Compositions and methods for performing magnetibuoyant separations
The methods of the invention employ targeted magnetic particles, preferably targeted nanomagnetic particles, and targeted buoyant particles such as buoyant microparticles and microbubbles. Among the benefits of the invention is the ability to combine targeted magnetic particles with differentially targeted buoyant particles to achieve separation of two or more specifically cell targeted populations during the same work flow.
1. A method of separating two or more target biomolecule species from a biological sample, comprising, wherein steps (b) and (c) are performed in order or performed simultaneously:
(a) in a reaction mixture, contacting a biological sample known or suspected to contain first and second biomolecule species of interest with a targeted magnetic particle species that targets the first biomolecule species of interest to form first target biomolecule/magnetic particle complexes, and contacting the biological sample with a targeted buoyant particle species, that targets the second biomolecule species of interest to form second target biomolecule/buoyant particle complexes;
(b) using a magnetic field to isolate the first biomolecule/magnetic particle complexes from the reaction mixture; and
(c) separating the second target biomolecule/buoyant particle complexes from the reaction mixture using buoyancy/floatation properties.
2. A method according to claim 1 wherein the targeted magnetic particle species is a targeted nanomagnetic particle species that comprises:
(i) a magnetic core particle;
(ii) a glass layer encapsulating the magnetic core particle;
(iii) a protein/polymer composite layer bound to the glass layer; and
(iv) a targeting moiety that targets the first biomolecule species of interest and comprises one member of a bioaffinity ligand pair bound to the protein/polymer composite layer.
3. A method according to claim 2 wherein molecules of the targeted nanomagnetic particle species have a diameter ranging from about 5 nm to about 500 nm.
4. A method according to claim 2 wherein the magnetic core particles of the targeted nanomagnetic particle species comprise magnetite (Fe 3 O 4 ) crystals, optionally wherein the magnetite crystals have a diameter ranging from about 5 nm to about 300 nm.
5. A method according to claim 2 wherein the glass layer of the targeted nanomagnetic particle species is a silane layer formed from organofunctional alkoxysilane molecules, optionally organofunctional alkoxysilane molecules that comprise a couplable end group, optionally a couplable end group selected from the group consisting of an amino, sulphydryl, carboxyl, and hydroxyl end group.
6. A method according to claim 2 wherein the protein/polymer composite layer of the targeted nanomagnetic particle species is covalently bound to the glass layer, optionally wherein the protein/polymer composite layer is comprised of serum albumin, optionally bovine or human serum albumin, dextran or casein and wherein optionally the protein/polymer composite layer is permanently bound by heating the composition from about 450° C. to about 850° C.
7. A method according to claim 2 wherein the targeting moiety of the targeted nanomagnetic particle species is selected from the group consisting of an antibody, an antigen-binding antibody fragment, a recombinant antibody, a cell surface receptor, a ligand-binding extracellular domain of a cell surface receptor, an aptamer, a nucleic acid, avidin, streptavidin, and biotin.
8. A method according to claim 1 wherein the targeted magnetic particles and/or the targeted buoyant particles each independently further comprise a detectable label.
9. A method according to claim 2 wherein the magnetic core particles of the targeted nanomagnetic particle species comprise a ferrous oxide, optionally Fe 3 O 4 or Fe 2 O 3 ; a chromium oxide, optionally CrO 3 ; or a stable metal oxide that comprises a substituted metal ion selected from the group consisting of Mn, Co, Ni, Zn, Gd, and Dy.
10. A method according to claim 1 wherein the targeted buoyant particle species comprises targeted buoyant microparticles, optionally targeted microbubbles, and a targeting moiety selected from the group consisting of an antibody, an antigen-binding antibody fragment, a recombinant antibody, a cell surface receptor, a ligand-binding extracellular domain of a cell surface receptor, an aptamer, a nucleic acid, avidin, streptavidin, and biotin.
11. A method according to claim 1 used to prepare an enriched cell population, wherein the cells of the enriched cell population express the first biomolecule species as a cell-surface antigen.
12. A method according to claim 1 wherein the first biomolecule species is a cell-surface antigen of a cell type useful for cell therapy, optionally human cell therapy.
13. A method of claim 1 , wherein the method further comprises:
(d) removing the magnetic field; and
(e) eluting the first target biomolecule/magnetic particle complexes.
14. A method according to claim 3 wherein molecules of the targeted nanomagnetic particle species have a diameter ranging from about 30 nm to about 300 nm.