• 专利附录
  • 专利说明
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  • 类似技术
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    • 专利摘要:
    The present invention relates to the use of a substrate for enhancing the fluorescence of a fluorescent molecule, the substrate comprising a solid polymeric support having a plurality of distinct recesses and the solid support being at least partially coated with at least one metal.
    公开号:AT517746A1
    申请号:T50793/2015
    申请日:2015-09-16
    公开日:2017-04-15
    发明作者:
    申请人:Fianostics Gmbh；Sony Dadc Biosciences Gmbh；
    IPC主号:
    专利说明:

    The present invention relates to the provision of nano-structured surfaces, which are particularly suitable for enhancing the fluorescence of suitable molecules as these molecules approach such surfaces. This effect is also known as metal-enhanced fluorescence (MEF) or surface-enhanced fluorescence (SEF). MEF and SEF are based on an electromagnetic interaction of the incident (mostly stimulating), mostly coherent (i.e., laser) light with the electron plasma of nano-metal structures. This results in an enhancement of the luminous efficacy of fluorescent molecules as they approach (e.g., bond) to a surface having such metal structures. As a result, surface-bound molecules shine more intensely because their fluorescence is amplified.
    By increasing the fluorescence, molecules bound to a surface can be measured in the lowest concentrations. For example, the binding of a fluorescently labeled antibody in the form of its binding kinetics can be followed directly.
    The extent of reinforcement depends on the shape, size and spacing of the nano-metal structures and on the type of metal used (e.g., Au, Ag, Al, etc.). For example, in the literature descriptions of spherical (often colloids, see, for example, B Yang et al., Small 6 (2010): 1038-43; Corrigan T et al J Fluorescence 15 (2005): 777-784) triangular or pyramidal ( see, eg, Pompa et al., Nature Nanotechnology 1 (2006): 126-130; Cade et al., Nanotechnology. 15 (2009): 20 (28)) or wire-and-wire. rod-shaped metal structures that are discontinuous and form so-called metal islands. However, the gain factors obtained vary greatly and the nano-metal structures are in most cases not reproducible.
    It is an object of the present invention to provide a substrate which is reproducibly producible and capable of enhancing the fluorescence of a fluorescent substance as soon as the fluorescent substance is brought close to the substrate (eg 10 nm or less), where these
    Substrates should allow above-average gain in MEF measurements.
    The present invention relates to a substrate and its use for enhancing the fluorescence of one or more fluorescent molecules, the substrate comprising a solid polymeric support having a plurality of distinct recesses and the solid support being at least partially coated with at least one metal.
    It has surprisingly been found that substrates with the inventive structure are able to significantly increase the fluorescence yield (quantum yield) of a fluorescent molecule or a fluorophore with and without the use of coherent light if the at least one fluorescent molecule or fluorophore in located near (metal-reinforced fluorescence, MEF). By "fluorescence yield" or "quantum yield" is meant the ratio between the number of emitted and absorbed photons.
    The fluorescence yield with the substrates according to the invention is even many times higher than the yield using previously known substrates, on the surface of which are usually metallic islands. This increase in fluorescence yield is surprising in that it has previously been assumed that the MEF effect can only occur on surfaces, the metallic islands in the form of deposited metal-containing colloids or other mutually insulated and metal-coated areas on a surface (Matveeva E. et al., Anal Biochem, 344 (2004): 303-11; Geddes CD, et al., J Fluoresc 12 (2002): 121-129). Substrates with a continuous metal layer or without elevations are known to exhibit no or very little MEF effect due to a fluorescence quenching effect of the metal surface itself (Pineda EC, et al., J. Chem. Phys., 83 (1985): 5330-5337, Barnes WL, J Mod Opt, 45 (1998): 661-699). For this reason, a person skilled in the art, without being aware of the present invention, would have selected a solid support with protrusions and not a support with recesses for coating with a metal.
    The substrate according to the invention is used to enhance the fluorescence of fluorophores. That the substrates according to the invention are used wherever the reinforcement of
    Fluorescence (i.e., an increase in fluorescence yield) is desired. Therefore, the substrate of the invention may be e.g. in immunoassays, any form of molecular diagnostics using nucleic acids (PCR, RT-PCR), cell-based bioassays (as commonly used in high-throughput screening, histological or cellular studies, multiplexing test systems (eg LUMINEX) if fluorescence is used to detect the target molecules.
    According to a preferred embodiment of the present invention, the amplification of the fluorescence is carried out at a distance of 0 to 50 nm, preferably from 1 to 50 nm, more preferably from 1 to 40 nm, even more preferably from 2 to 40 nm, even more preferably from From 1 to 30 nm, more preferably from 2 to 30 nm, even more preferably from 3 to 30 nm, even more preferably from 1 to 20 nm, even more preferably from 2 to 20 nm, even more preferably from 3 to 20 nm, even more preferably from 5 to 20 nm, more preferably from 5 to 15 nm, to the metal which is on the surface of the solid polymeric support.
    As used herein, "fluorescent molecules" as used herein includes molecules that spontaneously emit light upon excitation by electromagnetic waves, such as light of a particular wavelength. "Fluorophore" is an umbrella term and synonym of such molecules, and thus also includes molecules , which fluoresce or weakly fluoresce and are usually not referred to as fluorophores. Examples of such molecules are proteins or nucleic acids whose fluorescence ("intrinsic fluorescence") is mediated via aromatic structures (e.g., via the amino acids tryptophan or tyrosine).
    According to the invention, the "solid support" can consist of any desired polymeric material, provided that it can be coated with a metal and if recesses can be produced For example, the solid polymeric support comprises or consists of synthetic polymers such as polystyrene, polyvinyl chloride or polycarbonate, cycloolefin , Polymethyl methacrylate, polylactate or combinations thereof .. In principle, non-polymeric supports such as metals, ceramics or even glass could be used, provided that it can be coated with a metal and if recesses can be produced.
    The solid support comprises at least one material selected from the group consisting of the group of thermoplastic polymers and the polycondensates.
    According to a preferred embodiment of the present invention, the thermoplastic polymer is selected from the group consisting of polyolefins, vinyl polymers, styrenic polymers, polyacrylates, polyvinylcarbazole, polyacetal and fluoroplastics.
    The polycondensate is preferably selected from the group consisting of thermoplastic polycondensates, thermosetting polycondensates and polyadducts.
    According to a particularly preferred embodiment of the present invention, the material of the polymeric solid support comprises organic and / or inorganic additives and / or fillers, these being preferably selected from the group consisting of TiO 2, glass, carbon, colored pigments, lipids and waxes.
    Another aspect of the present invention relates to a process for producing a substrate for enhancing the fluorescence of a fluorophore comprising the step of coating a solid support according to the present invention with at least one metal.
    The solid supports according to the invention, including depressions, can basically be produced by various processes (see FIG. 15). (a) The solid supports are produced together with the depressions in one step (eg injection molding) (see FIG. 15 (a)) (b) The depressions are introduced into an existing solid support in further process steps (eg hot embossing, electron beam lithography or " Extreme Ultra Violet "(EUV) in conjunction with reactive ion etching or laser ablation) (see Fig. 15 (b)) (c) On a solid support, a thin patternable polymer layer is applied into which the recesses are introduced, such as in manufacture the BD-50 Blu-ray Disc (UV nanoimprint lithography) (see Fig. 15 (c)).
    Especially suitable for the preparation of these structures is also the so-called nanoimprint lithography (Chou S. et al., Nano-imprint lithography, Journal of Vacuum Science & Technology B Volume 14, No. 6, 1996, p. 4129-4133). The production of nanostructures by means of nanoprint lithography requires a positive, usually a monomer or polymer, as well as a nanostructured stamp ("master") .The stamp itself can be produced by nanolithography, which alternatively can also be prepared by etching A substrate is applied and then heated above the temperature of the glass transition, ie it is liquefied before the stamp is pressed in. In order to achieve controllable (and short-term) heating, laser or UV light is frequently used Positive on heating, the gaps of the stamp are completely filled with it.After cooling, the stamp is removed again.The positive, which is the solid support of the substrate according to the invention, is coated with metal by means of sputtering.
    The structuring of the lithographic stamps can again be done with nanoimprint. The materials used here are glass or light-transparent plastic.
    Particularly preferred is the preparation of the solid support including recesses by injection molding. The mold inserts for this purpose are typically drawn off from a lithographically produced Si wafer by means of Ni electroplating
    The solid support may basically have any shape (e.g., spherical, planar), with a planar shape being particularly preferred.
    A "well", as used herein, refers to the level of the surface of the solid support surrounding the well and extends into the support rather than as protruding or raised therefrom A well according to the present invention has a bottom The depth is thus the distance from the surface to a bottom of the depression The depressions on the solid support may have different shapes (eg round, oval, square, rectangular).
    A "plurality" of wells as used herein means that the solid support of the invention has at least one, more preferably at least two, even more preferably at least 5, even more preferably at least 10, even more preferably at least 20, even more preferably at least 30 more preferably at least 50, even more preferably at least 100, even more preferably at least 150, even more preferably at least 200, depressions, which depressions may be on an area of the solid support of 1000 pm 2, more preferably 500 pm 2, even more preferably 200 pm 2 even more preferably 100 pm.sup.2 Alternatively, the depressions can extend over a length of preferably 1000 .mu.m, more preferably of 500 .mu.m, even more preferably of 200 .mu.m, even more preferably of 100 .mu.m. "Separated from one another "Wells" as used herein means that the wells are separated by their side boundaries and no verbi Not on the surface of the solid support.
    According to a preferred embodiment of the present invention, the recesses of the solid support have a length and a width, wherein the ratio of the length to the width is 2: 1 to 1: 2, in particular about 1: 1.
    The recesses on the solid support can in principle have any shape. However, wells which have a length to width ratio of 2: 1 to 1: 2, preferably 1.8: 1, preferably 1.6: 1, preferably 1.5: 1, preferably 1.4: 1, are particularly preferred. preferably 1.3: 1, preferably 1.2: 1, preferably 1.1: 1, preferably 1: 1.8, preferably 1: 1.6, preferably 1: 1.5, preferably 1: 1.4, preferably 1: 1.3, before preferably 1: 1.2, preferably 1: 1.1, in particular 1: 1, have.
    According to a further preferred embodiment of the present invention, the length and the width of the recesses is 0.1 μm to 2 μm, preferably 0.2 μm to 2 μm, preferably 0.3 μm to 2 μm, preferably 0.1 μm to 1, 8 pm, preferably 0.2 pm to 1.8 pm, preferably 0.3 pm to 1.8 pm, preferably 0.1 pm to 1.5 pm, preferably 0.2 pm to 1.5 pm, preferably 0, 3 pm to 1.5 pm, preferably 0.1 pm to 1.2 pm, preferably 0.2 pm to 1.2 pm, preferably 0.2 pm to 1.2 pm, preferably 0.1 pm to 1 pm, preferably from 0.2 μm to 1 μm, preferably from 0.3 μm to 1 μm, preferably from 0.1 μm to 0.8 μm, preferably from 0.2 μm to 0.8 μm, preferably from 0.3 μm to 0.8 μm , preferably 0.1 pm to 0.6 pm, preferably 0.2 pm to 0.6 pm, preferably 0.3 pm to 0.6 pm, most preferably 0.2 pm to 0.6 pm.
    Particularly preferably, the depressions of the solid support according to the invention have a substantially round shape, wherein "essentially round" also includes oval and ellipsoidal shapes The shape of the depression can be seen in a plan view of the surface of the solid support.
    The depressions preferably have a depth of 0.1 μm to 5 μm, preferably of 0.1 μm to 4 μm, preferably of 0.1 μm to 3 μm, preferably of 0.1 μm to 2 μm, preferably of 0.1 pm to 1.5 pm, preferably from 0.1 pm to 1.2 pm, preferably from 0.1 pm to 1 pm, preferably from 0.1 pm to 0.9 pm, preferably from 0.1 pm to 0, 8 pm, preferably from 0.2 pm to 5 pm, preferably from 0.2 pm to 4 pm, preferably from 0.2 pm to 3 pm, preferably from 0.2 pm to 2 pm, preferably from 0.2 pm to 1.5 pm, preferably from 0.2 pm to 1.2 pm, preferably from 0.2 pm to 1 pm, preferably from 0.2 pm to 0.9 pm, preferably from 0.2 pm to 0.8 pm , preferably from 0.3 pm to 5 pm, preferably from 0.3 pm to 4 pm, preferably from 0.3 pm to 3 pm, preferably from 0.3 pm to 2 pm, preferably from 0.3 pm to 1, 5 pm, preferably from 0.3 pm to 1.2 pm, preferably from 0.3 pm to 1 pm, preferably from 0.3 pm to 0.9 pm, preferably from 0.3 pm to 0.8 pm , The depth of the recess is the distance from the surface of the solid metallized support to the bottom of the recess.
    According to a preferred embodiment of the present invention, the recesses have a distance ("period") to one another of from 0.2 μm to 2.5 μm, preferably from 0.3 μm to 1.4 μm, more preferably from 0.4 μm to 1 , 3 pm In a further preferred embodiment of the present invention, the depressions have a distance from one another of 0.2 μm to 2 μm, preferably from 0.2 μm to 1.8 μm, preferably from 0.2 μm to 1, 6 pm, preferably from 0.2 pm to 1.5 pm, preferably from 0.2 pm to 1.4 pm, preferably from 0.2 pm to 1.3 pm, preferably from 0.3 pm to 2.5 pm , preferably from 0.3 pm to 2 pm, preferably from 0.3 pm to 1.8 pm, preferably from 0.3 pm to 1.6 pm, preferably from 0.3 μm to 1.5 μm, preferably from 0 , 3 μπι to 1.3 μιη, preferably from 0.4 μιη to 2.5 μιη, preferably from 0.4 μιη to 2 μιη, preferably from 0.4 μιη to 1.8 μιη, preferably from 0.4 μιη to 1.6 μιη, preferably from 0.4 μιη to 1.5 ιη, preferably from 0.4 μιη to 1.4, preferably from 0.5 μιη to 2.5 μιη, preferably from 0.5 μιη to 2 μιη, preferably from 0.5 μιη to 1.8 μιη, preferably from 0 , 5 μιη to 1.6 μιη, preferably from 0.5 μιη to 1.5 μιη, preferably from 0.5 μιη to 1.4 μιη, preferably from 0.5 μιη to 1.3 μιη, preferably from 0.6 μιη to 2.5 μιη, preferably from 0.6 μιη to 2 μιη, preferably from 0.6 μιη to 1.8 μιη, preferably from 0.6 μιη to 1.6 μιη, preferably from 0.6 μιη to 1, 5 μm, preferably from 0.6 μm to 1.4 μm, preferably from 0.6 μm to 1.3 μm, preferably from 0.7 μm to 2.5 μm, preferably from 0.7 μm to 2 μm, preferably from 0.5 μm to 1.8 μm, preferably from 0.7 μm to 1.6 μm, preferably from 0.7 μm to 1.5 μm, preferably from 0.7 μm to 1.4 μm, preferably from 0 , 7 gm to 1.3 gm, with the pits being most preferably one Distance from 0.2 gm to 1.4 gm or 0.3 gm to 1.3 gm to each other. The distance between the pits ("period") is measured from the center of the pit.
    According to another preferred embodiment of the present invention, the metal layer on the solid support has a thickness of 10 nm to 200 nm, preferably 15 nm to 100 nm. The thickness of the metal layer on the solid support is particularly preferably from 10 nm to 190 nm, preferably from 10 nm to 180 nm, preferably from 10 nm to 170 nm, preferably from 10 nm to 160 nm, preferably from 10 nm to 150 nm, preferably from 10 nm to 140 nm, preferably from 10 nm to 130 nm, preferably from 10 nm to 120 nm, preferably from 10 nm to 110 nm, preferably from 10 nm to 100 nm, preferably from 10 nm to 90 nm, preferably from 10 nm to 80 nm, preferably from 10 nm to 70 nm, preferably from 10 nm to 60 nm, preferably from 10 nm to 50 nm, preferably 15 nm to 200 nm, preferably 15 nm to 190 nm, preferably from 15 nm to 180 nm, preferably from 15 nm to 170 nm, preferably from 15 nm to 160 nm, preferably from 15 nm to 150 nm, preferably from 15 nm to 140 nm, preferably from 15 nm to 130 nm, preferably from 15 nm to 120 nm, preferably from 15 nm to 110 nm, preferably from 15 nm to 90 nm, preferably from 15 nm to 80 nm, preferably from 15 nm to 70 nm, preferably from 15 nm to 60 nm, preferably from 15 nm to 50 nm, preferably from 20 nm to 200 nm, preferably from 20 nm to 190 nm, preferably from 20 nm to 180 nm, preferably from 20 nm to 170 nm, preferably from 20 nm to 160 nm, preferably from 20 nm to 150 nm, preferably from 20 nm to 140 nm, preferably from 20 nm to 130 nm, preferably from 20 nm to 120 nm, preferably from 20 nm to 110 nm , preferably from 20 nm to 100 nm, preferably from 20 nm to 90 nm, preferably from 20 nm to 80 nm, preferably from 20 nm to 70 nm, preferably from 20 nm to 60 nm, preferably from 20 nm to 50 nm.
    According to the invention, the solid polymeric support is "at least partially" coated with at least one metal. "At least in part" as used herein means that portion of the solid support in which the recesses are located at least 20%, preferably at least 30%. even more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90% more preferably at least 95%, more preferably at least 98%, especially 100%, is coated with at least one metal. Since the MEF effect requires a metallic surface, it is particularly preferred that the surface of the solid support is coated at least in the region of the recesses with at least one metal. In this case, the solid support may also comprise a plurality (for example at least two, at least three, at least four or at least five) superimposed metal layers of the same or different metals. An advantage to using multiple layers of metal on the solid support is that the first metal layer (e.g., chromium) applied directly to the support can improve the adhesion of the other metal layers.
    As used herein, the term "superimposed" means that one metal layer is disposed directly on another metal layer, resulting in a multilayer system of metal layers of the same metal or different metals.
    The metal layers are preferably continuous and unbroken. According to the invention, however, it has been found that the metal layer or layers on the solid polymeric support can also be interrupted without impairing the fluorescence-enhancing effect. The interrupted metal layer can be effected, for example, by a conductivity measurement of the surface of the substrate according to the invention. A lower or no conductivity means that the metal layer (s) are interrupted at the substrate surface. Broken metal layers can be made, for example, by contacting a substantially fully metal-coated substrate with a preferably saline solution, such as 10 mM phosphate buffer, with 150 mM NaCl for a period of time (10-90 minutes).
    The solid support of the present invention is "coated with at least one metal." Preferably, the metal layer comprises at least two, more preferably at least three, even more preferably at least four, even more preferably at least five, different metals Technically known methods are applied to the solid support, preferably sputtering (cathode sputtering) or thermal evaporation, electron beam evaporation, laser beam evaporation, arc evaporation, molecular beam epitaxy, ion beam assisted deposition and ion plating is used.
    According to a preferred embodiment of the present invention, the metal is selected from the group consisting of silver, gold, aluminum, chromium, indium, copper, nickel, palladium, platinum, zinc, tin and alloys comprising one or more of these metals.
    According to the invention, these metals or alloys thereof can be used for coating the solid support according to the invention. Particularly preferred is the coating of the solid support with silver or alloys comprising silver, since silver or its alloys show a particularly large reinforcing effect. Particularly preferred is an alloy comprising silver, indium and tin. The silver-containing alloys preferably have a silver content of more than 10%, more preferably more than 30%, even more preferably more than 50%, even more preferably more than 70%, even more preferably more than 80%. even more preferably more than 90%.
    According to a preferred embodiment, the substrate according to the invention is part of a capillary, a microtiter plate, a microfluidic chip, a test strip (for "lateral flow assays"), a carrier (eg microscope slide) for fluorescence microscopy, in particular for high-resolution methods such as confocal laser microscopy after Point scanner principle as well as 4Pi microscopes and STED (Stimulated Emission Depletion) microscopes, a sensor array or other optical detector array is.
    Particularly preferred is the use of the substrate according to the invention in microtiter plates, wherein the microtiter plates can comprise 6, 12, 24, 48, 96, 384 or 1536 wells. Microtiter plates are used for a variety of measurements and assays, which often also measure the fluorescence of samples. By providing the substrate according to the invention in the wells of microtiter plates, the fluorescence yield of the samples can be significantly increased. The substrates can be introduced and fixed in the wells by various methods. Preferably, the substrates are thereby fixed in the wells by means of gluing, welding techniques (for example laser welding) and thermal deposition.
    According to a particularly preferred embodiment of the present invention, the solid support comprises or consists of a cyclo-olefin copolymer or cyclo-olefin polymer and is part of a microtiter plate or part of the wells of a microtiter plate. COP 1060R (Zeo-nor® 1060R) has proven particularly suitable. The support is preferably coated with 10 to 60 nm, preferably up to 40 nm, of metal (for example silver).
    Certain measurements with fluorescent substances such as fluorophores are carried out in capillaries. Therefore, it is preferable to provide the substrates according to the invention in capillaries. An exemplary application is cytometry or flow cytometry, in which the number or also the type of fluorescent cells or fluorescence-marked cells is determined by means of a fluorescence measurement.
    Numerous fluorescence measurement applications occur in microfluidic chips (e.g., as a "lab-on-a-chip" application) where the substrates of the invention may be provided in the detection region of such chips.
    The substrates according to the invention can also be provided in conventional cuvettes. As a result, the fluorescence yield can also be significantly increased in fluorescence measurements, so that the smallest amounts of fluorescent substances in a sample can be measured. According to the invention, any cuvette shape can be used.
    Also in test strip systems ("lateral flow assays"), which are used for rapid tests or in-field tests (point of care), the substrates according to the invention (eg in the detection area ("detection line") can be used to control the fluorescence of a labeled To amplify analytes (eg a fluorescently labeled antibody) and thus to improve the sensitivity of the test.
    In a further preferred embodiment of the invention, the substrates according to the invention are applied to microscope slides as used in microscopy, in particular fluorescence microscopy. The fluorescence of fluorophores used to label cellular structures could thus be selectively enhanced and the optical resolution of the methods drastically improved, since less light intensity is needed, which would optimize the signal-to-noise ratio. Areas of application would be high-resolution methods such as point-scanner confocal laser microscopy and 4Pi microscopes and STED (Stimulated Emission Depletion) microscopes.
    According to another preferred embodiment of the present invention, the metal coating on the surface of the substrate at least partially comprises molecules for direct and / or indirect binding of fluorescent molecules.
    The substrates of the present invention can enhance the fluorescence of fluorescent molecules or fluorophores when the fluorophores are in close proximity (preferably less than 20 nm) of the substrates. The fluorophores or the fluorescent substances can thereby move freely in a liquid, the increase in fluorescence only taking place when these fluorophores or fluorescent molecules approach the substrate according to the invention. To increase the likelihood of the approach of the fluorophores or fluorescing molecules to the substrate, it is of particular advantage, if on the surface of the substrate (ie on the metal coating) molecules are irreversibly or reversibly bound, either the fluorophore or the fluorescent molecule itself ("direct binding") or a molecule to which a fluorophore or a fluorescent molecule (eg fluorescently labeled antibody; "indirect binding") is coupled. Methods for binding such molecules to metal structures are well known. In the simplest case, the binding occurs by physicochemical adsorption (mediated by ionic and hydrophobic interactions) of the proteins to the metal surface (e.g., Nakanishi K. et al J Biosci Bioengin 91 (2001): 233-244). Covalent methods for the immobilization of proteins after derivatization of the metal surfaces are also known (for example GB Sigal et al., Anal Chem 68 (1996): 490-7).
    Molecules for direct and / or indirect binding of fluorescent molecules or fluorophores are preferably selected from the group consisting of antibodies, antibody fragments, preferably Fab, F (ab) '2 or scFv fragments, nucleic acids, enzymes, lipids, virus particles, aptamers and combinations from that.
    On the one hand, these molecules are able to directly bind fluorophores or fluorescent molecules (for example antibodies and fragments thereof, nucleic acids, enzymes), on the other hand these molecules can also bind other molecules which are provided with a fluorophore or a fluorescent substance.
    Another aspect of the present invention relates to a capillary, a chip, preferably a microfluidic chip, a cuvette, a microtiter plate, a support for fluorescence microscopy or an optical detector array comprising a substrate according to the invention.
    Yet another aspect of the present invention relates to a kit comprising at least one microtiter plate, at least one capillary, at least one chip, preferably a microfluidic chip, at least one cuvette and / or at least one test strip comprising a substrate according to the present invention and one with a fluorophore analyte-binding
    A molecule or analyte-binding molecule labeled with an enzyme and a fluorescent substrate for the enzyme. "Fluorescent substrate for an enzyme" as used herein is a substrate capable of binding in or at the active site of the enzyme, whereby the substrate can acquire fluorescent properties, and of course the substrate may also have fluorescent properties prior to its attachment to the enzyme exhibit.
    The solid carriers having the wells defined above are then coated with one or more metals (e.g., two, three, four, or five metals). Methods for coating solid supports with metals are well known in the art, with particular preference being given to using PVD (Physical Vapor Deposition) methods, such as sputtering or vapor deposition methods.
    Thus, in accordance with a preferred embodiment of the present invention, the at least one metal is deposited on the surface of the solid support by sputtering or thermal evaporation, electron beam evaporation, laser beam evaporation, arc evaporation, molecular beam epitaxy, ion beam assisted deposition or ion plating.
    In order to enable the direct and / or indirect binding of fluorophores or other fluorescent substances on the surface of the substrate according to the invention, at least partially molecules for the direct and / or indirect binding of fluorophores via adsorptive or covalent chemical derivatives on the metal coating on the surface of the substrate -sierung the surface applied. "At least in part" as used herein means that at least 10%, preferably at least 30%, even more preferably at least 50%, even more preferably at least 70%, even more preferably at least 90%, even more preferably at least 90%, especially 100%, of the metal-coated solid support with molecules for the direct and / or indirect binding of fluorophores is provided.
    According to a preferred embodiment of the present invention, the molecules for direct and / or indirect binding of fluorophores are selected from the group consisting of
    Antibodies, antibody fragments, preferably Fab, F (ab) '2 or scFv fragments, nucleic acids, enzymes, lipids, virus particles, aptamers, and combinations thereof.
    A further aspect of the present invention relates to a method for the determination or quantification of at least one analyte in a sample comprising the steps: a) optional direct or indirect labeling of at least one analyte with at least one fluorophore, b) application of at least one labeled analyte from step a c) exciting at least one fluorophore by irradiating the substrate with light of suitable wavelength, and d) measuring the fluorescence to determine the presence of at least one analyte in the sample.
    The substrate according to the invention, which is able to significantly increase the fluorescence yield of fluorophores and other fluorescent molecules or substances, can be used for methods in which the fluorescence of samples is to be measured. By using the substrate according to the invention in such methods, the sensitivity of such methods can be significantly increased, so that not only the presence of very small amounts of analyte to be determined can be determined but also the quantification (small amounts) of analytes can be carried out more accurately.
    In a first step, the analytes to be determined or quantified in a sample are labeled directly or indirectly with a fluorophore or a fluorescent substance. In the case of a direct labeling of the analyte, the at least one fluorophore or the at least one fluorescent substance becomes covalent or noncovalent (for example by hydrogen bonding, electrostatic bonding, Van der Waals forces, hydrophobic interactions) to the substance to be determined or quantified Bound analyte. Indirect labeling introduces into the sample fluorescently labeled molecules (e.g., antibodies or fragments thereof) capable of binding to the analyte. This first process step is optional, since there are analytes to be determined or quantified which are themselves capable of fluorescence - with appropriate excitation. Samples comprising such analytes can be applied directly or after sample preparation to the substrate according to the invention (see step b) of the method according to the invention).
    After the at least one labeled analyte from step a) or the fluorescent analyte has been applied to the substrate according to the invention, the fluorophore or the fluorescing substance or fluorescent analyte is irradiated by irradiating the substrate with coherent or non-coherent light (eg laser or xenon flash lamp). suitable wavelength for fluorescence emission excited. As used herein, "light of suitable wavelength" means that the light used in the method of the invention has a wavelength suitable for inducing the fluorescence emission of a substance in contact For example, light having a wavelength of 485 is suitably the fluorescence emission of To induce fluorescein isothiocyanate (FITC).
    After excitation of the fluorescent substances by means of light, these substances emit light (fluorescence) of a specific wavelength. This emitted light of a defined wavelength is measured and can be used to quantify or determine the presence of an analyte in a sample. The emitted light can be measured by means of a detector (e.g., photomultiplier). Commercially available microtiter plate readers (Tecan F200pro, BioTek Synergy, Molecular Devices FilterMax or SpectraMax series etc.), flatbed fluorescence scanners (eg Tecan LS-Reloaded, fluorescence microscopes or any other proprietary analysis system (Roche COBAS, Abbot AxSYM, Behring Opus Plus ), when a corresponding fluorescence detector is integrated) arrive.
    According to a preferred embodiment of the present invention, the at least one fluorophore has an excitation wavelength in the range from 360 to 780 nm, preferably from 490 to 680 nm.
    According to a further preferred embodiment of the present invention, the at least one fluorophore has an emission wavelength in the range from 410 to 800 nm, preferably from 510 to 710 nm.
    The at least one fluorophore is preferably selected from the group consisting of methoxycoumarin, aminocoumarin,
    Cy2, Alexa Fluor 488, Fluorescein Isothiocyanate (FITC), Alexa Fluor 430, Alexa Fluor 532, Cy3, Alexa Fluor 555, 5-TAMRA, Alexa Fluor 546, Phycoerythrin (PE), Tetramethyl Rhodamine Isothiocyanate (TRITC), Cy3.5, Rhodamine, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Cy5, Alexa Fluor 660, Cy5.5, Alexa Fluor 680 and Cy7, preferably from the group consisting of fluorescein isothiocyanate (FITC), Cy3, phycoerythrin ( PE), tetramethyl rhodamine isothiocyanate (TRITC), Cy5 and Alexa Fluor 680.
    According to a preferred embodiment of the present invention, the analyte is indirectly labeled with at least one fluorophore by means of a fluorophore-labeled and analyte-binding molecule.
    According to another preferred embodiment of the present invention, the analyte-binding molecule is selected from the group consisting of antibodies, antibody fragments, preferably Fab, F (ab) '2 or scFv fragments, nucleic acids, enzymes, lipids, virus particles, aptamers, and combinations thereof.
    The present invention will be explained in more detail with reference to the following figures and examples, but without being limited thereto.
    Figure 1 shows a three dimensional AFM ("Atomic Force Microscope") image of a metal-coated planar solid support according to the invention (see Example 1).
    2 shows the MEF effect as a function of the fluorophore type and a silver layer thickness of 0, 20 and 50 nm Ag. The MEF effect manifests itself in the observed "relative increase", ie the ratio of the signal at the end of the measurement time after 600 seconds (t600) to the signal at the beginning of the measurement t (0) A relative increase of 1.0 means no signal change and the higher the relative increase, the stronger the MEF effect, and there is a general trend toward higher MEF with increasing metal layer thickness, but varying from fluorophore to fluorophore.
    FIG. 3 shows the dependence of the MEF on silver layer thickness in 5 nm steps for AlexaFlour 680 (see Example 2). From a layer thickness of 5 nm, a significant increase in the MEF effect can be observed.
    4 and 5 show AFM images of substrates according to the invention / structures comprising depressions of different periods.
    Fig. 6 shows the dependence of the MEF effect on the period (0.8 to 2.2 ym) of the structures.
    Fig. 7 shows the dependence of the MEF on the depth of the structures.
    Figures 8 and 9 show the obtained MEF gains compared to prior art colloid coated surfaces and MEF surfaces (PLASMONIX, Quanta-Wells 2, "Competitor Structure").
    Figure 10 shows MEF kinetics on nano-pillars (nano-columns, bumps) and inverted nano-pills (wells).
    11 shows the performance of an anti-rabbit IgG fluorescence immunoassay using a substrate according to the invention.
    Fig. 12 shows the substrate according to the invention comprising a solid support coated with a metal layer. The solid support has recesses with a depth, a width and a length. The recesses are located on the solid support at a certain distance (period) to each other.
    Fig. 13 shows the plan view (A) and a cross section (B) of a solid support according to the invention. The recesses on the solid support are characterized by a width, length and depth and have a certain distance (period) to each other.
    Fig. 14 shows the MEF effect using different buffers.
    Fig. 15 shows various methods with which the solid supports according to the invention including depressions can be produced. EXAMPLES:
    Example 1: Preparation of the substrate according to the invention
    Starting from the known state of the art (see, inter alia, Pompa et al., Nature Nanotechnology 1 (2006): 126-130, Cade et al., Nano-technology, 15 (2009): 20 (28), US 2009/0262640), it was attempted to As high as possible, slim columnar structures ("nano pillars") to produce by a large ratio (1: 2 to 1: 3) of diameter of the base to the height of the structure ("aspect ratio") to a thinning of the Metal layer during vapor deposition and so to the formation of the necessary literature for the MEF effect metal island structures to come. Therefore, "pillars" (columns) with different base diameter (250-550nm) and different height (250-850nm) were produced.
    For the production of the substrates, a special form of injection molding, namely injection-compression molding, was used. When injection-compression molding is introduced by thermoplastic Kunststoffschmelze in a slightly open tool while pressing (= embossing). The nanostructured stamp for injection molding was removed by means of nickel electroplating from a lithographically produced silicon master. Silicon masters are understood here to be a silicon wafer coated with positive lacquer, which was exposed by means of "laser lithography" and subsequently developed Surprisingly, only the metal-coated solid supports with the depressions (INPs) showed a clear MEF effect, whereas the substrates based on a solid support with bumps showed no or only a minimal MEF effect (see Fig. 10), therefore, the INP structures were further investigated.
    Example 2: Influence of the metal layer thickness
    In order to investigate the influence of the metal layer thickness on the surface of a solid support with wells with a diameter of about 450 nm, different layer thicknesses of silver were vapor-deposited.
    The direct adsorption of fluorescently labeled antibodies on a surface is the simplest way to compare differently structured surfaces in terms of sensitivity and amplification factor. The MEF effect was that unlike a surface without MEF, the binding kinetics ("MEF kinetics") of the antibody could be monitored directly in real time, allowing only the molecules near the surface to be stronger The solution containing the fluorescently labeled antibody was spotted onto the corresponding nano-structured surface and the change in the signal was monitored over time using a suitable fluorescence meter (Tecan 200F pro).
    Apart from the parameter "MEF kinetics", comparing the signal of a certain concentration of fluorescently labeled antibody on a surface with nano-metal structure with the signal of the same antibody on a surface without this structure can be used to define an amplification factor the effective occupancy densities, ie the actual amount of antibody on which the surfaces are equal.
    This can easily be done by detection of the bound antibody (Goat Anti-Rabbit FITC) with a labeled secondary antibody (an alkaline phosphatase-labeled Donkey anti-goat antibody) and did not give significant differences in antibody occupancy densities of the surfaces tested.
    In the case of the produced metal layer thickness variants, it now turned out that the MEF effect in the range of 0-50 nm Ag increases significantly independently of the fluorophore tested (see FIG. 2; rel. Increase of 1 means no MEF effect).
    Fig. 3 shows that a minimum layer thickness of 5 nm is needed to obtain an MEF. In addition, FIG. 3 shows that as the metal layer thickness is increased in steps of 5 nm, a continuous increase in the MEF effect can be observed.
    Example 3: Influence of the structure period
    The spacing of the pits ("period") could have an influence on the MEF effect of the substrate according to the invention Therefore, different solid supports with different periods were coated, for example, with silver:
    FIGS. 4 and 5 each show an AFM image of two substrates according to the invention with a period of 0.8 μm or 2.2 μm and a silver layer thickness of 50 nm.
    In order to demonstrate the MEF effect, 1 to 7 MEF kinetics of AlexaFlour 680 (13 nM in 10 mM PBS, pH 7.4) were prepared for all fields (see FIG. 6). It was found that the MEF effect was highest for a period of 0.8 and 1.0 pm. From a period of 1.2 μm, the MEF effect was much lower but still present.
    The following table shows the relative increases (signal t = 300s / signal t = 0s) of the MEF kinetics measurements of different fluorescently labeled antibodies for field 1 (0.8um) and 2 (1.0). The silver layer thickness on the INPs was 20 nm for these measurements, with a goat antibody labeled with the respective fluorophore being directed against rabbit IgG (diluted in 10 mM PBS pH 7.4, c = 13 nM):
    The MEF effect on the INPs could thus be demonstrated for a wide variety of fluorophores in the Ex / Em wavelength range from 485/520 (FITC) to 680/720 (AlexaFlour 680). The use of INPs is not limited to specific fluorophores.
    Example 4 Influence of the Depth of the Wells on the MEF Effect
    In order to investigate the influence of the depths of the wells (Inverted Nano-Pillars, INPs), solid supports with different depression depths (60 nm, 240 nm, 550 nm, 755 nm and 874 nm) were produced and evaporated with silver (20 nm layer thickness). ,
    Adsorption tests with fluorescently labeled antibodies ("MEF kinetics") showed that the MEF effect also increases with increasing depression depth. For solid supports with wells of less than 60 nm depth, an MEF effect was observed but was significantly lower compared to the other supports (see Figure 7).
    Example 5: Comparative Experiments
    The substrates according to the invention show an enhanced MEF effect in comparison with conventionally used structures. To prove this, microtiter plates were prepared according to a method known from the literature (direct monitoring of molecular recognition processes using fluorescence enhancement at colloid-coated microplates., C Lobmaier et al., Jul; 14 (4): 215-22) with silver Coated colloids and their amplification factors (defined as the ratio of the signals on the surface without and with silver colloids at the same antibody
    Surface concentration) in comparison to the structures according to the invention having recesses (20 nm Ag, 0.8 pm period). In addition, the only commercially available microtiter plate system (PLASMONIX, Quant-Wells 2) according to the manufacturer was investigated.
    The reinforcing factors of the substrate according to the invention, as shown in FIG. 9, were about 10 times as high as on colloidal plates or on plates from PLASMONIX. Apart from the significantly lower amplification factors, the PLASMONIX microtiter plates also do not show the typical MEF kinetics (see FIG. 9 in comparison with FIG. 7).
    Example 6: Anti-rabbit IgG fluorescence immunoassay
    The surfaces of a substrate according to the invention, a colloid-coated microtiter plate (MTP) and a standard microtiter plate from Greiner, as used in the prior art for immunoassays, were incubated with a solution of rabbit IgG (2 μg / ml) in PBS (10 mM Phosphate buffer with 150 mM NaCl pH 7.4) for 2 h at room temperature. Thereafter, the solution was removed, the surface washed with PBS containing 0.1% Triton X-100 and washed with a 5% Polyviniylpyro-lidone solution to block unspecific binding for 1 hr
    Brought in contact. After another wash with PBS / Triton X100, incubation was carried out with biotin-labeled anti-rabbit IgG antibodies of various concentrations for 1 h at RT. The binding of this anti-rabbit IgG antibody was finally detected after a final wash by MEF kinetics measurement with Cy3-labeled streptavidin over a period of 600 seconds (see FIG. 11). It can be clearly seen that no MEF kinetics occur on the standard microtiter plate and thus the immunoassay is also not feasible. The colloid-coated microtiter plate shows only a weak, the inventive substrate, however, a very pronounced MEF kinetics and thus an immunoassay with much steeper calibration curve. much higher sensitivity.
    The substrate according to the invention used in this example showed electrical conductivity prior to its coating with antibodies. After measuring the MEF kinetics no electrical conductivity of the substrate could be determined.
    This could be due to the formation of silver chloride on contact with PBS buffer.
    Example 7: MEF effect as a function of the buffer used
    In order to investigate the dependence of the MEF effect on the buffer used, as in Example 3, the MEF kinetics caused by the adsorption of a fluorescently labeled antibody (goat anti-rabbit antibody, labeled with Cy5) were observed, with a pure phosphate buffer instead of the PBS buffer (PB, 10 mM phosphate buffer), 1% (w / v) aqueous sodium citrate solution and diH 2 O. The tests were carried out on substrates with a period of lum (corresponding to field 2, see Example 3). As can be seen from FIG. 14, the adsorption from PBS provided the highest relative signal increase, but it was also possible to observe clear signals upon adsorption of the antibody from the other solutions. This could be a consequence of the possible formation of a silver chloride layer described in Example 6, which has a positive effect on the reinforcing effect.
    权利要求:
    Claims (46)
    [1]
    claims:
    Use of a substrate for enhancing the fluorescence of one or more fluorescent molecules, characterized in that the substrate comprises a solid polymeric support having a plurality of separate recesses and the solid support is at least partially coated with at least one metal.
    [2]
    2. Use according to claim 1, characterized in that the amplification of the fluorescence takes place at a distance of 0 to 50 nm to the metal.
    [3]
    3. Use according to claim 1 or 2, characterized in that the recesses of the solid support have a length and a width, wherein the ratio of the length to the width of 2: 1 to 1: 2, in particular 1: 1, is.
    [4]
    4. Use according to one of claims 1 to 3, characterized in that the recesses of the solid support have a length and a width, wherein the length and the width of the recesses is 0.1 pm to 2 pm.
    [5]
    5. Use according to one of claims 1 to 4, characterized in that the depressions have a substantially round shape.
    [6]
    6. Use according to one of claims 1 to 5, characterized in that the depressions have a depth of 0.1 pm to 5 pm.
    [7]
    7. Use according to one of claims 1 to 6, characterized in that the recesses have a distance from each other of 0.2 pm to 2.5 pm.
    [8]
    8. Use according to one of claims 1 to 7, characterized in that the solid support comprises at least partially one or more than one above the other arranged metal layers.
    [9]
    9. Use according to one of claims 1 to 8, characterized in that the metal layer on the solid support has a thickness of 10 nm to 200 nm.
    [10]
    10. Use according to one of claims 1 to 9, characterized in that the metal is selected from the group consisting of silver, gold, aluminum, chromium, indium, copper, nickel, palladium, platinum, zinc, tin and alloys comprising one or several of these metals.
    [11]
    11. Use according to claim 10, characterized in that the alloy comprises silver, indium and tin.
    [12]
    12. Use according to any one of claims 1 to 11, characterized in that the solid support comprises at least one material selected from the group consisting of the group of thermoplastic polymers and the polycondensates.
    [13]
    13. Use according to claim 12, characterized in that the thermoplastic polymer is selected from the group consisting of polyolefins, vinyl polymers, styrene polymers, polyacrylates, polyvinylcarbazole, polyacetal and fluoroplastics.
    [14]
    14. Use according to claim 12, characterized in that the polycondensate is selected from the group consisting of thermoplastic polycondensates, thermosetting polycondensates and polyadducts.
    [15]
    15. Use according to one of claims 12 to 14, characterized in that the material of the polymeric solid support comprises organic and / or inorganic additives and / or fillers, these are preferably selected from the group consisting of T1O2, glass, carbon, color pigments , Lipids and waxes.
    [16]
    16. Use according to one of claims 1 to 15, characterized in that the substrate is part of a capillary, a microtiter plate, a microfluidic chip, a test strip, a support for fluorescence microscopy, a sensor array or an optical detector array.
    [17]
    17. Use according to one of claims 1 to 16, characterized in that the metal coating on the surface of the substrate at least partially comprises molecules for the direct and / or indirect binding of fluorescent molecules.
    [18]
    18. Use according to claim 17, characterized in that the molecules for the direct and / or indirect binding of fluorescent molecules are selected from the group consisting of antibodies, antibody fragments, preferably Fab, F (ab) '2 or scFv fragments, nucleic acids, enzymes , Lipids, virus particles, aptamers and combinations thereof.
    [19]
    19. A substrate for enhancing the fluorescence of one or more fluorescent molecules, characterized in that the substrate comprises a solid polymeric support having a plurality of separate recesses and the solid support is at least partially coated with at least one metal.
    [20]
    20. A substrate according to claim 19, characterized in that the recesses of the solid support have a length and a width, wherein the ratio of the length to the width of 2: 1 to 1: 2, in particular 1: 1, is.
    [21]
    21. Substrate according to claim 19 or 20, characterized in that the recesses of the solid support have a length and a width, wherein the length and the width of the recesses is 0.1 pm to 2 pm.
    [22]
    22. Substrate according to one of claims 19 to 21, characterized in that the depressions have a substantially round shape.
    [23]
    23. Substrate according to one of claims 19 to 22, characterized in that the recesses have a depth of 0.1 pm to 5 pm.
    [24]
    24. Substrate according to one of claims 19 to 23, characterized in that the recesses have a distance from each other of 0.2 pm to 2.5 pm.
    [25]
    25. A substrate according to any one of claims 19 to 24, characterized in that the solid support comprises at least partially more than one superposed metal layers.
    [26]
    26. A substrate according to any one of claims 19 to 25, characterized in that the metal layer on the solid support has a thickness of 10 nm to 200 nm.
    [27]
    27. A substrate according to any one of claims 19 to 26, characterized in that the metal is selected from the group consisting of silver, gold, aluminum, chromium, indium, copper, nickel, palladium, platinum, zinc, tin and alloys comprising one or several of these metals.
    [28]
    28. A substrate according to claim 27, characterized in that the alloy comprises silver, indium and tin.
    [29]
    29. Substrate according to one of claims 19 to 28, characterized in that the solid support comprises at least one material selected from the group consisting of the group of thermoplastic polymers and the polycondensates.
    [30]
    30. A substrate according to claim 29, characterized in that the thermoplastic polymer is selected from the group consisting of polyolefins, vinyl polymers, styrene polymers, polyacrylates, polyvinylcarbazole, polyacetal and fluoroplastics.
    [31]
    31. A substrate according to claim 30, characterized in that the polycondensate is selected from the group consisting of thermoplastic polycondensates, thermosetting polycondensates and polyadducts.
    [32]
    32. A substrate according to any one of claims 29 to 31, characterized in that the material of the polymeric solid support comprises organic and / or inorganic additives and / or fillers, wherein these are preferably selected from the group consisting of T1O2, glass, carbon, Color pigments, lipids and waxes.
    [33]
    33. Substrate according to one of claims 19 to 32, characterized in that the metal coating on the surface of the substrate at least partially comprises molecules for direct and / or indirect binding of fluorescent molecules.
    [34]
    34. A substrate according to claim 33, characterized in that the molecules for direct and / or indirect binding of fluorescent molecules are selected from the group consisting of antibodies, antibody fragments, preferably Fab, F (ab) '2 or scFv fragments, nucleic acids, Enzymes, lipids, virus particles, apatamers and combinations thereof.
    [35]
    35. Capillary, chip, preferably microfluidic chip, cuvette, microtiter plate, a support for fluorescence microscopy or optical detector array comprising a substrate according to one of claims 19 to 34.
    [36]
    36. A set comprising at least one microtiter plate, at least one capillary, at least one chip and / or at least one cuvette and / or at least one test strip comprising a substrate according to one of claims 19 to 34 and an analyte-binding molecule labeled with a fluorescent molecule. the analyte-binding molecule labeled with an enzyme and a fluorescent substrate for the enzyme.
    [37]
    37. A method for producing a substrate for enhancing the fluorescence of a fluorophore comprising the step of at least partially coating a solid support as defined in claims 19 to 25 and 29 to 32 defined with at least one metal.
    [38]
    38. The method of claim 37, wherein the at least one metal is deposited on the surface of the solid support by a method selected from the group consisting of sputtering, thermal evaporation, electron beam evaporation, laser beam evaporation, arc vapor deposition, molecular beam epitaxy, ion beam assisted deposition, and ion plating becomes.
    [39]
    39. The method according to claim 37 or 38, characterized in that at least partially molecules for the direct and / or indirect binding of fluorophores are applied to the metal coating on the surface of the substrate.
    [40]
    40. The method according to claim 39, characterized in that the molecules for direct and / or indirect binding of fluorophores are selected from the group consisting of antibodies, antibody fragments, preferably Fab, F (ab) '2 or scFv fragments, nucleic acids, enzymes, Lipids, virus particles and combinations thereof.
    [41]
    41. Method for determining or quantifying at least one analyte in a sample comprising the steps: a) optional direct or indirect labeling of the at least one analyte with at least one fluorophore, b) application of the at least one labeled analyte from step a) or a fluorescent analyte to a substrate according to any one of claims 19 to 34, c) exciting the at least one fluorophore by irradiating the substrate with light of suitable wavelength, and d) measuring the fluorescence to determine the presence or quantification of the at least one analyte in the sample.
    [42]
    42. The method according to claim 41, characterized in that the at least one fluorophore has an excitation wavelength in the range from 360 to 780 nm, preferably from 490 to 680 nm.
    [43]
    43. The method of claim 41 or 42, characterized in that the at least one fluorophore has an emission wavelength in the range of 410 to 800 nm, preferably from 510 to 710 nm.
    [44]
    44. The method according to any one of claims 41 to 43, characterized in that the at least one fluorophore is selected from the group consisting of methoxy coumarin, aminocoumarin, Cy2, Alexa Fluor 488, fluorescein isothiocyanate (FITC), Alexa Fluor 430, Alexa Fluor 532, Cy3, Alexa Fluor 555, 5-TAMRA, Alexa Fluor 546, Phycoerythrin (PE), Tetramethyl Rhodamine Isothiocyanate (TRITC), Cy3.5, Rhodamine, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Cy5, Alexa Fluor 660, Cy5.5, Alexa Fluor 680 and Cy7, preferably selected from the group consisting of fluorescein isothiocyanate (FITC), Cy3, phycoerythrin (PE), tetramethyl rhodamine isothiocyanate (TRITC), Cy5 and Alexa Fluor 680.
    [45]
    45. The method according to any one of claims 41 to 44, characterized in that the indirect labeling of the analyte with at least one fluorophore by means of a fluorophore-labeled and analyte-binding molecule.
    [46]
    46. The method according to claim 45, characterized in that the analyte-binding molecule from the group consisting of antibodies, antibody fragments, preferably Fab, F (ab) '2 or scFv fragments, nucleic acids, enzymes, lipids, virus particles, Aptame ren and Combinations thereof is selected.
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    同族专利:
    公开号 | 公开日
    JP6968056B2|2021-11-17|
    PT3350597T|2021-11-15|
    EP3350597A1|2018-07-25|
    CN108351353B|2021-01-12|
    US11262297B2|2022-03-01|
    HRP20211723T1|2022-02-18|
    CN108351353A|2018-07-31|
    JP2018530754A|2018-10-18|
    WO2017046320A1|2017-03-23|
    EP3350597B1|2021-08-11|
    SI3350597T1|2022-01-31|
    US20180195956A1|2018-07-12|
    CA2998667A1|2017-03-23|
    DK3350597T3|2021-11-15|
    AT517746B1|2018-03-15|
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    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    ATA50793/2015A|AT517746B1|2015-09-16|2015-09-16|substratum|ATA50793/2015A| AT517746B1|2015-09-16|2015-09-16|substratum|
    CA2998667A| CA2998667A1|2015-09-16|2016-09-16|Substrate for the enhancement of fluorescence|
    US15/759,347| US11262297B2|2015-09-16|2016-09-16|Substrate for fluorescence amplification|
    CN201680064304.1A| CN108351353B|2015-09-16|2016-09-16|Substrate for enhanced fluorescence|
    HRP20211723TT| HRP20211723T1|2015-09-16|2016-09-16|Use of substrates for fluorescence amplification|
    EP16766311.1A| EP3350597B1|2015-09-16|2016-09-16|Use of substrates for fluorescence amplification|
    SI201631390T| SI3350597T1|2015-09-16|2016-09-16|Use of substrates for fluorescence amplification|
    PCT/EP2016/071953| WO2017046320A1|2015-09-16|2016-09-16|Substrate for fluorescence amplification|
    DK16766311.1T| DK3350597T3|2015-09-16|2016-09-16|USE OF FLUORESCENE REINFORCEMENT SUBSTRATE|
    JP2018514881A| JP6968056B2|2015-09-16|2016-09-16|Base material for enhancing fluorescence|
    PT167663111T| PT3350597T|2015-09-16|2016-09-16|Substrate for fluorescence amplification|
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