Abstract:
The invention features shaped articles containing a structure and a hydrogel coating thereon, the hydrogel coating containing alginic acid conjugated to a polyalkylene oxide and a binding moiety. The hydrogel coating on the structure is sized and shaped to fit in a well in a microtiter plate and the coating does not cover the entire exterior of the surface. The invention further features methods of capturing targets using shaped articles and methods of preparing shaped articles.

Description:
BACKGROUND 
       [0001]    The invention is generally directed to compositions and methods for separations and bioengineering. 
         [0002]    Cellular isolation techniques are an essential component in studying specific populations, allowing for growth, genomic, and proteomic investigations. The detachment of cells adhered to any surface requires the application of a force that is greater in magnitude to that of adhesion. Fluid shear forces have been shown to be a simple method for cell detachment. Although this is a local and simple method of cell release, excessive exposure to fluid shear results in cell damage and reduction in viability. An alternative approach is to cleave the protein ligand that is bound to the capture surface using enzymes, such as trypsin. However, enzymatic exposure can cause morphological changes due to a disruption of the cell membrane and glycocalyx, leading to losses in cellular activity. Furthermore, enzymatic digestion has been shown to directly affect both the behavior and chemical makeup of the cells themselves. 
         [0003]    These limitations illustrate the need to establish a general technique to capture and release biological materials, such as cells, without extensive physical or chemical perturbations to the cell environment. There remains a need for surfaces and gels that have high specificity for particular cells and that allow the release of captured cells without altering the behavior and makeup of the cells. 
         [0004]    In addition to biological separation, there is a critical need for biological purification technologies that also demonstrate high recovery and yield. Current automation platforms have limited recovery due to inherent limitations of the chemical composition within their process reagents and/or inefficiencies in the process workflows. These limited recoveries and yields have greatly hindered the study of rare cells, proteins, and nucleic acids within heterogeneous tissue samples. The purification of these targeted moieties would allow the advancement of new therapies, new fundamental biological understandings, and development of personalized theranostics. Although there has been some work on automation technologies that have been implemented in current workflows in an attempt to increase the purification yield, there is a need for a low non-specific binding chemistry within automation components. 
       SUMMARY OF THE INVENTION 
       [0005]    In general, the invention features shaped articles having a structure and a hydrogel coating that are useful in the separation of targets from a sample. 
         [0006]    In one aspect, the invention features a shaped article including a structure with a hydrogel coating. The hydrogel coating includes alginic acid conjugated to a polyalkylene oxide, e.g., polyethylene oxide (PEG), and a binding moiety. In specific embodiments, the hydrogel coating on the structure is sized and shaped to fit in a well in a microtiter plate, e.g., with 24, 96, 384, or 1536 wells, and the coating does not cover the entire exterior of the surface. In other embodiments, the structure includes a plurality of hydrogel coatings thereon, each of which may the same or different. In further embodiments, the structure includes a tubular portion, e.g., a capillary tube or a pipette tip, wherein the hydrogel coating is on the interior, exterior, or both of the portion. 
         [0007]    In embodiments of any of the above aspects, the hydrogel is gelled by crosslinking alginic acid molecules by a cation, e.g., Li + , Mg 2+ , Ca 2+ , Sr 2+ , Ba 2+ , Zn 2+ , Cu 2+ , or Al 3+ , preferably Ca 2+ . 
         [0008]    Examples of binding moieties include an antibody or antigen-binding fragment thereof, a peptide, an oligonucleotide, a receptor, or a ligand. A binding moiety that is an antibody or antigen binding fragment thereof may be a monoclonal antibody or antigen-binding fragment thereof, an Fab, a humanized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a monovalent antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a single-chain Fv molecule, a bispecific single chain Fv ((scFv&#39;) 2 ) molecule, a domain antibody, a diabody, a triabody, an affibody, a domain antibody, a SMIP, a nanobody, a Fv fragment, a Fab fragment, a F(ab&#39;) 2  molecule, and a tandem scFv (taFv) fragment. Specific binding moieties include an anti-CD4 antibody, anti-CD8 antibody, anti-CD15 antibody, anti-CD20 antibody, anti-CD24 antibody, anti-CD30 antibody, anti-CD31 antibody, anti-CD33 antibody, anti-CD34 antibody, anti-CD44 antibody, anti-CD45 antibody, anti-CD52 antibody, anti-CD90 antibody, anti-CD117 antibody, anti-CD133 antibody, anti-CD142 antibody, anti-CD146 antibody, anti-CD184 antibody, anti-CD200 antibody, anti-CD318 antibody, anti-A2B5 antibody, anti-c-Kit antibody, anti-EGFR antibody, anti-FGFR3 antibody, anti-FGFR4 antibody, anti-Flk1 antibody, anti-Frizzled-9 antibody, anti-GLAST antibody, anti-Glut1 antibody, anti-HER2 antibody, anti-α4 integrin antibody, anti-N-CAM antibody, anti-Notch-1 antibody, anti-Notch-2 antibody, anti-Sca1 antibody, anti-SIPRA antibody, anti-somatostatinR1 antibody, anti-somatostatinR2 antibody, anti-somatostatinR3 antibody, anti-somatostatinR4 antibody, anti-somatostatinR5 antibody, anti-SSEA-3 antibody, anti-SSEA-4 antibody, an anti-GCPR antibody, or anti-Stro-1 antibody, or antigen binding fragment thereof. A binding moiety that is a peptide may be TNF-α, IL-1β, IL-2, IL6, IL10, α4-integrin, CD15 or an extracellular fragment thereof, CD20 or an extracellular fragment thereof, CD30 or an extracellular fragment thereof, or VEGF. Other binding moieties include biotin, avidin, streptavidin, protein A, and protein G. 
         [0009]    Exemplary structures include a polymer, ceramic, glass, or metal. Examples of polymers include polyethylene, polypropylene, polybutene, polybutadiene, polystyrene, polyacrylonitrile, polycarbonate, PEEK, or a blend or a copolymer thereof. Examples of metals include aluminum, titanium, steel, copper, or zinc. 
         [0010]    In certain embodiments of any aspect of the invention, the structure includes a plurality of members, and the hydrogel coating is on each member. The hydrogel coatings may be the same or different, e.g., by including the same or different alginic acid, polyalkylene oxide, e.g., PEG, or binding moiety. In specific embodiments, the plurality of members is arranged for placement in individual wells of a microtiter plate, e.g., with 24, 96, 384, or 1536 wells, e.g., in a single row or column. 
         [0011]    In other embodiments of any aspect of the invention, the surface of the structure adjacent to the hydrogel coating is hydrophobic. 
         [0012]    In a related aspect, the invention features a method of capturing a target including providing a shaped article of the invention; contacting the hydrogel coating with a first liquid containing the target under conditions allowing for binding of the target to the binding moiety; and removing the hydrogel coating from contact with the first liquid, thereby capturing the target. The method may further include contacting the hydrogel coating with a second liquid containing a release agent that releases the target from the shaped article, e.g., H +  or OH −  (i.e., a pH change) or a chelating agent for a cation crosslinking the alginic acid, e.g., EDTA, EGTA, sodium citrate, BAPTA, crown ether, cryptand, phenanthroline sulfonate, dipyridyl sulfonate, dioxane, DME, diglyme, or triglyme. 
         [0013]    In certain embodiments, the shaped article is provided by contacting the structure with a third liquid containing alginic acid and optionally polyalkylene oxide, e.g., PEG, and binding moiety, so that the third liquid coats the structure, and contacting the structure coated with the third liquid with cations to crosslink the alginic acid to form the hydrogel coating. In this embodiment, the alginic acid in the third liquid may be conjugated to the polyalkylene oxide, e.g., PEG, and the binding moiety, or the polyalkylene oxide, e.g., PEG, and/or binding moiety may be conjugated to the alginic acid after gelation. 
         [0014]    In certain embodiment, the hydrogel coating is contacted with the first liquid in a well of a microtiter plate, e.g., with 24, 96, 384, or 1536 wells. In other embodiments, the first liquid is agitated during contact with the hydrogel coating, e.g., by movement of the shaped article. In other embodiments, the shaped article includes a lumen via which the first composition can be drawn in and expelled. 
         [0015]    In another aspect, the invention features a method of producing a shaped article by contacting a structure with a first liquid containing alginic acid and optionally polyalkylene oxide, e.g., PEG, and binding moiety, so that the first liquid coats the structure, and contacting the structure coated with the first liquid with cations to crosslink the alginic acid to form a hydrogel coating on the structure, thereby forming the shaped article. In certain embodiments, the two contacting steps occur in different wells of a microtiter plate, e.g., with 24, 96, 384, or 1536 wells. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0016]      FIGS. 1A-1E . 2  are schemes showing the sequence of coating a structure with alginic acid conjugated to PEG (A) and crosslinking the alginic acid (B); conjugation of a binding moiety, e.g., an antibody, to the hydrogel (C); contacting the hydrogel with a sample, e.g., a cell suspension, to separate target cells (D); and optionally releasing the target cells by pH change (E. 1 ) or chelation (E. 2 ). 
           [0017]      FIG. 2  is a scheme showing the formation and use of a shaped article of the invention in a sequence of steps carried out in a microtiter plate. 
           [0018]      FIG. 3  is a graph comparing the capture of cells using a hydrogel of the invention to Xantec polymer coatings. 
           [0019]      FIG. 4  is a micrograph of cells bound to a hydrogel of the invention coated on a wire. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0020]    The disclosure provides shaped articles having a structure with a hydrogel coating of alginic acid conjugated to a polyalkylene oxide, e.g., polyethylene oxide (PEG), and a binding moiety, methods of forming such articles, and methods of using such articles for separation of targets from a sample. The hydrogel is typically formed in the presence of cations. Such hydrogels are capable of easily dissolving in the presence of chelators. 
         [0021]    Alginic acid provides a foundation for an improved separation. In the presence of a cation, e.g., calcium, alginic acid crosslinks and solidifies. Polyalkylene oxide, e.g., PEG, provides nonspecific binding or other beneficial properties to the hydrogel. An article having an alginic acid hydrogel including binding moieties can be dipped or otherwise contacted with a sample, where target present in the sample will bind to the hydrogel. Once target binds, the hydrogel can be removed from the sample and dissolved using a chelator, e.g., to release the target or leave the uncoated structure available for reuse. In this context, the article can be used to purify a composition by positive or negative selection. For example, a desired target can bind to the binding moiety and be removed from the sample for further study or manipulation. 
         [0022]    Alternatively, the sample may be purified by using the hydrogel to remove a substance from the sample, leaving behind a purified sample. 
         [0023]    Shaped Articles 
         [0024]    The structure may be formed of any suitable material, such as a polymer, (e.g., polyethylene, polypropylene, polybutene, polybutadiene, polystyrene, polyacrylonitrile, polycarbonate, PEEK, or a blend or a copolymer thereof), ceramic, glass, or metal (e.g., aluminum, titanium, steel, copper, or zinc). The structure may also include combinations of these materials. The shape of the structure may be any suitable shape, such as elongated like a wire or tubular, i.e., having a lumen connectable to a pressure source. The hydrogel coating will typically be on the outer surface of the structure, although a structure having a lumen may include the coating on the interior of the lumen, the exterior of the lumen, or both. Suitable tubular structures include glass capillary tubes and micropipette tips. Structures or portions to be coated with the hydrogel may be textured or porous to allow greater surface area for adhesion of the hydrogel. Structures may also include larger voids, e.g., through holes, to increase the surface area for adhesion of the hydrogel. Smooth and solid structures are also of use in the invention. 
         [0025]    In preferred embodiments, the structure is sized and shaped so that the hydrogel coating fits in a well in a microtiter plate e.g., with 6, 24, 96, 384, or 1536 wells. In other embodiments, the structure of the shaped article is only partially coated with the hydrogel. In such embodiments, the structure may include a portion, e.g., having a hydrophobic coating, a different material, or a smooth surface texture, adjacent to the location where the hydrogel coating is placed and that is not wet by aqueous liquids. Such a portion can be used to define the location of the structure where the liquid including alginic acid will wet the structure and thus where the hydrogel will form. Structure may also include a plurality of members, e.g., to allow formation and/or use of multiple hydrogel coatings in one shaped article. Such members may be spaced, e.g., to fit into wells in one column or row of a microtiter plate, e.g., with 6, 24, 96, 384, or 1536 wells. In another embodiment, multiple hydrogels, e.g., distinguished by different binding moieties, can be disposed on a single structure, e.g., a single wire or tube. Structures with multiple hydrogels can be contacted with a single sample to separate multiple targets in spatially distinct locations on the shaped article. 
         [0026]    In certain embodiments, the structure is coated with hydrogel and stored prior to use in a separation. In other embodiments, the shaped article is formed in a sequence of steps preceding its use in separation. 
         [0027]    Methods 
         [0028]    A structure may be coated, e.g., by dipping, painting, or spraying, with a liquid alginic acid composition and then contacted with cations for crosslinking, e.g., by dipping, painting, or spraying. In producing the coating, the alginic acid may be conjugated to the polyalkylene oxide, e.g., PEG, and/or the binding moiety and in a liquid composition prior to coating. Alternatively, the polyalkylene oxide, e.g., PEG, and/or binding moiety may be conjugated to the alginic acid after the alginic acid is crosslinked on the structure. In certain embodiments, the alginic acid is conjugated to the polyalkylene oxide, e.g., PEG, prior to gelation, and the binding moiety is conjugated after gelation. Once coated with a liquid including the alginic acid, the structure can be removed from the liquid and contacted with cations to crosslink the hydrogel, e.g., by dipping in another liquid. Alternatively, cations can be added to the alginic acid liquid while the structure is in contact with it. Microtiter plates, e.g., with 6, 24, 96, 384, or 1536 wells, provide a commercially available platform for housing liquids for dipping structures in sequence to form hydrogels and also to contact the hydrogel formed with a sample. Other arrangements of containers can be used for this purpose 
         [0029]    Once formed, the hydrogel, or portion thereof, of a shaped article can be contacted with a sample to bind and separate a target (or multiple targets). 
         [0030]      FIG. 1  is a scheme of using shaped articles of the invention. In  FIG. 1A , a structure is dipped into a liquid including alginic acid conjugated to polyalkylene oxide, e.g., PEG. The liquid wets or adsorbs to the structure, which is then dipped in a solution of cation, e.g., Ca 2+ , to produce a crosslinked hydrogel on the structure ( 1 A). The hydrogel is then dipped in a solution of binding moiety, e.g., antibody, which conjugates to the hydrogel ( 1 B). The hydrogel is then dipped in a cell suspension, and target cells bind to the hydrogel ( 1 C). The hydrogel with bound cells is removed from the sample ( 1 D), and the cells can be released from the hydrogel by disrupting the binding to the binding moiety, e.g., by a pH change ( 1 E. 1 ), or by dissolving the gel ( 1 E. 2 ). 
         [0031]      FIG. 2  is a scheme showing the formation of a shaped article and the subsequent use of the article in a single sequence of steps. A series of structures (or a single structure with multiple members) is dipped into a row of wells in a microtiter plate, where the row contains a liquid alginic acid composition. The structure is then removed and dipped in the second row containing cations to crosslink the hydrogel and form a shaped article. The shaped article is then removed and dipped in the third row containing a binding moiety that conjugates to the hydrogel. The shaped article is then removed and dipped in the fourth row to rinse unbound reagents from the hydrogel. The shaped article is then dipped in the fifth row containing a sample containing targets that bind to the binding moiety. The shaped article may then be dipped in rows for rinsing, release of the target, contact with further samples, or release of target from the binding moiety or dissolution of the hydrogel. Additional reagents may also be employed. For example, prior to dipping in the liquid alginic acid composition, the structure or structures may be dipped in one or more reagents to clean or otherwise prepare the structure for adhesion of the hydrogel. Additional rows may also be employed to conjugate the polyalkylene oxide, e.g., PEG, with the alginic acid after gelation of the alginic acid. In addition, although illustrated with a microtiter plate, any collection of liquid containers may be employed, and such containers may or may not be physically connected to one another. The process shown in  FIG. 2  may be manual or fully automated. Use of the invention for separation or purification can be scaled to a production scale, e.g., to aid in purification of therapeutic biologics. 
         [0032]    The structure or shaped article may be agitated during the process of coating the structure, crosslinking the hydrogel, conjugating a polyalkylene oxide, e.g., PEG, and/or binding moiety, contacting the hydrogel with sample, or contacting the hydrogel with an agent to release the target or dissolve the hydrogel. Such agitation may be performed by any suitable method known in the art. For example, liquids into which the structure or shaped article is dipped can be stirred (e.g., with a magnetic stir bar), sonicated, shaken, or aerated to provide stirring. Alternatively, the structure or shaped article can be moved, e.g., vertically, horizontally, rotationally, or combinations thereof, in the liquid to agitate it. When a structure has a lumen connected to a suction source, e.g., a pipettor, liquid can be drawn in and out of the lumen to agitate the liquid. 
         [0033]    Hydrogel 
         [0034]    Hydrogels of the invention are formed from alginic acid conjugated to a polyalkylene oxide, e.g., PEG, and a binding moiety, e.g., as generally described in WO 2012/106658. A reference to alginic acid is also a reference to a salt form, e.g., sodium alginate, unless otherwise noted. Suitable alginic acid is 20 kDa medium viscosity. Polyalkylene oxides, e.g., PEG and polypropylene oxide, are known in the art. Linear or branched, e.g., 4-arm or 8-arm, polyalkylene oxides, e.g., PEG, may be employed. The polyalkylene oxide, e.g., PEG, preferably has a molecular weight between 10 kDa and 20 kDa. An exemplary ratio of polyalkylene oxide, e.g., PEG, to alginic acid is 1:2 by weight. 
         [0035]    Alginic acid naturally possess multiple carboxyl groups that provide convenient groups for conjugation to polyalkylene oxide, e.g., PEG, and/or binding moieties. The polyalkylene oxide, e.g., PEG, and binding moiety will naturally possess or be modified to possess an appropriate group to conjugate to a carboxyl group. Suitable groups include amine groups, which are often found in binding moieties that include amino acids or can be introduced into binding moieties and polyalkylene oxides, e.g., PEG. For example, amine-terminated polyalkylene oxide, e.g., PEG, can be employed. In other embodiments, a linker may be use to conjugate appropriate groups on the polyalkylene oxide, e.g., PEG, or binding moiety to carboxyl groups on the alginic acid. In the hydrogel, a single polyalkylene oxide, e.g., PEG, may be conjugated to one or more alginic acid molecules. When a polyalkylene oxide binds to more than one alginic acid, the number of such crosslinks in the composition may or may not be sufficient to form a gel. The binding moiety can bind to either the alginic acid directly or to a polyalkylene oxide, e.g., PEG, bound to alginic acid. 
         [0036]    The hydrogel forms by crosslinking of the alginic acid with a cation, e.g., Li + , Mg 2+ , Ca 2+ , Sr 2+ , Ba 2+ , Zn 2+ , Cu 2+ , or Al 3+ . A preferred cation is Ca 2+ . Gelation of hydrogels of the invention may be reserved by contact with a chelator for the cation, e.g., EDTA, EGTA, sodium citrate, BAPTA, crown ether, cryptand, phenanthroline sulfonate, dipyridyl sulfonate, dioxane, DME, diglyme, or triglyme. 
         [0037]    Binding Moiety 
         [0038]    Binding moieties are those that bind to a particular target with specificity. Suitable binding moieties, as well as methods of conjugating such moieties to alginic acid or polyalkylene oxide, e.g., PEG, are known in the art. Examples of binding moieties include an antibody or antigen-binding fragment thereof, a peptide, a polynucleotide (e.g., an aptamer or specific sequence for hybridization), a receptor, a ligand, or a charged polymer. Typically, the target of the binding moiety will naturally be part of the target, but targets may be modified for binding to a binding moiety. For example, biotin-avidin or streptavidin or histidine tags and nickel or cobalt ions bound to a chelator may be used. Other binding moieties include protein A and protein G for antibodies. In certain embodiments, the binding interaction between the binding moiety and the target may be disrupted, e.g., by a change in pH or salt concentration or addition of a denaturant, e.g., a detergent, reducing agent, or oxidizing agent, or cleaving agent, e.g., protease or nuclease. 
         [0039]    Preferred targets are specific cells or fragments thereof, viruses or fragments thereof, nucleic acids, proteins, lipids, and metabolites. 
         [0040]    An antibody or antigen binding fragment thereof is, for example, a monoclonal antibody or antigen-binding fragment thereof, an Fab, a humanized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a monovalent antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a single-chain Fv molecule, a bispecific single chain Fv ((scFv&#39;) 2 ) molecule, a domain antibody, a diabody, a triabody, an affibody, a domain antibody, a SMIP, a nanobody, a Fv fragment, a Fab fragment, a F(ab&#39;) 2  molecule, and a tandem scFv (taFv) fragment. Specific antibodies include an anti-CD4 antibody, anti-CD8 antibody, anti-CD15 antibody, anti-CD20 antibody, anti-CD24 antibody, anti-CD30 antibody, anti-CD31 antibody, anti-CD33 antibody, anti-CD34 antibody, anti-CD44 antibody, anti-CD45 antibody, anti-CD52 antibody, anti-CD90 antibody, anti-CD117 antibody, anti-CD133 antibody, anti-CD142 antibody, anti-CD146 antibody, anti-CD184 antibody, anti-CD200 antibody, anti-CD318 antibody, anti-A2B5 antibody, anti-c-Kit antibody, anti-EGFR antibody, anti-FGFR3 antibody, anti-FGFR4 antibody, anti-Flk1 antibody, anti-Frizzled-9 antibody, anti-GLAST antibody, anti-Glut1 antibody, anti-HER2 antibody, anti-α4 integrin antibody, anti-N-CAM antibody, anti-Notch-1 antibody, anti-Notch-2 antibody, anti-Sca1 antibody, anti-SIPRA antibody, anti-somatostatinR1 antibody, anti-somatostatin R2 antibody, anti-somatostatin R3 antibody, anti-somatostatin R4 antibody, anti-somatostatinR5 antibody, anti-SSEA-3 antibody, anti-SSEA-4 antibody, anti-GCPR antibody, or anti-Stro-1 antibody. Antigen binding fragments of such antibodies are also suitable binding moieties. Examples of peptides include TNF-α, IL-1β, IL-2, IL6, IL10, α4-integrin, CD15 or an extracellular fragment thereof, CD20 or an extracellular fragment thereof, CD30 or an extracellular fragment thereof, or VEGF. 
         [0041]    A single hydrogel may include multiple binding moieties, e.g., when one or more targets are desired in a single sample. In embodiments, where the structure includes a plurality of hydrogels, individual hydrogels may have the same or different binding moieties conjugated to them. 
         [0042]    Samples 
         [0043]    Hydrogels of the invention may be used to separate targets from any suitable liquid sample (or sample that has been dissolved or suspended in liquid). Samples may include bodily fluids, e.g., blood, plasma, serum, urine, saliva, lymph, spinal fluid, bile, mucus, tears, and amniotic fluid. Other samples include cell or viral culture and environmental samples (e.g., air, surface, or water samples). 
       EXAMPLES 
       [0044]    Stainless steel wires were coated with an alginate—PEG solution and crosslinked with a 1 mg/mL solution of Ca 2+ . The divalent cations ionically bound the alginic acid and formed a solid coating around the wire (as shown in  FIG. 4 ). The resulting coating was 200-500 microns in thickness homogenously around the wire. Antibodies against EpCAM proteins (100 μg/mL) on circulating tumor cells were chemical conjugated to the hydrogel for the purposes of selectively binding MCF-7 cancer cells. The hydrogel coating of the invention was compared against a propriety polymer coating (Xantec) ( FIG. 3 ). Both the hydrogel coated and Xantec polymer coated stainless steel wires were placed in a homogenous suspension of MCF-7 breast cancer cells in phosphate buffer solution for 30 minutes with slow rotational mixing. The wires were removed and rinsed prior to staining for cell nuclei via DAPI staining. As shown in  FIG. 4 , cells can be visualized and counted under a fluorescence microscope. The hydrogel coating of the invention provided significantly higher recoveries versus the Xantec polymer coating as shown in  FIG. 3 . 
         [0045]      FIG. 3 : Comparative recovery data of capture MCF-7 cells of polymer coating technology (Xantec) against the hydrogel coating of the invention. The purifications from the suspension containing a known concentration of MCF-7 cells were performed with an anti-EpCAM (PK) coating and a un-functionalized (NK) bare hydrogel coating. 
         [0046]      FIG. 4 : Fluorescent image demonstrating compositions of mentioned hydrogel bound to a medical grade stainless steel wire. Compositions were functionalized with anti-EpCAM and captured MCF-7 cells to the surface stained with DAPI. 
       Other Embodiments 
       [0047]    All publications, patents, and patent applications mentioned in the above specification are hereby incorporated by reference. Various modifications and variations of the described device and methods of use of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention. 
         [0048]    Other embodiments are in the claims.