Stock material or miscellaneous articles – Coated or structually defined flake – particle – cell – strand,... – Particulate matter
Reexamination Certificate
2001-04-06
2004-08-10
Le, H Thi (Department: 1773)
Stock material or miscellaneous articles
Coated or structually defined flake, particle, cell, strand,...
Particulate matter
C428S407000, C428S900000
Reexamination Certificate
active
06773812
ABSTRACT:
2. FIELD OF THE INVENTION
This invention relates to magnetically-responsive particles, magnetically-responsive fluorescently-tagged particles, and the fabrication of such particles. The methods described can be used to fabricate particle populations that are distinguishable from each other by fluorescence and/or magnetic response. Multiple particle populations thus constructed will find utility in a number of fields, including clinical biological assays.
3. BACKGROUND OF THE INVENTION
Microspheres have long been used as substrates on which to perform chemical reactions.
Catalysts for chemical modifications, such as hydrogenation or hydroformylation, can be attached to polymer beads to facilitate separation of the catalyst from the reaction products (U.S. Pat. No. 4,506,030, incorporated herein by reference). It is also a well known technique to couple biological molecules to the surface of a microsphere to assay the presence or absence of a reacting species in a biological sample; an example of such an assay would be an antigen-antibody reaction (U.S. Pat. No. 5,948,627, incorporated herein by reference).
This assay system can be improved by conducting the reaction on a microsphere (or other carrier particle) that has been labeled with a fluorescent material. The use of fluorescent labels on or in the microspheres allows preparation of numerous distinguishable sets of microspheres, based on different dye emission spectra or signal intensity. In the case of their use in a biological assay, these particles can then be analyzed on a flow cytometer to classify the size and fluorescence of the particles, as well as the fluorescence associated with the assay system being studied (e.g., a fluorescently labeled antibody in a “capture sandwich” assay)(U.S. Pat. No. 5,948,627, incorporated herein by reference). This concept has been developed even further by incorporating multiple dyes in a particle, and creating distinguishable sets by using varying concentrations of these dyes. In this manner, hundreds, or even thousands, of different microsphere sets can be produced. In an assay, each different microsphere set would be associated with a different target, thus allowing numerous tests to be conducted on a single sample in a single container (U.S. Pat. No. 5,981,180, incorporated herein by reference).
Another method of modifying the particle is to incorporate a magnetically responsive substance, such as Fe
3
O
4
, into the structure. Such particles may be manufactured in a number of ways. For example, by adding the magnetic substance to the reaction vessel prior to initializing the polymerization that forms the particle (U.S. Pat. No. 4,339,337, incorporated herein by reference). In this case the magnetically responsive material is dispersed throughout the polymeric particle. A variation of this concept involves encapsulation of one or more magnetic particles in a hydrophobic polymeric shell (U.S. Pat. No. 5,356,713, incorporated herein by reference). Another method utilizes pH-induced precipitation of metal salts onto and into the pores of a polymeric microsphere (U.S. Pat. No. 4,774,265, incorporated herein by reference). The resulting particle may then be further coated with polymer to fix the magnetic material into the final particle. No attempt has been made to make particles with different magnetic response so that different populations would be discernable.
Magnetic particles such as these have found a number of uses in biomedical research and diagnostics. Antibodies targeted to specific cells can be coupled to magnetic microspheres, then, upon exposure to a biological sample, these cells can be selectively removed and collected by applying a magnetic field, towards which the target cell-microsphere pair will migrate (U.S. Pat. No. 4,230,685, incorporated herein by reference). Similarly, immunoassays may be performed on the surface of magnetic beads, and the magnetic field is applied to immobilize the microparticle during a wash step.
Magnetically-responsive fluorescent particles have also been made (EP 0463144B1). Magnetic particles may be directly dyed by either covalently coupling a dye to the surface of the particle, or by absorbing a hydrophobic dye into the particle. The former option has the disadvantage of the dye being exposed to the surrounding environment, and it is known that varying this solvent environment often results in spectral changes for the dye. When the fluorescent intensity must be tightly controlled, these spectral changes can not be tolerated. Absorption of the dye into the particle allows the dye to be exposed to a consistent environment, thus providing consistent spectral properties, but the dyeing process involves the use of organic solvents, which are often incompatible with the polymer used to form the particle. A polymer such as polystyrene can be used if it also contains a cross-linker, such as divinylbenzene. However, the layered construction of many magnetic particles results in a microsphere, that, upon immersion in organic solvents which leads to a swelling of the polymer, often loses its sphericity or some of its magnetic component. Depending on the desired use, this may or may not be acceptable.
Another approach is to add the magnetic material to a dyed particle. This can be accomplished through the salt precipitation technique described earlier, and, if the dye in the particle is not sensitive to the reaction conditions, results in an adequate particle. It may, however, be necessary to add another layer of polymeric material to the particle in order to immobilize the magnetic component. This is usually accomplished with a free radical initiated polymerization, which often causes dye decomposition.
4. SUMMARY OF THE INVENTION
This invention relates to particles, and methods of making particles, with a desired magnetic response. The term “particle” refers to a core particle, for example a microsphere or bead, associated with at least one magnetic material. The core particle may also, for example, be associated with reactants, fluorescent tags, and other materials known in the art to be useful with core particles. The term “magnetic,” as used hereinafter, includes all types of materials that respond to magnetic fields, such as, but not limited to, ferromagnetic, paramagnetic, and superparamagnetic materials. The term “magnetic material” encompasses any material having at least some magnetic content and therefore includes material having an amount of magnetic material ranging from greater than 0% to 100%. In some embodiments, the magnetic material is a polymeric magnetic material. Polymeric magnetic material includes for example, material in which the magnetic material is mixed with polymeric material and magnetic material which is coated with polymeric material. The term “reactant” encompasses any substance capable of associating with at least one other substance, and which is used to identify or quantitate an analyte in solution. For example, antibodies and antigens can both be reactants. Surface reactive moieties such as amines, thiols, carboxylic acids, hydrazines, halides, alcohols, and aldehydes are also non-limiting examples of reactants. The term “analyte” refers to any substance suspected of being present in a sample.
In one aspect, the present invention provides particles having a desired magnetic response. The phrase “magnetic response” refers to attractive or repulsive forces as a result of the application of a magnetic field. The magnetic response can be, for example, migration rate or retention time in response to a magnetic field. “Desired” implies that the amount and type of magnetic material associated with the core particle are chosen by the skilled artisan to provide a core particle having a magnetic response suitable to achieve the skilled artisan's preferred end result. The migration rate need not be quantitative (e.g. distance/time), but may be qualitative (e.g., faster or slower than another population of particles). So too, retention time may be quantitative or qualitative. The phrase “population of particles” refer
Chandler Don J.
Herren Michael A.
Conley & Rose, P.C.
Huston Charles D.
Le H Thi
Luminex Corporation
Mewherter Ann Marie
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