Solid phases optimized for chemiluminescent detection
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 .