CN105339791A - Molecular nets on solid phases - Google Patents

Molecular nets on solid phases Download PDF

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Publication number
CN105339791A
CN105339791A CN201480025377.0A CN201480025377A CN105339791A CN 105339791 A CN105339791 A CN 105339791A CN 201480025377 A CN201480025377 A CN 201480025377A CN 105339791 A CN105339791 A CN 105339791A
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molecular network
capture molecules
solid phase
devices
molecular
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CN201480025377.0A
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CN105339791B (en
Inventor
艾米丽·斯坦
布鲁斯·菲尔普斯
罗伯特·普莱斯
迪娜·尤兹丽
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Innovitch Co.,Ltd.
Senna Cancer Diagnosis Co.
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SEVIDENT Inc
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Priority claimed from US13/938,055 external-priority patent/US9910040B2/en
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Priority to CN201910037871.3A priority Critical patent/CN110068677A/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent

Abstract

Disclosed is a covalently-linked multilayered three-dimensional matrix comprising capture molecules, linkers and spacers (referred to as a molecular net) for specific and sensitive analyte capture from a sample. Also disclosed herein is a molecular net comprising covalently-linked multilayered three-dimensional matrix comprising more than one type of capture molecule and more than one type of linker and may comprise one or more spacer for specific and sensitive capture of more than one type of analyte from a sample. A molecular net may comprise a pseudorandom nature. Use of various capture molecules, linkers and spacers in a molecular net may confer unique binding properties to a molecular net. Porosity, binding affinity, size exclusion abilities, filtration abilities, concentration abilities and signal amplification abilities of a molecular net may be varied and depend on the nature of components used in its fabrication. Uses of a molecular net may include analyte capture, analyte enrichment, analyte purification, analyte detection, analyte measurement and analyte delivery. Molecular nets may be used in liquid phase or on solid phases such as nanomaterials, modified metal surfaces, nanospheres, microspheres, microtiter plates, slides, pipettes, cassettes, cartridges, discs, probes, lateral flow devices, microfiuidics devices, microfiuidics devices, optical fibers and others.

Description

Molecular network in solid phase
The cross reference of related application
This application claims the U.S.Provisional Serial 61/783 submitted on March 14th, 2013, the rights and interests of 189, and be the U.S. Patent Application Serial Number 13/511 submitted on March 22nd, 2012, the U.S. Patent Application Serial Number 13/938 that on July 9th, 364 and 2013 submits to, the part continuation application of 055, the full content of above application is combined in this by reference.
Background technology
The current strategies use of catching for solid phase assays thing, analyzing quality testing survey and analysis measurement of existence is adsorbed to or covalently bonded senses to the individual layer capture molecules on surface for directly real-time, or be combined with secondary detection step with indirect detection mode, these strategies are well-known in the art.Direct and indirect method all illustrates the limitation on sensitivity, specificity, signal to noise ratio (S/N ratio) and/or cost.
There are the needs to the analysis thing capture technique for solid phase surface or device, this technology can when seldom or do not have optionally to catch from complex sample when sample preparation and analyze thing and located to maximize the mode of catching analysis measurement and/or detection by the analysis thing of described selection, and its mode is and most of technical compatibility.
Summary of the invention
Describe for catching the device analyzing thing.In one embodiment, a device can comprise a solid phase and a molecular network, and this molecular network is attached to going up at least partially of the surface of this solid phase.This molecular network can comprise multiple capture molecules of at least one type, and these capture molecules are coupled to each other by polytype multiple connection molecule, to form a covalently bound multi-layer three-dimension matrix.These capture molecules can be arranged in conjunction with this analysis thing.
Also describe a kind of manufacture for catching the method for the device analyzing thing.In one embodiment, a kind of method can comprise provides a solid phase, and a molecular network is placed on going up at least partially of the surface of this solid phase.This molecular network can comprise multiple capture molecules of at least one type, and these capture molecules are coupled to each other by polytype multiple connection molecule, to form a covalently bound multi-layer three-dimension matrix.These capture molecules can be arranged in conjunction with this analysis thing.
Also describe the method for the amount of the analysis thing measured in sample.In one embodiment, a kind of method can comprise provides one or more device, and each device comprises a solid phase and a molecular network, and this molecular network covers the surface of this solid phase at least partially.This molecular network can comprise multiple capture molecules of at least one type, and these capture molecules are coupled to each other by polytype multiple connection molecule, to form a covalently bound multi-layer three-dimension matrix.These capture molecules are arranged in conjunction with this analysis thing.The method also comprises these devices is exposed to this sample, and allow this analysis thing at least partially in conjunction with the capture molecules of the molecular network of these devices.
Accompanying drawing explanation
Fig. 1 illustrates comparing of in IgG purifying traditional conjugated microparticle and molecular network microparticle;
Fig. 2 illustrates the conjugated and corresponding analysis measurement ability of traditional capture molecules and microparticle;
Fig. 3 illustrates the validity of molecular network microparticle in analyte;
Fig. 4 illustrates the validity of molecular network in analyte with topological structure;
Fig. 5 illustrates comparing of in TauELISA traditional conjugated microparticle and molecular network microparticle;
Fig. 6 illustrates comparing of in TSHLuminex sandwich immunoassay traditional conjugated microparticle and molecular network microparticle;
Fig. 7 illustrates the example molecule net on particle;
Fig. 8 illustrates the example molecule net topology feature on particle;
Fig. 9 illustrates the example molecule net sent for analyzing thing.
Embodiment
U.S. Patents Serial numbers 61/281,991,61/337,257,61/340,287,61/343,467,61/410,837,61/489,646 and 61/489, structure has been shown in 648 and use a covalently bound pseudorandom multi-layer three-dimension matrix to make it possible to fast and catch specifically from the protein of untreated samples, nucleic acid, carbohydrates, lipid, cell or other analyze thing, and illustrate that this molecular network of use may be the obvious improvement to conventional analysis thing associated methods, each in these patents is combined in this by reference.The Design and manufacture of molecular network
The characteristic of molecular network can by following imparting: select for the capture molecules used (example of capture molecules can comprise antibody, nucleic acid probe, enzyme, recombinant protein, peptide and other); The gained specificity that described capture molecules is given; The size and number of the capture molecules selected; The placement of capture molecules in this or these molecule stratum reticulare and interval; The combination of capture molecules; The order of capture molecules can be used; And use capture molecules and the ratio being connected molecule and spacer molecule.
The characteristic of molecular network can also by following imparting: select for the connection molecule used (example connecting molecule comprises with bifunctional, different bifunctional, three functions and multi-functional type); Connect the chemical specificity of molecule; Connect the angstrom length of molecule; Connect the combination of molecule; The order connecting molecule can be used; And use capture molecules and the ratio being connected molecule and spacer molecule.
The characteristic of molecular network can also by following imparting: select for the spacer molecule used (example of spacer molecule comprise PEG, polymkeric substance, nucleic acid, albumin, Fc district, peptide and other); The chemical characteristic of spacer molecule; The size and number of spacer molecule; The order of spacer molecule can be used; And use spacer molecule and the ratio being connected molecule and capture molecules.
Capture molecules, the placement connecting molecule and spacer molecule and interval can: give the feature topology on molecular network surface; Give the characteristic density in each layer of a molecular network; Give the feature porosity of molecular network; Eliminate space constraint and thus sterically hindered; Improve binding ability; Reduce non-specific binding; Make it possible to the analysis thing (such as, simultaneously catching the analysis thing of the analysis thing of degraded, complete analysis thing and compound) in conjunction with various ways, and other.
Hole in molecular network can be random, pseudorandom or irregular distribution.The hole of molecular network may be used for filtered sample; May be used for by size exclusion distinguish in sample in conjunction with gesture molecule; May be used for realize due to sterically hindered minimizing, the combination of large molecule or cell or other.The hole of molecular network comprises multiple capture molecules, multiple connection molecule, and can comprise multiple spacer molecule.The classic method that solid phase produces hole relates to modifies the machinery of solid phase surface, and adopts as laser ablation, lamination, offset printing, laser printing or other method produce hole, hole or other structures in solid surface.Prepare this solid surface subsequently for the conjugated capture molecules of reception.The needs that the machinery using molecular network to eliminate effects on surface is modified, and be therefore more cost-efficient.In addition, classic method is still subject to the obstruction of the problem of high non-specific binding, and need be bonded to machinery modify solid phase on catch chemicals, this is not an improvement.In addition, compared with catching form with tradition, due to the size exclusion characteristic given by the porosity set up in each layer of molecular network, flexibility can be imparted in molecular network.In some layers, pore diameter and the degree of depth can be similar or can vary depending on the application.In some layers, pore size can change, and the change of pore size can depend on application.
The hole that can be endowed on a molecular network can include, but are not limited to hole skin, nano-pore, micropore, filter opening, sieve aperture, bag or other.By selecting specificity capture molecules, connection molecule and spacer molecule and the method incorporated them in each layer of molecular network, hole can be imparted in molecular network.Hole can also be imparted in molecular network for the manufacture of the specificity capture molecules of successive layers, the method that connects molecule and spacer molecule by selecting and being incorporated to.
Based on the character of the capture molecules used in layer, connexon and introns, the diameter range of molecular network hole can from about 6 dusts to being greater than about 1um.In some cases, the hole of molecular network can comprise a pore diameter scope.Exemplary diameter scope can be from about 5nm to about 50nm, from about 10nm to about 100nm, from about 50nm to about 200nm, from about 250nm to about 500nm, from about 500nm to about 1um and from about 800nm to about 1.5um.
In some cases, capture molecules may be used for producing hole in molecular network.In these examples, before capture molecules is incorporated to molecule stratum reticulare, capture molecules can be connected to each other in advance.In some cases, connexon can be selected based on the angstrom length of spacerarm.In some instances, extender can be used to be connected on second connexon by first connexon, to produce a long Multifunctional linker.In some cases, introns may be used for producing hole in molecular network.Also before introns are incorporated to molecule stratum reticulare, introns can be connected to each other in advance.In other instances, inertia physics plug may be used for constructing hole, and each whereby physics plug can be placed on the layer of a constructed earlier, the layer that structure one is new simultaneously.After hardening, these physics plugs can be removed, therefore leave the hole of special diameter.
The flexible nature of molecular network makes it possible to use polytype capture molecules.In some instances, molecular network comprises the capture molecules of single type.In other instances, molecular network comprises polytype capture molecules.In some instances, use in the manufacture process of molecular network and make described molecular network can the more than one epi-position of bound analyte more than a kind of monoclonal antibody.Use more than the epitope specificity capture molecules of a type can realize the improvement that molecular network analysis thing is caught, and relevant to the performance of this molecular network.In some instances, the use more than a kind of nucleotide sequence can in the fabrication process for generation of can the molecular network of more than one epi-position of bound analyte.Useful example depends on the use of molecular network, and can comprise when being used in test, in the performance of the floor level detected, sensitivity, positive predictive value, negative predictive value, the ability worked to degraded sample, the ability worked to various different groups and otherwise improvement; Can comprise when being used as tools for purification, in binding ability, purity, binding kinetics, target analytes exhausts and other improve easily performance; Or other.
In some instances, can be used in molecular network for the use of the capture molecules of the analysis thing mutually confirmed and relevant to the performance of this molecular network.The use of the capture molecules mutually confirmed in molecular network can use in a kind of mode of confirmation, analyzes catching of thing and can provide statistically more significant positive findings whereby more than a kind of; More stable test result can be provided; Other information about sample can be provided; And other.The use of the capture molecules mutually confirmed in molecular network can also be used for limiting sample, or tester can be used as in testing, or may be used for the more than one correlation molecule variable measuring related disease states, or may be used for the more than one correlation molecule variable measuring diseases related treatment.
Molecular network can the example of the manufactured mutual confirmation analysis thing for catching from sample simultaneously can comprise: gene order and corresponding protein (the cancer related SNP such as, in BRCA1 and BRCA1 albumen); MRNA and corresponding protein (such as, mankind's lactase mRNA and lactase albumen); Gene order and corresponding mRNA product (the disease association SNP such as, in LMNA and pre-montage or the Lamin A/C mRNA of montage); MiRNA and relevant mRNA or protein (miR9 and REST or CoRESTmRNA or miR9 and REST albumen); Small-molecule drug and drug targets (holder method is for Buddhist nun and Janus kinases); Epitope specificity biopreparate and corresponding target (such as anti-TNF antibody and circulation TNF cell factor); (such as, Anti-DNA autoantibody, the Anti-DNA CD4 such as epitope-specific antibodies, epitope specific T-cells and/or epitope specificity B cell +t cell and/or Anti-DNA B cell); Or other.Useful example depend on use and can to the diagnosis in measurement sensitivity, positive predictive value, negative predictive value, specificity, disease, the ability that the sample experiencing genetic drift is worked, relevant in response to the performance of the measurement capability for the treatment of, the ability measuring therapeutic agent validity or otherwise improvement.
In an example, molecular network can manufacture by catching the mode analyzing thing with positioning combination, and its mode is the intensity strengthening detectable signal or the detection that can strengthen bound analyte, as when using in the test utilizing optical detection.By pre-aligned layering capture molecules, the analysis thing of catching is placed in a hierarchical fashion and can be carried out quick detect analytes by signal enhancement.The example of the signal enhancement realized by molecular network can relate to fluorescence, FRET (fluorescence resonance energy transfer), absorbance, luminescence, light scattering, surface plasma body resonant vibration, optical heterodyne detect or other.
Demand to the method time-consuming again of the costliness for super sensitivity detection can be substituted by molecular network described in Design and manufacture, these methods as PCR, branched DNA or signal amplify needed for multistep detection method.Can also substitute the expensive and demand of the analytical equipment of complexity by molecular network described in Design and manufacture.
Usually, the quantity being incorporated to the capture molecules in three-dimensional molecular net matrix is less than or equal to the use conventional method conjugated quantity to the capture molecules on surface in two dimensions.Two dimension capture molecules surface conjugates can depend on to use single type connexon or can depend on and use 2 kinds of connexons continuously by conjugated for capture molecules on solid surface.In the process of layer manufacturing molecular network, polytype connexon simultaneously for capture molecules being connected to the capture molecules of a new layer, and in the introns that the capture molecules of the connection of new layer is connected to previous layer or capture molecules.Molecular network can be manufactured in the solution before being placed on solid surface.The molecular network manufactured in advance can be adsorbed or is covalently attached on solid surface.Also molecular network directly successively can be fabricated onto on solid surface.Can use non-covalent (electrostatic, Van der Waals force or other) or covalent approach described molecular network is placed on a solid surface.In some instances, polystyrene, polyurethane, tygon or treated surface as PLL coating surface, comprise-COOH, NHS, amine or other modification of surfaces can available from commercial sources (example of supplier can comprise Sai Mo company (Thermo), Millipore Corp. (Millipore), Lu Ming Ces Co., Ltd (Luminex) and other companies) and be used as molecular network place solid phase surface.In other instances, solid phase surface can pass through chemicals (as acid) and carry out pre-service, so that activated surface part and therefore produce attachment point between solid phase surface and the reactive part of molecular network.In some instances, connexon can be utilized to carry out pre-service to solid phase, solid phase surface is covalently attached to molecular network.
Design and manufacture for the molecular network used on solid phase surface can produce the covalently bound multi-layer three-dimension matrix of of the capture molecules of being fixed by the covalent linker in each layer.Design and manufacture can occur in a sequential manner, and wherein manufacture a ground floor and manufacture succeeding layer in a sequential manner, each whereby layer can interconnect by the mode of covalency, so that lift structure integrality, topological structure, porosity and/or stability.Independent capture molecules, connexon and introns can be selected to facilitate one or more characteristics of molecular network.Characteristic can comprise analyze thing specificity, thermal stability, layer thickness, pore diameter, absorbance, spectrum, emission spectrum, solid phase compatibility or other.
Known length may be used for producing different topological structures on the surface of molecular network with the use being connected molecule and introns with the capture molecules of width.The topological characteristic that can be imparted on molecular network can include but not limited to ripple, pit, site, hole, mound shape protuberance, branch, long filament, fiber, slight crack, protruding section or other, and can to arrange with random, pseudorandom or irregular mode in molecular network.
The topological characteristic of molecular network can by using capture molecules and connexon; Capture molecules, connexon and introns; Or connexon and introns produce.In some cases, capture molecules may be used for the topological characteristic producing molecular network.In these examples, before capture molecules covalency is incorporated to molecule stratum reticulare, capture molecules can be connected to each other in advance.In some cases, connexon can be selected based on the angstrom length of spacerarm.In some instances, introns can be used to be connected on second connexon by first connexon, to produce a long Multifunctional linker.In some cases, introns may be used for producing topological structure in molecular network.Also before introns are incorporated to molecule stratum reticulare, introns can be connected to each other in advance or be connected to capture molecules.
Can Design and manufacture molecular network such as affinity, size exclusion, filtration, fluorescence and other feature are given in each layer of molecular network.Can based on size, length, diameter, thickness, optical characteristics, chemical characteristic or other select specificity capture molecules, connect molecule and spacer molecule, feature to be given in molecular network in the fabrication process.
Can manufacture molecular network in one way, one or more whereby capture molecules can provide structure function in covalently bound multi-layer three-dimension matrix, can provide the effect that structure function and analysis thing are caught.Some examples of capture molecules can catch effect for structure and/or analysis thing in molecular network.
By for the diameter of the capture molecules used in the manufacture process of each layer, connexon and introns, width and/or length, partly can determine the distance between the capture molecules in each layer of molecular network, molar relationship whereby between each connection-catch-spacer molecule can be similar or can be different, and the selection of described molecule can depend on the size of analysis thing to be captured and/or shape, for measuring the method for the analysis thing of catching and/or desired purposes.
Can Design and manufacture molecular network in one way, that can have equivalence or the non-equivalent mol ratio of each capture molecules, connexon and introns component in a layer of described molecular network whereby.The mol ratio between described component can be changed every now and then to produce hole or other topological characteristics in each layer.Described hole and topological characteristic can have a diameter range and can have a depth range be associated.The change of the mol ratio between molecular network component may occur in the single layer of molecular network, or may occur in the more than one layer of molecular network, and depends on the desired use of molecular network.
Table 1. has the example of the molecular network structural constituent analyzing thing capture ability
Some examples of the analysis thing size may considered in Design and manufacture process are provided in table 2.Molecular network surface chemistry, pore diameter, topological structure, layering or other Design and manufacture can based on analysis thing shapes; Analyze thing structure, analyze thing hypotype, analyze thing load, there are the formation of the analyte complex of other molecules and other forms.In addition, molecular network can be designed and manufacture for combining and catching described analysis thing, or can be designed and manufacture for getting rid of described analysis thing.The example analyzing thing and analysis thing size can see in table 2.
The example of thing and their size analyzed by table 2.
The molecular network comprising structural constituent and catch component can relate to and catch one or more by the arrangement of covalently bound three-dimensional (3D) substrates multilayer and analyze thing, these one or more analyze one or more relevant to following characteristics of thing: surface chemistry; Analyze thing shape; Analyze thing structure; Analyze thing hypotype; Analyze thing load; Posttranslational modification; Chemical modification; Active; Or other.
Molecular network can comprise structural constituent, and these structural constituents also to work with the relevant mode of catching analyzing thing, and can be arranged in the interconnection 3D substrates multilayer of molecular network by covalently bound son.Molecular network can also comprise introns described structural molecule/capture molecules to be interconnected, and its mode is for maximizing structure enhancing, stability and/or specific analyte capture ability.Comprise the molecular network example of catching composition/structure component, connexon and introns to present in table 3.
The manufacture of molecular network is unique, because capture molecules is fixed in 3D matrix by covalent linker molecules.In large quantifier elimination, prove that molecular network has the thermal stability of improvement, and extended storage life and exceed traditional capture technique.
The example of table 3. molecular network and their purposes
In some instances, solid phase can be diameter range from about 2nm to the particle of about 200mm, and molecular network can be attached on the surface of described particle.Particle can comprise: polystyrene, tygon, silicon dioxide, compound substance, nylon, PVDF, nitrocellulose, cellulosic material, carbon, or can be magnetic, paramagnetic, fluorescence, bar code or other.
Molecular network can be adsorbed or is covalently attached on the surface of particle, and its mode is make in the individual layer of described molecule king net or produce pseudorandom or orderly hole in all layers.In the most basic form, particle can be coated with a molecule stratum reticulare at first, and this molecule stratum reticulare can be connected on a second layer, and this second layer can be attached in a third layer.Molecule stratum reticulare can comprise the identical capture molecules of same concentrations or variable concentrations in each layer.With multi-layer phase ratio before, molecular network particle can also comprise different capture molecules in each layer, and can manufacture by the mode of the capture molecules being incorporated to identical or different concentration.
In some instances, molecular network can be attached on particle surface, and its mode is make to produce the asymmetric particle with predetermined polarity.This particle can utilize the initiation layer comprising the structural molecule with major diameter, width and/or length to carry out Design and manufacture, and can be connected on particle so that polarization by asymmetric mode.A second layer can be connected on a ground floor, and a third layer can be connected on a second layer, etc.The quantity of the layer in molecular network particle can change according to use.
In some instances, molecular network can be attached on a section of particle, to produce the asymmetric particle with predetermined polarity.This particle is fabricated, this initiation layer is applied on a section of particle whereby, and a second layer is connected to this initiation layer in the same sector of described particle whereby, and a third layer is connected to this second layer in the same sector of described particle whereby, and whereby one the 4th layer be connected to this third layer in the same sector of described particle.
In some instances, molecular network can on the particle surface of passive adsorption to non-functionalized.In other instances, particle surface can be functionalized and may need before the attachment to activate.In other instances, particle surface can be activated before functionalization, and when functionalization, molecular network can be attached.But in other instances, molecular network can by direct construction at the surface of the particles.Molecular network is attached to the physics and/or chemical feature that particle can change described particle.In some instances, molecular network can comprise the multiple pseudorandom topological characteristics be placed on particle surface.In some instances, molecular network particle can comprise multiple topological characteristic, and these topological characteristics comprise capture molecules and connexon, and can comprise introns.The example of different topological characteristics can comprise: annex, spike, highly, plane, mound shape protuberance, slight crack, thin skin, site, groove, hole and other, and can by the one or more layers being connected directly between molecular network and/or the component of catching be connected directly on one or more layers of molecular network form.
Other examples of topological characteristic can comprise bag, post, projection, branch, protuberance, ridge, crack, grating texture, thin slice, bead, spheroid or other.Topological characteristic can be preshaped in the solution, and be connected on molecular network, or can be formed when structure each layer.
Molecular network on particle can comprise capture molecules heterogeneous at one or more layers of molecular network.The benefit of uneven design can with single particle has kinds of surface chemical substance multiple analytes catch relevant.The non-homogeneous capture molecules being incorporated to molecular network in the fabrication process can throughout each layer of stochastic distribution; Can throughout each layer of stratification; Or other, this depends on purposes.
Molecular network can be attached on particle, to increase the surface area of described particle.Molecular network can also for increasing particle diameter.Except analyzing thing capture ability, the topological characteristic of the molecular network on particle can also be relevant with the grain size increased.
In some instances, a first molecule stratum reticulare can be attached on particle surface, to change physics and/or the chemical characteristic of particle.In many commercial particle, " microballon effect " or " surface effect " can hinder result and still fail to understand well.The conjugated technology of tradition producing 2D conjugates and the conjugated surface of 2D is often limited by surface effect.Molecular network may be used for minimizing or offset microballon effect, so as to minimize in mensuration non-specific binding in bead surface, microballon autofluorescence, microballon interference or other.In some instances, molecular network particle can give the analysis thing binding ability of increase, and the blocking-up can given undesirable analysis thing non-specific binding so that increase the purity of signal to noise ratio (S/N ratio), yield and purity analysis thing in measuring or other.
In some instances, in yet another aspect, feature of the present invention is the molecular network comprising more than one layer on particle, wherein each layer comprises the capture molecules for analyzing thing, wherein each layer comprises the different capture molecules analyzing thing for difference, and wherein different layers can analyze thing for difference can catch a kind of analysis thing or multiple analytes.
In yet another aspect, the molecular network be placed on solid phase surface may be used for the purity increasing one or more analysis things reclaimed from sample.Compared with business 2D functionalized surface, the non-specific binding of undesirable analysis thing can be reduced in the surface of molecular network coating.
In some instances, the molecular network be placed on particle obviously can increase the analysis thing capture ability of particle.The other layering of molecular network can increase the quantity of each particle bound analyte further, and may be used for recovery or the yield of the analysis thing improving sample, and may be used for one or more analysis things exhausting sample.Use the benefit of molecular network
Molecule and cell tests strategy adopt that substance or Multiple immunizations measure, PCR measures, sequencing technologies of future generation or other to identify in sample one or more existence analyzing things or measure these one or more analyze the amount of thing.
In multiple assay, multiple reaction can spatially be separated, or can be incorporated in in a single test reaction, and the solid phase comprising unique identification device can be adopted to provide information.Some examples of unique identification device can comprise different bar codes, different fluorescent emission, different chemical substances, different ordered nucleus thuja acid labels or other use.
Solid phase can use in substance and multiple assay, can depend on the specific binding of target analytes, to produce measurable change in measurable signal or signal, and solid phase can use or can use in indirect determination in directly measuring.Can measuring-signal can from the positive test produce, and can comprise electricity, thermal and magnetic, light, vibration, isotope or other can measure feature.
Use current strategies to realize sensitive and in reproducible measurement many difficulties and cause high non-specific binding, lower sensitivity, low signal-to-noise ratio, and therefore need the sample handling procedure of upstream from sample, remove non-specific component as much as possible, add outward and use to determine high sensitivity reading technology required for real signal and complicated algorithm from noise, this makes them be difficult to be transformed into almost real-time, easy-to-use molecular diagnosis and analysis measurement instrument.
Molecular network may be used for replacing Current commercial method, and the specificity that can realize from sample and sensitive analysis thing are caught, detect and measured.Present in Fig. 1-Fig. 6 with the example that molecular network replaces current method to catch obtained result for analyzing thing.By replacing with molecular network current 2D method to catch for analyzing thing and measure, can realize measuring sensitivity, analyzing the floor level of quality testing survey and the improvement of other features.The minimizing of ground unrest can realize by replacing with molecular network current 2D method, and may be used for improving analysis thing purifying, analyzing thing purity and measure sensitivity.
The benefit of molecular network presents in table 4, and can comprise: to a kind of in primary sample, several or different kinds of molecules and cell analysis thing fast Acquisition; When producing the ability of sensitive and specific signal when relating to indirectly and using in the test of direct detecting method; Produce the ability with the signal of the fluorescence intensity of enhancing; Make the ability that bound analyte is concentrated; To reduce the ability that the sterically hindered mode analyzed between thing and/or between detection molecules is spatially separated bound analyte; The stability strengthened; Reduce background and other.
The attested benefit of table 4. molecular network
Molecular network and their purposes
Molecular network may be used for wherein analyzing thing joint efficiency, analyzes thing binding kinetics, analyzes thing binding ability, analyzes quality testing surveys, analysis measurement, enrichment of analytes, analysis thing purifying and analysis thing are sent may be important multiple application.Molecular network can use or can be attached in solid phase in the liquid phase.
Molecular network can be attached to one by absorption or covalency process and accept on the surface.The example of solid phase includes but not limited to: nanotube, metal, particle, microtiter plate, microslide, box, probe, crossing current test vessel, support, conduit, valve, blood vessel, pin, solid phase apparatus or other.The example of chemical substance of different solid phases that can be compatible for molecular network attachment includes but not limited to: plastics, other polymkeric substance, film, colloidal metal, silicon dioxide, carbon nano-tube, protein, carbohydrates, lipid, nucleic acid, cell, tissue or other.
Molecular network can be attached to solid phase apparatus on the surface so as to catch from sample, purifying or exhaust one or more analyze thing.Present in Fig. 1 and use molecular network to catch from sample and/or an example of purity analysis thing.May be used for some other examples of catching from sample with the molecular network of purity analysis thing is: for the microballoon of albumin A, Protein G or the albumen L net coating that immunoglobulin class is caught; For the microballoon of the Streptavidin net coating that biotin is caught; For the microballoon of the TNF net coating that anti-TNF biology is caught; For the microballoon of the IL6 net coating that anti-IL6 biology is caught; For the microballoon of the IgM net coating that RNA virus is caught; For the microballoon of the IgFc net coating that complement is caught; For the microballoon that the antigen net of antigen specific immune globulin applies; Antigen specific immune cell capture; And other.Molecular network can use in chromatography, and for catching from sample with purifying, one or more analyze thing.
Molecular network may be used for from sample, catch analysis thing, carries out the analysis measurement in downstream for passing through independently method (referred to here as sample preparation).Independently the example of method can comprise: mass spectrophotometry, immunoassays, PCR, order-checking of future generation, qRT-PCR, digital pcr, microscopy, fluorescence, flow cytometry, microballon cell art or other.
In yet another aspect, the present invention improves signal to noise ratio (S/N ratio) when being used for measuring.
Molecular network can be attached to solid phase apparatus on the surface, to measure one or more existence analyzing thing, disappearance, modification or concentration.The example using molecular network to survey for analyzing quality testing and/or measure is presented in Fig. 3 and Fig. 4.In some other examples, molecular network may be used for detecting in direct or indirect mode simultaneously and measuring two or more specific analytes.Catch Main Analysis thing by the specificity capture molecules that can relate to by molecular network of indirectly catching of molecular network, this makes it possible to the detection carrying out one or more the relevant second analyte be associated to the Main Analysis thing of catching.Molecular network can be used as the discovering tool of catching Main Analysis thing from sample, and makes it possible to identify, detect or measure by associating the second analyte of catching.Molecular network can in this way for drug discovery, approach mapping, and proteomics, transcription group, sugar group, iipidomic, metabolomic research, functional genomics, diet group, nutrition, pharmacology, toxicology and other in use.
In some instances, molecular network may be used for detecting the resistance to the action of a drug in cell.Cell can be tumour cell, immunocyte, microbial cell or other cells.Molecular network for these application can comprise the capture molecules of the one or more specific characteristics for a kind of cell type.Molecular network can manufacture with the mode of catching relevant surface topology of intact cell to give in addition.
In other instances, molecular network can be manufactured and measure for: immune cell responses; Immune response is monitored; Immune response is classified; Immunoglobulin titer; Biotinylated molecules is caught; Multiple immunizations measures; Substance immunoassays; Sequencing reaction of future generation; PCR; Microorganism-capturing; Microorganism finds; MRNA and encoding proteins are measured; SNP (single nucleotide polymorphism) maps; SNP detects; Disease marker sample preparation; MiRNA catches and/or measures; Posttranslational modification finds and/or catches and/or measure; Kinase activity is measured; Or other.
Molecular network can have give in the fabrication process multiple and measure feature, and can be used as sensor by direct or indirect mode.The measured change that business method comes in one or more features of detection molecules net sensor can be used, these business methods adopt that optics sensing, electrochemical sensing, electromagnetism sense, electrical impedance or other.In an example, molecular network sensor may be used for catching and bound analyte.Analyze thing and combine measurable change that can cause in the feature of molecular network sensor.The binding events or modification event that relate to about molecular network sensor can be monitored within a period of time, and can be detected by device, relaying and the change of collecting in molecular network sensor characteristics.Molecular network is used as other examples of sensor can comprise analyze thing binding events, enzymatic reaction, analysis thing modify event, Cell Differentiation, cell-ECM interact or other.
The example of measurable feature includes, but are not limited to: physical form, highly, density, fluorescence intensity, wavelength shift (FRET or FRAP), vibration frequency, absorbance, flexibility, refractivity, conductance, impedance, resistance, melt temperature, denaturation temperature, freezing temperature and other.
Can be that compatible measurement mechanism can comprise for using together with molecular network sensor: photon multichannel analyzer, spectrometer, magnetic resonance imager, magnetic field detector, optical fiber, glass pipet, circuit, photofluorometer, optical spectrum analyser, flow cytometer, ccd video camera, microscope, sound chamber, loudspeaker, photometer and other.These measurement mechanisms may be used for measuring following change: thickness, topological structure, load, insulation, electric capacity, voltage, color, tonequality, vibration, magnetic, enzymatic activity or be used as other features of molecular network of sensor.
In addition, molecular network can use in flexible circuit, and capture molecules and/or structural molecule can be connected on electroconductive molecule whereby.Molecular network circuit can single-sided flexible circuit, two contact (naked back flexible circuit), engraving flexible circuit, double-sided flex circuit, multi-layer flexible circuit, ridged flexible circuit, ridged flexible board, polymer thick film flexible circuit or other in use.Most of flexible circuit is the passive wire structures for being interconnected by multiple electronic unit (as integrated circuit, resistor, capacitor etc.), but some flexible circuits only for directly or by means of connector manufacture interconnecting between other electronic packages.For using in circuit or can being made up of synthesis component for the molecular network as circuit block, or can be made up of biological chemistry capture molecules and/or cell, or can manufacture by a kind of mode needing to be used in flexible circuit.Molecular network circuit can also be used as sensor.
In some instances, molecular network circuit can have certain electric chemical characteristic, and may be used for monitoring the different parameters in electrochemical cell/electrolytic cell, as pH, electric current, voltage, impedance or other.The contingent binding events of molecular network and modification event can be measurable, and can be reflected by the change of conductance, electric current or voltage.More properly, can monitor introducing containing the sample analyzing thing by the change of the electrochemical properties of molecular network and/or surrounding environment, this analysis thing has specific binding affinity to the parts in molecular network circuit or to its responding property.
The example that binding events in circuit on molecular network used occurs can comprise: the noncovalent interaction that antibody-antigene interacts, nucleic acid-nucleic acid interacts, enzyme-substrate interacts, medicine-target interacts, enzyme-co-factor interacts, part-cell interaction or any other specific surfaces chemistry drive.The analysis thing of molecular network is caught and can be determined by the change of the pH in electrochemical cell/electrolytic cell, curtage.The measurement of the change of molecular network feature can also derive from one or more components of betiding in molecular network or betides one or more modification event of catching and analyzing thing or modify event in a large number.The example of modification event can comprise: enzymatic lysis; Posttranslational modification (as phosphorylation, sulfonation, glycosylation, methylating or other); The removal (as dephosphorylation) of posttranslational modification; Or other similar modifications.Modification event can be determined by the change of the pH in electrochemical cell/electrolytic cell, curtage, and this change is owing to the change in molecular network feature or in surrounding buffer system.
Determine that the method for the change of the electrochemical properties of molecular network used in circuit can comprise the nano-pore that uses scan ion electric current microscope, Nanofluid diode, display voltage-gated ion electric current or nanochannel, ion nanometer gate, sensing platform based on nano-pore, and for by the flow of Medium Measurement flow of charge or the additive method of change.More properly, when transadmittance metre hole applies bias voltage, the intrinsic sensitivity of many solid nano hole sensors is the selection permeability of electrolyte or gas current.Molecular network can be applied on the surface of nano-pore, and can monitoring current, change in voltage and impedance.
Molecular network can also be applied on the surface of carbon nano-tube, and this molecular network can build to produce the size exclusion for analyte sensing and affinity needs by a kind of mode whereby.
Bio-layer interferometry (BLI) is the unmarked technology for measuring bio-molecular interaction.This is a kind of optical analysis technique, and this technical Analysis is from the interference figure of the white light of following two surface reflections: an immobilized molecules stratum reticulare biosensor tips and an internally reflective layer.Any change being bonded to the quantity of the molecule in this biosensor tips causes the displacement of interference figure, and this displacement can be measured in real time.Be fixed on molecular network coating biosensor tips on part and the analysis thing in solution between combination cause the optical thickness at this biosensor tips place to increase, this generates a wavelength shift Δ λ, this wavelength shift is directly measuring of the variation in thickness of biostrome.Interaction can be measured in real time, thus provides accurately and the ability of monitor binding specificity, association and dissociation rate or concentration.Only be bonded to molecular network biology sensor or interference figure can be made to be shifted and to produce response curve from the molecule that molecular network biology sensor dissociates.Non-binding molecule can change the refractive index of surrounding medium, or can change flow velocity, but will not affect interference figure.This is a specific characteristic of BLI, and extend its for analyze thing-capture molecules combine, quantitatively, the ability that performs in the crude samples that uses in affinity and dynamic (dynamical) application.
Molecular network particle can also be used for active agent delivery.Activating agent can be loaded previously in the capture molecules of the one or more layers being arranged in molecular network.Activating agent can comprise: medicine, therapeutic agent, toxin, virus, anaphylactogen, vaccine component, antigen, immunomodulator, surfactant, microorganism, oligonucleotides, nutrient or other.Molecular network particle may be used for medicine or therapeutic agent delivery, vaccine delivery, biofermentation or other.Molecular network can comprise one or more target agent on surperficial exposed surface, to promote by described molecular network particle target specific cells type, organization type, organ type or the specificity in other.Target agent can be the capture molecules of molecular network.Target agent can comprise: antibody, acceptor, part, anti-part or other.Target agent in molecular network can by the capture molecules, connexon and the introns that are covalently attached in surperficial exposed surface.Target agent can also contribute to the topological characteristic of molecular network.
Example
Example 1. is for analyzing the comparison of conventional 2D and the 3D molecular network microparticle of thing purifying.
Use in the mill by monomeric protein G be connected Protein G and crosslinking agent B S 3, EMCS, EGS, BMPH and other composition molecular network.0.8-10um magnetic polystyrene microparticle and 45um nitrocellulose microparticle surfaces carry out molecular network manufacture in real time.In some instances, capture molecules (Protein G) is used to originate as only support structure.In some instances, the Protein G connected in advance and monomeric protein G are mixed to be used as the other support structure of some layers manufacturing molecular network.But in some other examples, the ground floor comprising the molecular network in Protein G and IgFc district is used as the support structure of the extra play manufacturing this molecular network.In some instances, Protein G molecules net comprises 2 layers, and in other instances, Protein G molecules net comprises 3 layers.Last one deck of molecular network comprises multiple topological characteristic, analyzes the combination of thing (being IgG in this case) and the recovery from sample to strengthen.Fig. 1 is compared with business Protein G microparticle, the example of the data using Protein G molecules net microparticle to obtain.In brief, IgG-Alexa647 is added (1ug/ pipe) in serum human.Uncoated microparticle, business Protein G microparticle and Protein G molecules net particle are at room temperature hatched together with spiked sample 15-60 minutes (100,000 particle (uncoated contrast), 100,000 particle (business) and 25,000 particle (molecular network)).Magnetic resolution is used to be separated from sample by particle and particle is washed three times in PBST.Particle is suspended in 2xLSB, boils and be loaded on SDS-PAGE.Compared with input contrast, measured the IgG reclaimed by Coomassie-stained band light densitometry.What describe in Fig. 1 is the recovery of the input of each purification types.Use optimization molecular network can reduce the ground unrest in mensuration and increase visible signal.
The validity that the conventional 2D conjugates of example 2. is surveyed for analyzing quality testing.
The classic method be covalently attached to by capture antibody on a surface is two-dimension method (X and Y plane), because there is not the extra play on the surface of the connection antibody be added on a surface.For by capture antibody, the classic method be covalently attached on a surface relates to the connexon of single type, such as EDC, NHS, sulfo group-NHS or other.Occasionally, use second connexon to be fixed to by capture antibody on a surface, but relate to the first connexon remove and for other height or layering are added in the conjugated surface of antibody.In an example, according to the instruction of manufacturer, Anti-Human's class neurofilament is pressed down protein antibodies and is attached on Luminex particle (microballon district #54) by connexon.Subsequently by particle quencher, close and wash, use afterwards.Particle is pressed down in albumen at serum+neurofilament clean in advance and hatches 15 minutes with the concentration range of (0-1ng/mL).To press down together with albumen (10ng/mL) in conjunction with particle washing and with biotin-anti-neurofilament and hatch 15 minutes.Continue neurofilament to be pressed down Protein Detection in 15 minutes by avidin-PE (30ng/mL) visual.The particle of washing is analyzed subsequently, each sample collection 100 particles on Luminex100.What present in Fig. 2 is the median fluorescence intensity (FI) exceeding background fluorescence intensity under each dilutability.
Fig. 3. the effect validity of molecular network microparticle in analyte.
Manufacture and press down protein antibodies (as Fig. 2) and connexon sulfo group-NHS, EMCS, EGS, BMPH and other molecular networks formed, to provide 3-dimensional multi-layered (X, a Y and Z plane) matrix by identical Anti-Human's class neurofilament.Subsequently these molecular networks can be covalently attached on Luminex microparticle (microballon district #54).The mensuration performance utilizing 4 layers of molecular network is presented in Fig. 3.3-dimensional multi-layered molecular network is used to observe the mensuration MFI of improvement.
Fig. 4. there is the validity of molecular network in analyte of topological structure.
Manufacture and press down protein antibodies (as Fig. 2 and Fig. 3) and connexon sulfo group-NHS, EMCS, EGS, BMPH and other molecular networks formed, to provide 3-dimensional multi-layered (X, a Y and Z plane) matrix by identical Anti-Human's class neurofilament.Subsequently these molecular networks can be covalently attached on Luminex microparticle (microballon district #54).The mensuration performance utilizing 5 layers of molecular network is presented in Fig. 4.The 3-dimensional multi-layered molecular network being used in skin the topological structure with reinforcement observes the mensuration MFI of improvement.
Fig. 5. the conjugated microparticle of the tradition in ELISA compares with molecular network microparticle.
Use by for the monoclonal antibody of the mankind-TauF and crosslinking chemical sulfo group-NHS, EMCS, EGS, BMPH and other molecular networks formed in the mill.Molecular network manufacture is carried out in real time on the surface at the magnetic corpuscular of 0.5um, 6.3um and 10um.In some instances, use capture molecules, anti-Tau monoclonal antibody are originated as only support structure.In some instances, in a ground floor, introns albumin is mixed with the mol ratio of 1.5:1.0 (albumin: anti-Tau antibody) with anti-Tau monoclonal antibody, to be used as the other support structure manufacturing this ground floor.In some instances, use the second capture molecules and mankind's tubulin, it provides support structure and catches effect in molecular network.Fig. 5 is compared with business Tau microparticle (gametophyte LV) ELISA (identical condition determination, identical antibody equity), the example of the data using anti-Tau molecular network (LV.P6Cap-TECH) to obtain.Fig. 5 uses molecular network to the ground unrest reduced in mensuration and the example of the visible signal increased in ELISA.
Fig. 6. use the conjugated microparticle of tradition of Luminex and comparing of molecular network microparticle.
Use by for the monoclonal antibody of the mankind-thyrotropic hormone and crosslinking chemical EDC, BS (PEG) in the mill 9, EMCS, EGS, BMPH and other composition molecular network.Molecular network manufacture is carried out in real time on the surface at Luminex magnetic corpuscular.In some instances, use capture molecules, anti-TSH monoclonal antibody are originated as only support structure.In some instances, in a ground floor, introns (PEG, thermal denaturation lysozyme and other) are mixed with the mol ratio of 1.0:2.0 (introns: anti-TSH antibody) with anti-TSH monoclonal antibody, to be used as the other support structure manufacturing this ground floor.In some instances, an anti-TSH molecular network comprises 4 layers, and in other instances, an anti-TSH molecular network comprises 6 layers, and wherein last one deck comprises multiple topological structure so that the analysis thing strengthened in Luminex combines and performance.Fig. 6 A uses molecular network to increase the example of overall MFI in Luminex measures.Fig. 6 B is the example data obtained in Luminex measures, the floor level detected in measuring for increasing Luminex.
Fig. 7. the example molecule net on particle.
Fig. 7 depicts some examples, and wherein molecular network can be placed on a particle surface.In some instances, molecular network is placed on (Fig. 7 A on a particle surface in a manner circumferentially, 1001), the molecular network whereby with X, Y and Z spatial orientation can be quite symmetrical, and wherein each layer (example of 3 layers, 1002,1003 and 1004) is added in the Z plane of particle.In some instances, molecular network can be placed on (Fig. 7 B on a particle surface by asymmetric mode, 1005), the molecular network whereby with X, Y and Z spatial orientation can be placed on the surface of particle by the mode of the polarity producing particle, and wherein each layer (example of 3 layers, 1006,1007 and 1008) with layer independently mode (such as, some layers can have less height and other layers can have higher height) be added in the Z plane of particle.In other instances, molecular network can be placed on (Fig. 7 C in a part for particle surface by asymmetric mode, 1009), the molecular network whereby with X, Y and Z spatial orientation can be placed on the surface of particle by the mode of the polarity producing particle, and wherein each layer (example of 3 layers, 1010,1011 and 1012) with layer independently mode (such as, some layers can have the specificity of analyzing thing for a kind of and other layer can have the specificity analyzing things for other) be added in the Z plane of particle.
Fig. 8. the example molecule net topology feature on particle.
Fig. 8 depicts some examples, and wherein molecular network can be placed on a particle surface.In some instances, molecular network is placed on (Fig. 8 A on a particle surface in a manner circumferentially, 2001), the molecular network whereby with X, Y and Z spatial orientation can be quite symmetrical in a layer (2002), and wherein each layer (example of 3 layers, 2002,2003 and 2004) be added in the Z plane of particle in different and asymmetric mode (such as, producing topological structure).In some instances, molecular network can be placed on (Fig. 8 B on a particle surface by asymmetric mode, 2005), the molecular network whereby with X, Y and Z spatial orientation can be placed on the surface of particle by the mode producing architectural feature (2008) in the whole layer 1 (2006), 2 (2007) and 3 (2008) of particle, and wherein each layer with layer independently mode (such as, some layers can have less height and other layers can have higher height) be added in the Z plane of particle.In addition, the structural detail in each layer can also play the effect analyzed thing and catch in molecular network.In other instances, molecular network can be placed on (Fig. 8 C in a part for particle surface by asymmetric mode, 1009), the molecular network whereby with X, Y and Z spatial orientation can be placed on the surface of particle by the mode of the polarity producing particle, and wherein each layer (example of 4 layers, 2010,2011,2012 and 2013) with layer independently mode be added in the Z plane of particle, and each whereby layer can play structure and analyze the thing effect of catching.Such as, some layers can be analyzed thing based on size to one and have specificity (such as, Fig. 8 C, 2010), and outer (such as, Fig. 8 C, 2013) can have specificity to the analysis thing of size.
Fig. 9. for analyzing the example molecule net that thing is sent.
Fig. 9 depicts some examples, and wherein molecular network can be placed on a particle surface, to catch or target analytes is sent for analysis thing.In some instances, molecular network is placed on (Fig. 9 A on a particle surface in a manner circumferentially, 3001), the molecular network whereby with X, Y and Z spatial orientation can be quite symmetrical in a layer (3002), and wherein each layer (example of 3 layers, 3002,3003 and 3004) be added in the Z plane of particle in different and asymmetric mode (such as, producing topological structure).In some instances, analyzing thing goods (3003) can by the capture molecules that is loaded previously in one or more layers of molecular network.In skin, different capture molecules can be connected in a molecular network, is used for produce topological structure and/or the affinity that different target analyzes thing.In some instances, pre-loaded analysis thing can comprise medicine, therapeutic agent, siRNA, miRNA, dsRNA, virus, toxin, immunogene or other.Pre-loaded goods can be the capture molecules noncovalent associations with one or more types in molecular network layer.In some instances, the different capture molecules (3004) of molecular network can be arranged in the skin of molecular network, and can play topological structure and analyze thing catch effect.Different capture molecules can have for one or more different specificitys analyzing thing, this analysis thing can comprise antibody, anti-part, part, acceptor, antigen or other, and one or more structures and/or affinity and/or targeting can be played.
In some instances, molecular network can be placed at the surface of the particles (Fig. 9 B, 3005) by a kind of mode, and analyzing thing goods (3006) whereby can be loaded previously in all layers of molecular network.In some layers, capture molecules may be used for producing the topological characteristic (3008) playing particle targeting.In some instances, the skin of molecular network particle can make described particle target specific cells, tissue, organ or other.
Figure 10. for the molecular network of purity analysis thing from sample
Molecular network can use (providing example in Figure 10) in sample purification process.In some instances, be designed and manufacture the molecular network analyzing thing for exhausting one or more to may be used for processing sample, to carry out analysis material consumption to the greatest extent.Illustrative methods can be included in batch slurry or hatch about 15 minutes to about 24 hours in the chromatography column by molecular network together with sample.Can sample supernatant be collected according to method for optimizing or flow through liquid.Can molecular network be collected and make differently to analyze existence and the amount of the analysis thing of catching.The sample of molecular network process can be collected and make differently to carry out analyzing to determine to analyze in sample the residual existence of thing, or other analysis things in sample can be analyzed.
Figure 11. for the molecular network of detection and analyte from sample
Molecular network can use (providing example in Figure 11) in analysis measurement instrument or diagnostic tool.In some instances, be designed and manufacture the molecular network analyzing thing for catching one or more to may be used for processing sample, survey to carry out analysis quality testing and measure.Illustrative methods can be included in batch slurry, box, microslide, microtiter plate or other in molecular network is hatched about 15 minutes to about 2 hours together with sample.Can sample supernatant be collected according to method for optimizing or flow through liquid.Can molecular network be collected and make differently to analyze existence and the amount that one or more of catching analyze things.The sample of molecular network process can also be collected and make differently to carry out analyzing to measure other analysis things.Method for measuring the change of molecular network feature can comprise optics, electrophoresis, electricity, magnetic, chemistry, heat or additive method.
Although describe the present invention with reference to specific embodiment of the present invention, those skilled in the art should be understood that, can make different changes without departing from the present invention and multiple equivalent can be replaced.In addition, many amendments can be made to adapt to particular case, material, material composition, process, process steps or step, to realize without departing from the present invention by benefit provided by the invention.All this amendments are all intended to be in the scope of claims.
Be combined in this by reference at this whole publication quoted and patent documentation, all pointed out particularly and to be respectively combined in this by reference as every a such publication or document.It is all relevant prior aries that the quoting of publication and patent documentation there is no the bright any such file of expectation, do not form yet in it perhaps any of date admit.

Claims (52)

1., for catching the device analyzing thing, this device comprises:
A solid phase; And
A molecular network, this molecular network is attached to going up at least partially of the surface of this solid phase, this molecular network comprises the capture molecules of at least one type, these capture molecules are attached to each other by polytype connection molecule, to form a covalently bound multi-layer three-dimension matrix, these capture molecules are arranged in conjunction with this analysis thing.
2. device as claimed in claim 1, wherein this solid phase is made up of one or more in plastics, polymkeric substance, film, colloidal metal, silicon dioxide, carbon nano-tube, protein, carbohydrates, lipid, nucleic acid, cell and tissue.
3. device as claimed in claim 1, wherein this solid phase comprises one or more in nano material, the metal surface of modification, nanosphere, microballoon, microtiter plate, microslide, transfer pipet, box, cylinder, disk, probe, lateral flow device, microfluidic device and optical fiber.
4. device as claimed in claim 1, wherein this molecular network manufactured in advance and be adsorbed to this solid phase this on the surface.
5. device as claimed in claim 1, wherein this molecular network by be covalently attached to this solid phase this on the surface.
6. device as claimed in claim 1, wherein this molecular network by be built directly in this solid phase this on the surface.
7. device as claimed in claim 1, wherein these capture molecules comprise one or more in antibody, nucleic acid probe, enzyme, recombinant protein and peptide.
8. device as claimed in claim 1, wherein these capture molecules comprise the multiple monoclonal antibody for the multiple epi-positions in conjunction with this analysis thing.
9. device as claimed in claim 1, wherein these capture molecules are the analysis things for multiple mutual confirmation.
10. device as claimed in claim 1, wherein these connection molecules comprise with one or more in bifunctional, different bifunctional, three functions and multi-functional type.
11. devices as claimed in claim 1, wherein when these capture molecules are in conjunction with this analysis thing, this molecular network has the feature measured of at least one experience change.
12. devices as claimed in claim 11, wherein this can measure feature comprise physical form, highly, density, fluorescence intensity, wavelength shift, vibration frequency, absorbance, flexibility, refractivity, conductance, impedance, resistance, melt temperature, one or more in denaturation temperature and freezing temperature.
13. devices as claimed in claim 1, wherein these capture molecules are also attached to each other by polytype spacer molecule.
14. devices as claimed in claim 13, wherein these spacer molecules comprise one or more in PEG, polymkeric substance, nucleic acid, albumin, Fc district and peptide.
15. devices as claimed in claim 13, wherein these spacer molecules and a certain amount of spacer molecule are selected for and give this molecular network one or more desired physical characteristics.
16. devices as claimed in claim 15, it is one or more that the physical characteristics wherein desired by these comprises in porosity, CHARGE DISTRIBUTION and topological characteristic.
17. 1 kinds of methods manufactured for catching the device analyzing thing, the method comprises:
A solid phase is provided; And
A molecular network is placed on going up at least partially of the surface of this solid phase, this molecular network comprises the capture molecules of at least one type, these capture molecules are attached to each other by polytype connection molecule, to form a covalently bound multi-layer three-dimension matrix, these capture molecules are arranged in conjunction with this analysis thing.
18. methods as claimed in claim 17, wherein put a molecular network comprise manufacture this molecular network in advance and this molecular network is adsorbed onto this solid phase this on the surface.
19. methods as claimed in claim 17, wherein put a molecular network comprise this molecular network is covalently attached to this solid phase this on the surface.
20. methods as claimed in claim 17, wherein put a molecular network comprise this molecular network is built directly in this solid phase this on the surface.
21. 1 kinds of methods measuring the amount of the analysis thing in sample, the method comprises:
One or more device is provided, each device comprises a solid phase and a molecular network, this molecular network covers the surface of this solid phase at least partially, this molecular network comprises the capture molecules of at least one type, these capture molecules are attached to each other by polytype connection molecule, to form a covalently bound multi-layer three-dimension matrix, these capture molecules are arranged in conjunction with this analysis thing;
These devices are exposed to this sample; And
Allow this analysis thing at least partially in conjunction with these capture molecules of these molecular networks of these devices.
22. methods as claimed in claim 21, comprise the change of measuring in this sample further.
23. methods as claimed in claim 22, wherein measure this change and comprise that to use in photon multichannel analyzer, spectrometer, magnetic resonance imager, magnetic field detector, optical fiber, glass pipet, circuit, photofluorometer, optical spectrum analyser, potentiostat, calorimeter, electrophoresis, flow cytometer, ccd video camera, microscope, sound chamber, loudspeaker and photometer one or more.
24. methods as claimed in claim 21, wherein when these capture molecules are in conjunction with this analysis thing, this molecular network has the feature measured of at least one experience change.
25. methods as claimed in claim 24, comprise the change in this feature measured measuring these molecular networks further.
26. methods as claimed in claim 25, wherein measure this change and comprise that to use in photon multichannel analyzer, spectrometer, magnetic resonance imager, magnetic field detector, optical fiber, glass pipet, circuit, photofluorometer, optical spectrum analyser, potentiostat, calorimeter, electrophoresis, flow cytometer, ccd video camera, microscope, sound chamber, loudspeaker and photometer one or more.
Analyze the device of thing for catching for 27. 1 kinds, this device comprises:
A solid phase; And
Multiple layer, the plurality of layer is attached to going up at least partially of the surface of this solid phase, these layers comprise a molecular network, this molecular network comprises the capture molecules of at least one type, these capture molecules are attached to each other by polytype connection molecule, to form a covalently bound multi-layer three-dimension matrix, these capture molecules are arranged in conjunction with this analysis thing.
28. devices as claimed in claim 27, wherein this solid phase is made up of one or more in plastics, polymkeric substance, film, colloidal metal, silicon dioxide, carbon nano-tube, protein, carbohydrates, lipid, nucleic acid, cell and tissue.
29. device as claimed in claim 27, wherein this solid phase comprises one or more in nano material, the metal surface of modification, nanosphere, microballoon, microtiter plate, microslide, transfer pipet, box, cylinder, disk, probe, lateral flow device, microfluidic device and optical fiber.
30. devices as claimed in claim 27, wherein this molecular network manufactured in advance and be adsorbed to this solid phase this on the surface.
31. devices as claimed in claim 27, wherein this molecular network by be covalently attached to this solid phase this on the surface.
32. devices as claimed in claim 27, wherein this molecular network by be built directly in this solid phase this on the surface.
33. devices as claimed in claim 27, wherein these capture molecules comprise one or more in antibody, nucleic acid probe, enzyme, recombinant protein and peptide.
34. devices as claimed in claim 27, wherein these capture molecules comprise the multiple monoclonal antibody for the multiple epi-positions in conjunction with this analysis thing.
35. devices as claimed in claim 27, wherein these capture molecules are the analysis things for multiple mutual confirmation.
36. devices as claimed in claim 27, wherein these connection molecules comprise with one or more in bifunctional, different bifunctional, three functions and multi-functional type.
37. devices as claimed in claim 27, wherein when these capture molecules are in conjunction with this analysis thing, this molecular network has the feature measured of at least one experience change.
38. devices as claimed in claim 37, wherein this can measure feature comprise physical form, highly, density, fluorescence intensity, wavelength shift, vibration frequency, absorbance, flexibility, refractivity, conductance, impedance, resistance, melt temperature, one or more in denaturation temperature and freezing temperature.
39. devices as claimed in claim 27, wherein these capture molecules are also attached to each other by polytype spacer molecule.
40. devices as claimed in claim 39, wherein these spacer molecules comprise one or more in PEG, polymkeric substance, nucleic acid, albumin, Fc district and peptide.
41. devices as claimed in claim 39, wherein these spacer molecules and a certain amount of spacer molecule are selected for and give this molecular network one or more desired physical characteristics.
42. devices as claimed in claim 41, it is one or more that the physical characteristics wherein desired by these comprises in porosity, CHARGE DISTRIBUTION and topological characteristic.
43. 1 kinds of methods manufactured for catching the device analyzing thing, the method comprises:
A solid phase is provided; And
Multiple layer is placed on going up at least partially of the surface of this solid phase, these layers comprise a molecular network, this molecular network comprises polytype capture molecules, these capture molecules are attached to each other by polytype connection molecule, to form a covalently bound multi-layer three-dimension matrix, these capture molecules are arranged in conjunction with this analysis thing.
44. methods as claimed in claim 43, wherein put multiple layer comprise manufacture these layers in advance and these layers are adsorbed onto this solid phase this on the surface.
45. methods as claimed in claim 43, wherein put multiple layer comprise these layers are covalently attached to this solid phase this on the surface.
46. methods as claimed in claim 43, wherein put multiple layer comprise these layers are built directly in this solid phase this on the surface.
47. 1 kinds of methods measuring the amount of the analysis thing in sample, the method comprises:
One or more device is provided, each device comprises a solid phase and multiple layer, these layers cover the surface of this solid phase at least partially, these layers comprise a molecular network, this molecular network comprises the capture molecules of at least one type, these capture molecules are attached to each other by polytype connection molecule, to form a covalently bound multi-layer three-dimension matrix, these capture molecules are arranged in conjunction with this analysis thing;
These devices are exposed to this sample; And
Allow this analysis thing at least partially in conjunction with these capture molecules of these molecular networks of these devices.
48. methods as claimed in claim 47, comprise the change of measuring in this sample further.
49. methods as claimed in claim 48, wherein measure this change and comprise that to use in photon multichannel analyzer, spectrometer, magnetic resonance imager, magnetic field detector, optical fiber, glass pipet, circuit, photofluorometer, optical spectrum analyser, potentiostat, calorimeter, electrophoresis, flow cytometer, ccd video camera, microscope, sound chamber, loudspeaker and photometer one or more.
50. methods as claimed in claim 47, wherein when these capture molecules are in conjunction with this analysis thing, this molecular network has the feature measured of at least one experience change.
51. methods as claimed in claim 50, comprise the change in this feature measured measuring these molecular networks further.
52. methods as claimed in claim 51, wherein measure this change and comprise that to use in photon multichannel analyzer, spectrometer, magnetic resonance imager, magnetic field detector, optical fiber, glass pipet, circuit, photofluorometer, optical spectrum analyser, potentiostat, calorimeter, electrophoresis, flow cytometer, ccd video camera, microscope, sound chamber, loudspeaker and photometer one or more.
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