Stock material or miscellaneous articles – Coated or structually defined flake – particle – cell – strand,... – Particulate matter
Reexamination Certificate
1999-08-02
2002-07-23
Le, Hoa T. (Department: 1773)
Stock material or miscellaneous articles
Coated or structually defined flake, particle, cell, strand,...
Particulate matter
C428S407000, C524S202000
Reexamination Certificate
active
06423410
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a paramagnetic polymer particle and method of making.
More specifically, the present invention relates to an ultrasonically generated composite paramagnetic polymer particle including a plurality of paramagnetic polymer bead aggregates.
BACKGROUND OF THE INVENTION
A typical method for measuring the binding of antigens or ligands to proteins is particle agglutination or precipitation. In such a method a protein bound suspension of beads or particles reacts with some antigen or ligand causing the particles to flocculate or precipitate. Quantitation is accomplished through the use of spectrophotometry to measure the change of light transmission through the medium. Another method which is far more sensitive is the use of radioactivity in the measurement. In such a method, either the ligand or the particle bound protein is radiolabeled and complexed with either unlabeled protein or ligand. The uncomplexed material is then separated from the bound complex by simple filtration and then counted through the process of scintillation. Filtration of insoluble particles incorporating a protein-ligand complex is efficient when large numbers of particles are employed, yet can be difficult when small numbers of particles are used.
In cases where small numbers of particles are used, other insoluble supports are utilized, such as paramagnetic particles. U.S. Pat. No. 5,750,412 discloses exposing antibody-bound paramagnetic particles to a magnetic field in order to separate antibody-bound antigen from unbound antigen in immunoassays. Such materials are attracted to a magnetic field and this attraction is used to separate the bound protein-ligand complex from the surrounding liquid medium. Magnetic separation methods have also been applied successfully in cell sorting. A definite advantage that magnetic separation has over simple filtration is the ability to separate out small numbers of particles from small reaction volumes. Another advantage magnetic separation has over simple filtration is the ease to which one is able to automate the simultaneous washing and separation of hundreds of individual solid phase complexation reactions.
In the pharmaceutical industry, compound libraries for biological testing are routinely synthesized through the use of solid phase organic chemistry. Solid phase organic synthesis allows for the quick separation of products from unreacted starting material as well as reagents and side-products that are not originally bound to the support. The physical separation of the support from the solubilized components of the reaction mixture has primarily been accomplished by filtration through a glass or polymer filter. Although filtration has been the method of choice in solid phase organic synthesis, it has limitations that warrant the development of newer approaches. One such limitation is the difficulty in automating the simultaneous washing and filtration of hundreds of small scale solid-phase reactions.
U.S. Pat. No. 5,684,130 discloses the use of magnetic separation in the field of solid-supported organic chemistry has recently been demonstrated in the synthesis of peptides, non-peptides compounds and in the affinity chromatography of compound mixtures using a paramagnetic support in organic solvent. Clearly, paramagnetic supports have many useful applications, including in biology and chemistry which warrant the development of improved paramagnetic supports and/or better methods for their manufacture.
U.S. Pat. No. 4,358,388 discloses magnetite crystals encapsulated in polystyrene by the suspension polymerization of styrene in the presence of an organosoluble initiator, a suspending agent, magnetite (Fe
3
O
4
) and an emulsifying agent in water. The mixture was homogenized to give organic phase droplets ranging from 0.03-5 microns in diameter. The homogenized mixture was then polymerized to give spherically shaped magnetic-polymer latex particles. The resulting particles consist of a core of polystyrene polymer with magnetite crystals located as inclusions along the outer most periphery of the particles.
U.S. Pat. No. 5,091,206 also discloses a process for producing paramagnetic particles with a uniform spherical surface. Magnetite crystals are coated on the surface of pre-made uniformly spherical polystyrene particles (2-6 microns in diameter) to form a non-covalent composite or “seed”. A vinyl monomer is then polymerized on the surface of the polymer particles-magnetite seeds to produce spherical paramagnetic particles having an average particle diameter of between 4-7 micrometers. The polymerization process involved heating together in a rotating reaction flask small quantities of polymer particles-magnetite seeds, vinyl monomer, water soluble initiator and an anionic surfactant in water. The percent magnetite incorporation of the particles is increased by taking the polymer coated magnetite particles and successively adding more magnetite to them to form new “seeds” which are again polymerized together. This process of successively adding magnetite followed by polymer coating the resulting aggregate layers can enhance the percent magnetite incorporation of the final paramagnetic bead. However, among other disadvantages, in order to achieve relatively high levels of magnetite incorporation repeated polymerization and separation steps are required making this method both very costly and time consuming to perform.
Many variables contribute toward the ultimate size, shape and thickness of polymer coatings. The amount and types of surfactant used, the reaction temperature and the stirring speed and/or the agitation method can all have a direct effect on structural properties. For example, power ultrasound has been reported to enhance the uniformity of polymer coverage around titanium dioxide particles under emulsion polymerization conditions and to aid in the formation of nanometer sized iron colloid particles. See J. P. Lorimer et al, 269 Colloid. Polym. Sci. 393-397 (1991); K. S. Suslick et al, 118 J. Am. Chem. Soc. 11960-61 (1996).
There are many drawbacks to prior art paramagnetic polymer particles and methods of making. Current polymer coated paramagnetic particles are relatively expensive to produce. For example, making polymer coated magnetite particles exhibit high levels of magnetite incorporation is both costly and time consuming. Because magnetite is hydrophilic by nature, it is difficult to stabilize in the hydrophobic environment of a monomer droplet. A number of different and sometimes exotic surfactants, co-surfactants and suspending agents are usually required in order to allow the magnetite to be closely associated with the monomer droplets. U.S. Pat. No. 5,232,782 discloses metal oxides made more hydrophobic by coating them with an organosilating reagent. However making the magnetite more hydrophobic can produce other problems that can be difficult to predict let alone control. For example, due to an increase in the number of hydrophobic components in the polymer reaction mixture, more surfactant is required to stabilize the magnetite incorporated monomer droplet. However, too much surfactant can force the separation of the hydrophobic magnetite and the monomer droplet itself, resulting in a lowering in the magnetite incorporation. Too little surfactant can result in rapid aggregation of the magnetite itself causing the hydrophobic magnetite to fall out of suspension. It can also cause the coalescence of the monomer droplets into larger droplets containing less magnetite. Another problem is the expense and time required to make very small and uniform magnetite crystals having a diameter of less than 1 micron in size. Lastly, it is difficult to make polymer coated paramagnetic particles be stable both in an aqueous as well as an organic solvent environment.
SUMMARY OF THE INVENTION
It is a primary object of the present invention to provide an improved paramagnetic polymer particle.
It is another primary object of the invention to provide a composite paramagnetic particle having high levels of mag
Lee Jun Young
Sucholeiki Irving
Sung Nak-Ho
Le Hoa T.
MDS Proteomics Inc.
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