function reagent concentration 0.1-1.0mg/ml during point sample), the positive least concentration that HBs Ab probe can detect, containing on the functional areas that contain amination activation directional nano convex body on the functional areas that the organosilicon bag is activated the directional nano convex body than it lowly more than 50%, the positive least concentration that HCV Ag probe can detect then differs not obvious on two functional areas.This explanation, activation structural unit of the present invention compound, effect with obvious raising reaction total sensitivity.
Embodiment 12: the application that the third nanostructured of the present invention is formed
In the present embodiment, many space structures unitary reactor that chip used being respectively: embodiment 6 prepares and the contrast chip 1 that contains single space structure unitary reactor, contrast chip 2 and contrast chip 3.Wherein: the chip that contains many space structures unitary reactor is selected from table 4; Contrast chip 1 only contains functionalization non-nano structural area (probe points), contains activation slide, the use identical function reagent, prepared under identical point batten spare of identical activation structure for use; Contrast chip 2 only contains functionalization non-directional nanostructured district (probe points), for use contain identical activation structure the activation slide, use the functionalized nano particle contain identical function reagent, prepared under identical point batten spare; Contrast chip 3 only contains functionalized directional nanostructured zone (probe points), contains activation oriented nano structure carrier, the use identical function reagent, prepared under identical point batten spare of identical activation structure for use.
In the present embodiment, chip of the present invention and each contrast chip is relatively: 1). under preferred function reagent concentration (for example function reagent concentration 0.1-0.2mg/ml during point sample), when using negative sample, the average marking signal reading of the catches of probe under the same scan condition (for example antigen probe), on functionalization non-directional nanostructured district that compares on the functionalized directional nanostructured zone and functionalization non-nano structural area, high by 50% respectively, more than 80%, the average marking signal reading of the negative sample catches of HBs Ad probe then differs not obvious on three functional areas.Thereby many space structures unitary reactor of the present invention can be at difference in functionality reagent, different probe detection method (for example sandwich method, indirect method, or the like) and different sample, and provides preferred version more neatly, the sensitivity of Ti Gaoing on the whole.This also illustrates, preferred space structure of the present invention compound, the effect with obvious raising reaction total sensitivity.
Embodiment 13: the application that the 4th kind of nanostructured of the present invention formed
In the present embodiment, chip used being respectively: the chip that contains at least one overactivity structural response device and the contrast chip that are selected from embodiment 7.Wherein, the difference of contrast chip is that activated group density is less than 0.5 μ mol/ μ m
2
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 0.1-0.2mg/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.Thereby nanostructured chip of the present invention has the reaction sensitivity of obvious raising.
Embodiment 14: the application that the 5th kind of nanostructured of the present invention formed
In the present embodiment, chip used being respectively: the chip and the contrast chip that are selected from containing of embodiment 8 of multiple class reaction kinetic device.Wherein, the contrast chip contains single kind reaction kinetic device, and a kind of is antigen chip, and a kind of is antibody chip, and other structure is identical.
In the present embodiment, the 4th kind of nanostructured of the present invention formed (nanostructured chip for example of the present invention) and formed (for example contrasting chip) relatively with contrast, just can obtain the result who obtains at two contrast chip reactors on a reactor.This also illustrates, preferred probes kind of the present invention compound has the integrated level of obvious raising.
Embodiment 15: the application that the 6th kind of nanostructured of the present invention formed
With chip agent box relatively is example.In the present embodiment, chip used kit is respectively: be selected from many nanometers structure kit of embodiment 9 preparations and single nanostructured kit in contrast.Single nanostructured kit is respectively contrast agents box 1 that contains nano chips and conventional label and the contrast agents box 2 that contains conventional chip and nanometer label, wherein nano chips and nanometer label respectively with many nanometers structure kit in nano chips identical with the nanometer label.
In the present embodiment, chip agent box of the present invention and each contrast chip agent box is relatively: 1). and under preferred function reagent concentration (for example function reagent concentration 0.1-0.2mg/ml during point sample), the average signal reading when using identical positive under the same scan condition is high more than 75%.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 1 times respectively.This explanation, preferred functionalized nano structure of the present invention compound, effect with obvious raising reaction sensitivity.Other nanostructured of the present invention is formed (for example nano enzyme target kit, nano-sensor kit), and the result identical with chip agent box of the present invention also should be arranged.
Embodiment 16: the application that the 7th kind of nanostructured of the present invention formed
In the present embodiment, used groups of nanostructures becomes to be respectively: the nanostructured that multiplicity is higher is formed and the contrast nanostructured is formed.Wherein: the nanostructured that multiplicity is higher is formed the prepared product that is selected from embodiment 10, and the contrast nanostructured is formed the relevant nanometer structure that is selected among the embodiment 4-9 and formed.In the present embodiment, the nanostructured that multiplicity is higher is formed with the contrast nanostructured and is formed relatively, provides more confession preferred condition, thereby higher application degree of freedom is arranged.
Claims (13)
1. a nanostructured that contains oriented nano structure functional zone is formed,
Wherein:
Described oriented nano structure functional zone comprises at least and aligns nanometer convex body and the wherein said distribution density of nanometer convex body that aligns greater than 10/μ m on function reagent and the carrier
2With
Described function reagent is meant that it comprises in order to give the reagent of described nanostructured with reactivity:
A. at least one many nanometers structural unit reactor, described many nanometers structural unit reactor is meant and contains the reactor that at least one comprises the oriented nano structure functional zone that aligns nanometer convex body and at least a other nano structured unit;
B. at least one many activation structural response device, described many activation structural response devices are meant that containing at least two comprises the functional zone of different activation structures and wherein at least one is the reactor of oriented nano structure functional zone;
C. at least one many space structures reactor, described many space structures reactor are meant that containing at least two comprises the functional zone of different spaces structure and wherein at least one is the reactor of oriented nano structure functional zone;
D. at least one overactivity structural response device, described overactivity structural response device is meant and contains at least one activated group density greater than 0.5 μ mol/ μ m
2The reactor of oriented nano structure functional zone;
E. at least one multiple class reaction kinetic device, described multiple class reaction kinetic device are meant that containing at least two comprises the functional zone of variety classes function reagent and wherein at least one is the reactor of oriented nano structure functional zone; Or/and
F. at least one many nanometers structure kit, described many nanometers structure kit is meant: the kit that contains at least one reactor that contains oriented nano structure functional zone and at least a other nanostructured reactant.
2. the described nanostructured of claim 1 is formed, and wherein said many nanometers structural unit reactor contains one of the following kind or the combination of multiple nano structured unit:
A. align the nanometer convex body and align functionalized nano particle fixing on the nanometer convex body;
B. align functionalized nano particle fixing on nano particle fixing on the nanometer convex body and the nano particle;
C. align functionalized reagent fixing on nano particle fixing on the nanometer convex body and the nano particle.
3. the described nanostructured of claim 2 is formed, and the distribution density of wherein said functionalized nano particle in the many nano structure functional zones of described orientation is greater than 50/μ m
2
4. the described nanostructured of claim 1 is formed, and wherein said many activation structural response devices contain following activation textural association: hydrophobic coating and covalent bond activated group.
5. the described nanostructured of claim 4 is formed, and the surface hydrophobicity angle of wherein said hydrophobic coating is greater than 60 degree.
6. the described nanostructured of claim 4 is formed, and wherein said activated group comprises following one or more groups: amino acid based, synthetic peptidyl, amino, aldehyde radical, epoxy radicals, amino hydrazine, ethyloic, sulfonic acid propyl group, mercapto ethyl pyridinyl, mercapto.
7. the described nanostructured of claim 1 is formed, and wherein said many space structures reactor contains one of the following kind or the combination of multiple functional zone:
A. functionalization non-nano structural area and described functionalized directional nanostructured zone;
B. functionalization non-directional nanostructured district and described functionalized directional nanostructured zone;
C. functionalization non-nano structural area, functionalization non-directional nanostructured district and described functionalized directional nanostructured zone.
8. the described nanostructured of claim 1 is formed, and wherein said multiple class reaction kinetic device contains the following function agent combination: antigen and antibody.
9. the described nanostructured of claim 8 is formed, and wherein said antibody is in described oriented nano structure functional zone, and antigen is in other functional zone.
10. the described nanostructured of claim 8 is formed, and wherein said antibody comprises the hepatitis B surface antibody.
11. the described nanostructured of claim 8 is formed, wherein said antigen comprises hepatitis C virus antigen or/and hiv antigen.
12. the described nanostructured of claim 1 is formed, wherein said other nanostructured reactant comprises the nanometer separator or/and the nanometer label.
13. the described nanostructured of claim 1 is formed, wherein said nanometer convex body comprises nano wire, nanotube, nanocone.
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