WO2017035419A1

WO2017035419A1 - Composites for separating rare cells

Info

Publication number: WO2017035419A1
Application number: PCT/US2016/048801
Authority: WO
Inventors: Aihua Fu; Hua Zhou
Original assignee: NVIGEN Inc
Current assignee: NVIGEN Inc
Priority date: 2015-08-25
Filing date: 2016-08-26
Publication date: 2017-03-02
Anticipated expiration: 2018-02-25

Abstract

The present invention generally relates to composites for capturing rare cells in samples, the preparation and the use thereof. The composite comprises 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 3000 nm. The composite may capture rare cells with high cell capture yield, low non-specific capture, high cell viability and low clump formation.

Description

COMPOSITES FOR SEPARATING RARE CELLS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional patent application no.

62/209,758, filed August 25, 2015, the disclosure of which is incorporated herein by reference.

FIELD OF THE DISCLOSURE

[0002] The present invention generally relates to composites for capturing rare cells in samples.

BACKGROUND OF THE DISCLOSURE

[0003] 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. When 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. However, 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. BRIEF SUMMARY OF THE DISCLOSURE

[0004] 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.

[0005] In one aspect, 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.

[0006] In some embodiments, the PEG compound is maleimide-PEG or the derivative thereof. [0007] In some embodiments, the rare cells are present in the sample at less than 100 cells/ml. In some embodiments, the rare cells are circulating tumor cells (CTCs). In some embodiments, the CTCs can be directly separated from whole blood sample. In other embodiments, 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. In some embodiments, the comprehensive circulating markers (e.g., cells, protein, nucleic acids) together can provide better diagnostic and therapeutic guidance value.

[0008] In some embodiments, the bead has a diameter ranging from about 50 to about

3000 nm.

[0009] In some embodiments, the bead comprises at least one magnetic nanoparticle.

In some embodiments, 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. In some embodiments, the magnetic nanoparticle has a diameter ranging from about 1 nm to about 100 nm.

[0010] In some embodiments, 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.

[0011] In some embodiments, 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.

[0012] In some embodiments, 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.

[0013] In some embodiments, the analyte-capturing member in the composite provided herein is an antibody. In some embodiments, the antibody is anti-EpCAM antibody.

[0014] In some embodiments, the analyte-capturing member in the composite provided herein is a ligand of the cell surface marker. In some embodiments, the ligand is a peptide, a small molecule, an aptamer, a hormone, a drug, a toxin or a neurotransmitter.

[0015] In some embodiments, 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.

[0016] In some embodiments, 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.

[0017] In some embodiments, 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.

[0018] In another aspect, 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.

[0019] In some embodiments, 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.

[0020] In some embodiments, 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 μg/mg conjugate.

[0021] In some embodiments, 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 μg/mg conjugate, in particular about 140 μg/mg conjugate.

[0022] In some embodiments, 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 μg/mg conjugate, in particular about 8 μg/mg conjugate.

[0023] In a further aspect, 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.

[0024] In some embodiments, 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. [0025] In some embodiments, the steps of separating and detecting are engineered to be automatic with robotic liquid handlers or specially designed flow devices.

[0026] In some embodiments, 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. In some embodiments, 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). In some embodiments, different cell contents can be identified when cells are alive, fixed, intact or after cell lysis.

[0027] In some embodiments, the rare cells are immune cells or circulating cells that can indicate the presence of a disease or the response to a treatment. In some embodiments, 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. In some embodiments, the treatment is immunotherapy.

[0028] In some embodiments, the presence of the rare cells in the sample and/or the identification of the rare cells is indicative of a disease. In some embodiments, the disease is tumor, inflammation, infectious disease, autoimmune disease, or neurodegenerative disease.

BRIEF DESCRIPTION OF THE FIGURES

[0029] FIG. 1 shows a general procedure for cell capture using the composite according to the embodiments of the present disclosure.

[0030] 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.

[0031] FIG. 3 shows the capture yield for spike-in CTC capture from whole blood samples for two different cell lines.

[0032] FIG. 4 shows the capture yield at different bead volume.

[0033] FIG. 5 shows the capture yield in cell capture assay using different incubation times.

[0034] FIG. 6 shows the capture yield of the composite of the present disclosure in comparison to the beads from other vendors.

[0035] FIG. 7 A and 7B shows the cell capture yield of two different batches of

EpCAM bead and the composites treated with N-ethyl maleimide, maleimide-PEG and maleimide-PEG amine. [0036] 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.

[0037] 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.

[0038] FIG. 10 shows image of cell captured by fluorescent composite of the present disclosure viewed under a fluorescent microscope.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0039] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0040] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0041] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

[0042] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0043] 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.

[0044] Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible.

[0045] It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" includes a plurality of compounds. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

[0046] Composite

[0047] 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. [0048] Bead

[0049] The term "bead" as used herein, 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.). In some embodiments, the bead comprises at least one magnetic nanoparticle.

[0050] In some embodiments, the magnetic nanoparticle of the bead provided herein may comprise a superparamagnetic iron oxide (SPIO) nanoparticle. The SPIO nanoparticle is an iron oxide nanoparticle, either maghemite (y-Fe₂0₃) or magnetite (Fe₃0₄), 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,

Superparamagnetic Iron Oxide Nanoparticles: Synthesis, Surface Engineering, Cytotoxicity and Biomedical Applications, published by Nova Science Pub Inc, 2011). In one

embodiment, 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). In another embodiment, 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). In another embodiment, 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). In certain embodiments, the SPIO nanoparticles are those disclosed in US patent application PG Pub 20100008862.

[0051] In some embodiments, 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

(poly(lactic-co-glycolic acid) (see, e.g., Minsoung Rhee et al, Adv. Mater. 23, H79-H83 (2011)), PCL (polycaprolactone) (see, e.g., Marianne Labet et al, Chem. Soc. Rev. 38, 3484- 3504 (2009)), PEG (poly ethylene glycol) or other polymers); siliceous nanoparticles; and non-SPIO magnetic nanoparticles (e.g., MnFe204 (see, e.g., Jae-Hyun Lee et al, Nature

Medicine 13, 95-99 (2006)), synthetic antiferromagnetic nanoparticles (SAF) (see, e.g., A. Fu et al, Angew. Chem. Int. Ed. 48, 1620-1624 (2009)), and other types of magnetic

nanoparticles). In certain embodiments, the non-SPIO nanoparticle is a colored nanoparticle, for example, a semiconductor nanoparticle such as a quantum dot.

[0052] 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. For example, gold nanoparticles can be made by reduction of chloroaurate solutions (e.g., HAuCl₄) by a reducing agent such as citrate, or acetone dicarboxulate. For another example, CdS semiconductor nanoparticle can be prepared from Cd(C10₄)₂ and Na₂S on the surface of silica particles. For another example, 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). 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. For another example, 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. For another example, 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. For another example, SAFs, a non- SPIO magnetic nanoparticle, can be prepared by depositing a ferromagenetic layer on each of the two sides of a nonmagnetic space layer (e.g., ruthenium metal), along with a chemical etchable copper release layer and protective tantalum surface layers, using ion-bean deposition in a high vacuum, and the SAF

nanoparticle can be released after removing the protective layer and selective etching of copper.

[0053] The diameter of the magnetic nanoparticles ranges from about 1 nm to about

100 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. For example, to control the size of nanoparticles, 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. For another example, 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. To obtain nanoparticles within a desired size range, 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).

[0054] The shape of the magnetic nanoparticles can be spherical, cubic, rod shaped

(see, e.g., A. Fu et al, Nano Letters, 7, 179-182 (2007)), tetrapod-shaped (see, e.g., L. Manna et al, Nature Materials, 2, 382-385 (2003)), pyramidal, multi-armed, nanotube, nanowire, nanofiber, nanoplate, or any other suitable shapes. 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). For example, when the nanoparticles are prepared by the bottom-up process (i.e. from molecule to nanoparticle), a shape controller which adsorbs strongly to a specific crystal plane may be added to control the growth rate of the particle.

[0055] 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

substantially the same materials. A cluster of heterogeneous mini-nanoparticles refers to a pool of particles having different features or characteristics or consisting of substantially different materials. For example, 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. When the nanoparticles are associated with a coating (as described below), different nanoparticles in a heterogeneous nanoparticle pool do not need to associate with each other at first, but rather, they could be individually and separately associated with the coating.

[0056] In some embodiments, a bead provided herein comprises a plurality of magnetic nanoparticles. For example, 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.

[0057] 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. For example, a magnet (e.g., magnetic grid) can be placed in the proximity of the beads so as to attract the magnetic beads.

[0058] In some embodiments, 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 π-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) and non covalent bonds (e.g., hydrophobic, hydrophilic, charge-charge, or π-stacking interactions, van der Waals force, London dispersion force, Mechanical bond, Halogen bond, Aurophilicity, Intercalation, Stacking, Entropic force, and chemical polarity).

[0059] In some embodiments, 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).

[0060] As used herein, 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). (A. Vincent, et. al., J. Phys. Chem. C, 2007, 111, 8291- 8298; J. E. Lee, et. al, J. Am. Chem. Soc, 2010, 132, 552-557; Y. -S. Lin, et. al, J. Am. Chem. Soc, 2011, 133, 20444-20457; Z. Lu, Angew. Chem. Int. Ed., 2010, 49, 1862-1866.)

[0061] In certain embodiments, 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/cc to 600 mg/cc, from 0.01 mg/cc to 500 mg/cc, from 0.1 mg/cc to 800 mg/cc, from 0.1 mg/cc to 700 mg/cc, from 0.1 mg/cc to 1000 mg/cc, from 1 mg/cc to 1000 mg/cc, from 5 mg/cc to 1000 mg/cc, from 10 mg/cc to 1000 mg/cc, from 20 mg/cc to 1000 mg/cc, from 30 mg/cc to 1000 mg/cc, from 30 mg/cc to 1000 mg/cc, from 30 mg/cc to 900 mg/cc, from 30 mg/cc to 800 mg/cc, or from 30 mg/cc to 700 mg/cc).

[0062] 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. In helium pycnometry method, dry powders of the testing 3-D structure are filled with helium, and the helium pressure produced by a variation of volume is studied to provide for the density. 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 3-D structure is fully extended in a buffer solution. In certain embodiments, 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. For example, 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. Similarly, the 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.

[0063] In certain embodiments, the beads can be dispersed as a plurality of large nanoparticles coated with the 3-D structure in an aqueous solution. In such case, 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. For each individual large nanoparticle, 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.

[0064] The number of large nanoparticles for a given core mass can be calculated using any suitable methods. For example, an individual large nanoparticle may be composed of a plurality of small nanoparticles which are visible under TEM. In such case, 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. By dividing the core mass with the average mass of a small nanoparticle, the total number of small nanoparticles can be estimated. For an individual large nanoparticle, 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.

[0065] In certain embodiments, the low density, porous 3-D structure is highly porous. For example, 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.

[0066] The porosity of the 3-D structure can be characterized by the Gas/Vapor adsorption method. In this technique, usually 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. 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.

[0067] In some embodiments, 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

TEM. The bead (e.g., nanoparticle(s) coated with or embedded in a low density, porous 3-D structure) can be observed or measured by ways known in the art. For example, 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. In certain embodiments, the thickness of the 3-D structure is measured as 10s, 100s, 1000s nanometer range by DLS, but cannot be readily determined under TEM. For example, when the beads provided herein are observed under TEM, the nanoparticles can be identified, however, the low density, porous 3-D structure cannot be obviously observed, or is almost transparent. This distinguishes the low density, porous 3-D structures from those reported in the art that comprise nanoparticles coated with crosslinked and size tunable 3-D structure, including the mesoporous silica nanoparticles or coating (see, e.g., J. Kim, et. al, J. Am. Chem. Soc, 2006, 128, 688-689; J. Kim, et. al, Angew. Chem. Int. Ed., 2008, 47, 8438-8441). This feature also indicates that the low density, porous 3-D structure has a much lower density and/or is highly porous in comparison to other coatings known in the art. 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)). As 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. For example, when 3-D structure is loaded with organic fiuorophores such as Rhodamin, over 105 Rhodamin molecules can be loaded to 3-D structure of one nanoparticle.

[0068] In some embodiments, 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).

[0069] In certain embodiments, the silane-containing molecule comprises an organosilane, which is also known as silane coupling agent. Organosilane has a general formula of R_xSiY_4-x, wherein 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 OCH₃, OC₂H₅, and OC₂H₄OCH₃. For example, 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 trimethoxysilane, 3- glycidoxy-propyltriethoxysilane, 3-isocyanatopropyltriethoxysilane, and 3- cyanatopropyltriethoxy silane. [0070] In some embodiments, the bead provided herein can be colored or non-colored.

"Colored" as used herein, means that the bead is capable of generating a color signal under a suitable condition. For example, 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.

[0071] In certain embodiments, the bead is bar-coded or associated with a detectable agent. The term "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" as used herein, refers to a molecule capable of generating a detectable signal that distinguishes one bar-coded bead from another. For example, the colored bead may comprise a colored nanoparticle (e.g. a quantum dot) which emits a detectable color signal at a known wave length.

[0072] In certain embodiments, the characteristics or the identity of a bar-coded bead is based on multiplexed optical coding system as disclosed in Han et al, Nature

Biotechnology, Vol. 19, pp: 631-635 (2001) or US Pat. Appl. 10/185, 226. Briefly, multicolor semiconductor quantum-dots (QDs) are embedded in the bead. For each QD, there is a given intensity (within the levels of, for example 0-10) and a given color

(wavelength). 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 (m¹¹-¹).

[0073] In certain embodiments, 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.

[0074] Examples of 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-Amino-6-chloro-2- methoxyacridine; ABQ; Acid Fuchsin; ACMA (9-Amino-6-chloro-2-methoxyacridine);

Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin Feulgen SITSA; Aequorin (Photoprotein); AFPs— AutoFluorescent Protein— (Quantum Biotechnologies); Alexa® Fluor 350; Alexa® Fluor 405; Alexa® Fluor 500; Alexa Fluor 430™; Alexa Fluor 488™; Alexa Fluor 532™; Alexa Fluor 546™; Alexa Fluor 568™; Alexa Fluor 594™;

Alexa Fluor 633™; Alexa Fluor 647™; Alexa Fluor 660™; Alexa Fluor 680™; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; AMCA

(Aminomethylcoumarin); AMCA-X; Aminoactinomycin D; Aminocoumarin;

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- TAG™ CBQCA; ATTO-TAG™ 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; BOBO™-l; BOBO™- 3; Bodipy 492/515; Bodipy 493/503; Bodipy 500/510; Bodipy 505/515; Bodipy 530/550; Bodipy 542/563; Bodipy 558/568; Bodipy 564/570; Bodipy 576/589; Bodipy 581/591;

Bodipy 630/650-X; Bodipy 650/665-X; Bodipy 665/676; Bodipy Fl; Bodipy FL ATP;

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-PRO™- 1; BO- PRO™-3; Brilliant Sulphoflavin FF; BTC; BTC-5N; Calcein; Calcein Blue; Calcium Crimson™;

Calcium Green; Calcium Green- 1 Ca 2+ Dye; Calcium Green-2 Ca 2+ ; Calcium Green-5N Ca 2+ ; Calcium Green-C18 Ca 2+ ; Calcium Orange; Calcofluor White; Carboxy-X- rhodamine (5-ROX); Cascade Blue™; Cascade Yellow; Catecholamine; CCF2 (GeneBlazer); CFDA; Chlorophyll; Chromomycin A; Chromomycin A; CL- ERF; CMFDA; Coumarin Phalloidin; C-phycocyanine; CPM Methylcoumarin; CTC; CTC Formazan; Cy2™; Cy3.1 8; Cy3.5™; Cy3™; Cy5.1 8; Cy5.5™; Cy5™; Cy7™; cyclic AMP Fluorosensor (FiCRhR); Dabcyl; Dansyl; Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DFIPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3' DCFDA; DCFH (Dichlorodihydro fluorescein Diacetate); DDAO; DHR (Dihydrorhodamine 123); Di-4-A EPPS; Di-8- A EPPS (non-ratio); DiA (4-Di-16-ASP); Dichlorodihydro fluorescein Diacetate (DCFH); DiD-Lipophilic Tracer; DiD (DiIC18(5)); DIDS; Dihydrorhodamine 123 (DHR); Dil

(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;

Erythrosin ITC; Ethidium Bromide; Ethidium homodimer-1 (EthD-1); Euchrysin; EukoLight;

Europium (III) chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FIF (Formaldehyd

Induced Fluorescence); FITC; Flazo Orange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald; FluoroGold (Hydroxystilbamidine); Fluor-Ruby; Fluor X; FM 1-

43™; FM 4-46; Fura Red™ (high pH); Fura Red™/Fluo-3; Fura-2; Fura-2/BCECF; Genacryl

Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow 5GF;

GeneBlazer (CCF2); Gloxalic Acid; Granular blue; Haematoporphyrin; Hoechst 33258;

Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine

(FluoroGold); Hydroxytryptamine; Indo-1, high calcium; Indo-1, low calcium;

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;

Calcein/Ethidium homodimer; LOLO-1; LO-PRO-1; Lucifer Yellow; Lyso Tracker Blue; Lyso Tracker Blue -White; Lyso Tracker Green; Lyso Tracker Red; Lyso Tracker Yellow;

LysoSensor Blue; LysoSensor Green; LysoSensor Yellow/Blue; Mag Green; Magdala Red

(Phloxin B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-Indo-1; Magnesium Green;

Magnesium Orange; Malachite Green; Marina Blue; Maxilon Brilliant Flavin 10 GFF;

Maxilon Brilliant Flavin 8 GFF; Merocyanin; Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane; Monobromobimane

(mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); BD; NBD

Amine;Nile Red; Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; Nuclear Yellow;

Nylosan Brilliant lavin E8G; Oregon Green; Oregon Green 488-X; Oregon Green™; Oregon

Green™ 488; Oregon Green™ 500; Oregon Green™ 514; Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE- TexasRed [Red 613]; Phloxin

B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R;

PhotoResist; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA;

Pontochrome Blue Black; POPO-1; POPO-3; PO— PRO-1; PO-PRO-3; Primuline; Procion

Yellow; Propidium lodid (PI); PYMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Red 613 [PE-TexasRed] ; Resorufm; RH 414;

Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G;

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

Red B; Sevron Orange; Sevron Yellow L; SITS; SITS (Primuline); SITS (Stilbene

Isothiosulphonic Acid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein; SNARF1;

Sodium Green; SpectrumAqua; SpectrumGreen; SpectrumOrange; Spectrum Red; SPQ (6- methoxy-N-(3- sulfopropyl)quinolinium); Stilbene; Sulphorhodamine B can C;

Sulphorhodamine Extra; SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO 15; SYTO 16;

SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO 22; SYTO 23; SYTO 24; SYTO 25;

SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO 59; SYTO 60;

SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO 82; SYTO 83;

SYTO 84; SYTO 85; SYTOX Blue; SYTOX Green; SYTOX Orange; Tetracycline;

Tetramethylrhodamine (TRITC); Texas Red™; Texas Red-X™ conjugate;

Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thio flavin 5; Thioflavin S;

Thioflavin TCN; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofiuor White); TMR; TO-

PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; Tricolor (PE-Cy5); TRITC

TetramethylRodaminelsoThioCyanate; True Blue; TruRed; Ultralite; Uranine B; Uvitex SFC;

WW 781; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; YO-PRO-1; YO-

PRO-3; YOYO-1; YOYO-3, Sybr Green, Thiazole orange (interchelating dyes), fluorescent semiconductor nanostructures, lanthanides or combinations thereof.

[0075] Examples of radioisotopes include, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹1, ³⁵S, ³H, ^mIn, ¹¹²In, ¹⁴C, ⁶⁴Cu, ⁶⁷Cu, ⁸⁶Y, ⁸⁸Y, ⁹⁰Y, ¹⁷⁷Lu, ²¹¹At, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, ³²P, ¹⁸F, ²⁰¹T1, ⁶⁷Ga, ¹³⁷Cs and other radioisotopes.

[0076] Examples of 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.

[0077] In some embodiments, the bead in the composite provided herein carries payload. In some embodiments, 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.

[0078] In some embodiments, 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. For example, during the silanization process, precursors containing such functional groups can be added. These functional groups may also be introduced after the formation of the 3-D structure. For example, 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.

[0079] Analyte-capturing member

[0080] The bead of the composite provided herein is operably linked to an analyte- capturing member.

[0081] The term "operably linked" as used herein, 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. 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.

[0082] In some embodiments, 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 (k₀ff/k_on) when the binding between the two molecules reaches equilibrium. 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 k₀ff) 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)). [0083] Examples of 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) or a guest molecule (e.g., prostaglandin, itraconazole).

[0084] 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

human .gamma.1, .gamma.2, or .gamma.4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Similarly, protein G is recommended for specific binding to antibodies of all mouse isotypes and for antibodies based on human .gamma.3 heavy chains (Guss et al., EMBO J. 5: 1567 1575 (1986)). Avidin (or streptavdin) and biotin can specifically bind to each other to form strong and specific non-covalent association. 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.

[0085] In some embodiments, the analyte-capturing member can be an antibody. In some embodiments, the antibody is against epithelial cell adhesion molecule (EpCAM).

[0086] In some embodiments, the analyte-capturing member can be a ligand of the cell surface marker. In some embodiments, the ligand can be a peptide, small molecule, hormone, a drug, toxin, or neurotransmitter.

[0087] 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. In some embodiments, 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. In such embodiments, 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. In some embodiments, 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.

[0088] Polyethylene glycol (PEG) compound

[0089] The bead of the composite provided herein is also operably linked to PEG compound.

[0090] The term "PEG compound", as used herein, refers to compounds containing PEG chains that operably linked to the bead provided herein. In some embodiments, the PEG compounds are operably linked to the bead such that the bead is coated with the PEG compounds. In some embodiments, a methoxy-group can be at the polymer end. In other embodiments, a hydroxyl group can be at the polymer end.

[0091] In some embodiments, 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. In some embodiments, the PEG compound used herein comprises maleimide functional group. For example, the PEG compound used herein can be maleimide-PEG or maleimide-PEG-amine.

[0092] In principle, 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

2 and 50 monomer units, between 2 and 40 monomer units, between 3 and 40 monomer units, between 4 and 40 monomer units and so on. The longer PEG chains are, the larger diameter the resulting composite has. In some embodiments, depending on the PEG compound used, 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. In some embodiments, the maleimide-PEG or

maleimide-PEG-amine used as the PEG compound of the present disclosure may have a weight average molecular weight (Mw) of about 5000 Da.

[0093] 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.

[0094] Methods for Preparing the Bead comprising a low density, porous 3-D structure

[0095] In some embodiments, 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. In certain embodiments, 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.

[0096] 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).

[0097] 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.

[0098] In some embodiments, 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, FeCl₃, A1C1₃, ZnCl₂, and ZnBr₂). 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). During the silanization process, one or more silane-containing molecules can be added at different stages of the process. For example, in the priming stage, organosilanes such as aminopropyl trimethoxyl silane or mercaptopropyl trimethoxyl silane can be added to initiate the silanization on the core nanoparticle surface. For another example, silane molecules having fewer alkoxy groups (e.g., only 2 alkoxy groups) can be added to the reaction at the growth stage of silanization. For another example, at the ending 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. Alternatively, 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).

[0099] To control the formation of the low density siliceous structure, 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. Without being bound to theory, it is believed that these procedures can help make the reaction medium homogeneous, well dispersed and promote the formation of low density porous 3-D structure with increased voids or porosity. In certain embodiments, 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. For example, 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. In certain

embodiments, sonicating is applied in each stage of the silanization process.

[00100] In certain embodiments, the density reducing procedures comprise introducing at least one alcohol to the reaction. In certain embodiments, the alcohol has at least 3 (e.g., at least 4, at least 5 or at least 6) carbon atoms. For example, the alcohol may have 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more carbon atoms. In certain embodiments, the alcohol can be

monohydric alcohols, or polyhydric alcohols. Illustrative examples of monohydric alcohols include, propanol, butanol, pentanol, hexyl alcohol, etc. [00101] Illustrative examples of polyhydric alcohols include, propylene glycol, glycerol, threitol, xylitol, etc. In certain embodiments, the alcohol can have a saturated carbon chain or an unsaturated carbon chain. An alcohol having a saturated carbon chain can be represented as C_nH(₂n+2₎0 in chemical formula. In certain embodiments, n is no less than 3, or no less than 4, or no less than 5 (e.g., n=3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more). Alcohol with an unsaturated carbon chain has a double or a triple bond between two carbon atoms. In certain embodiments, the alcohol can be a cyclic alcohol, for example, cyclohexanol, inositol, or menthol.

[00102] In certain embodiments, 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). In certain

embodiments, 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%>). In certain embodiments, 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%>,). In certain embodiments, the density reducing procedure comprises introducing air bubbles to the reaction. In certain embodiments, 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.

[00103] Other 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.

[00104] 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.

[00105] 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.

[00106] After the low-density, porous 3-D structure is formed on the surface of the magnetic nanoparticle(s), 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. In some embodiments, the bead can have a diameter of about 500 nm. In some embodiments, the bead can have a diameter of about 100 nm. In some embodiments, the bead can have a diameter of about 50 nm.

[00107] In certain embodiments, 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.

[00108] In certain embodiments, the obtained bead can be further purified. The purification may include use of dialysis, tangential flow filtration, diafiltration, or

combinations thereof.

[00109] 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.

[00110] Methods for Preparing the Composite [00111] Another aspect of the present disclosure relates to methods of preparing the composite provided herein. In some embodiments, 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. In some embodiments, 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.

[00112] In some embodiments, the analyte-capturing member is conjugated to the bead via covalent linkage to form the conjugate. 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.

[00113] In some embodiments, 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.

[00114] After the formation of the conjugate, the conjugate is further treated with a PEG compound. During the treatment, 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. In some embodiments, the PEG compound comprises maleimide functional group. For example, the PEG compound can be maleimide-PEG or maleimide-PEG-amine.

[00115] In some embodiments, to produce the composite of the present disclosure, the PEG compound can be added at a concentration of about 5-200 μg/mg conjugate, for example, 10-200 μg/mg conjugate, 20-200 μg/mg conjugate, 30-200 μg/mg conjugate, 40-200 μg/mg conjugate, 50-200 μg/mg conjugate, 60-200 μg/mg conjugate, 70-200 μg/mg conjugate, 80- 200 μg/mg conjugate, 90-200 μg/mg conjugate, 100-200 μg/mg conjugate, 110 μg/mg conjugate, 120 μg/mg conjugate, 130 μg/mg conjugate, 140 μg/mg conjugate, 150 μg/mg conjugate, 160 μg/mg conjugate, 170 μg/mg conjugate, 180 μg/mg conjugate, 190 μg/mg conjugate and the like.

[00116] In some embodiments, in addition to PEG, 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 μg/mg conjugate, in particular about 140 μg/mg conjugate. [00117] In some embodiments, in addition to PEG, 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 μg/mg conjugate, in particular about 8 μg/mg conjugate.

[00118] Compared with the conjugate without the treatment of PEG compound, 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.

[00119] 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. In some embodiments, to provide high rare cell capture yield while keeping low non-specific binding and low clump formation, 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. On the other hand, if the size of the composite is larger than 3000 nm, 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. Moreover, 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.

[00120] Products by Process

[00121] 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. Optionally, 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.

[00122] Methods of Using the Composite

[00123] Another aspect of 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. [00124] In some embodiments, the rare cells are CTCs. In some embodiments, the CTCs can be directly separated from whole blood sample. In other embodiments, 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. In some embodiments, the comprehensive circulating markers (e.g., cells, protein, nucleic acids) together can provide better diagnostic and therapeutic guidance value.

[00125] In some embodiments, the composite comprises magnetic nanoparticles in the bead. In such embodiments, after the rare cells are captured by the analyte-capturing member of the composite, 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. In some embodiment, 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. In some embodiments, 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.

[00126] In some embodiments, the presence of rare cells in the sample can be identified according to the barcode of the composite that captures the rare cells. For example, the rare cells can be captured by fluorescent composite and viewed under a fluorescent microscope, which allows a simultaneous rare cell isolation and identification.

[00127] In some embodiments, 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.

[00128] In some embodiments, the captured rare cells are subject to further analysis (e.g., FACS, microscopy) for identification. For example, the rare cells can be lyzed to detect the analyte in the cytosol or the nucleus of the cells.

[00129] In some embodiments, 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. [00130] In some embodiments, 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.

EXAMPLE 1

[00131] This example illustrates the preparation of bead comprising nanoparticles of gold and semiconductor quantum dots and low density siliceous structure.

[00132] The low density siliceous structure is a versatile and flexible platform for making biocompatible nanoparticles. For example, to incorporate gold nanoparticles into the siliceous structure, 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. Then the sample was sonicated for a few hours with constant blowing of air bubbles, afterwards, PEG-silane, mercaptopropyltrimethoxysilane and aminopropyltrimethoxysilane were added, the sample was sonicated for additional 2-3 hours. Afterwards, mixture of chlorotrimethylsilane, methanol, and TMAOH or other silane molecules that only have one alkoxyl group connecting with the silicon atom were added to react with surface siloxyl groups presented on the surface of the already grown siliceous structure. After additional sonicating and aging, stable 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.

EXAMPLE 2.

[00133] This example illustrates the preparation of bead comprising nanoparticles of semiconductor quantum dots and low density siliceous structure

[00134] As another example, semiconductor quantum dots in the form of individual nanocrystal or nanocrystal clusters could also be incorporated within the highly porous/low density siliceous structure. For example, 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

aminopropyltrimethoxysilane or mercaptopropyltrimethoxysilane. Afterwards, tetramethyl ammonium hydroxide was added. Then the reaction solvent was adjusted using a higher number alcohol, such as butanol or proponol. After sonicating the sample for 1-4 hours and blowing air bubbles, small amount of aminopropyltrimethoxysilane,

mercaptopropyltrimethoxysilane, polyethyleneoxidesilane and water was subsequently added, and the sample then underwent sonication for another 1 to 4 hours. Then, mixture of chlorotrimethylsilane, methanol, and TMAOH or other silane molecules that only have one alkoxyl group connecting with the silicon atom were added. This sample was then sonicated for another 1-4 hours, followed by overnight aging under mild shaking or vibration. The resulting nanoparticles with low density/highly porous siliceous structure were transferred into physiological buffer solutions by centrifugal filtering, centrifugation, dialysis or any other solution exchange methods. 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.

EXAMPLE 3.

[00135] This example illustrates the preparation and characterization of bead comprising magnetic nanoparticles and low density siliceous structure

[00136] Preparation of the bead

[00137] 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.

[00138] Characterization of density of the coating:

[00139] To calculate the density of the coating, both the dry mass and the volume of the coating were characterized.

[00140] Since 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 μΐ 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. To measure the mass of the particles before coating, 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.

Table 1

[00141] 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

3 3

coating of each large particle was calculated as 4/3 χ π (R - R ), in which the R

with coating core with coating of an individual larg <_^<e nanoparticle was measured using dynamic light scattering (DLS) technique, and the R of the large core particle was directly imaged and measured using TEM.

Table 2

[00142] 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

nanoparticle. 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.

Table 3

[00143] The density of the coating was calculated using the mass of the coating divided by the total volume of the coating, i.e., 0.06 mg/0.1875 x l0^"6m³=0.32 mg/cm³.

[00144] The density of the low density siliceous structure prepared herein is only 0.32 mg/cm³, 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 10⁴ denser than the siliceous structure provided herein.

[00145] Characterization of porosity using BET method:

[00146] Large magnetic nanoparticles after coating were captured to the side of the vial and dried. 2 samples of 65 mg (sample 1) and 45 mg (sample 2) dry mass were prepared for the BET measurement.

[00147] Surface pore sizes were measured using BET method for the dry mass of the coated nanoparticles. The results are shown in the below Tables.

Table 4 Characterization for Sample 1

Table 5 Characterization for Sample 2

[00148] 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 10⁴ lower than those reported in the art.

[00149] 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.

EXAMPLE 4.

[00150] This example illustrates the preparation of EpCAM bead (bead conjugated with anti -EpCAM antibody) and the composite of the present disclosure.

[00151] 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.

[00152] The EpCAM beads were prepared by incubating 1 mg of streptavidin-beads together with 40 μg 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.

EXAMPLE 5.

[00153] This example illustrates the cell capture by composite of the present disclosure. [00154] 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.

[00155] Healthy human blood was centrifuged to remove plasma. Then it was diluted with equal volume of PBS buffer containing 0.2% BSA and 4mM EDTA. 100 of CFSE- stained H1650 cells and 12.5 μΐ of Composite 2 was then added to 1 ml of diluted blood for cell capture assay. After 1 hr of incubation at 4°C, the tube was put against a magnet for about 5 min. Then the supernatant was removed, and composite/cell pellet was washed with PBS for 3 times. After the last wash, the composite/cell pellet was re-suspended with 100 μΐ of PBS buffer. Cells were counted using a microscope. The experiment was performed in duplicate. FIG. 2 showed that 6 lots of Composite 2 made from 3 batches of beads have a cell capture yield of 85% on average. Non-specific cell binding is low, at about 0.03% of white blood cells from per ml of whole blood sample.

EXAMPLE 6.

[00156] 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.

[00157] Two cancer cell lines, H1650 non-small cell lung cancer cells and MDA-MB-

231 breast cancer cells, were tested for 100 cell spike-in 3 ml of whole blood experiments. In the experiment, after cell spike-in, Ficoll® was gently added to allow centrifugation separation of plasma and buffy coat layers. The plasma part was transferred to a new tube for cfDNA extraction. The buffy coat layer was transferred to another new tube, and 0.5-2 ml of RBC lysis buffer was added to lyse RBC. After washing, cell pellet was re-suspended to 1 ml of PBS with 0.2% BSA and 4mM EDTA, followed by addition of 12.5 μΐ of Composite 2 in buffer at a concentration of 1 mg/ml and incubation for 1 hr. After washing, the captured cells were redispersed in PBS buffer and a little drop was counted under a fluorescence microscope. As shown in FIG. 3, capture yield of 90% was achieved for both cell lines. The procedure described above not only provides high capture yield for rare cell capture, but also allows separation of plasma for cfDNA capture from the same blood sample.

EXAMPLE 7.

[00158] This example illustrates the effect of the amount of the composite on cell capture yield. [00159] Different volumes of suspension comprising Composite 2 at a concentration of

1 mg/ml were used to capture 100 HI 650 cells from 1 ml of whole blood. As shown in FIG. 4, the cell capture yield firstly increased with increasing volume of composite, and then decreased after the saturation volume point. The volume of 12.5 μΐ provides the highest capture yield.

EXAMPLE 8.

[00160] This example illustrates the effect of incubation time on cell capture yield by composite of the present disclosure.

[00161] 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.

EXAMPLE 9.

[00162] This example compared the cell capture yield using the composite of the present disclosure and other beads.

[00163] Streptavidin conjugated magnetic beads from different vendors (Dynabeads,

Seradyne beads, Ocean Nanotech beads, respectively) were preincubated with 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.

[00164] 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

FIG. 6, compared with the comparative beads, Composite 2 of the present disclosure shows the highest capture yield of greater than 80%.

EXAMPLE 10.

[00165] This example compared the cell capture using the composite of the present disclosure and EpCAM bead without any treatment.

[00166] Two batches of beads comprising streptavidin were used to produce the composite of the present disclosure using the procedure described in Example 4. Beads from both batches comprise sulphur-containing group, nitrogen-containing group and phosphorus- containing group on the surface, and beads from batch 1 have 20% more sulphur-containing groups and nitrogen-containing groups on beads' surface than those from batch 2. The composites obtained from the two batches were used to carry out cell capture assay as described in Example 5.

[00167] For composites obtained from batch 1, as shown in FIG. 7A, 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.

[00168] For composites obtained from batch 2, as shown in FIG. 7B, 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. 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.

EXAMPLE 11.

[00169] This example illustrates the cell capture by composite obtained from the treatment of EpCAM bead with two different doses of maleimide-PEG.

[00170] EpCAM beads from batch 2 in Example 10 were treated with 2 different doses of maleimide-PEG: 138.9 μg and 194.4 μg 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.

[00171] As illustrated in FIG. 8, compared to the EpCAM bead without any treatment, the composites obtained from the maleimide-PEG treatment at the two different doses increased the cell capture yield by 35% and 20%, respectively.

EXAMPLE 12.

[00172] This example illustrates the cell capture by composite obtained from the treatment of EpCAM bead with four different doses of maleimide-PEG.

[00173] The 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.

[00174] As illustrated in FIG. 9, compared to the EpCAM bead without any treatment, maleimide-PEG treatment at a dose between 6 μg/mg beads and 16 μg/mg beads increased the cell capture yield by 20-40%. Maleimdie-PEG treatment at a dose of about 8 μg maleimide PEG /mg beads might be optimal for EpCAM beads with SNEP surface. EXAMPLE 13.

[00175] This example illustrates the simultaneous CTC isolation and identification using the composite of the present disclosure.

[00176] 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.

[00177] Breast cancer cells SKBR3 were pre-stained with CFSE (green cells) and spiked into a buffer with 106 PBMC cells. The composite was added and incubated for 15 min at 4°C. After washing, cells were re-suspended into PBS buffer and viewed under a fluorescent microscope, as shown in FIG. 10. It can be seen that the fluorescent composite of the present disclosure allows simultaneous CTC isolation and fluorescent identification.

Claims

WHAT IS CLAIMED IS:

1. 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 3000 nm.

2. The composite of claim 1, where the PEG compound is maleimide-PEG or the derivative thereof.

3. The composite of claim 1, wherein the rare cells is present in the sample at less than 100 cells/ml.

4. The composite of claim 1, wherein the bead has a diameter ranging from about 50 to about 3000 nm.

5. The composite of claim 1, wherein the bead comprises at least one magnetic nanoparticle.

6. The composite of claim 5, wherein 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.

7. The composite of claim 5, wherein the magnetic nanoparticle has a diameter ranging from about 1 nm to about 100 nm.

8. The composite of claim 5, wherein 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.

9. The composite of claim 8, wherein the low density, porous 3-D structure has a thickness ranging from about 1 nm to about 2500 nm.

10. The composite of claim 8, wherein the low density, porous 3-D structure has a density of <1.0 g/cc.

11. The composite of claim 1, wherein the bead 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, carbon-containing group, phosphorus- containing group, and epoxy-containing group.

12. The composite of claim 1, wherein the analyte-capturing member is an antibody.

13. The composite of claim 12, wherein the antibody is anti-EpCAM antibody.

14. The composite of claim 1, wherein the analyte-capturing member is a ligand of the cell surface marker.

15. The composite of claim 14, wherein the ligand is a peptide, a small molecule, an aptamer, a hormone, a drug, a toxin or a neurotransmitter.

16. The composite of claim 1, wherein the rare cells are circulating tumor cells (CTCs).

17. The composite of claim 1, wherein the analyte-capturing member is operably linked to the bead through covalent linkage or non-covalent linkage.

18. The composite of claim 17, wherein the analyte-capturing member is operably linked to the bead through biotin-streptavidin interaction, protein A or G-antibody interaction or DNA-protein interaction.

19. The composite of any of claims 1-18, wherein the bead 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.

20. The composite of any of claims 1-19, wherein the bead 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.

21. 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.

22. The method of claim 21, wherein the bead comprises at least one magnetic

nanoparticle, and 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.

23. The method of claim 21, wherein the bead 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, carbon-containing groups,

phosphorus-containing group, epoxy-containing group or combination thereof.

24. The method of claim 21, where the PEG compound is maleimide-PEG or the derivative thereof.

25. The method of claim 24, where the PEG compound is maleimide-PEG-amine.

26. The method of claim 24, wherein the maleimide-PEG is present at a concentration of about 5-200 μg/mg conjugate.

27. The method of claim 26, wherein the bead further comprises nitrogen-containing group, sulfur-containing group and phosphorus-containing group within or on the surface, and the maleimide-PEG is present at a concentration of about 50-200 μg/mg conjugate.

28. The method of claim 27, wherein the maleimide-PEG is present at a concentration of about 140 μg/mg conjugate.

29. The method of claim 26, wherein the bead further comprises nitrogen-containing group, sulfur-containing group, carbon-containing group, phosphorus-containing group and epoxy-containing group within or on the surface, and the maleimide-PEG is present at a concentration of about 5-20 μg/mg conjugate.

30. The method of claim 29, wherein the maleimide-PEG is present at a concentration of about 8 μg/mg conjugate.

31. A method for capturing rare cells in a sample, comprising:

mixing the composite of any of claims 1-20 with the sample; and

detecting the rare cells binding to the composite.

32. The method of claim 31, wherein the presence of the rare cells in the sample and/or the identification of the rare cells is indicative of a disease.

33. The method of claim 31, wherein the rare cells are immune cells or circulating cells that can indicate the presence of a disease or the response to a treatment.

34. The method of claim 33, wherein the rare cells are selected from the group consisting of stem cells, cancer stem cells, T-cells, B-cells, K cells, CAR-T cells and CTCs.

35. The method of claim 31, further comprising 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.

35. The method of claim 34, wherein the steps of separating and detecting are engineered to be automatic with robotic liquid handlers, microfluidic flow cells, or a magnetic sifter or patterned magnetic structure containing devices.

36. The method of claim 31, further comprising determining a treatment according to the presence of the rare cells in the sample and/or the identification of the rare cells.

37. The method of claim 32, wherein the disease is tumor, inflammation, infectious disease, autoimmune disease, or neurodegenerative disease

38. The method of claim 33, where the treatment is immunotherapy.