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  Assay particles and methods of useRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Antigen-antibody Binding, Specific Binding Protein Assay Or Specific Ligand-receptor Binding AssayThe Patent Description & Claims data below is from USPTO Patent Application 20070037215. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] The present application claims the benefit of priority to U.S. Provisional Patent Application Ser. No. 60/707,492, which was filed on Aug. 11, 2005. BACKGROUND [0002] The scintillation proximity assay (SPA) is an approach for assay development and biochemical screening that allows rapid and sensitive measurement of molecular interactions in a homogeneous system, obviating the need for separation and washing steps. In theory, all that is required in the assay is mixing and measurement. The technology has proven useful in radiometric screening since its adoption in about 1992, with hundreds of PubMed literature references citing SPA applications to date. SPA is considered to be convenient, cost effective and safer than radioactive filter binding assays, providing fewer handling steps, no need for filters and scintillation cocktails as well as reduced disposal costs. The signal detection for SPA can be performed using any photomultiplier tube-based scintillation counter or CCD camera imager. SPA has enabled advances in high throughput screening, being both automation-friendly and requiring minimal hands-on involvement. It has been estimated that the assay provides a 30-fold increase in productivity relative to typical filtration assays. [0003] Turning to the technical aspects of SPA, binding reactions can be assayed without the washing or filtration procedures normally used to separate bound from free fractions. Assays are typically performed using radioactive labels that emit electrons with only a short range (about 10 um) in water. When bound close to a solid scintillator surface by the binding reaction the radioactive labels are able to transfer electron energy to the scintillator to produce photons detectable with a scintillation counter. Electrons emitted from labeled molecules not bound close to the surface dissipate their energy in the medium and are not detected. The amount of light (photons) generated is proportional to the amount of radiolabeled molecules bound to the solid scintillant. Thus the bound fraction is detected specifically without separation of the solution from the support. [0004] SPA beads are microscopic beads which contain a scintillant that can be stimulated to emit light. As is indicated above, this stimulation event only occurs when radiolabeled molecules of interest are bound to the surface of the bead, then blue light is emitted that can be detected on standard scintillation counters. Another type of SPA beads, often referred to as SPA imaging beads, emit red light that can be detected on standard CCD cameras. Assay plates coated with scintillant have also been used for SPA methods. [0005] Further applications, formats, materials and procedures for performing SPA and SPA-like technologies are expected to contribute further to high throughput screening capability as well as to advances in bioanalytical and biomedical sciences. BRIEF DESCRIPTION OF THE DRAWINGS [0006] FIG. 1 shows exemplary assay particles encompassed within the technology described herein. The drawings exemplify cross-sections of assay particles having (A) a core and a shell portion; (B) core, shell and coating portions; (C) core, shell, coating and outer layer portions; and (D) a core portion containing an exemplary type of encoding element. [0007] FIG. 2 shows results from experiments in which TiO.sub.2-coated assay particles were used to prepare a sample enriched in phosphorylated molecules. Shown in FIG. 2A are mass spectra of eluted and unbound samples of .alpha.-casein tryptic digest fractionated on TiO.sub.2-coated magnetic beads. Shown in FIG. 2B are mass spectra of eluted, and unbound and control samples of a-casein tryptic digest fractionated on TiO.sub.2-coated glass assay particles as well as the negative control mass spectrum of a-casein tryptic digest eluted from uncoated glass assay particles. [0008] FIG. 3 shows a schematic diagram depicting an exemplary homogenous protein kinase scintillation proximity assay using a tritiated peptide substrate and an air-filled glass assay particle coated with an inorganic phosphor that binds selectively to phosphorylated molecules, according to an embodiment of the technology described herein. [0009] FIG. 4 is a schematic diagram of an exemplary homogenous protein kinase time-resolved fluorescence assay using a cyanine 5 dye-labeled peptide substrate and an air-filled glass assay particle coated with an inorganic phosphor that binds selectively to phosphorylated molecules, according to an embodiment of the technology described herein. SUMMARY [0010] The invention provides assay particles useful, for example, for detecting analytes and molecular interactions. One type of assay particle includes a core portion encased by a shell portion, wherein the shell portion comprises an inorganic phosphor that binds selectively to a target molecule. Another type of an assay particle includes a core portion encased by a shell portion, and a coat portion covering the shell portion, wherein the coat portion comprises an inorganic phosphor that binds selectively to a target molecule. A further type of assay particle includes a core portion encased by a shell portion, and a coat portion covering the shell portion, wherein the coat portion comprises an inorganic phosphor and a target selective binding moiety, and wherein the assay particle is buoyant in aqueous media. An additional type of assay particle includes a core portion encased by a shell portion, and a coat portion covering the shell portion, wherein the shell portion comprises an inorganic phosphor and the coat portion comprises a target selective binding moiety, and wherein the assay particle is buoyant in aqueous media. In an embodiment, the core portion of an assay particle can include a material selected from the group of a gas, a liquid, a solid and a mixture thereof, such as a material selected from the group of air, organic solvent and organic polymer. In an embodiment, an assay particle can be buoyant in aqueous media. In an embodiment, the target molecule is a phosphorylated molecule. inorganic phosphor is selected from the group of rare-earth ion-doped yttrium oxide, rare-earth ion-doped zirconium oxide, rare-earth ion-doped yttrium oxysulfide and rare-earth ion-doped yttrium aluminum garnet. The rare-earth dopant can be selected from, for example, the group of terbium (III), europium (III), dysprosium (III), samarium (III), ruthenium (II), rhenium (I) and a combination thereof. In an embodiment, the assay particle has a density of less than 1 g/cm3. 1. In some embodiments, the target selective binding agent can be, for example, selected from the group of antibody, aptamer, protein A, protein G, streptavidin, avidin, captavidin, neutravidin, metal chelate, siderophore, lectin, and an oligonucleotide. [0011] The invention provides methods for detecting a target molecule using an assay particle described herein. In one aspect, a method involves contacting a sample suspected of containing the target molecule with an assay particle, wherein (i) the target molecule comprises a moiety capable of emitting radiation, and (ii) the assay particle comprises a shell portion comprising an inorganic phosphor capable of binding selectively to target molecules; wherein binding of the target molecule to the inorganic phosphor produces a light signal, whereby the target molecule is detected. DETAILED DESCRIPTION [0012] The technology described herein provides assay particles and related methods and kits for detecting analytes and performing protein interaction and enzyme assays, such as protease, kinase, phosphatase, receptor binding, and molecular interaction assays, in scintillation and luminescence proximity assay formats, as well as fluorescence assay formats. [0013] Assay beads have become an important tool for performing high throughput assays in biomedical research and drug development. Various commercial SPA methods involve using plastic beads (for example, polystyrene, polyvinyltoluene or polyethyleneimine) containing an organic scintillant, such as 2,4-diphenyloxazole (PPO) or anthracene. Other SPA methods use beads made from the inorganic scintillators yttrium silicate or yttrium oxide. Various inositol phosphates and phosphorylated lipids, most particularly sphingosine phosphate, have previously been detected and quantified by SPA using commercially available solid yttrium silicate and solid yttrium oxide particles (Brandish et al, 2003; 2004; Noremant et al, 2002). [0014] The technology provided herein includes assay particles that can be used in proximity assays of various types, including SPA. One type of assay particle provided herein has a shell portion, which is made from an inorganic phosphor material, and which encases a core portion. The particular inorganic phosphor material used for this assay particle has the ability to bind to target molecules. As an example, the inorganic phosphor material can be a hydrated metal oxide, such as TiO.sub.2, which can bind selectively to phosphorylated molecules. Exemplary constructions of such assay particles include TiO.sub.2 formed into hollow microspheres (the shell) surrounding a core of air or another gas, and TiO.sub.2 layered as a shell onto a polymer core. In either case, the resulting assay particle can have a low density relative to the assay solution, if desired. Another type of assay particle provided herein also has a shell and core, but further has a coating portion. The coating portion in this case contains an inorganic phosphor that has the ability to bind to target molecules. Carrying on with the TiO.sub.2 example, an exemplary construction of an assay particle having a coating portion include a hollow microsphere (the shell and core) coated with TiO.sub.2. A further type of assay particle provided herein has shell, core and coat portions, with either or both the shell and coat portions containing an inorganic phosphor and a target selective binding agent. This type of particle is composed of materials that allow it to be buoyant in aqueous media. Each of the assay particles can be relatively lightweight in comparison to typical metal oxide SPA beads, such as those referenced above, which are solid, and can be buoyant in aqueous media. [0015] Therefore, the technology provides an assay particle, comprising a core portion encased by a shell portion, wherein the shell portion comprises an inorganic phosphor that binds selectively to a target molecule. In another embodiment, an assay particle of the invention includes a core portion encased by a shell portion, and a coat portion covering the shell portion, wherein the coat portion comprises an inorganic phosphor that binds selectively to a target molecule. In a further embodiment, an assay particle of the invention includes a core portion encased by a shell portion, and a coat portion covering the shell portion, wherein the coat portion comprises an inorganic phosphor and a target selective binding moiety, and wherein the assay particle is buoyant in aqueous media. In yet another embodiment, an assay particle of the invention includes a core portion encased by a shell portion, and a coat portion covering the shell portion, wherein the shell portion comprises and inorganic phosphor and the coat portion comprises a target selective binding moiety, and wherein the assay particle is buoyant in aqueous media. The invention provides assay particles useful, for example, for detecting analytes and binding molecule interactions. One type of assay particle includes a core portion encased by a shell portion, wherein the shell portion comprises an inorganic phosphor that binds selectively to a target molecule. Another type of an assay particle includes a core portion encased by a shell portion, and a coat portion covering the shell portion, wherein the coat portion comprises an inorganic phosphor that binds selectively to a target molecule. A further type of assay particle includes a core portion encased by a shell portion, and a coat portion covering the shell portion, wherein the coat portion comprises an inorganic phosphor and a target selective binding moiety, and wherein the assay particle is buoyant in aqueous media. An additional type of assay particle includes a core portion encased by a shell portion, and a coat portion covering the shell portion, wherein the shell portion comprises an inorganic phosphor and the coat portion comprises a target selective binding moiety, and wherein the assay particle is buoyant in aqueous media. In an embodiment, the core portion of an assay particle can include a material selected from the group of a gas, a liquid, a solid and a mixture thereof, such as a material selected from the group of air, organic solvent and organic polymer. In an embodiment, an assay particle can be buoyant in aqueous media. In an embodiment, the target molecule is a phosphorylated molecule. The inorganic phosphor can be selected from the group of rare-earth ion-doped yttrium oxide, rare-earth ion-doped zirconium oxide, rare-earth ion-doped yttrium oxysulfide and rare-earth ion-doped yttrium aluminum garnet. The rare-earth dopant can be selected from, for example, the group of terbium (III), europium (III), dysprosium (III), samarium (III), ruthenium (II), rhenium (I) and a combination thereof. In an embodiment, the assay particle has a density of less than 1 g/cm3. [0016] As used herein, the term "core portion" when used in reference to an assay particle of the invention means the innermost part of the particle, which is surrounded by a shell. The core portion can be composed of any substance containable within the shell portion of the assay particle, including one or more of a gas, solid, matrix, gel, colloid and liquid, and mixtures thereof. The composition of the core portion can be selected to impart certain physical properties to the assay particle, such as a particular magnetic, density or buoyancy property. Exemplary gases suitable for a core portion of an assay particle include air, nitrogen and oxygen. Exemplary liquids suitable for a core portion of an assay particle include oils, organic solvents, aqueous solutions and mixtures thereof. Exemplary solids and matrices, suitable for a core portion of an assay particle include organic materials such as cellulose, polyethyleneimine, dextran, agarose, polyacrylamide, polyvinyltoluene, Trisacryl, hydroxyalkyl methacrylate, poly(vinylacetate-co-ethylene), oxirane acrylate, polyethylene, polypropylene, poly (vinyl chloride), poly (methyl methacrylate), phenol resin, poly (vinylidene difluoride), poly (ethylene terephthalate), polyvinylpyrrolidone, polycarbonate and starch, and inorganic materials such as glass, ceramic, metal, glass, alumina, silica, zirconia, a ferromagnetic material and a paramagnetic material. [0017] In an embodiment, an assay particle of the invention can be buoyant in an assay medium, typically an aqueous medium. An assay particle can have a low density relative to an assay medium or a density similar to an assay medium, for example, to render the assay particle buoyant in aqueous media. Therefore, a core portion can be selected to have a low density relative to one or both the shell portion and the coat portion (if present) of an assay particle, and also to have a density lower or similar to an assay medium. A core portion can therefore have a density of less than 1 g/cm.sup.3, such as less than 0.5 g/cm.sup.3 and 0.1 g/cm.sup.3, although a more dense core portion can be used, for example, to obtain a buoyancy characteristic in a particular medium, such as a viscous medium. As a specific example of a core portion of an assay particle, described below is preparation of an assay particle having a gas core portion (Example 1). Assay particles having lower density than the aqueous medium, upon standing for a period of time, will float to the surface of the media, allowing their imaging above the assay container, which can reduce background in comparison to imaging particles in solution. In addition, larger particles having inorganic phosphor coatings become feasible to employ in SPA due to reduced settling of particles associated with the high density of inorganic particles or crystals. Larger particles or particles are readily imaged using less sophisticated CCD camera-based optical imaging techniques. [0018] The term "shell portion" when used in reference to an assay particle of the invention means a skin or thickness of material surrounding or enveloping the core portion of the assay particle. The composition of the shell portion generally is selected to be compatible with the core portion of the assay particle. Thus, a relatively non-porous material is useful for the shell when the underlying core is a gas or liquid, whereas a non-porous or porous material can be suitable when the underlying core is, for example, a solid or matrix. The shell portion generally has a thickness of about 1 nm to about 500 nm, such as from about 1 nm to about 200 nm. A thickness can be selected, for example, based on the composition of the core, and to obtain an assay particle having a particular physical characteristic such as weight, strength, durability and the like. Exemplary materials for assay particle shells include glass, ceramic, metal, metal oxides, alumina, silica, and zirconia. Specific examples of shell materials are hollow glass and ceramic microspheres, which typically have relatively high strength to weight ratio. Commercially available glass and ceramic micropheres range in density from 0.16 to 0.7 g/cm.sup.3, depending upon the specific product, with sizes typically ranging from 15 to 200 .mu.m. Several varieties of bubbles and microspheres are available commercially from 3M.TM., including Scotchlite.TM. Glass Bubbles, Scotchlite.TM. Glass Bubbles Floated Series and Z-Light Spheres.TM. Ceramic microspheres. These micropheres range in size from 20 to 60 .mu.m. Procedures for preparing micropheres and assay particles based on microspheres are described herein below. [0019] The term "coat portion," when used in reference to an assay particle means a layer of material that covers the surface of the shell portion of the assay particle. A coat portion generally has a thickness of less than 3000 .mu.m, such as less than 2000 .mu.m and less than 1000 .mu.m, depending on the coating material. The coat portion can be continuously or discontinuously present on the surface of the shell potion of an assay particle. The texture of the coat portion can vary from smooth to coarse, depending on the materials used. For example, the coat portion can have a surface of fine or coarse crystalline material, particulate material, proteinacious material, gel material, organic material and the like. [0020] An assay particle described herein includes an "inorganic phosphor," present in the shell portion or the coat portion. As used herein, the term "phosphor" means a substance that emits light when excited by radiation, such as ultraviolet light, electron bombardment and electrical fields. A phosphor useful for an assay particle described herein is capable of converting radiation from a substance in a sample, such as an analyte or target molecule, into light energy that can be detected using a photomultiplier tube, CCD camera or the like. An inorganic phosphor useful for the assay particles described herein is typically capable of emitting light under conditions of a scintillation proximity assay (SPA). Continue reading... Full patent description for Assay particles and methods of use Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Assay particles and methods of use patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. 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