High capacity assay platforms

Abstract

A high capacity assay platform capable of binding target molecules includes a substrate and a polymer matrix attached to the substrate. The polymer matrix comprises a plurality of polymer molecules where at least some of the polymer molecules are covalently attached directly to the substrate and at least some of which molecules are crosslinked to other polymer molecules. Some of the polymer molecules have at least one binding ligand covalently attached thereto, and the density of the polymer matrix on the substrate is at least 2 μg/cm 2 .

1 . A method of preparing an assay platform comprising a substrate and a polymer matrix attached to the substrate, wherein the polymer matrix is capable of binding target molecules comprising contacting the substrate with a polymer composition comprising a plurality of polymer molecules having repeating units, wherein at least some of the polymer molecules have at least one reactive group covalently attached thereto, wherein at least some of the polymer molecules have at least one binding ligand covalently attached thereto, wherein the polymer molecules have an average molecular weight of at least 100 kDa, and wherein at least 25% of the polymer molecules have at least one reactive group and at least one binding ligand covalently attached thereto; and activating the reactive groups to covalently bind at least some of the polymer molecules directly to the substrate and to induce cross-linking between polymer molecules to form a polymer matrix attached to the substrate.  
     
     
         2 . The method according to  claim 1  wherein all of the polymer molecules have at least one binding ligand covalently attached thereto and wherein at least some of the polymer molecules have no reactive group covalently attached thereto.  
     
     
         3 . The method according to  claim 1  further comprising drying the polymer composition on the substrate prior to activating the reactive groups.  
     
     
         4 . The method according to  claim 3  further comprising derivatizing the binding ligand in the formed polymer matrix by attaching thereto a different binding ligand.  
     
     
         5 . The method according to  claim 1  wherein the reactive groups are covalently attached to the polymer molecules through a spacer.  
     
     
         6 . The method according to  claim 5  wherein the spacer comprises a lysine molecule.  
     
     
         7 . The method according to  claim 5  wherein the spacer further comprises an aminocaproic acid molecule.  
     
     
         8 . The method according to  claim 1  wherein the reactive groups are α, β unsaturated ketone photo-reactive groups and wherein the photo-reactive groups are activated by exposure to light at from about 1,000 mwatts/cm 2  to about 5,000 mwatts/cm 2 .  
     
     
         9 . The method according to  claim 1  wherein the reactive groups are α, β unsaturated ketone photo-reactive groups and wherein the photo-reactive groups are activated by exposure to light at from about 1,000 mwatts/cm 2  to about 3,000 mwatts/cm 2 .  
     
     
         10 . The method according to  claim 1  wherein the reactive groups are α, β unsaturated ketone photo-reactive groups and wherein the photo-reactive groups are activated by exposure to light at from about 1,500 mwatts/cm 2  to about 2,500 mwatts/cm 2 .  
     
     
         11 . The method according to  claim 1  wherein the reactive groups are α, β unsaturated ketone photo-reactive groups and wherein the photo-reactive groups are activated by exposure to light at from about 3 Joules/cm 2  to about 6 Joules/cm 2 .  
     
     
         12 . The method according to  claim 1  wherein the reactive groups are benzophenone groups and wherein the photo-reactive groups are activated by exposure to light for 0.5 sec/cm 2  to 5.0 sec/cm 2 .  
     
     
         13 . The method according to  claim 1  wherein an amount of the polymer composition is contacted with the substrate to provide a polymer matrix having a density of 4 μg/cm 2  to 30 μg/cm 2 .  
     
     
         14 . The method according to  claim 1  wherein an amount of the polymer composition is contacted with the substrate to provide a polymer matrix having a density of 6 μg/cm 2  to 15 μg/cm 2 .  
     
     
         15 . The method according to  claim 1  wherein an amount of the polymer composition is contacted with the substrate to provide a polymer matrix having a binding ligand density of at least 1 nanomole/cm 2 .  
     
     
         16 . The method according to  claim 1  wherein an amount of the polymer composition is contacted with the substrate to provide a polymer matrix having a binding ligand density of 1.2 nanomoles/cm 2  to 185 nanomoles/cm 2 .  
     
     
         17 . The method according to  claim 1  wherein an amount of the polymer composition is contacted with the substrate to provide a polymer matrix having a binding ligand density of 1.5 nanomoles/cm 2  to 90 nanomoles/cm 2 .  
     
     
         18 . The method according to  claim 1  wherein an amount of the polymer composition is contacted with the substrate to provide a polymer matrix having a binding ligand density of 1.8 nanomoles/cm 2  to 15 nanomoles/cm 2 .  
     
     
         19 . The method according to  claim 1  wherein the polymer molecules having reactive groups covalently attached thereto have less than 1 reactive group per 200 repeating units.  
     
     
         20 . The method according to  claim 1  wherein the polymer molecules having reactive groups covalently attached thereto have less than 1 reactive group per 600 repeating units.  
     
     
         21 . The method according to  claim 1  wherein the polymer molecules having binding ligand covalently attached thereto have from 1 binding ligand per 1 repeating unit to 1 binding ligand per 100 repeating units.  
     
     
         22 . The method according to  claim 1  wherein the polymer molecules having binding ligand covalently attached thereto have from 1 binding ligand per 1 repeating unit to 1 binding ligand per 20 repeating units.  
     
     
         23 . The method according to  claim 1  wherein the polymer molecules have an average molecular weight of 300 kDa to 6,000 kDa.  
     
     
         24 . The method according to  claim 1  wherein the polymer molecules have an average molecular weight of 400 kDa to 3,000 kDa.  
     
     
         25 . The method according to  claim 1  wherein the polymer molecules have an average molecular weight of 500 kDa to 2,000 kDa.  
     
     
         26 . The method according to  claim 1  wherein the percentage of polymer molecules having both reactive groups and binding ligand covalently attached thereto is from 25% to 80%.  
     
     
         27 . The method according to  claim 26  wherein the percentage of polymer molecules having both reactive groups and binding ligand covalently attached thereto is from 40% to 75%.  
     
     
         28 . The method according to  claim 27  wherein the percentage of polymer molecules having both reactive groups and binding ligand covalently attached thereto is from 50% to 60%.  
     
     
         29 . The method according to  claim 28  wherein the percentage of polymer molecules having both reactive groups and binding ligand covalently attached thereto is approximately 50%.  
     
     
         30 . The method according to  claim 1  wherein the binding ligand is covalently attached to the polymer molecules through a spacer.  
     
     
         31 . The method according to  claim 30  wherein the spacer comprises a lysine molecule.  
     
     
         32 . The method according to  claim 30  wherein the spacer further comprises an aminocaproic acid molecule.  
     
     
         33 . The method according to  claim 1  wherein the substrate is a multiwell plate.  
     
     
         34 . The method according to  claim 33  wherein the multiwell plate is selected from the group consisting of a 96 well polystyrene plate, a 96 well polypropylene plate, a 384 well polystyrene plate and a 384 well polypropylene plate.  
     
     
         35 . The method according to  claim 1  wherein the substrate is a MALDI plate.  
     
     
         36 . The method according to  claim 1  wherein the substrate is glass.  
     
     
         37 . The method according to  claim 1  wherein the substrate is plastic.  
     
     
         38 . The method according to  claim 1  wherein the substrate is a multiwell polystyrene plate, wherein the polymer molecules are dextran polymers, wherein the binding ligand is a nickel chelate, and wherein an amount of the polymer composition is contacted with the substrate to provide a polymer matrix having a binding ligand density of 1.5 nanomoles/cm 2  to 7.5 nanomoles/cm 2 .  
     
     
         39 . The method according to  claim 1  wherein the substrate is a multiwell polystyrene plate, wherein the polymer molecules are dextran polymers, wherein the binding ligand is a Gallium or Iron chelate, and wherein an amount of the polymer composition is contacted with the substrate to provide a polymer matrix having a binding ligand density of 1.5 nanomoles/am 2  to 7.5 nanomoles/cm 2 .  
     
     
         40 . The method according to  claim 1  wherein the substrate is a multiwell polystyrene plate, wherein the polymer molecules are dextran polymers, wherein the binding ligand is glutathione, and wherein an amount of the polymer composition is contacted with the substrate to provide a polymer matrix having a binding ligand density of 1.5 nanomoles/cm 2  to 7.5 nanomoles/cm 2 .  
     
     
         41 . The method according to  claim 1  wherein the substrate is a multiwell polypropylene plate, wherein the polymer molecules are dextran polymers, wherein the binding ligand is an oligo dT, and wherein an amount of the polymer composition is contacted with the substrate to provide a polymer matrix having a binding ligand density of 1.5 nanomoles/cm 2  to 7.5 nanomoles/cm 2 .  
     
     
         42 . The method according to  claim 1  wherein the substrate is a multiwell polystyrene plate or a multiwell polypropylene plate, wherein the polymer molecules are dextran polymers, wherein the binding ligand is streptavidin, and wherein an amount of the polymer composition is contacted with the substrate to provide a polymer matrix having a binding ligand density of 1.5 μg/cm 2  to 7.5 μg/cm 2 .  
     
     
         43 . The method according to  claim 1  wherein the substrate is a multiwell polystyrene plate, wherein the polymer molecules are dextran polymers, wherein the binding ligand is selected from the group consisting of protein A, protein G, protein L, or a mixture thereof and wherein an amount of the polymer composition is contacted with the substrate to provide a polymer matrix having a binding ligand density of 1.5 μg/cm 2  to 7.5 μg/cm 2 .  
     
     
         44 . An assay platform produced by the method of  claim 1 .  
     
     
         45 . An assay platform produced by the method of  claim 2 .  
     
     
         46 . An assay platform produced by the method of  claim 3 .  
     
     
         47 . An assay platform produced by the method of  claim 19 .  
     
     
         48 . An assay platform produced by the method of  claim 20 .  
     
     
         49 . An assay platform produced by the method of  claim 21 .  
     
     
         50 . An assay platform produced by the method of  claim 22 .  
     
     
         51 . An assay platform produced by the method of  claim 23 .  
     
     
         52 . An assay platform produced by the method of  claim 24 .  
     
     
         53 . An assay platform produced by the method of  claim 35 .  
     
     
         54 . An assay platform produced by the method of  claim 36 .  
     
     
         55 . An assay platform produced by the method of  claim 37.