MULTIFUNCTIONAL BEADS AND METHODS OF USE FOR CAPTURING RARE CELLS
Described are multi-functional beads and methods to capture rare cells directly from low-volume biological samples and perform both functional and genomic assays from those cells. This is accomplished using a multifunctional capture bead that allows co-localization of both the single cell capture element and the molecular assay components. When combined with a digital microfluidic platform this enables encoding and/or barcoding of specific single cells.
1 . A method of performing an assay on rare cells captured from a biological sample comprising:
contacting a solution containing the biological sample with a multifunctional bead, the multifunctional bead comprising;
a microsphere between 0.1 and 100 μm in size;
a cell capture element, on the surface of the microsphere, capable of binding to a protein or cell specific marker on the surface of a rare cell; and
a biomolecular capture element, on the surface of the microsphere, capable of binding to biomolecular components contained within or produced by the rare cell;
incubating the multifunctional beads with a biological sample containing the rare cells and binding the multifunctional bead to the rare cells through the surface capture element to create a bead-bound rare cells;
capturing biomolecules contained within or produced by the rare cells through the biomolecular capture element; and
assaying the rare cells by analyzing the biomolecules captured by the biomolecular capture element.
2 . A method of claim 1 where the cell capture element is a MHC tetramer capable of binding to antigen specific T cells.
3 . The method of claim 2 where the capture element is an antibody capable of binding to a cell surface marker on the target cell.
4 . A method of claim 1 where the biomolecular capture element, are antibodies, nucleic acids, oligonucleotides, artificially synthesized bioactive polymers, fluorescent conjugates, metal conjugates or a combination thereof.
5 . The method of claim 4 where the biomolecular capture element is an antibody.
6 . The method of claim 1 where the analyzing the biomolecules comprises, nucleic acid sequencing, cytokine secretion analysis, quantification of gene expression, quantifying the amount of rare cells in the biological sample, quantifying the functional activity of the rare cells in the biological sample or a combination thereof
7 . The method of claim 1 where the analyzing the biomolecules comprises encoding and/or barcoding of selected rare cells.
8 . The method of claim 1 further comprising the step of transforming the bead-bound rare cells into droplets prior to capturing biomolecules.
9 . A method of capturing rare cells from a biological sample comprising:
contacting a solution containing the biological sample with a multifunctional bead, the multifunctional bead comprising;
a microsphere between 0.1 and 100 μm in size;
a cell capture element, on the surface of the microsphere, capable of binding to a protein or cell specific marker on the surface of a rare cell; and
a biomolecular capture element, on the surface of the microsphere, capable of binding to biomolecular components contained within or produced by the rare cell;
binding the multifunctional bead to the rare cells through the surface capture element to create bead-bound rare cells;
flowing the solution containing the bead-bound rare cells through a microfluidic device, the microfluidic device having microfluidic compartments;
partitioning the bead-bound rare cells of the solution into at least one of the microfluidic compartments;
contacting a biomolecular capture element with bead-bound rare cells; and
capturing biomolecules contained within or produced by the rare cells through the biomolecular capture element.
10 . A method of claim 9 where the cell capture element is a WIC tetramer capable of binding to antigen specific T cells.
11 . The method of claim 10 where the capture element is an antibody capable of binding to a cell surface marker on the target cell.
12 . A method of claim 9 where the biomolecular capture element is antibodies, nucleic acids, oligonucleotides, fluorescent conjugates, metal conjugates, artificially synthesized bioactive polymers or a combination thereof.
13 . The method of claim 12 where the biomolecular capture element is an antibody.
14 . The method of claim 9 further comprising the step of assaying the rare cells by analyzing the biomolecules captured by the biomolecular capture element.
15 . The method of claim 14 where the analyzing the biomolecules comprises nucleic acid sequencing cytokine secretion analysis, quantification of gene expression, a, quantifying the amount of rare cells in the biological sample, quantifying the functional activity of the rare cells within the sample or a combination thereof
16 . The method of claim 14 where the analyzing the biomolecules comprising encoding and/or barcoding of selected rare cells.
17 . The method of claim 9 where the multifunctional bead is magnetic and partitioning the bead-bound rare cells comprises magnetic trapping of the bead-bound rare cells on magnetized pillars of the microfluidic compartments.
18 . The method of claim 9 where the multifunctional bead is placed in the digital microfluidic device prior to contact with the biological solution.
19 . The method of claim 18 where one or more of the microfluidic compartments contain a biomolecular capture element.
20 . The method of claim 19 where different biomolecular capture elements are contained in different microfluidic compartments.
21 . A multifunctional bead for capturing rare cells, the multifunctional bead comprising;
a microsphere between 0.1 and 100 μm in size.
a cell capture element, on the surface of the microsphere, capable of binding to a protein or cell specific marker on the surface of a rare cell; and
a biomolecular capture element, on the surface of the microsphere, capable of binding to biomolecular components contained within or produced by the rare cell.
22 . The multifunctional bead of claim 21 where the microsphere is ceramics, glass, polymer, metals, or a combination thereof.
23 . The multifunctional bead of claim 22 where the microsphere is polyethylene, polystyrene, or a combination thereof.
24 . The multifunctional bead of claim 21 wherein the microsphere is magnetic.
25 . The multifunctional bead of claim 21 where the cell capture element is a MHC tetramer capable of binding to antigen specific T cells.
26 . The multifunctional bead of claim 21 where the capture element is an antibody capable of binding to a cell surface marker on the target cell.
27 . The multifunctional bead of claim 21 where the biomolecular capture element is antibodies, nucleic acids, oligonucleotides, fluorescent conjugates, metal conjugates, artificially synthesized bioactive polymers or a combination thereof.
28 . The multifunctional bead of claim 21 where the bead is a magnetic microsphere, the cell capture element is a WIC tetramer, and the capture element is an antibody.