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WO2017070570A1 - Compositions texturées, systèmes, et procédés pour une fluorescence améliorée - Google Patents

Compositions texturées, systèmes, et procédés pour une fluorescence améliorée Download PDF

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Publication number
WO2017070570A1
WO2017070570A1 PCT/US2016/058262 US2016058262W WO2017070570A1 WO 2017070570 A1 WO2017070570 A1 WO 2017070570A1 US 2016058262 W US2016058262 W US 2016058262W WO 2017070570 A1 WO2017070570 A1 WO 2017070570A1
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Prior art keywords
microstructures
textured surface
microfeatures
metal nanoparticles
textured
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Alan Gordon Goodyear
Barry Beroth
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Individual
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    • 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
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • 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
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/553—Metal or metal coated

Definitions

  • the present invention relates to substrates for analytical procedures such as diagnostic tests, wherein the substrates feature metal nanoparticles disposed thereon.
  • the substrates are textured surfaces, surfaces with microfeatures (and optionally smaller microstructures in and/or between microfeatures) that increase the surface area of the substrate.
  • the metal nanoparticles may be disposed on the surface of the microfeatures and/or microstructures.
  • biomolecules e.g., "probes”
  • probes e.g., "probes”
  • Each spot on the substrate may contain thousands of identical probes attached to its surface. In many cases, each spot has a different probe and represents a different test. Fluid containing unknown biomolecules (e.g., "targets”) is flooded over the probes so the targets can chemically bind to the probes on each spot. After washing, only strongly bound biomolecules remain.
  • the probes, the targets, or both the probes and targets have been previously labeled by attaching tags (e.g., detection moieties, e.g., fluorescent dyes or the like) for detection purposes.
  • tags e.g., detection moieties, e.g., fluorescent dyes or the like
  • a laser illuminates the substrate, generating colored spots (e.g., signals) from the illuminated tags of bound biomolecules.
  • a scanner measures the emitted light intensity and these results are analyzed by computer software.
  • the total strength of the signal emanating from a spot on the substrate can largely depend upon the amount of target samples binding to the probes present on the spot.
  • the sensitivity and specificity of the test is related to the signal strength. Stronger signals result in superior performance. Weak signals may result in incorrect interpretation of results (a false negative) or detection limits too close to properly tell apart (a false positive or a false negative).
  • substrates or sections of substrates are typically substantially flat, e.g., two-dimensional (2-D).
  • 2-D substrates are not sensitive enough, not specific enough, and not consistent enough for widespread diagnostic tests because they emit insufficient signal.
  • substrates with textured surfaces e.g., surfaces comprising microfeatures that increase the surface area of the substrates.
  • smaller microstructures are disposed in and/or between the microfeatures, wherein the microstructures further increase the surface area of the substrate (see, for example, U.S. Pat. No. 7,195,872 and EP No. 1 ,451 ,584, the disclosures of which are incorporated in their entirety herein by reference).
  • the increase in surface area allows for binding of more biomolecule probes, which in turn can help increase signal intensity.
  • the present invention features substrates with textured surfaces wherein the microfeatures and/or microstructures of the textured surfaces comprise metal nanoparticles (e.g., the metal nanoparticles are attached to the microfeatures and/or microstructures, the metal nanoparticles are contained within the microfeatures and/or microstructures, etc.).
  • the metal nanoparticles can enhance fluorescence of a nearby fluorescent molecule (e.g., fluorescent label (detection moiety) of a biomolecule), thereby increasing the signal intensity for each bound target to probe.
  • U.S. Pat. No. 5,837,552 discloses metal-enhanced analytical procedures wherein a surfaced article includes a substrate surface, metal islands, a spacing/coupling agent layer, and binding biomolecules that bond with biomolecule targets. Spaced apart metal islands are formed on the substrate and have at least some interconnections formed between them. A continuous layer coats the islands and all surfaces between the islands. The continuous layer includes a coupling agent or linker that immobilizes the first binding biomolecules. The first biomolecules bond to the coupling agent and the second binding biomolecules bind to the first binding biomolecules to allow detection of presence or concentration of the binding biomolecules.
  • 5,866,433 discloses a metal-enhanced fluorescence sensor with a biorecognitive layer for measuring the concentration of one or more analytes in a sample. At least one island layer is applied on a sensor substrate. The islands of the island layer are in the form of electrically conductive material, the biorecognitive layer being directly applied on the island layer or bound via a spacer film.
  • an analyte-specific fluorescent compound is provided which may be added to the sample or is provided in the sensor itself.
  • the biorecognitive layer can bind the analyte to be measured directly or by means of analyte-binding molecules, the originally low quantum yield of the fluorescent compound increasing strongly in the vicinity of the island layer.
  • U.S. Pat. No. 5,837,552 specifies a continuous layer including a coupling agent or linker that coats the metal islands and all surfaces between the metal islands.
  • U.S. Pat. No. 5,866,433 specifies a biorecognitive layer being directly applied on the nanometric particles or islands of electrically conductive material or bound to the island layer via a spacer film.
  • a continuous or biorecognitive layer is not used to coat the metal islands or nanometric particles. Instead the metal particles are embedded into or sit on top of the textured surface.
  • U.S. Pat. No. 8,075,956 and U.S. Pat. No. 8,182,878 teach a method for making metal-enhanced fluorescence devices on plastic substrates by modifying a polymeric surface for subsequent deposition of metallic particles, wherein the polymeric surface is modified by increasing hydroxyl and/or amine functional groups thereby providing an activated polymeric surface for deposition of metallic particles onto a substantially smooth surface to form a fluorescence-sensing device.
  • the present invention differs from U.S. Pat. No. 8,075,956 and U.S. Pat. No. 8,182,878 because the present invention uses the textured surface as a means to separate (limit the space between) the metal nanoparticles with respect to the labeled biomolecule (biomolecule with detection moiety), whereas the state of the art had no control over the spacing.
  • the dimensions of the texture and aspect ratio of the texture defines and limits the separation of the nanoparticles.
  • the benefits of the present invention include the increase in surface area allowing for increased probe and metal nanoparticle binding, the ability to space metal nanoparticles using the features of the texture (and allowing the metal nanoparticles to be in close proximity to the fluorescent molecule), and the increase in fluorescence of fluorescent molecules due to the presence of the metal nanoparticles.
  • the interaction of the all the metal nanoparticles contained within any one element (e.g., microfeature and/or microstructure) of the texture with incident electromagnetic radiation results in a further amplified emission from the detection molecule attached to the biomolecule.
  • the critical surface density may be a value (or a range of values) where the majority of the metal nanoparticles are not touching their neighboring metal nanoparticles (though some will touch). Up to some critical surface density, the enhancement effect is expected to increase. Different shapes of nanoparticles may exist, e.g., spheres, spheroids, prisms, rods, wires, the like, combinations thereof, etc. Note that the present invention does not feature a continuous metallic film.
  • the present invention features textured surfaces for analytical tests (e.g., diagnostic assays, e.g., microarrays) wherein the microfeatures and/or microstructures of the textured surface comprise metal nanoparticles (e.g., the metal nanoparticles are attached to the microfeatures and/or microstructures, the metal nanoparticles are contained within the microfeatures and/or microstructures, etc.).
  • the textured surfaces may also comprise labeled biomolecules (e.g., biomolecules labeled with a detection moiety) attached to the microfeatures and/or microstructures of the textured surface.
  • the metal nanoparticles are attached to the surface of the microfeatures and/or microstructures of the textured surface.
  • the metal nanoparticles are embedded within the microfeatures and/or microstructures of the textured surface.
  • the metal nanoparticles When stimulated by electromagnetic radiation, the metal nanoparticles interact with the detection moieties in a manner that allows for increased fluorescence signaling.
  • the microfeatures and microstructures of the textured surface space the metal nanoparticles at particular distances and can help position the metal nanoparticles in particular proximity to the labeled biomolecules.
  • the fluorescence signal intensity is amplified due to the textured surface allowing for an increase in biomolecule binding and metal nanoparticle binding (and metal nanoparticle spacing), as well as the interactions of the metal nanoparticles and fluorescent molecules.
  • the present invention features a textured surface comprising a plurality of metal nanoparticles on or within microstructures of the textured surface.
  • the textured surface may be a feature of a microarray substrate (e.g., the microarray substrate comprises the textured surface), or the textured surface may be independent of a microarray substrate or a feature of a different type of substrate.
  • the microstructures in the textured surface provide an increase in surface area as compared to a surface without microstructures. At least a portion of the microstructures comprise (e.g., display, hold, etc.) metal nanoparticles.
  • the microarray further comprises a biomolecule (e.g., an oligonucleotide, an amino acid, a peptide such as an antibody or fragment thereof, a carbohydrate, a lipid, or a combination thereof) attached to a microstructure of the textured surface.
  • the biomolecule may be bound to the microstructure directly or bound via a linker molecule (e.g. a silane molecule).
  • the biomolecule comprises a detection moiety, e.g., a detection moiety adapted for intrinsic fluorescence, extrinsic fluorescence, chemiluminescence or phosphorescence.
  • the microarray further comprises microfeatures that are larger features than the microstructures.
  • the microfeatures provide an increase in surface area as compared to a surface without microfeatures.
  • the microstructures of the textured surface may be disposed in and/or between the microfeatures.
  • Metal nanoparticles may be further disposed on or contained within microfeatures, and a biomolecule (as described above) may be attached to a microfeature.
  • the textured surface comprises microfeatures and not necessarily microstructures.
  • the textured surface with microfeatures may be a feature of a microarray substrate (e.g., the microarray substrate comprises the textured surface), or the textured surface with microfeatures may be independent of a microarray substrate or a feature of a different type of substrate.
  • the metal nanoparticles are adapted to enhance fluorescence signal intensity of the biomolecule and/or the detection moiety of the biomolecule as compared to fluorescence signal intensity without the metal nanoparticles.
  • the metal nanoparticles are disposed on the microstructures of the textured surface such that the metal nanoparticles are not a continuous layer on the microstructures of the textured surface. The dimensions of the microstructures limit the spacing between adjacent metal nanoparticles.
  • the nanoparticles may be less than 50 nm in their largest dimension.
  • the nanoparticles may be spherical, oblate or prolate spheroids, triangular prisms, rectangular or rod-like shapes, or combinations thereof.
  • the textured surface comprises a polymer material, a glass, a ceramic, a metal, a cellulosic material, or a combination thereof. Attachment of the metal nanoparticles to the textured surface may be according to the methods described herein.
  • the present invention features a method of preparing a textured substrate comprising metal nanoparticles disposed on or within microstructures (and/or microfeatures) of the textured surface.
  • the method comprises exposing a textured substrate (comprising a plurality of microstructures and/or microfeatures) to a source of metallic atoms, wherein the source of metallic atoms deposits metal nanoparticles on or within the microstructures and/or microfeatures of the textured surface.
  • Metal nanoparticles may comprise atoms of silver, gold, platinum, copper, aluminum, zinc, chromium, nickel, iron, tin, the like, or combinations thereof.
  • the method may further comprise depositing biomolecules (e.g., biomolecules with detection moieties) on the microstructures and/or microfeatures.
  • FIG. 1A shows an example of a textured surface comprising microfeatures (1 10).
  • FIG. 1 B shows an example of a textured surface comprising microfeatures (1 10) and smaller microstructures (120) disposed in and between the microfeatures.
  • FIG. 2A shows a well (105) with microstructures (120) disposed on the bottom surface of the well.
  • a well may refer to a well in a microarray substrate or any other appropriate well such as a larger well of a multi-well plate.
  • FIG. 2B shows a well (105) with both microfeatures (1 10) and microstructures (120) disposed on the bottom surface of the well.
  • FIG. 3A shows a detailed view of microstructures (120) (but may also represent microfeatures (1 10)).
  • FIG. 3B shows the microstructures/microfeatures (1 10/120) of FIG. 3A displaying (comprising or holding) metal nanoparticles (210).
  • FIG. 3C shows the microstructures/microfeatures (1 10/120) and metal nanoparticles (210) of FIG. 3B with biomolecules (310) (e.g., oligonucleotides) (with detection moieties (320)) bound to the microstructures/microfeatures (1 10/120).
  • biomolecules e.g., oligonucleotides
  • detection moieties 320
  • a biomolecule may refer to an oligonucleotide (e.g., DNA, RNA), protein or peptide (e.g., antibody, antigen), lipid, carbohydrate or other molecule found in biological entities (e.g., a peptide nucleic acid, a fatty acid, a vitamin, a cofactor, a purine, a pyrimidine, formysin, a phytochrome, a phyyofluor, or phycobiliprotein, etc.) or entire biological entities (e.g., a virus, a phage, a prion a bacteria, etc.).
  • biological entities e.g., a peptide nucleic acid, a fatty acid, a vitamin, a cofactor, a purine, a pyrimidine, formysin, a phytochrome, a phyyofluor, or phycobiliprotein, etc.
  • biological entities e.g., a peptid
  • a substrate may refer to a material on which processing is conducted to produce new film or layers of material such as deposited coatings or deposited molecules (e.g., probes, particles, etc.). These substrates exist in a variety of formats, including microarrays, micro-well plates, slides, and test strips. Microarrays may be based on the laboratory microscope slide. Micro-well plates (sometimes called multi-well plates or microtiter plates or microplates) usually comprise a series of spatially discrete wells (usually 96 wells, 384 wells but sometimes lower or higher) contained within plate-like geometry. A test strip is a strip of material used in tests.
  • a microarray is a general term for a typically smooth, flat surface of a substrate wherein a plurality of distinct and separate locations upon the surface of the substrate is established at the start of the analysis by populating each of the locations with biomolecules (probes) of a known composition.
  • the probes are fixed to their unique locations on the flat substrate by an attachment layer that prevents detachment of the probes from the substrate.
  • the entire microarray is flooded with biomolecules of unknown composition (targets).
  • targets biomolecules of unknown composition
  • a microarray may feature a plurality of small wells (e.g., as described in Examples 1 -5 below), e.g., wells that are microns in width (e.g., 10 microns, 15 microns, 20 microns, 30 microns, etc.). These are distinct from the wells in a multi-well plate.
  • a textured surface or a textured substrate refers to a surface with microfeatures and/or microstructures (described below) that increase the surface area as compared to a surface without the microfeatures and/or microstructures, e.g., a flat, non-textured surface.
  • a textured surface may also have smaller microstructures in the microfeatures (e.g., on the sides of, on top of, both on the sides or on top of, etc.) and/or between the microfeatures.
  • the microstructures further increase the surface area of the surface (see FIG. 1A, FIG. 1 B, FIG. 2A, FIG. 2B, FIG. 3A).
  • a textured surface may be part of a microarray substrate.
  • a textured surface is advantageous because the textured surface has a substantial increase in surface area as compared to a flat, non-textured surface.
  • the three-dimensional nature of the textured surface can take many forms.
  • the microfeatures may exhibit large values of height to width (aspect ratio).
  • the high aspect ratio may be an indicator to performance of the structured surface, e.g., the higher the aspect ratio, the more area that is made available.
  • microfeatures and/or microstructures may comprise a pit, a trench, a pillar, a cone, a wall, a micro-rod, a tube, a channel, the like, or a combination thereof.
  • the present invention is not limited to the patterning described therein. In some examples, the microstructures may comprise even smaller features.
  • the present invention features a textured surface (e.g., a slide or a microarray substrate comprising a textured surface), wherein the textured surface comprises a plurality of microfeatures and/or smaller microstructures that comprise metal nanoparticles (e.g., metal nanoparticles are disposed on the microfeatures and/or microstructures, the metal nanoparticles are contained within the microfeatures and/or microstructures, the metal nanoparticles are embedded in the microfeatures and/or microstructures, etc.).
  • the microfeatures provide an increase in surface area (of the textured surface) as compared to a surface without microfeatures.
  • the smaller microstructures may be disposed in at least a portion of the microfeatures.
  • the smaller microstructures may be disposed between at least a portion of the microfeatures.
  • the microstructures also provide an increase in surface area, e.g., an increase in surface area of the microfeatures, and an increase in surface area of the textured surface as compared a surface without microstructures.
  • At least a portion of the microfeatures and microstructures display (comprise) nanoparticles adapted to bind a biomolecule.
  • the metal nanoparticles are attached to the surface of the microfeatures and/or microstructures.
  • the metal nanoparticles are contained within the microfeatures and/or microstructures.
  • the microfeatures may be distributed uniformly or randomly on the textured surface.
  • the microfeatures may have a height from 0.1 ⁇ to 100 ⁇ , e.g., 100 ⁇ , 90 ⁇ , 80 ⁇ , 70 ⁇ , 60 ⁇ , 50 ⁇ , 40 ⁇ , 30 ⁇ , 20 ⁇ , 10 ⁇ , 1 ⁇ , 0.5 ⁇ , 0.1 ⁇ , etc.
  • the microfeatures have a cross section (or an average cross section) from 0.01 ⁇ 2 to 500 ⁇ 2 .
  • the microfeatures may be at least 1 micron apart. In some embodiments, the microfeatures may be at least 5 microns apart. In some embodiments, the microfeatures are at least 10 microns apart.
  • the microfeatures are at least 15 microns apart. In some embodiments, the microfeatures are at least 20 microns apart. In some embodiments, the microfeatures are at least 50 microns apart. In some embodiments, the microfeatures are at least 100 microns apart. In some embodiments, the microfeatures are less than 500 microns apart. In some embodiments, the microfeatures are less than 100 microns apart. In some embodiments, the microfeatures are less than 50 microns apart. In some embodiments, the microfeatures are less than 20 microns apart. In some embodiments, the microfeatures are less than 10 microns apart. In some embodiments, the microfeatures are less than 5 microns apart.
  • the microfeatures may exhibit large values of aspect ratio (height to width). Also, the aspect ratios of the various microfeatures may differ. In some embodiments, the microfeatures have an average aspect ratio of less than 25 (height divided by an average cross sectional width), or the microfeatures may each have an aspect ratio of less than 25. In some embodiments, the microfeatures have an average aspect ratio of less than 10 (height divided by an average cross sectional width), or the microfeatures may each have an aspect ratio of less than 10. In some embodiments, the microfeatures have an average aspect ratio of less than 5 (height divided by an average cross sectional width), or the microfeatures may each have an aspect ratio of less than 5. In some embodiments, the microfeatures have an average aspect ratio of less than 1 (height divided by an average cross sectional width), or the microfeatures may each have an aspect ratio of less than 1. The present invention is not limited to the aforementioned aspect ratio values.
  • the present invention is not limited to textured surfaces with microfeatures: in some embodiments the textured surface comprises only the smaller microstructures (e.g., see FIG. 2A). In some embodiments, the microstructures may be disposed in the microfeatures and/or between the microfeatures (e.g., see FIG. 1 B, FIG. 2B).
  • the microstructures may have a height less than 5 ⁇ , e.g., 5 ⁇ , 4 ⁇ , 3 ⁇ , 2 ⁇ , etc. In some embodiments, the microstructures have a height less than 1 ⁇ . In some embodiments, the microstructures have a height from 5 ⁇ to 0.1 ⁇ . In some embodiments, the microstructures have an average aspect ratio of less than 25 (height divided by an average cross sectional width), or the microstructures may each have an aspect ratio of less than 25. In some embodiments, the microstructures have an average aspect ratio of less than 10 (height divided by an average cross sectional width), or the microstructures may each have an aspect ratio of less than 10.
  • the microstructures have an average aspect ratio of less than 5 (height divided by an average cross sectional width), or the microstructures may each have an aspect ratio of less than 5. In some embodiments, the microstructures have an average aspect ratio of less than 1 (height divided by an average cross sectional width), or the microstructures may each have an aspect ratio of less than 1.
  • the present invention is not limited to the aforementioned aspect ratio values.
  • the microstructures may be less than 500 nm apart. In some embodiments, the microstructures are less than 100 nm apart. In some embodiments, the microstructures are less than 50 nm apart. In some embodiments, the microstructures less than 20 nm apart. The microstructures may be at least 20 nm apart. In some embodiments, the microstructures may be at least 50 nm apart. In some embodiments, the microstructures are at least 100 nm apart. In some embodiments, the microstructures are at least 500 nm apart.
  • the presence of the microfeatures may increase the surface area of the textured surface at least 10% as compared to a surface without the microfeatures.
  • the presence of the microfeatures may increase the surface area of the textured surface at least 50% as compared to a surface without the microfeatures.
  • the presence of the microfeatures may increase the surface area of the textured surface at least 100% as compared to a surface without the microfeatures.
  • the presence of the microfeatures may increase the surface area of the textured surface at least 200% as compared to a surface without the microfeatures.
  • the presence of the microstructures may increase the surface area of the textured surface at least 10% as compared to a surface without the microstructures.
  • the presence of the microstructures may increase the surface area of the textured surface at least 50% as compared to a surface without the microstructures.
  • the presence of the microstructures may increase the surface area of the textured surface at least 100% as compared to a surface without the microstructures.
  • the presence of the microstructures may increase the surface area of the textured surface at least 200% as compared to a surface without the microstructures.
  • microfeatures and/or microstructures do not reduce the surface area of the microfeatures, microstructures, and/or textured surface.
  • the microfeatures may form cavities, wherein microstructures are disposed in the cavities.
  • the cavities may be adapted to retain liquid.
  • the textured surface may be a part of a microarray substrate.
  • the textured surface may be a feature of a spherical surface, a rod surface, a flexible film surface, a foil surface, or other appropriate surface.
  • the textured surface is a part of a slide, a plate, a well (e.g., a well of a multi-well plate, a well of a microarray substrate), or any other appropriate surface for attaching biomolecules.
  • the textured surface may cover a portion of the microarray substrate (or other surface on which the texture surface lies). In some embodiments, the textured surface covers all of the microarray substrate (or other surface on which the texture surface lies).
  • the textured surface may be constructed from a material comprising a polymer material, a glass, a ceramic, a metal, the like, or a combination thereof.
  • Polymers may include but are not limited to cyclic olefin copolymer (COC), polyethylene terephthalate (PET), polymethyl methacrylate (PMMA), polystyrene, the like, or a combination thereof.
  • the present invention also features methods for producing the textured substrates with metal nanoparticles.
  • the metal nanoparticles may be attached to the microfeatures and/or microstructures.
  • the metal nanoparticles are contained within the microfeatures and/or microstructures, e.g., not necessarily attached to the surface but within the microfeatures and/or microstructures.
  • the metal nanoparticles may have average sizes of less than 30 nm, less than 20 nm, less than 10 nm, less than 5 nm, from 5 to 10 nm, from 5 to 20 nm, from 5 to 30 nm, from 10 to 20 nm, from 0 to 30 nm, etc.
  • the methods of the present invention feature attaching metal nanoparticles to the microfeatures and/or microstructures of the textured surface (or embedding metal nanoparticles in the microfeatures and/or microstructures).
  • the metal nanoparticles are not attached to (or embedded in) the microfeatures and/or microstructures of the textured surface in the form of a continuous layer of metal nanoparticles; instead, the attachment/embedding is in the form of a layer that is not continuous (e.g., in the form of a layer that is nearly but not completely a continuous layer).
  • Metallic nanoparticles can be produced by a number of methods (e.g., coating as described in Examples 1 -5 below). Many methods, e.g., methods involving wet chemistry, are well studied and available in the literature. These methods usually result in a suspension of the metal nanoparticle in some solvent. Depositing these metal nanoparticles from the suspension is difficult and usually driven by diffusion.
  • An alternative method involves making the metal nanoparticles as a vapor (e.g., in a vacuum chamber). Two well-known methods are sputtering and metal vapor evaporation (from a ceramic crucible or electrically heated ceramic boat). In both cases, a metal atom deposited on a surface from the vapor is quite energetic and moves rapidly around the surface.
  • the atoms tend to nucleate (e.g., the metal atoms adhere to each other and slowly aggregate in a small cluster of metallic atoms).
  • these clusters are quasi-crystalline in nature. The clusters continue to grow as more atoms adhere to the exterior. Often the clusters take specific shapes; spheres, prisms, triangular and rod-like shapes have been observed. All of these possibilities are incorporated in this invention and are referred herein after as metal nanoparticles.
  • the present invention may incorporate the metal nanoparticles into microfeatures and/or microstructures by attaching the metal nanoparticles on the surfaces of the microfeatures and/or microstructures or by containing the metal nanoparticles within the microfeatures and/or microstructures (e.g., not necessarily attaching to the surface).
  • the metal nanoparticles may be embedded in the microfeatures and/or microstructures (e.g., in some embodiments, the metal nanoparticles are embedded in the polymer or material of the textured surface.)
  • a suitable solvent within the microfeatures and/or microstructures may allow the metal nanoparticles to become free from the walls of the microfeatures and/or microstructures.
  • the present invention allows for more than one metal nanoparticle being attached/adhered to or being contained in a microfeatures and/or microstructure.
  • the textured surface has a plurality of metal nanoparticles inside substantially all of the microfeatures and/or microstructures of the textured surface.
  • the size of the metal nanoparticles may be a small fraction of the width of the microfeatures and/or microstructures of the textured surface.
  • Metal nanoparticles may be derived from various metals or combinations of metals, for example silver, gold, platinum, copper, aluminum, zinc, chromium, nickel, iron, tin, the like, or combinations thereof.
  • the metals may be selected from a group of materials known to enhance the emission of electromagnetic radiation from the detection moiety.
  • the present invention is not limited to the aforementioned examples.
  • atoms of different metals are present in the microfeatures and/or microstructures of the textured surface, there may be several outcomes: (a) the metal atoms may mix freely to form an alloy composition whereby the atoms of the different metals are freely disbursed with the nanoparticles; (b) the metal atoms do not freely intermix and in fact form individual nanoclusters - each microfeature and/or microstructure of the textured surface may contain several different nanoclusters; and/or (c) some microfeatures and/or microstructures of the textured surface may contain a particular proportion of atoms of more than one metal while other microfeatures and/or microstructures may contain a different proportion atoms of other metals.
  • biomolecules are attached to the microfeatures and/or microstructures of the textured surface.
  • Biomolecules may include but are not limited to oligonucleotides, peptides/proteins or amino acids (e.g., antibodies, fragments thereof, etc.), carbohydrates, lipids, etc.
  • the biomolecules may comprise a detection moiety, e.g., a fluorescent dye or other fluorescent molecule.
  • the present invention is not limited to a fluorescent dye or molecule; in some embodiments, the moiety is a luminescence moiety, a phosphorescence moiety, etc.
  • the attachment of the detection moiety may be at the end of the biological molecule, e.g., an end of the biomolecule that is not attached to the substrate.
  • the purpose of the detection moiety is to react to incoming electromagnetic radiation by absorbing a photon and transitioning to an excited state. After a certain time, the detection moiety is able to de-excite at a slightly different energy level and thus emit electromagnetic radiation at a different frequency. This phenomenon is referred to as fluorescence. Other forms of non-radiative de-excitation exist.
  • Some biological molecules are capable of self-fluorescence.
  • the present invention may feature stimulating the intensity of this self-fluorescence by the interaction with the metal nanoparticles, wherein a detection moiety may not be necessary.
  • the binding of the biological molecules to the substrate can be achieved by several means, e.g., covalently, electrostatically etc. Attachment methods are well known to those skilled in that art.
  • the shape and size of the microfeatures and/or microstructures of the textured surface can help ensure that the metal nanoparticles are in close proximity to the detection moieties of the biomolecules (e.g., the dimension of the microfeatures and/or microstructures can help specify and limit the distance between detection moiety and metal nanoparticle.)
  • the detection moiety of the biomolecule is generally greater than some minimum distance from the metal nanoparticle within the microfeatures and/or microstructures of the textured surface. If the spacing is less than this minimum distance, an effect known as quenching suppresses the emission of electromagnetic radiation. On the other hand, if the detection moiety is too far from the metal nanoparticles, the emission of electromagnetic radiation is diminished. Thus, there is a range of distances that optimize the emissions. This effect may define upper and lower limits on the diameter of the microfeatures and/or microstructures of the textured surface.
  • the labeled biomolecule e.g., biomolecule with detection moiety
  • the labeled biomolecule is located between two adjacent metal nanoparticles (e.g., separated by some distance). Under these circumstances, the amplification of the emitted electromagnetic radiation may be markedly enhanced.
  • the present invention enhances the intensity of the electromagnetic emissions from the label (e.g., detection moiety) of the biological molecule through a combination of: the increase of the topological surface density (e.g., increased probe binding and increased metal nanoparticle binding due to the textured surface), spacing of metal nanoparticles, and the interaction of the detection moiety with the metal nanoparticles.
  • Examples 1 -5 describe non-limiting examples of measuring signal intensity using textured surfaces of the present invention.
  • the result of the interaction between the detection moiety (label) on the biological molecule and the nearby metal nanoparticle is that the intensity of the emissions is increased (e.g., when contrasted with emissions from a similar coated smooth substrate).
  • the proximity of the metal nanoparticle to the detection moiety acts to reduce the residence time the detection moiety spends in its excited state. For example, when the nanoparticle is within a certain distance to the detection moiety, the detection moiety de- excites at a faster rate. Once de-excited, the detection moiety is available for further excitation. Overall result is an increase in the number of photons emitted per unit time. This in turn translates to a higher level of signal into the detector. This manifests itself as increased sensitivity to the diagnostic test.
  • the interaction between the metal nanoparticle and the detection moiety may be based solely on the location of both entities within the microfeatures and/or microstructures of the textured surface.
  • each microfeatures and/or microstructures of the textured surface will contain a multiple of metal nanoparticles and in this case the interaction between the nanoparticles further enhances the emissions from the detection moiety.
  • this is related to mechanisms explained by quantum cavity electrodynamics and is considered distinct from the mechanisms associated with metal enhanced fluorescence.
  • This invention applies to any surface upon which a diagnostic test is to be performed that involves the binding of a biological molecule.
  • Such surfaces include but are not limited to microarrays (made of glass, polymer, cellulosic or metal materials), slides, multi-well plates (of all sizes and number of wells), etc.
  • the invention also is applicable when applied to surfaces within microfluidic diagnostic tests.
  • microfeatures and/or microstructures of the textured surface are closed at their lowest points within the substrate and this feature distinguishes these elements from porous structures often used to achieve an increase in surface area.
  • internal features e.g., the pores
  • Example 1 describes the preparation of a glass microscope slide according to the present invention.
  • the present invention is not limited to the methods and compositions herein.
  • a glass microscope slide is anisotropically etched using Reactive Ion Etching using a fluorine-based etchant. The depth of the etch is approximately 1 micron. After cleaning with acids, the surface is repeatedly washed in de-ionized water followed by an extended immersion on reagent grade ethanol. After removing excess ethanol, the slide is placed in a vacuum oven (at 200C and 1 milltorr) for several hours. Half of the slide surface is masked using metal foil. The remaining surface is flash coated with a thin coat of silver. The silver coating is approximately 5nm. The protective foil is removed. The two surfaces are returned to the vacuum oven where they are coated with an amino-silane.
  • the amino silane is evaporated from a petri dish with the oven set to about 60C and a vacuum pressure of 1 millitorr.
  • the slide is removed from the oven and allowed to cool.
  • the surface is loosely covered to prevent atmospheric contaminants from falling onto the activated surface.
  • the slide is printed with a Cy3 dye-labeled 20mer oligomer in a regular pattern in both the metal-coated section of the slide and the uncoated section of the slide. Several concentrations are used.
  • the printed slide is hybridized with a complementary oligomer that has a Cy 5 dye labeled attached. After washing and drying, the slide is scanned using a GenePix scanner and the relative intensities of the Cy 5 are measured.
  • the silver coated section of the slide displayed intensities that ranged from 3x to 50x when compared to the side not coated with metal.
  • Example 2 describes the preparation of a polymer film according to the present invention.
  • the present invention is not limited to the methods and compositions herein.
  • a polymer film is laminated to a rigid sheet.
  • the surface of the polymer film is nano-embossed with a suitable tool featuring a nano-scale texture.
  • the tool impresses the texture into a thin liquid coating of a UV-curable acrylate.
  • the textured film is cured by exposure to UV.
  • the tool is removed from the surface.
  • the textured surface is cleaned repeatedly with easily evaporable solvents (such as 99.9% ethanol) and finally dried in a vacuum oven for at least 1 hour at about 1 millitorr and 60C.
  • Half of the textured surface is coated with silver as described in Example 1.
  • the treated slide is subjected to the printing and hybridization process also described in Example 1 .
  • a similar improvement in intensity was observed with the textured surface when compared to the non-textured surface.
  • Example 3 describes the preparation of a molded plastic slide according to the present invention.
  • the present invention is not limited to the methods and compositions herein.
  • a molded plastic slide comprised of wells approximately 20 microns square covering the surface of the substrate.
  • the wells comprise a textured surface (e.g., microfeatures) covering the bottom of the each well.
  • the textured surface is coated with silver nanoparticles by flash evaporation of silver in a high vacuum chamber.
  • the silver atoms are deposited on the textured surface and ripen into nanoparticles in the elements (microstructures) of the texture. In this case, the duration of the coating step is minimized since too much silver will result in the formation of a continuous coat.
  • the treated slide is subjected to the printing and hybridization process also described in Example 1. A similar improvement is intensity was observed.
  • Example 4 describes the preparation of a polymer film according to the present invention.
  • the present invention is not limited to the methods and compositions herein.
  • a polymer film is laminated to a rigid sheet.
  • the surface of the polymer film is nano-embossed with a suitable tool featuring a nano-scale texture.
  • the tool delivers the texture into a thin coating of a UV-curable acrylate.
  • the textured film is cured by exposure to UV.
  • the tool is removed from the surface.
  • the textured surface is cleaned repeatedly with suitable solvents and finally dried in a vacuum oven.
  • Half of the textured surface is coated with silver nanoparticles.
  • Two methods were used to deliver the silver nanoparticles to the elements (e.g., microstructures) of the textured surface.
  • the first method involved a chemical synthesis of silver nanoparticles.
  • the textured slide is immersed in the suspension of silver nanoparticles.
  • Example 5 describes preparation of polymer films according to the present invention.
  • the present invention is not limited to the methods and compositions herein.
  • Two polymer films are laminated to a both sides of a plate wherein the polymer films are in tension during the lamination process.
  • the overall thickness is set to 1 mm in order to be compatible with current printing and array readers. This configuration results in an enhanced stiffness when compared with a single plate of the same thickness and composition.
  • the exterior faces of the polymer films will become the textured coated surface described in this invention.
  • the film face can be textured prior to lamination. Alternatively, the texture can be applied to the laminate. Both film faces may be textured.
  • the process by which metal and labeled moieties (detection moieties) are deposited are similar to that described in other examples. Similar improvements in signal intensity were observed from the nanotextured surface when compared to the non-textured surface.
  • references to the inventions described herein using the phrase “comprising” includes embodiments that could be described as “consisting of, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase “consisting of is met.

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Abstract

La présente invention concerne des surfaces texturées pour tests analytiques tels que les microréseaux, les microcaractéristiques et microstructures de la surface texturée comprenant des nanoparticules métalliques, les biomolécules marquées avec les fractions de détection pouvant en outre être fixées aux microcaractéristiques et microstructures. Lorsque stimulées par rayonnement électromagnétique, les nanoparticules métalliques interagissent avec les fractions de détection d'une manière qui permet une signalisation accrue de fluorescence. Les microcaractéristiques et microstructures de la surface texturée espacent les nanoparticules sous certaines distances et peuvent aider à placer les nanoparticules à proximité des biomolécules marquées. L'intensité du signal de fluorescence est amplifiée due à la surface texturée permettant une augmentation de la liaison des biomolécules et de la liaison des nanoparticules métalliques, ainsi que les interactions des nanoparticules métalliques et des molécules fluorescentes.
PCT/US2016/058262 2015-10-22 2016-10-21 Compositions texturées, systèmes, et procédés pour une fluorescence améliorée Ceased WO2017070570A1 (fr)

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JP2022529148A (ja) * 2019-04-12 2022-06-17 ストライカー・コーポレイション 医療処置中に廃棄物を収集するための廃棄物収集カート

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US20030059820A1 (en) * 1997-11-26 2003-03-27 Tuan Vo-Dinh SERS diagnostic platforms, methods and systems microarrays, biosensors and biochips
US20030115987A1 (en) * 2001-12-20 2003-06-26 Aveka, Inc. Process for the manufacture of metal nanoparticle
US20030148401A1 (en) * 2001-11-09 2003-08-07 Anoop Agrawal High surface area substrates for microarrays and methods to make same
US20100285972A1 (en) * 2003-05-05 2010-11-11 Nanosys, Inc. Nanofiber surfaces for use in enhanced surface area applications
US20130123118A1 (en) * 2010-04-30 2013-05-16 Pharmaseq, Inc. Metal Nanoparticle Structures For Enhancing Fluorescence-Based Arrays

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Publication number Priority date Publication date Assignee Title
US20030059820A1 (en) * 1997-11-26 2003-03-27 Tuan Vo-Dinh SERS diagnostic platforms, methods and systems microarrays, biosensors and biochips
US20030148401A1 (en) * 2001-11-09 2003-08-07 Anoop Agrawal High surface area substrates for microarrays and methods to make same
US20030115987A1 (en) * 2001-12-20 2003-06-26 Aveka, Inc. Process for the manufacture of metal nanoparticle
US20100285972A1 (en) * 2003-05-05 2010-11-11 Nanosys, Inc. Nanofiber surfaces for use in enhanced surface area applications
US20130123118A1 (en) * 2010-04-30 2013-05-16 Pharmaseq, Inc. Metal Nanoparticle Structures For Enhancing Fluorescence-Based Arrays

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2022529148A (ja) * 2019-04-12 2022-06-17 ストライカー・コーポレイション 医療処置中に廃棄物を収集するための廃棄物収集カート
JP7510953B2 (ja) 2019-04-12 2024-07-04 ストライカー・コーポレイション 医療処置中に廃棄物を収集するための廃棄物収集カート

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