WO2017035419A1 - Composites destinés à la séparation de cellules rares - Google Patents

Composites destinés à la séparation de cellules rares Download PDF

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Publication number
WO2017035419A1
WO2017035419A1 PCT/US2016/048801 US2016048801W WO2017035419A1 WO 2017035419 A1 WO2017035419 A1 WO 2017035419A1 US 2016048801 W US2016048801 W US 2016048801W WO 2017035419 A1 WO2017035419 A1 WO 2017035419A1
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WIPO (PCT)
Prior art keywords
composite
bead
peg
cells
containing group
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PCT/US2016/048801
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English (en)
Inventor
Aihua Fu
Hua Zhou
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Nvigen, Inc.
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Publication of WO2017035419A1 publication Critical patent/WO2017035419A1/fr

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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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
    • B01J20/0203—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising compounds of metals not provided for in B01J20/04
    • B01J20/0233—Compounds of Cu, Ag, Au
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
    • B01J20/0203—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising compounds of metals not provided for in B01J20/04
    • B01J20/0274—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising compounds of metals not provided for in B01J20/04 characterised by the type of anion
    • B01J20/0285—Sulfides of compounds other than those provided for in B01J20/045
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
    • B01J20/06—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising oxides or hydroxides of metals not provided for in group B01J20/04
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
    • B01J20/10—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate
    • B01J20/103—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate comprising silica
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
    • B01J20/28004—Sorbent size or size distribution, e.g. particle size
    • B01J20/28007—Sorbent size or size distribution, e.g. particle size with size in the range 1-100 nanometers, e.g. nanosized particles, nanofibers, nanotubes, nanowires or the like
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
    • B01J20/28009—Magnetic properties
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
    • B01J20/28011—Other properties, e.g. density, crush strength
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3204—Inorganic carriers, supports or substrates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3244—Non-macromolecular compounds
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B01J20/3257—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one of the heteroatoms nitrogen, oxygen or sulfur together with at least one silicon atom, these atoms not being part of the carrier as such
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/3272—Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/3272—Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • B01J20/3274—Proteins, nucleic acids, polysaccharides, antibodies or antigens
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3289—Coatings involving more than one layer of same or different nature
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3291—Characterised by the shape of the carrier, the coating or the obtained coated product
    • B01J20/3293—Coatings on a core, the core being particle or fiber shaped, e.g. encapsulated particles, coated fibers
    • G01N30/48—
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2220/00—Aspects relating to sorbent materials
    • B01J2220/40—Aspects relating to the composition of sorbent or filter aid materials
    • B01J2220/49—Materials comprising an indicator, e.g. colour indicator, pH-indicator

Definitions

  • the present invention generally relates to composites for capturing rare cells in samples.
  • High yield capture of rare cells such as circulating tumor cells (CTC) is critical for using CTC as a diagnostic and prognostic method for cancer patients.
  • CTC circulating tumor cells
  • the separation uses immuno-magnetic interactions, that is, using antibody conjugated beads to capture CTC through specific antibody-CTC surface marker interaction, under the same external magnet field gradient, the larger the magnetic beads, the larger the magnetic forces hence the higher the CTC capture yield.
  • larger sizes of magnetic beads limit their uses, for example, difficulty in manipulation inside microfluidic flow channels due to higher chances of clump formation, or higher non-specific binding that lowers captured CTC purity. Accordingly, there is need to prepare magnetic-beads antibody conjugates having high rare cell capture yield while keeping low clump formation.
  • the present disclosure provides a composite for capturing rare cells that presents higher rare cell capture yield, while keeping low non-specific binding and low clump formation.
  • the present disclosure provides a composite for capturing rare cells in a sample, comprising: a bead operably linked to polyethylene glycol (PEG) compound; an analyte-capturing member operably linked to the bead, said analyte-capturing member specifically binding to a surface marker of the rare cells, wherein the composite has a diameter ranging from about 100 to about 2000 nm.
  • PEG polyethylene glycol
  • the PEG compound is maleimide-PEG or the derivative thereof.
  • the rare cells are present in the sample at less than 100 cells/ml.
  • the rare cells are circulating tumor cells (CTCs).
  • CTCs can be directly separated from whole blood sample.
  • the blood sample can be partitioned first to separate out plasma from cells, so that plasma can be used to detect other biomarkers, for example proteins or nucleic acids.
  • the comprehensive circulating markers e.g., cells, protein, nucleic acids
  • together can provide better diagnostic and therapeutic guidance value.
  • the bead has a diameter ranging from about 50 to about
  • the bead comprises at least one magnetic nanoparticle.
  • the magnetic nanoparticle comprises a superparamagnetic iron oxide (SPIO) nanoparticle, or a non-SPIO nanoparticle, or a combination of SPIO nanoparticle and non-SPIO nanoparticle.
  • the magnetic nanoparticle has a diameter ranging from about 1 nm to about 100 nm.
  • the bead further comprises a low density, porous 3-D structure, wherein the at least one magnetic nanoparticle is embedded in the 3-D structure.
  • the low density, porous 3-D structure has a thickness ranging from about 1 nm to about 2500 nm. In some embodiments, the low density, porous 3-D structure has a density of ⁇ 1.0 g/cc.
  • the bead further comprises one or more functional groups within or on the surface, the functional groups can be selected from the group consisting of nitrogen-containing group, sulfur-containing group, phosphorus-containing group, carbon-containing group, and epoxy-containing group.
  • the analyte-capturing member in the composite provided herein is an antibody.
  • the antibody is anti-EpCAM antibody.
  • the analyte-capturing member in the composite provided herein is a ligand of the cell surface marker.
  • the ligand is a peptide, a small molecule, an aptamer, a hormone, a drug, a toxin or a neurotransmitter.
  • the analyte-capturing member is operably linked to the bead through covalent linkage or non-covalent linkage. In some embodiments, the analyte-capturing member is operably linked to the bead through biotin-streptavidin interaction, protein A or G-antibody interaction or DNA-protein interaction.
  • the bead in the composite provided herein is bar-coded with or associated with a detectable agent selected from the group consisting of a fluorescent molecule, a chemo-luminescent molecule, a bio-luminescent molecule, a radioisotope, a MRI contrast agent, a CT contrast agent, an enzyme-substrate label, a coloring agent, and any combination thereof.
  • a detectable agent selected from the group consisting of a fluorescent molecule, a chemo-luminescent molecule, a bio-luminescent molecule, a radioisotope, a MRI contrast agent, a CT contrast agent, an enzyme-substrate label, a coloring agent, and any combination thereof.
  • the bead in the composite provided herein carries payload selected from the group consisting of a targeting moiety, a binding partner, a detectable agent, a biological active agent, a drug, a therapeutic agent, a radiological agent, a chemological agent, a small molecule drug, a biological drug, and any combination thereof.
  • the present disclosure provides a method of producing composites for capturing rare cells in a sample, comprising: conjugating an analyte-capturing member to a bead to form a conjugate; and treating the conjugate with a PEG compound, wherein the bead comprises PEG within or on the surface.
  • the bead used in the method further comprises one or more functional groups within or on the surface, the functional groups are selected from the group consisting of nitrogen-containing group, sulfur-containing group, phosphorus- containing group, carbon-containing group, epoxy-containing group or combination thereof.
  • the PEG compound for treating the conjugate is maleimide-PEG or the derivative thereof. In some embodiments, the PEG compound is maleimide-PEG-amine. In some embodiments, the maleimide-PEG for treating the conjugate is present at a concentration of about 5-200 ⁇ g/mg conjugate.
  • the bead used in the method further comprises nitrogen-containing group, sulfur-containing group and phosphorus-containing group within or on the surface, and the maleimide-PEG for treating the conjugate is present at a concentration of about 50-200 ⁇ g/mg conjugate, in particular about 140 ⁇ g/mg conjugate.
  • the bead used in the method further comprises nitrogen-containing group, sulfur-containing group, phosphorus-containing group and epoxy- containing group within or on the surface, and the maleimide-PEG for treating the conjugate is present at a concentration of about 5-20 ⁇ g/mg conjugate, in particular about 8 ⁇ g/mg conjugate.
  • the present disclosure provides a method for capturing rare cells in a sample, comprising: mixing the composite provided herein with the sample; and detecting the rare cells binding to the composite.
  • the method further comprises separating the captured rare cell using a permanent magnet, a magnetic column, a magnetic material patterned structure or device, or a magnetic sifter before detecting the rare cells.
  • the steps of separating and detecting are engineered to be automatic with robotic liquid handlers or specially designed flow devices.
  • the method further comprises determining a treatment according to the presence of the rare cells in the sample and/or the identification of the rare cells.
  • the identification of the cells includes identifying the contents of cells (such as protein components, nucleic acid component such as genotyping or mRNA expression, or other components such as hormones, metabolites, other small molecules or intracellular vehicles).
  • different cell contents can be identified when cells are alive, fixed, intact or after cell lysis.
  • the rare cells are immune cells or circulating cells that can indicate the presence of a disease or the response to a treatment.
  • the rare cells are selected from the group consisting of stem cells, cancer stem cells, T-cells, B-cells, NK cells, CAR-T cells and CTCs.
  • the treatment is immunotherapy.
  • the presence of the rare cells in the sample and/or the identification of the rare cells is indicative of a disease.
  • the disease is tumor, inflammation, infectious disease, autoimmune disease, or neurodegenerative disease.
  • FIG. 1 shows a general procedure for cell capture using the composite according to the embodiments of the present disclosure.
  • FIG. 2 shows the cell capture yield of 6 lots of composite made from 3 batches of bead for spike-in CTC capture from whole blood samples.
  • FIG. 3 shows the capture yield for spike-in CTC capture from whole blood samples for two different cell lines.
  • FIG. 4 shows the capture yield at different bead volume.
  • FIG. 5 shows the capture yield in cell capture assay using different incubation times.
  • FIG. 6 shows the capture yield of the composite of the present disclosure in comparison to the beads from other vendors.
  • FIG. 7 A and 7B shows the cell capture yield of two different batches of
  • FIG. 8 shows the cell capture yield of EpCAM beads and the composites obtained from the treatment of EpCAM beads with maleimide-PEG at two different doses, wherein the EpCAM beads comprise nitrogen-containing groups, sulfur-containing groups, and phosphorus-containing groups.
  • FIG. 9 shows the cell capture yield of EpCAM bead and the composites obtained from the treatment of EpCAM beads with maleimide-PEG at four different doses, wherein the EpCAM beads comprise nitrogen-containing groups, sulfur-containing groups, phosphorus-containing groups, and epoxy-containing groups.
  • FIG. 10 shows image of cell captured by fluorescent composite of the present disclosure viewed under a fluorescent microscope.
  • Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of chemistry, solid state chemistry, inorganic chemistry, organic chemistry, physical chemistry, analytical chemistry, materials chemistry, biochemistry, biology, molecular biology, recombinant DNA techniques, pharmacology, imaging, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.
  • One aspect of the present disclosure provides a composite for capturing rare cells in a sample, comprising a bead operably linked to polyethylene glycol (PEG) compound; an analyte-capturing member operably linked to the bead, said analyte-capturing member specifically binding to a surface marker of the rare cells, wherein the composite has a diameter ranging from about 100 to about 2000 nm.
  • PEG polyethylene glycol
  • bead refers to a bead having a diameter ranging from about 50 nm to about 3000 nm (e.g. 50-2500 nm, 50-2000 nm, 50-1500 nm, 50-1000 nm, 50-900 nm, 50-800 nm, 50-700 nm, 50-600 nm, 50-500 nm, 50-400 nm, 50-300 nm, 50- 200 nm, 50-100 nm, 60 nm, 70 nm, 80 nm, 90 nm etc.).
  • the bead comprises at least one magnetic nanoparticle.
  • the magnetic nanoparticle of the bead provided herein may comprise a superparamagnetic iron oxide (SPIO) nanoparticle.
  • SPIO nanoparticle is an iron oxide nanoparticle, either maghemite (y-Fe 2 0 3 ) or magnetite (Fe 3 0 4 ), or nanoparticles composed of both phases.
  • the SPIO nanoparticle can be synthesized with a suitable method and dispersed as a colloidal solution in organic solvents or water. Methods to synthesize the SPIO nanoparticles are known in the art (see, for example, Morteza Mahmoudi et al,
  • the SPIO nanoparticles can be made through wet chemical synthesis methods which involve co-precipitation of Fe and Fe salts in the presence of an alkaline medium. During the synthesis, nitrogen may be introduced to control oxidation, surfactants and suitable polymers may be added to inhibit agglomeration or control particle size, and/or emulsions (such as water-in-oil microemulsions) may be used to modulate the physical properties of the SPIO nanoparticle (see, for example, Jonathan W. Gunn, The preparation and characterization of superparamagnetic nanoparticles for biomedical imaging and therapeutic application, published by ProQuest, 2008).
  • emulsions such as water-in-oil microemulsions
  • the SPIO nanoparticles can be generated by thermal decomposition of iron pentacarbonyl, alone or in combination with transition metal carbonyls, optionally in the presence of one or more surfactants (e.g., lauric acid and oleic acid) and/or oxidatants (e.g., trimethylamine-N-oxide), and in a suitable solvent (e.g., dioctyl ether or hexadecane) (see, for example, US patent application PG Pub 20060093555).
  • the SPIO nanoparticles can also be made through gas deposition methods, which involves laser vaporization of iron in a helium atmosphere containing different concentrations of oxygen (see, Miller J.S. et al., Magnetism: Nanosized magnetic materials, published by Wiley- VCH, 2002).
  • the SPIO nanoparticles are those disclosed in US patent application PG Pub 20100008862.
  • the magnetic nanoparticle of the bead provided herein may comprise a non-SIPO nanoparticle.
  • the non-SPIO nanoparticles include, for example, metallic nanoparticles (e.g., gold or silver nanoparticles (see, e.g., Hiroki Hiramatsu, F.E.O., Chemistry of Materials 16, 2509-2511 (2004)), semiconductor nanoparticles (e.g., quantum dots with individual or multiple components such as CdSe/ZnS (see, e.g., M. Bruchez, et al, science 281, 2013-2016 (1998))), doped heavy metal free quantum dots (see, e.g., Narayan Pradhan et al, J. Am. chem. Soc. 129, 3339-3347 (2007)) or other semiconductor quantum dots); polymeric nanoparticles (e.g., particles made of one or a combination of PLGA
  • the non-SPIO nanoparticle is a colored nanoparticle, for example, a semiconductor nanoparticle such as a quantum dot.
  • the non-SPIO nanoparticles can be prepared or synthesized using suitable methods known in the art, such as for example, sol-gel synthesis method, water-in-oil micro- emulsion method, gas deposition method and so on.
  • gold nanoparticles can be made by reduction of chloroaurate solutions (e.g., HAuCl 4 ) by a reducing agent such as citrate, or acetone dicarboxulate.
  • CdS semiconductor nanoparticle can be prepared from Cd(C10 4 ) 2 and Na 2 S on the surface of silica particles.
  • II- VI semiconductor nanoparticles can be synthesized based on pyrolysis of organometallic reagents such as dimethyl cadmium and trioctylphosphine selenide, after injection into a hot coordinating solvent (see, e.g., Gunter Schmid, Nanoparticles: From Theory to Application, published by John Wiley & Sons, 2011).
  • organometallic reagents such as dimethyl cadmium and trioctylphosphine selenide
  • Doped heavy metal free quantum dots for example Mn-doped ZnSe quantum dots can be prepared using nucleation-doping strategy, in which small-sized MnSe nanoclusters are formed as the core and ZnSe layers are overcoated on the core under high temperatures.
  • polymeric nanoparticles can be prepared by emulsifying a polymer in a two-phase solvent system, inducing nanosized polymer droplets by sonication or homogenization, and evaporating the organic solvent to obtain the nanoparticles.
  • siliceous nanoparticles can be prepared by sol-gel synthesis, in which silicon alkoxide precursors (e.g., TMOS or TEOS) are hydrolyzed in a mixture of water and ethanol in the presence of an acid or a base catalyst, the hydrolyzed monomers are condensed with vigorous stirring and the resulting silica nanoparticles can be collected.
  • silicon alkoxide precursors e.g., TMOS or TEOS
  • SAFs a non- SPIO magnetic nanoparticle
  • a nonmagnetic space layer e.g., ruthenium metal
  • a chemical etchable copper release layer and protective tantalum surface layers using ion-bean deposition in a high vacuum
  • nanoparticle can be released after removing the protective layer and selective etching of copper.
  • the diameter of the magnetic nanoparticles ranges from about 1 nm to about
  • nm for example, 1-90 nm, 1-80 nm, 1-70 nm, 1-60 nm, 1-50 nm, 1-40 nm, 1-30 nm, 1- 20 nm, 1-10 nm, 2-40 nm, 5-20 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, etc.).
  • the size of magnetic nanoparticles can be controlled by selecting appropriate synthesis methods and/or systems.
  • synthesis of nanoparticles can be carried out in a polar solvent which provides ionic species that can adsorb on the surface of the nanoparticles, thereby providing electrostatic effect and particle-particle repulsive force to help stabilize the nanoparticles and inhibit the growth of the nanoparticles.
  • the nanoparticles can be synthesized in a micro-heterogeneous system that allows compartmentalization of nanoparticles in constrained cavities or domains.
  • Such a micro-heterogeneous system may include, liquid crystals, mono and multilayers, direct micelles, reversed micelles, microemulsions and vesicles.
  • the synthesis conditions may be properly controlled or varied to provide for, e.g., a desired solution concentration or a desired cavity range (a detailed review can be found at, e.g., Vincenzo Liveri, Controlled synthesis of nanoparticles in microheterogeneous systems, Published by Springer, 2006).
  • the shape of the magnetic nanoparticles can be spherical, cubic, rod shaped
  • tetrapod-shaped see, e.g., L. Manna et al, Nature Materials, 2, 382-385 (2003)
  • Methods are known in the art to control the shape of the nanoparticles during the preparation (see, e.g., Waseda Y. et al., Morphology control of materials and nanoparticles: advanced materials processing and characterization, published by Springer, 2004).
  • a shape controller which adsorbs strongly to a specific crystal plane may be added to control the growth rate of the particle.
  • a single bead may comprise a single nanoparticle or a plurality of mini- nanoparticles (A. Fu et al, J. Am. chem. Soc. 126, 10832-10833 (2004), J. Ge et al, Angew. Chem. Int. Ed. 46, 4342-4345 (2007), Zhenda Lu et al, Nano Letters 11, 3404-3412 (2011).).
  • the mini-nanoparticles can be homogeneous (e.g., made of the same composition/materials or having same size) or heterogeneous (e.g., made of different compositions/materials or having different sizes).
  • a cluster of homogeneous mini-nanoparticles refers to a pool of particles having substantially the same features or characteristics or consisting of
  • a cluster of heterogeneous mini-nanoparticles refers to a pool of particles having different features or characteristics or consisting of substantially different materials.
  • a heterogeneous mini-nanoparticle may comprise a quantum dot in the center and a discrete number of gold (Au) nanocrystals attached to the quantum dot.
  • Au gold
  • a bead provided herein comprises a plurality of magnetic nanoparticles.
  • the bead comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 100s or 1000s magnetic nanoparticles.
  • the magnetic nanoparticles in the bead impart magnetic property to the bead, which allows the bead and thus the composite to be pulled or attracted to a magnet or in a magnetic field.
  • Magnetic property can facilitate manipulation (e.g., separation, purification, or enrichment) of the bead and thus the composite using magnetic interaction.
  • the beads can be attracted to or magnetically guided to an intended site when subject to an applied magnetic field, for example a magnetic field from high-filed and/or high-gradient magnets.
  • a magnet e.g., magnetic grid
  • a magnet e.g., magnetic grid
  • the beads provided herein further comprise a coating.
  • the at least one nanoparticle can be embedded in or coated with the coating.
  • Any suitable coatings known in the art can be used, for example, a polymer coating and a non-polymer coating.
  • the coating may interact with the nanoparticle(s) through 1) intra-molecular interaction such as covalent bonds (e.g., Sigma bond, Pi bond, Delta bond, Double bond, Triple bond, Quadruple bond, Quintuple bond, Sextuple bond, 3c-2e, 3c-4e, 4c-2e, Agostic bond, Bent bond, Dipolar bond, Pi backbond, Conjugation, Hyperconjugation, Aromaticity, Hapticity, and Antibonding), metallic bonds (e.g., chelating interactions with the metal atom in the core nanoparticle), or ionic bonding (cation ⁇ -bond and salt bond), and 2) inter- molecular interaction such as hydrogen bond (e.g., Dihydrogen bond, Dihydrogen complex, Low-barrier hydrogen bond, Symmetric hydrogen bond
  • the coating includes a low density, porous 3-D structure, as disclosed in U.S. Prov. Appl. 61/589, 777 and U.S. Pat. Appl. 12/460,007 (all references cited in the present disclosure are incorporated herein in their entirety).
  • the low density, porous 3-D structure refers to a structure with density much lower (e.g., 10s times, 20s times, 30s times, 50s times, 70s times, 100s times) than existing mesoporous nanoparticles (e.g., mesoporous nanoparticles having a pore size ranging from 2 nm to 50 nm).
  • mesoporous nanoparticles e.g., mesoporous nanoparticles having a pore size ranging from 2 nm to 50 nm.
  • the low density, porous 3-D structure refers to a structure having a density of ⁇ 1.0 g/cc (e.g., ⁇ 100mg/cc, ⁇ 10mg/cc, ⁇ 5mg/cc, ⁇ lmg/cc,
  • ⁇ 0.5mg/cc, ⁇ 0.4mg/cc, ⁇ 0.3mg/cc, ⁇ 0.2mg/cc, or ⁇ 0.1mg/cc) for example, from 0.01 mg/cc to 10 mg/cc, from 0.01 mg/cc to 8 mg/cc, from 0.01 mg/cc to 5 mg/cc, from 0.01 mg/cc to 3 mg/cc, from 0.01 mg/cc to 1 mg/cc, from 0.01 mg/cc to 1 mg/cc, from 0.01 mg/cc to 0.8 mg/cc, from 0.01 mg/cc to 0.5 mg/cc, from 0.01 mg/cc to 0.3 mg/cc, from 0.01 mg/cc to 1000 mg/cc, from 0.01 mg/cc to 915 mg/cc, from 0.01 mg/cc to 900 mg/cc, from 0.01 mg/cc to 800 mg/cc, from 0.01 mg/cc to 700 mg/cc, from 0.01 mg
  • the density of 3-D structure can be determined using various methods known in the art (see, e.g., Lowell, S. et al, Characterization of porous solids and powders: surface area, pore size and density, published by Springer, 2004). Exemplary methods include, Brunauer Emmett Teller (BET) method and helium pycnometry (see, e.g., Varadan V. K. et al., Nanoscience and Nanotechnology in Engineering, published by World Scientific, 2010). Briefly, in BET method, dry powders of the testing 3-D structure is placed in a testing chamber to which helium and nitrogen gas are fed, and the change in temperature is recorded and the results are analyzed and extrapolated to calculate the density of the testing sample.
  • BET Brunauer Emmett Teller
  • the density of the 3-D structure can be determined using the dry mass of the 3- D structure divided by the total volume of such 3-D structure in an aqueous solution.
  • dry mass of the core particles with and without the 3-D structure can be determined respectively, and the difference between the two would be the total mass of the 3-D structure.
  • volume of a core particle with and without the 3-D structure in an aqueous solution can be determined respectively, and the difference between the two would be the volume of the 3-D structure on the core particle in an aqueous solution.
  • the beads can be dispersed as a plurality of large nanoparticles coated with the 3-D structure in an aqueous solution.
  • the total volume of the 3-D structure can be calculated as the average volume of the 3-D structure for an individual large nanoparticle multiplied with the number of the large nanoparticles.
  • the size (e.g., radius) of the particle with 3-D structure can be determined with Dynamic Light Scattering (DLS) techniques, and the size (e.g., radius) of the particle core without the 3-D structure can be determined under Transmission Electron Microscope (TEM), as the 3-D structure is substantially invisible under TEM. Accordingly, the volume of the 3-D structure on an individual large nanoparticle can be obtained by subtracting the volume of the particle without 3-D structure from the volume of the particle with the 3-D structure.
  • DLS Dynamic Light Scattering
  • TEM Transmission Electron Microscope
  • the number of large nanoparticles for a given core mass can be calculated using any suitable methods.
  • an individual large nanoparticle may be composed of a plurality of small nanoparticles which are visible under TEM.
  • the average size and volume of a small nanoparticle can be determined based on measurements under TEM, and the average mass of a small nanoparticle can be determined by multiplying the known density of the core material with the volume of the small particle.
  • the total number of small nanoparticles can be estimated.
  • the average number of small nanoparticles in it can be determined under TEM. Accordingly, the number of large nanoparticles for a given core mass can be estimated by dividing the total number of small nanoparticles with the average number of small nanoparticles in an individual large nanoparticle.
  • the low density, porous 3-D structure is highly porous.
  • the low density, porous 3-D structure can be a structure having 40%-99.9% (preferably 50% to 99.9%) of empty space or pores in the structure, where 80% of the pores having size of 1 nm to 500 nm in pore radius.
  • the porosity of the 3-D structure can be characterized by the Gas/Vapor adsorption method.
  • nitrogen at its boiling point, is adsorbed on the solid sample.
  • the amount of gas adsorbed at a particular partial pressure could be used to calculate the specific surface area of the material through the Brunauer, Emmit and Teller (BET) nitrogen adsorption/desorption equation.
  • BET Brunauer, Emmit and Teller
  • the pore sizes are calculated by the Kelvin equation or the modified Kelvin equation, the BJH equation (see, e.g., D. Niu et al, J. Am. chem. Soc. 132, 15144-15147 (2010)).
  • the porosity of the 3-D structure can also be characterized by mercury porosimetry (see, e.g., Varadan V. K. et al, supra). Briefly, gas is evacuated from the 3-D structure, and then the structure is immersed in mercury. As mercury is non-wetting at room temperature, an external pressure is applied to gradually force mercury into the sample. By monitoring the incremental volume of mercury intruded for each applied pressure, the pore size can be calculated based on the Washburn equation.
  • the low density, porous 3-D structure has a porous structure (except to the core nanoparticle or core nanoparticles) which could not be obviously observed or substantially transparent under TEM, for example, even when the feature size of the 3-D structure is in the 10s or 100s nanometer range.
  • the term "obviously observed” or “substantially transparent” as used herein means that, the thickness of the 3-D structure cannot be readily estimated or determined based on the image of the 3-D structure under
  • the bead e.g., nanoparticle(s) coated with or embedded in a low density, porous 3-D structure
  • the bead can be observed or measured by ways known in the art.
  • the size (e.g., radius) of the bead with the 3-D structure can be measured using DLS methods, and the size (e.g., radius) of the core particle without the 3-D structure can be measured under TEM.
  • the thickness of the 3-D structure is measured as 10s, 100s, 1000s nanometer range by DLS, but cannot be readily determined under TEM.
  • the nanoparticles can be identified, however, the low density, porous 3-D structure cannot be obviously observed, or is almost transparent.
  • the porosity of the 3-D structure can be also evaluated by the capacity to load different molecules (see, e.g., Wang L. et al, Nano Research 1, 99-115 (2008)).
  • the 3-D structure provided herein has a low density and high porosity, it is envisaged that more payload can be associated with the 3-D structure than with other coatings.
  • organic fiuorophores such as Rhodamin
  • Rhodamin over 105 Rhodamin molecules can be loaded to 3-D structure of one nanoparticle.
  • the low density, porous 3-D structure is made of silane- containing or silane-like molecules (e.g., silanes, organosilanes, alkoxysilanes, silicates and derivatives thereof).
  • silane-like molecules e.g., silanes, organosilanes, alkoxysilanes, silicates and derivatives thereof.
  • the silane-containing molecule comprises an organosilane, which is also known as silane coupling agent.
  • Organosilane has a general formula of R x SiY 4-x , wherein R group is an alkyl, aryl or organic functional group.
  • R group is an alkyl, aryl or organic functional group.
  • Y group is a methoxy, ethoxy or acetoxy group, x is 1, 2 or 3.
  • the R group could render a specific function such as to associate the organosilane molecule with the surface of the core nanoparticle or other payloads through covalent or non-covalent interactions.
  • the Y group is hydrolysable and capable of forming a siloxane bond to crosslink with another organosilane molecule.
  • Exemplary R groups include, without limitation, disulphidealkyl, aminoalkyl, mercaptoalkyl, vinylalkyl, epoxyalkyl, and methacrylalkyl, carboxylalkyl groups.
  • the alkyl group in an R group can be methylene, ethylene, propylene, and etc.
  • Exemplary Y groups include, without limitation, alkoxyl such as OCH 3 , OC 2 H 5 , and OC 2 H 4 OCH 3 .
  • the organosilane can be amino-propyl-trimethoxysilane, mercapto-propyl- trimethoxysilane, carboxyl-propyl-trimethoxysilane, amino-propyl-triethoxysilane, mercapto-propyl- triethoxysilane, carboxyl-propyl-triethoxysilane, bis-[3-(triethoxysilyl) propyl]- tetrasulfide, bis-[3-(triethoxysilyl) propyl]- disulfide, aminopropyltriethoxysilane, N-2-(aminoethyl)-3 - amino propyltrimethoxysilane, vinyltrimethoxysilane, vinyl-tris(2-methoxyethoxy)silane, 3- methacryloxypropyltrimethoxy silane, 2-(3,4-epoxycyclohexy)-ethyl
  • the bead is capable of generating a color signal under a suitable condition.
  • the colored bead may emit a fluorescent color signal upon excitation with a light of a certain wavelength.
  • the bead may alternatively be non-colored. A non-colored bead does not emit a color signal when subject to a condition that would otherwise induce a color signal for a colored bead.
  • the bead is bar-coded or associated with a detectable agent.
  • bar-coding or “bar-coded” means that the bead is associated with a known code or a known label that allows identification of the bead.
  • Code refers to a molecule capable of generating a detectable signal that distinguishes one bar-coded bead from another.
  • the colored bead may comprise a colored nanoparticle (e.g. a quantum dot) which emits a detectable color signal at a known wave length.
  • the characteristics or the identity of a bar-coded bead is based on multiplexed optical coding system as disclosed in Han et al, Nature
  • QDs multicolor semiconductor quantum-dots
  • the bead For each single color coding, the bead has different intensity of QDs depending on the number of QDs embedded therein. If QDs of multiple colors (n colors) and multiple intensity (m levels of intensity) are used, then the bead may have a total number of unique identities or codes, which is equal to m to the exponent of n less one (m 11"1 ). In addition, since the porous structure can be associated with additional payloads (e.g., fluorescent organic molecules), if there are Y number of additional fluorescent colors available, the total number of code can be Yx (m 11 - 1 ).
  • additional payloads e.g., fluorescent organic molecules
  • the bead (with or without bar-coding) is colored by being operably linked to a detectable agent.
  • a detectable agent can be a fluorescent molecule, a chemo-luminescent molecule, a bio-luminescent molecule, a radioisotope, a MRI contrast agent, a CT contrast agent, an enzyme-substrate label, and/or a coloring agent etc.
  • fluorescent molecules include, without limitation, fluorescent compounds (fluorophores) which can include, but are not limited to: 1,5 IAEDANS; 1,8-ANS; 4-Methylumbelliferone; 5-carboxy-2,7-dichlorofiuorescein; 5- Carboxyfluorescein (5-FAM); 5 -Carboxynaptho fluorescein; 5- Carboxytetramethylrhodamine (5-TAMRA); 5-FAM (5- Carboxyfluorescein); 5-HAT (Hydroxy Tryptamine); 5 -Hydroxy Tryptamine (HAT); 5-ROX (carboxy-X- rhodamine); 5-TAMRA (5-Carboxytetramethylrhodamine); 6- Carboxyrhodamine 6G; 6-CR 6G; 6- JOE; 7-Amino-4-methylcoumarin; 7- Aminoactinomycin D (7-AAD); 7- Hydroxy-4-methylcoumarin; 9-
  • Aminomethylcoumarin (AMCA); Anilin Blue; Anthrocyl stearate; APC (Allophycocyanin); APC-Cy7; APTRA-BTC; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO- TAGTM CBQCA; ATTO-TAGTM FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); Berberine Sulphate; Beta Lactamase; Bimane; Bisbenzamide; Bisbenzimide (Hoechst); bis-BTC; Blancophor FFG; Blancophor SV; BOBOTM-l; BOBOTM- 3; Bodipy 492/515; Bodipy 493/503; Bodipy 500/510; Bodipy 505/515; Bodipy 530/550; Bodip
  • Bodipy Fl-Ceramide Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy
  • TMR-X, SE Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PROTM- 1; BO- PROTM-3; Brilliant Sulphoflavin FF; BTC; BTC-5N; Calcein; Calcein Blue; Calcium CrimsonTM;
  • DiIC18(3) Dinitrophenol; DiO (DiOC18(3)); DiR; DiR (DiIC18(7)); DM-NERF (high pH); D P; Dopamine; DTAF; DY-630- HS; DY-635- HS; ELF 97; Eosin; Erythrosin;
  • FITC Induced Fluorescence
  • Flazo Orange Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald; FluoroGold (Hydroxystilbamidine); Fluor-Ruby; Fluor X; FM 1-
  • Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO-JO- 1; JO- PRO- 1; LaserPro; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF;
  • Leucophor SF Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B;
  • LysoSensor Blue LysoSensor Green; LysoSensor Yellow/Blue; Mag Green; Magdala Red
  • PhotoResist Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA;
  • Rhodamine B Rhodamine B 200; Rhodamine B extra; Rhodamine BB; RhodamineBG;
  • Rhodamine Green Rhodamine Phallicidine; Rhodamine Phalloidine; Rhodamine Red;
  • Rhodamine WT Rose Bengal; R-phycocyanine; R-phycoerythrin (PE); S65A; S65C; S65L; S65T; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron Brilliant Blue
  • SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein; SNARF1;
  • Sulphorhodamine Extra SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO 15; SYTO 16;
  • SYTO 40 SYTO 41; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO 59; SYTO 60;
  • SYTO 84 SYTO 85; SYTOX Blue; SYTOX Green; SYTOX Orange; Tetracycline;
  • TTC Tetramethylrhodamine
  • Texas RedTM Texas Red-XTM conjugate
  • DiSC3 Thiadicarbocyanine
  • Thiazine Red R Thiazole Orange
  • Thio flavin 5 Thioflavin S;
  • PRO-1 PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; Tricolor (PE-Cy5); TRITC
  • PRO-3 PRO-3; YOYO-1; YOYO-3, Sybr Green, Thiazole orange (interchelating dyes), fluorescent semiconductor nanostructures, lanthanides or combinations thereof.
  • radioisotopes examples include, 123 I, 124 I, 125 I, 131 1, 35 S, 3 H, m In, 112 In, 14 C, 64 Cu, 67 Cu, 86 Y, 88 Y, 90 Y, 177 Lu, 211 At, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 18 F, 201 T1, 67 Ga, 137 Cs and other radioisotopes.
  • enzyme-substrate labels include, luciferases (e.g., firefly luciferase and bacterial luciferase), luciferin, 2,3-dihydrophthalazinedionesm, alate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like.
  • luciferases e.g., firefly luciferase and bacterial luciferase
  • luciferin 2,3-di
  • the bead in the composite provided herein carries payload.
  • the payload can be selected from the group consisting of a targeting moiety, a binding partner, a detectable agent, a biological active agent, a drug, a therapeutic agent, a radiological agent, a chemological agent, a small molecule drug, a biological drug, and any combination thereof.
  • the bead in the composite provided herein also comprises one or more functional groups within or on the surface.
  • These functional groups may be introduced within or on the surface of the bead during the formation of the 3-D structure.
  • precursors containing such functional groups can be added.
  • These functional groups may also be introduced after the formation of the 3-D structure.
  • the surface of the bead may be chemically modified by reacting precursors containing the functional groups with the reactive groups on the surface of the bead.
  • These functional groups include nitrogen-containing group, sulfur-containing group, carbon-containing group, phosphorus-containing group, epoxy-containing group and the like.
  • Examples of the functional groups include, but are not limited to amino, mercapto, carboxyl, phosphonate, biotin, streptavidin, avidin, hydroxyl, alkyl or other hydrophobic molecules, polyethylene glycol, oligos, peptides, saccharides, phospholipids, PNAs, or other hydrophilic or hydrophobic molecules etc.
  • the bead of the composite provided herein is operably linked to an analyte- capturing member.
  • operably linked includes embedding, incorporating, integrating, binding, attaching, combining, cross-linking, mixing, and/or coating the analyte- capturing member to the bead.
  • the analyte-capturing member can be operably linked to the bead through non-covalent linkage (e.g. hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interaction) or covalent linkage.
  • the analyte- capturing member is operably linked to the bead through biotin-streptavidin interaction, protein A or G-antibody interaction or DNA-protein interaction.
  • the analyte-capturing members are molecules capable of capturing or specifically binding to an analyte.
  • Capture or “specifically bind” as used herein, means that a non-random binding interaction between two molecules.
  • the specific binding can be characterized by binding affinity (Kd), which is calculated as the ratio of dissociation rate to association rate (k 0 ff/k on ) when the binding between the two molecules reaches equilibrium.
  • Kd binding affinity
  • the dissociation rate (k off ) measured at the binding equilibrium may also be used when measurement of k on is difficult to obtain, for example, due to aggregation of one molecule.
  • the antigen-binding affinity e.g.
  • K D or k 0 ff can be appropriately determined using suitable methods known in the art, including, for example, Biacore (see, for example, Murphy, M. et al, Current protocols in protein science, Chapter 19, unit 19.14, 2006) and Kinexa techniques (see, for example, Darling, R. J., et al, Assay Drug Dev. Technol., 2(6): 647-657 (2004)).
  • Biacore see, for example, Murphy, M. et al, Current protocols in protein science, Chapter 19, unit 19.14, 2006
  • Kinexa techniques see, for example, Darling, R. J., et al, Assay Drug Dev. Technol., 2(6): 647-657 (2004).
  • analyte-capturing members include Protein A; Protein G;
  • antigen-binding members e.g., antibodies or fragments thereof; nuclei acid (or a fragment of nuclei acid, an oligo nucleotide); or a protein/peptide binding specifically to a molecule such as another protein/peptide, an antibody, a piece of nuclei acid (DNA or RNA), carbohydrate, lipid, a polymer, or a small organic molecule such as a drug; a ligand (e.g., a peptide, small molecule, hormone, a drug, toxin, neurotransmitter) that specifically binds to a receptor, or a receptor that specifically binds to a ligand, a chemical in a supermolecular structure (e.g., host-guest chemistry complex such as a p-xylylenediammonium bound within a cucurbituril) whereas the chemical is a host molecule (e.g.
  • cyclodextrins calixarenes, cucurbiturils, porphyrins , metallacrowns, crown ethers, zeolites, cyclotriveratrylenes, cryptophanes and carcerands
  • a guest molecule e.g., prostaglandin, itraconazole
  • Protein A is an affinity ligand for an antibody having an immunoglobulin Fc domain, and can be useful in purification of antibodies that are based on
  • An antigen binding member can be an antibody, an antibody fragment or an antibody memetics, such as, for example, scFV, Fab, Fab', Fv, single domain antibody, diabody, nanobody, domain antibody, dsFv, or canelized antibody.
  • the antibodies or fragments can be polyclonal, monoclonal, of animal origin (e.g., murine, rabbit, camel), of human origin (e.g., fully human), chimeric, humanized, variable regions, CDRs, ScFv, bispecific, diabody, or other forms of antibodies with antigen- binding capabilities.
  • the analyte-capturing member can be an antibody.
  • the antibody is against epithelial cell adhesion molecule (EpCAM).
  • the analyte-capturing member can be a ligand of the cell surface marker.
  • the ligand can be a peptide, small molecule, hormone, a drug, toxin, or neurotransmitter.
  • the bead can be operably linked to multiple analyte-capturing members targeting multiple cell surface markers, thereby improving the cell capture yield of the resulting composite.
  • the bead can be bar-coded with or associated with a detectable agent such as a fluorescent molecule, a chemo-luminescent molecule, a bio- luminescent molecule, a radioisotope, a MRI contrast agent, a CT contrast agent, an enzyme- substrate label, or a coloring agent.
  • the barcode of the bead can be used to identify the rare cells and/or the cell surface markers and quantify the expression level of the cell surface markers.
  • the bead is both magnetic and barcoded, which facilitates the capture of the rare cells and identification of the rare cells/cell surface markers simultaneously.
  • the bead of the composite provided herein is also operably linked to PEG compound.
  • PEG compound refers to compounds containing PEG chains that operably linked to the bead provided herein.
  • the PEG compounds are operably linked to the bead such that the bead is coated with the PEG compounds.
  • a methoxy-group can be at the polymer end.
  • a hydroxyl group can be at the polymer end.
  • the PEG compound comprises functional groups which may be reacted with the groups on the surface of the bead so that the PEG compound is operably linked to the bead provided herein.
  • the functional group may include, but are not limited to a maleimide functional group, an ester functional group, an amine functional group, and a hydroxyl functional group.
  • the PEG compound used herein comprises maleimide functional group.
  • the PEG compound used herein can be maleimide-PEG or maleimide-PEG-amine.
  • the PEG chain of the PEG compounds can have a length between
  • 1 and 100 monomer units for example, between 1 and 90 monomer units, between 1 and 80 monomer units, between 1 and 70 monomer units, between 1 and 60 monomer units, between
  • the thickness of the coating of PEG compounds is up to 20 nm, up to 19 nm, up to 18 nm, up to 17 nm, up to 16 nm, up to 15 nm, up to 14 nm, up to 13 nm, up to 12 nm, up to 11 nm, up to 10 nm, up to 9 nm, up to 8 nm, up to 7 nm, up to 6 nm, up to 5 nm, up to 4 nm, up to 3 nm, up to 2 nm, up to 1 nm and the like.
  • maleimide-PEG-amine used as the PEG compound of the present disclosure may have a weight average molecular weight (Mw) of about 5000 Da.
  • the modification of the surface of the bead with PEG compounds may result in composites having a narrower size distribution. Compared with the beads without any treatment, the composites obtained from the treatment of beads with PEG compounds show improved cell capture yield while maintaining low non-specific cell capture in cell capture assays.
  • the composite provided herein comprises a bead comprising at least one magnetic nanoparticle embedded in or coated with a low-density, porous 3-D structure.
  • the bead is formed by coating or surrounding at least one magnetic nanoparticle with a low density, porous 3-D structure such that the nanoparticle(s) is or are embedded in the 3-D structure.
  • the low-density, porous 3-D structure is formed by the depositing, or covering of the surface of the magnetic nanoparticle(s) through the assembly or cross-linking of silane- containing or silane-like molecules.
  • the low density porous 3-D structure can be prepared by a silanization process on the surface of the magnetic nanoparticle(s).
  • Silanization process includes, for example, the steps of crosslinking silicon- containing or silane-like molecules (e.g., alkoxysilanes such as amino-propyl - trimethoxysilane, mercapto-propyl-trimethoxysilane, or sodium silicate) under acidic or basic conditions.
  • silicon- containing or silane-like molecules e.g., alkoxysilanes such as amino-propyl - trimethoxysilane, mercapto-propyl-trimethoxysilane, or sodium silicate
  • an acidic or a basic catalyst is used in the crosslinking.
  • exemplary acid catalyst includes, without limitation, a protonic acid catalyst (e.g., nitric acid, acetic acid and sulphonic acids) and Lewis acid catalyst (e.g., boron trifluoride, boron trifluoride monoethylamine complex, boron trifluoride methanol complex, FeCl 3 , A1C1 3 , ZnCl 2 , and ZnBr 2 ).
  • exemplary basic catalysts include, an amine or a quaternary ammonium compound such as tetramethyl ammonium hydroxide and ammonia hydroxide.
  • the silanization process may include one or more stages, for example, a priming stage in which the 3-D structure starts to form, a growth stage in which a layer of siliceous structure is readily formed on the core nanoparticle and more are to be formed, and/or an ending stage in which the 3-D structure is about to be completed (e.g., the outer surface of the 3-D structure is about to be formed).
  • a priming stage in which the 3-D structure starts to form
  • a growth stage in which a layer of siliceous structure is readily formed on the core nanoparticle and more are to be formed
  • an ending stage in which the 3-D structure is about to be completed (e.g., the outer surface of the 3-D structure is about to be formed).
  • one or more silane-containing molecules can be added at different stages of the process.
  • organosilanes such as aminopropyl trimethoxyl silane or mercaptopropyl trimethoxyl silane can be added to initiate the silanization on the core nanoparticle surface
  • silane molecules having fewer alkoxy groups can be added to the reaction at the growth stage of silanization.
  • organosilane molecules with one or a variety of different functional groups may be added.
  • These functional groups can be amino, carboxyl, mercapto, or phosphonate group, which can be further conjugated with other molecules, e.g., hydrophilic agent, a biologically active agent, a detectable label, an optical responsive group, electronic responsive group, magnetic responsive group, enzymatic responsive group or pH responsive group, or a binding partner, so as to allow further modification of the 3-D structure in terms of stability, solubility, biological compatibility, capability of being further conjugation or derivation, or affinity to payload.
  • hydrophilic agent e.g., hydrophilic agent, a biologically active agent, a detectable label, an optical responsive group, electronic responsive group, magnetic responsive group, enzymatic responsive group or pH responsive group, or a binding partner, so as to allow further modification of the 3-D structure in terms of stability, solubility, biological compatibility, capability of being further conjugation or derivation, or affinity to payload.
  • the functional groups can also be a group readily conjugated with other molecules (e.g., a group conjugated with biologically active agent, a thermal responsive molecule, an optical responsive molecule, an electronic responsive molecule, a magnetic responsive molecule, a pH responsive molecule, an enzymatic responsive molecule, a detectable label, or a binding partner such as biotin or avidin).
  • a group conjugated with biologically active agent e.g., a thermal responsive molecule, an optical responsive molecule, an electronic responsive molecule, a magnetic responsive molecule, a pH responsive molecule, an enzymatic responsive molecule, a detectable label, or a binding partner such as biotin or avidin.
  • the preparation further includes density reducing procedures such as introducing air bubbles in the reaction or formation, increasing reaction temperature, microwaving, sonicating, vertexing, labquakering, and/or adjusting the chemical composition of the reaction to adjust the degree of the crosslinking of the silane molecules.
  • density reducing procedures such as introducing air bubbles in the reaction or formation, increasing reaction temperature, microwaving, sonicating, vertexing, labquakering, and/or adjusting the chemical composition of the reaction to adjust the degree of the crosslinking of the silane molecules.
  • the density reducing procedure comprises sonicating the reaction or formation mixture.
  • the conditions of the sonicating procedure (e.g., duration) in the silanization process can be properly selected to produce a desired porosity in the resulting low density porous 3-D structure.
  • the sonicating can be applied throughout a certain stage of the silanization process.
  • the duration of sonicating in a silanization stage may last for, e.g., at least 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours.
  • sonicating is applied in each stage of the silanization process.
  • the density reducing procedures comprise introducing at least one alcohol to the reaction.
  • the alcohol has at least 3 (e.g., at least 4, at least 5 or at least 6) carbon atoms.
  • the alcohol may have 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more carbon atoms.
  • the alcohol can be
  • Alcohol with an unsaturated carbon chain has a double or a triple bond between two carbon atoms.
  • the alcohol can be a cyclic alcohol, for example, cyclohexanol, inositol, or menthol.
  • the alcohol can have a straight carbon chain (e.g., n- propyl alcohol, n-butyl alcohol, n-pentyl alcohol, n-hexyl alcohol, etc) or a branched carbon chain (e.g., isopropyl alcohol, isobutyl alcohol, tert-butyl alcohol, etc).
  • a straight carbon chain e.g., n- propyl alcohol, n-butyl alcohol, n-pentyl alcohol, n-hexyl alcohol, etc
  • a branched carbon chain e.g., isopropyl alcohol, isobutyl alcohol, tert-butyl alcohol, etc.
  • the alcohol is present in a volume fraction of about 30% to about 70%> (e.g., about 30% to about 70%, about 30% to about 60%, about 30% to about 55%, about 40% to about 70%), about 45%> to about 70%>, about 40%> to about 60%>).
  • the alcohol is present in volume fraction of around 50%>) (e.g., around 45%>, around 46%>, around 47%), around 48%>, around 49%>, around 50%>), around 51%>, around 52%>, around 53%>, around 54%), around 55%>, around 56%>, around 57%>, around 58%>, around 59%>, or around 60%>,).
  • the density reducing procedure comprises introducing air bubbles to the reaction.
  • the air bubbles can be in constant presence during the reaction process.
  • the air bubbles can be introduced to the reaction through any suitable methods, for example, by blowing bubbles to the reaction, or by introducing a gas-producing agent to the reaction mixture.
  • experimental conditions can also be optimized to provide for formation of a desired low density porous 3-D structure.
  • Such experimental conditions include, for example, the concentration of the core nanoparticles, the concentration of the catalyst, the ratio of the concentration of the catalyst to the core nanoparticle, the temperature at which the low density siliceous structure is formed, or the molecular structure of the organosilanes.
  • the thickness of the low density, porous 3-D structure which directly correlates to the size of the bead, could be controlled (e.g., from 1 nm to 2500 nm) by, for example, modifying the quantity of the silane-containing molecules (e.g., trialkoxysilane or sodium silicate), the reaction time, and time lapse between reaction steps and such kind of reaction parameters.
  • the silane-containing molecules e.g., trialkoxysilane or sodium silicate
  • the thickness of the 3-D structure can be about 1 to 2500 nm thick. In certain embodiments, the thickness can be about 1 to 10 nm thick. In certain embodiments, the thickness can be about 1 to 20 nm thick. In certain embodiments, the thickness can be about 1 to 30 nm thick. In certain embodiments, the thickness can be about 1 to 40 nm thick. In certain embodiments, the thickness can be about 1 to 50 nm thick. In certain embodiments, the thickness can be about 1 to 60 nm thick. In certain embodiments, the thickness can be about 1 to 100 nm thick. In certain embodiments, the thickness can be about 1 to 500 nm thick. In certain embodiments, the thickness can be about 1 to 1000 nm thick. In certain embodiments, the thickness can be about 1 to 2000 nm thick.
  • the magnetic nanoparticle(s) is or are embedded in the 3-D structure.
  • the resulting bead can have a thickness (e.g., the longest dimension of the bead or a diameter if the bead is a sphere) of about 50 nm to about 3000 nm, about 50 nm to about 2000 nm, about 50 to 1000 nm, 50 to 500 nm, or 50 to 100 nm.
  • the bead can have a diameter of about 500 nm.
  • the bead can have a diameter of about 100 nm.
  • the bead can have a diameter of about 50 nm.
  • one or more functional groups can be introduced within or on the surface of the bead.
  • the functional groups may be introduced during the formation of the coating. For example, during the crosslinking process, precursors containing such functional groups can be added, in particular, during the ending stage of the cross- linking process.
  • the functional groups may also be introduced after the formation of the bead, for example, by introducing functional groups to the surface of the bead by chemical modification. In certain embodiments, the functional groups are inherent in the bead or in the coating.
  • Examples of the functional groups include, but are not limited to amino, mercapto, carboxyl, phosphonate, biotin, streptavidin, avidin, hydroxyl, alkyl or other hydrophobic molecules, polyethylene glycol or other hydrophilic molecules, and photo cleavable, thermo cleavable or pH responsive linkers.
  • the obtained bead can be further purified.
  • the purification may include use of dialysis, tangential flow filtration, diafiltration, or
  • the bead having a low density, porous 3-D structure prepared herein may be operably linked to one or more analyte-capturing members and one or more PEG compounds, using methods described herein and/or conventional methods known in the art.
  • the method can comprise conjugating an analyte-capturing member to a bead provided herein to form a conjugate; and treating the conjugate with a PEG compound, wherein the bead comprises PEG within or on the surface.
  • the bead further comprises one or more functional groups selected from the group consisting of nitrogen-containing group, sulfur-containing group, phosphorus- containing group, epoxy-containing group or combination thereof.
  • the analyte-capturing member is conjugated to the bead via covalent linkage to form the conjugate.
  • the analyte-capturing member is operably linked to the bead through biotin-streptavidin interaction, protein A or G- antibody interaction or DNA-protein interaction.
  • the analyte-capturing member is conjugated to the bead via non-covalent linkage, such as hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interaction.
  • the conjugate is further treated with a PEG compound.
  • the functional group in PEG compound reacts with the groups on the surface of the conjugate, such that the surface of the conjugate is further coated with PEG compound.
  • the PEG compound comprises maleimide functional group.
  • the PEG compound can be maleimide-PEG or maleimide-PEG-amine.
  • the PEG compound can be added at a concentration of about 5-200 ⁇ g/mg conjugate, for example, 10-200 ⁇ g/mg conjugate, 20-200 ⁇ g/mg conjugate, 30-200 ⁇ g/mg conjugate, 40-200 ⁇ g/mg conjugate, 50-200 ⁇ g/mg conjugate, 60-200 ⁇ g/mg conjugate, 70-200 ⁇ g/mg conjugate, 80- 200 ⁇ g/mg conjugate, 90-200 ⁇ g/mg conjugate, 100-200 ⁇ g/mg conjugate, 110 ⁇ g/mg conjugate, 120 ⁇ g/mg conjugate, 130 ⁇ g/mg conjugate, 140 ⁇ g/mg conjugate, 150 ⁇ g/mg conjugate, 160 ⁇ g/mg conjugate, 170 ⁇ g/mg conjugate, 180 ⁇ g/mg conjugate, 190 ⁇ g/mg conjugate and
  • the bead used for producing the composite of the present disclosure further comprises nitrogen-containing group, sulfur- containing group, carbon-containing group and phosphorus-containing group within or on the surface. In such situations, after conjugating an analyte-capturing member to the bead, the maleimide-PEG is added at a concentration of about 50-200 ⁇ g/mg conjugate, in particular about 140 ⁇ g/mg conjugate.
  • the bead used for producing the composite of the present disclosure further comprises nitrogen-containing group, sulfur- containing group, phosphorus-containing group and epoxy-containing group within or on the surface. In such situations, after conjugating an analyte-capturing member to the bead, the maleimide-PEG is added at a concentration of about 5-20 ⁇ g/mg conjugate, in particular about 8 ⁇ g/mg conjugate.
  • the composite obtained after the treatment of conjugate with PEG compound can provide an enhanced cell capture yield by 10-60%, for example, by 20-60%, 20-50%, 20-40%, 20-30%, 20%, 30% and the like.
  • the size of the composite thus obtained may depend on the size of the bead (the size and the number of the magnetic nanoparticles in the bead), the thickness of the optional low density porous 3-D structure, and the thickness of the PEG compound coating.
  • the diameter of the composite ranges from about 100 to 3000 nm. If the size of the composite is less than 100 nm, the magnetic property of the composite may be insufficient to provide efficient direct magnetic manipulation of the composite.
  • the composite may settle too fast in the solution when mixing with the sample containing the rare cells, which may lead to inhomogeneous capture of representative rare cells.
  • the magnetic moment and magnetic force exerted on each rare cell will be larger with larger sizes of beads, which may affect more of cell viability.
  • Another aspect of the present disclosure relates to composite prepared by the methods provided herein.
  • the composite prepared in the present disclosure can be further characterized for the low density, porous 3-D structure, such as density, porosity, surface areas, thickness etc. of the 3-D structure.
  • the analyte-capturing members and optional payload in the composite may be characterized as well, such as the amount of the analyte-capturing member or the payload.
  • the present disclosure provides a method for capturing rare cells in a sample by mixing the composite provided herein with the sample, and detecting the rare cells binding to the composite.
  • the composite of the present disclosure may provide high capture specificity and high capture yield.
  • the rare cells are CTCs.
  • the CTCs can be directly separated from whole blood sample.
  • the blood sample can be partitioned first to separate out plasma from cells, so that plasma can be used to detect other biomarkers, for example proteins or nucleic acids.
  • the comprehensive circulating markers e.g., cells, protein, nucleic acids
  • together can provide better diagnostic and therapeutic guidance value.
  • the composite comprises magnetic nanoparticles in the bead.
  • the composite-rare cell conjugate may be enriched or separated by applying a magnetic field using a permanent magnet, a magnetic column, a magnetic material patterned structure or device, or a magnetic sifter.
  • the composite-rare cell conjugate may be enriched or separated using a magnetic sifter such as those described in US patent Nos. US7615382B2 and US8481336B2, the content of which is incorporated herein in their entirety.
  • the enriched composite-rare cell conjugate may be further washed in the presence of magnetic field and then collected. This provides composite-rare cell conjugate which does not need to be processed to remove the composite before the captured rare cell is used for further investigation or application.
  • the presence of rare cells in the sample can be identified according to the barcode of the composite that captures the rare cells.
  • the rare cells can be captured by fluorescent composite and viewed under a fluorescent microscope, which allows a simultaneous rare cell isolation and identification.
  • the composite-rare cell conjugate is further processed via methods known in the art, including dissolution, lyze, de-paraffin, filtering, centrifuge, vacuum, dispersing, flowing, condensation, or a combination thereof, to remove the composite.
  • the captured rare cells are subject to further analysis (e.g., FACS, microscopy) for identification.
  • the rare cells can be lyzed to detect the analyte in the cytosol or the nucleus of the cells.
  • separating and detecting of the captured cell can be engineered to be automatic with robotic liquid handlers, microfluidic flow cells, or specially designed flow devices for example a magnetic sifter or patterned magnetic structure containing devices.
  • the presence of the rare cells and/or the identification of the rare cells is indicative of a disease (for example, tumor) or can help a doctor to choose a treatment of for the disease.
  • This example illustrates the preparation of bead comprising nanoparticles of gold and semiconductor quantum dots and low density siliceous structure.
  • the low density siliceous structure is a versatile and flexible platform for making biocompatible nanoparticles.
  • Au nanoparticles synthesized in either water solution or organic solutions could be utilized. Briefly, Au was precipitated out at the sample vial bottom after centrifuge at 13k rpm for 15min, then silane molecules such as aminopropyltrimethoxysilane and TMAOH was added. The reaction solvent was adjusted using a higher number alcohol, such as butanol or proponol.
  • nanoparticles with the highly porous siliceous structure were collected and stored within physiological buffer solutions through centrifugal filtering, centrifugation, dialysis or any other solution exchange methods.
  • the resulting Au bead was observed under TEM.
  • the nanoparticle core size was about 20 nm and hydrodynamic size was about 60 nm.
  • the siliceous coating was not obvious from the TEM.
  • semiconductor quantum dots in the form of individual nanocrystal or nanocrystal clusters could also be incorporated within the highly porous/low density siliceous structure.
  • CdSe/ZnS nanoparticles in organic solvents such as chloroform, Toluene, or Hexane could be precipitated out by adding methanol and then through centrifugation. The nanocrystal pellet was then re-dispersed in
  • the resulting CdSe/ZnS bead was observed under TEM, and an exemplary TEM image was shown in Figure 2.
  • the nanoparticle core size was about 10 nm and hydrodynamic size was about 200 nm.
  • the siliceous coating was not obvious from the TEM.
  • Magnetic nanoparticles were formed by clustering multiple small particles and then were coated. The clustering happened with the addition of a worse solvent for generating dispersed nanoparticles, such as butanol or isopropanol, followed by the addition of the silanization reagents to form the low density porous 3-D structure under constant blowing of air bubbles.
  • the bead as prepared was observed under TEM. As shown in TEM image, each large core nanoparticle comprised a cluster of small nanoparticles, and the coating was substantially invisible under TEM.
  • the magnetic particles had high magnetic response that they could be directly captured using a magnet. This allowed generation of dry particles to measure the mass of the material.
  • the dry mass of particles before and after coating was quantified as follows. 200 ⁇ of the coated particle solution was pipetted out into a centrifugal vial whose mass was pre-measured. Coated magnetic nanoparticles were captured to the side of the vial wall, and the supernatant was removed. The captured particles were washed with water. At the end, the particles absorbed to the side wall were left to dry in the open vial under a fume hood. The mass of the vial with the dry coated particles were measured.
  • the dry coated particle mass was calculated by subtraction of the mass of the vial from the mass of the vial with the dry coated particles inside.
  • uncoated particles corresponding to the same amount of the magnetic material as in the coated nanoparticles, assuming an 80% coating processing yield, was captured to the side of the vial, and dried.
  • the dry mass of the particles before coating was measured by subtraction of the mass of the vial from the mass of the vial with the dry uncoated particles inside.
  • the mass of the coating was equal to the mass of the dry coated particles minus the dry mass of particles before coating.
  • the total volume of the coating was calculated using the number of large particles in the above mass multiplied by the volume of the coating of each individual large nanoparticles.
  • the particles were suspended in an aqueous solution, and the volume of the
  • the number of large particles in the mass was calculated by dividing the total number of small nanoparticles by the number of small nanoparticles in each large
  • the total number of small nanoparticles was estimated by dividing the mass of total magnetic material by the mass of an individual small nanoparticle (i.e. calculated using the size and density of the small nanoparticle). The number of small nanoparticles in each individual large particle was counted from the TEM micrograph. Hence, the total volume of the coating can be calculated as the volume of coating of a large nanoparticle multiplied by the total number of the large nanoparticles.
  • the density of the low density siliceous structure prepared herein is only 0.32 mg/cm 3 , which is significantly lower than the density of some reported silica coatings, for example, those reported in Vincent et al (Vincent, A. et al, J. Phys. Chem. C 2007, 1 1 1, 8291- 8298), that have a density of 1-2 g/cc and are 10 4 denser than the siliceous structure provided herein.
  • the surface area and the pore volume of the porous bead were measured with dry mass of the porous bead. If measured with a porous bead sample suspended in an aqueous solution, the pore volume and the surface area are expected to be much higher than the measurements with the dry mass, as the density of the coating has been shown to be at least 10 4 lower than those reported in the art.
  • the measured density based on the dry power samples does not reflect the real density of the 3-D structure because of the ultralow density of the 3-D structure, the framework easily collapses during the drying process, hence providing much smaller numbers in the porosity measurement than when the 3-D structure is fully extended, for example, like when the porous bead is fully extended in a buffer solution.
  • This example illustrates the preparation of EpCAM bead (bead conjugated with anti -EpCAM antibody) and the composite of the present disclosure.
  • the beads comprising superparamagnetic iron oxide nanoparticles coated with low density 3D structure were prepared as shown in Example 3.
  • the beads were adjusted to about 1 mg/ml and covalently conjugated to 0.3 mg/ml streptavidin through a crosslinker Sulfo-SMCC after overnight incubation.
  • Anti-EpCAM antibody was biotinylated using commercial biotinylation kit following standard protocol.
  • EpCAM beads were prepared by incubating 1 mg of streptavidin-beads together with 40 ⁇ g of biotinylated anti -EpCAM antibody overnight at 4°C. After washing and blocking, the EpCAM beads were resuspended in buffer at a concentration of 1 mg/ml. The EpCAM beads were then treated with 6 uM of N-ethyl maleimide, maleimide-PEG and maleimide-PEG-amine, respectively to give the Composite 1, 2 and 3.
  • Composite 2 as prepared in Example 4 was suspended in buffer at a concentration of 1 mg/ml and was used to carry out cell capture assay. The general procedure for cell capture is shown in FIG. 1.
  • This examples illustrates the cell capture by composite of the present disclosure for two cancer cell lines HI 650 non-small cell lung cancer cells and MDA-MB- 231 breast cancer cells.
  • the cell capture assay was carried out as described in Example 5, except that the incubation time was varied from 15 min to 90 min. As shown in FIG. 5, an incubation time of 60 min achieves the highest cell capture yield.
  • Seradyne beads Ocean Nanotech beads, respectively
  • biotinylated anti-EpCAM antibody overnight at 4°C followed by washing and blocking, thereby providing streptavidin-anti EpCAM magnetic beads serving as the comparative beads.
  • Composite 2 of the present disclosure and the comparative beads were used to capture 100 HI 650 cells from 1 ml of whole blood, as described in Example 5. As shown in
  • the composite obtained from the treatment of EpCAM bead with maleimide-PEG increased the cell capture yield by about 10% compared to the EpCAM bead without any treatment.
  • the composites obtained from the treatment of EpCAM bead with N-ethyl maleimide or maleimide-PEG- amine showed no increase in cell capture yield compared to the EpCAM bead without any treatment.
  • the composite obtained from the treatment of EpCAM bead with maleimide-PEG increased the cell capture yield by about 30% compared to the EpCAM bead without any treatment.
  • the composite obtained from the treatment of EpCAM bead with N-ethyl maleimide decreased the cell capture yield, and the composite obtained from the treatment of EpCAM bead with maleimide-PEG-amine showed similar cell capture yield.
  • This example illustrates the cell capture by composite obtained from the treatment of EpCAM bead with two different doses of maleimide-PEG.
  • EpCAM beads from batch 2 in Example 10 were treated with 2 different doses of maleimide-PEG: 138.9 ⁇ g and 194.4 ⁇ g maleimide-PEG /mg beads, to form the composites of the present disclosure, which were further used in cell capture assays as described in Example 5.
  • the EpCAM bead without any treatment was also used to carry out the cell capture for comparison.
  • the composites obtained from the maleimide-PEG treatment at the two different doses increased the cell capture yield by 35% and 20%, respectively.
  • This example illustrates the cell capture by composite obtained from the treatment of EpCAM bead with four different doses of maleimide-PEG.
  • EpCAM beads comprising streptavidin as well as sulphur-containing group, nitrogen-containing group, phosphorus-containing group and epoxy-containing group on the surface were used to prepare EpCAM beads, which were treated with 4 different doses of maleimide-PEG to form the composites of the present disclosure, as described in Example 4.
  • the composites obtained were used to carry out cell capture assay as described in Example 5.
  • maleimide-PEG treatment at a dose between 6 ⁇ g/mg beads and 16 ⁇ g/mg beads increased the cell capture yield by 20-40%.
  • Maleimdie-PEG treatment at a dose of about 8 ⁇ g maleimide PEG /mg beads might be optimal for EpCAM beads with SNEP surface.
  • the beads were prepared as in Example 3, except that both magnetic nanoparticles and quantum dots were coated, so that the resulting beads are both magnetic and fluorescent.
  • the beads were further used to prepare the composite of the present disclosure as in Example 4, in which maleimide-PEG was used to treat the EpCAM beads.

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Abstract

La présente invention concerne d'une manière générale des composites pour la capture de cellules rares dans des échantillons, leur préparation et leur utilisation. Le composite comprend un grain lié de manière fonctionnelle à un composé de polyéthylène glycol (PEG) ; un élément de capture d'analyte lié de manière fonctionnelle au grain, ledit élément de capture d'analyte se liant de façon spécifique à un marqueur de surface des cellules rares, le composite ayant un diamètre allant d'environ 100 à environ 3000 nm. Le composite peut capturer des cellules rares avec un haut rendement de capture de cellules, une capture non-spécifique faible, une viabilité cellulaire élevée et une faible formation de flocons.
PCT/US2016/048801 2015-08-25 2016-08-26 Composites destinés à la séparation de cellules rares WO2017035419A1 (fr)

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