Solid phases optimized for chemiluminescent detection

Abstract

Solid supports for chemiluminescent assays are provided. The solid support includes a plurality of probes covalently or physically attached to the support surface and a chemiluminescent enhancing moiety incorporated onto the surface or into the bulk of the support. The solid support can be a multi-layered support including an upper probe binding layer (e.g., an azlactone polymer layer or porous functional polyamide layer) adjacent to a cationic microgel layer. The azlactone-functional polymer can be a copolymer of dimethylacrylamide and vinylazlactone crosslinked with ethylenediamine. The cationic microgel layer can be a cross-linked quaternary onium salt containing polymer. A method and a kit for conducting chemiluminescent assays using the solid supports is also provided. The kit comprises a dioxetane substrate, a biopolymer probe-enzyme complex, and a solid support. The solid support can be an azlactone functional polymer layer adjacent to a cationic microgel layer; a porous polyamide functional layer adjacent to a cationic microgel layer; or a quaternized azlactone functional polymer layer.

1 - 31 . (canceled) 
     
     
         32 . A solid support for chemiluminescent assays comprising a functional polymer layer adjacent to a layer comprising a cationic microgel, wherein the functional polymer layer comprises an azlactone polymer layer or a porous polyamide layer. 
     
     
         33 . The solid support of  claim 32 , wherein the functional polymer layer comprises a copolymer of dimethylacrylamide and vinylazlactone crosslinked with ethylenediamine. 
     
     
         34 . The solid support of  claim 33 , wherein the layer comprising a cationic microgel comprises a cross-linked quaternary onium salt containing polymer. 
     
     
         35 . The solid support of  claim 32 , further comprising a backing material in contact with the layer comprising a cationic microgel. 
     
     
         36 . The solid support of  claim 35 , wherein the backing material comprises an oriented polymeric layer. 
     
     
         37 . The solid support of  claim 34 , wherein the cross-linked quaternary onium salt containing polymer comprises a quaternized azlactone functional polymer comprising azlactone functionalities quaternized with an amino functional quaternary onium salt or quaternized benzyl halide repeating units. 
     
     
         38 . The solid support of  claim 32 , further comprising a probe for a biopolymer target and/or a fluorescent moiety bonded to an exposed surface of the functional polymer layer. 
     
     
         39 . A method of conducting a chemiluminescent assay wherein the presence or amount of one or more components of an analyte is determined, the method comprising steps of:
 contacting the analyte with a solid support;   treating the analyte on the solid support with a biopolymer probe-enzyme complex;   incubating the enzyme complex treated analyte with an enzyme-cleavable 1,2-dioxetane, wherein the enzyme-cleavable 1,2-dioxetane can be cleaved by an enzyme to yield a chemiluminescent dioxetane reporter molecule; and   measuring the degree of chemiluminescence obtained;   wherein the solid support comprises an azlactone polymer layer adjacent to a layer comprising a cationic microgel or a porous polyamide layer adjacent to a layer comprising a cationic microgel; and   wherein the analyte is contacted with an exposed surface of the azlactone polymer layer or the porous polyamide layer opposite the cationic microgel layer.   
     
     
         40 . The method of  claim 39 , further comprising a step of washing the solid support surface after the treating step. 
     
     
         41 . The method of  claim 39 , wherein the functional polymer layer comprises an azlactone polymer layer and wherein the dioxetane reporter molecule has a half-life that is sufficiently long to allow the reporter molecule to diffuse through the functional polymer layer and become sequestered in the layer comprising a cationic microgel. 
     
     
         42 . The method of  claim 41 , wherein the dioxetane reporter molecule has a half-life of from about 2 seconds to about 60 minutes. 
     
     
         43 . The method of  claim 39 , further comprising a step of covalently bonding the probe to a surface of the functional polymer layer. 
     
     
         44 . The method of  claim 39 , wherein the biopolymer probe is an antibody, the method further comprising a step of binding an antigen target to the antibody. 
     
     
         45 . The method of  claim 39 , wherein the contacting step further comprises fixing the analyte on the exposed surface. 
     
     
         46 . The method of  claim 39 , wherein the reporter molecule is a dioxetane phenolate anion. 
     
     
         47 . A method of conducting a chemiluminescent assay wherein the presence or amount of one or more components of an analyte is determined, comprising steps of:
 contacting the analyte with an exposed surface of a solid support;   treating the analyte on the solid support with a biopolymer probe-enzyme complex;   incubating the enzyme complex treated analyte with an enzyme-cleavable 1,2-dioxetane, wherein the enzyme-cleavable 1,2-dioxetane can be cleaved by the enzyme to yield a chemiluminescent reporter molecule; and   measuring the degree of chemiluminescence obtained;   wherein the solid support comprises a quaternized azlactone functional polymer.   
     
     
         48 . The method of  claim 47 , wherein the quaternized azlactone functional polymer comprises azlactone repeating units quaternized with amino-functional quaternary onium compounds. 
     
     
         49 . The method of  claim 47 , wherein the azlactone functional polymer comprises quaternized benzyl halide repeating units. 
     
     
         50 . The method of  claim 47 , further comprising a step of covalently bonding the biopolymer probe to the solid support surface. 
     
     
         51 . The method of  claim 47 , wherein the contacting step further comprises fixing the analyte on the exposed surface. 
     
     
         52 . A kit for conducting chemiluminescent assays to determine the presence or absence of a component of an analyte, comprising:
 a) a dioxetane substrate bearing an enzyme-labile protecting group which, when cleaved, yields a chemiluminescent reporter molecule;   b) a biopolymer probe-enzyme complex, wherein the biopolymer probe is specific for the component being assayed, and wherein the enzyme is capable of cleaving the enzyme-labile protecting group; and   c) a solid support;   wherein the solid support comprises: an azlactone functional polymer layer adjacent to a cationic microgel layer; a porous polyamide layer adjacent to a cationic microgel layer; or a quaternized azlactone functional polymer layer.   
     
     
         53 - 58 . (canceled) 
     
     
         59 . A method of making a solid support for chemiluminescent assays having high feature density, the method comprising:
 providing a shrinkable backing material;   applying a solid support to the shrinkable backing material;   applying a chemiluminescent quantum yield enhancing material to an exposed surface of the solid support;   applying a plurality of probes for a biopolymer target to an exposed surface of the solid support; and   shrinking the backing material.   
     
     
         60 . The method of  claim 59 , wherein the quantum yield enhancing material and/or the probes are applied to the exposed surface of the solid support in a plurality of spaced, discrete regions. 
     
     
         61 . The method of  claim 59 , wherein the quantum yield enhancing material and/or the probes are covalently attached to the exposed surface of the solid support. 
     
     
         62 . The method of  claim 60 , wherein the quantum yield enhancing material and/or the probes are covalently attached to the exposed surface of the solid support. 
     
     
         63 . The method of  claim 59 , wherein the solid support comprises an azlactone functional layer or a polyamide functional layer. 
     
     
         64 . The method of  claim 59 , wherein the solid support comprises an azlactone functional layer or a porous polyamide functional layer adjacent to a layer comprising a cationic microgel. 
     
     
         65 . The method of  claim 59 , wherein the shrinkable backing material comprises an oriented polymer layer. 
     
     
         66 . A solid support made by the method of  claim 59 . 
     
     
         67 . A solid support made by the method of  claim 64 .