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 . A solid support for chemiluminescent assays comprising a chemiluminescent quantum yield enhancing material and a plurality of probes for a biopolymer target, wherein the probes are covalently, ionically or physically attached to a surface of the solid support. 
     
     
         2 . The solid support of  claim 1 , wherein the chemiluminescent quantum yield enhancing material comprises a quaternary onium polymer having the general formula: 
       
         
           
           
               
               
           
         
         wherein n is a positive integer; 
         wherein R 1 , R 2  and R 3  are independently: a straight or branched chain alkyl group having from 1 to 20 carbon atoms optionally substituted with one or more hydroxy, alkoxy, aryloxy, amino or substituted amino, amido, ureido, fluoroalkane or fluoroaryl groups; a monocycloalkyl group having from 3 to 12 carbon ring carbon atoms optionally substituted with one or more alkyl, alkoxy or fused benzo groups; a polycycloalkyl group having 2 or more fused rings, each ring having from 5 to 12 carbon atoms optionally substituted with one or more alkyl, alkoxy or aryl groups; an aryl, alkaryl or aralkyl group having at least one ring and from 6 to 20 carbon atoms optionally substituted with one or more alkyl, aryl, fluorine or hydroxy groups; 
         wherein at least two of R 1 , R 2  and R 3 , together with the quaternary nitrogen atom to which they are bonded, can form a saturated or unsaturated, unsubstituted or substituted nitrogen-containing, nitrogen and oxygen-containing or nitrogen and sulfur-containing ring having from 3 to 5 carbon atoms and 1 to 3 heteroatoms and which may be benzoannulated; 
         wherein M is a nitrogen or a phosphorous atom; and 
         wherein X −  represents a counter ion. 
       
     
     
         3 . The solid support of  claim 1 , wherein the chemiluminescent quantum yield enhancing material comprises a quaternary onium polymer having the general formula: 
       
         
           
           
               
               
           
         
         wherein n is a positive integer and each R is an n-pentyl group. 
       
     
     
         4 . The solid support of  claim 1 , wherein the quantum yield enhancing material is coated onto the surface of the solid support. 
     
     
         5 . The solid support of  claim 1 , wherein the quantum yield enhancing material is covalently attached to the surface of the solid support. 
     
     
         6 . The solid support of  claim 5 , wherein the probes are covalently attached to the quantum yield enhancing material. 
     
     
         7 . The solid support of  claim 1 , wherein the probes are probes for a nucleic acid target. 
     
     
         8 . The solid support of  claim 1 , wherein the probes are probes for a protein target. 
     
     
         9 .- 19 . (canceled) 
     
     
         20 . The solid support of  claim 1 , wherein the probes are covalently attached to the surface of the solid support in a plurality of discrete regions. 
     
     
         21 . The solid support of  claim 20 , wherein the quantum yield enhancing material is only present in the plurality of discrete regions where the probes are attached to the surfaced of the solid support. 
     
     
         22 .- 24 . (canceled) 
     
     
         25 . A method of modifying the surface of a solid support to enhance the quantum yield of chemiluminescent emissions, the method comprising reacting a functional group on a quantum yield enhancing compound with functional groups on the solid support surface to covalently attach chemiluminescent enhancing moieties to the solid support surface. 
     
     
         26 . The method of  claim 25 , wherein the functional groups on the solid support surface comprise azalactone groups. 
     
     
         27 . The method of  claim 25 , wherein the quantum yield enhancing compound comprises a quaternary onium polymer or a quaternary onium compound having the general formula: 
       
         
           
           
               
               
           
         
         wherein Q is N or P; [LINK] is a divalent linker moiety; R 1 , R 2  and R 3  are, independently, an alkyl group, an aryl group or a nitrogen heterocycle; R is hydrogen, an alkyl group, or an aryl group; and X −  is a counterion; and wherein the step of covalently bonding the enhancing moiety to the support surface comprises reacting an amino group on the quaternary onium polymer or the amino group on the quaternary onium compound with functional groups on the support surface. 
       
     
     
         28 .- 52 . (canceled) 
     
     
         53 . A solid support for chemiluminescent assays comprising a polymer having a repeating unit defined by the formula: 
       
         
           
           
               
               
           
         
         wherein n is a positive integer and each R is an n-pentyl group. 
       
     
     
         54 .- 56 . (canceled) 
     
     
         57 . A kit for conducting chemiluminescent assays to determine the presence or amount 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) the solid support of  claim 53 .   
     
     
         58 . The kit of  claim 57 , wherein the biopolymer probe is selected from the group consisting of an antibody, a nucleic acid or an avidin probe. 
     
     
         59 .- 67 . (canceled)