Color-produing diagnostic systems, reagents and methods

Abstract

Provided herein are novel reagents and their use in color-producing detection systems for performing diagnostic tests and analytical assays.

1 . A composition of hydrogen donor polymer conjugates for dye formation in an oxidative coupling reaction with participation of electron acceptor/coupler, peroxide and compounds with peroxidative activity, comprising any of the following:
 a) Water soluble hydrogen donor conjugates prepared by covalent linking of hydrogen donors to water soluble carrier polycarboxylic polymer together with additional uncharged hydrophilic polymer;   b) Water soluble hydrogen donor conjugates prepared by covalent linking to water soluble carrier polycarboxylic polymer together with additional uncharged hydrophilic polymer and bio-affinity partner;   c) Water soluble hydrogen donor conjugates prepared by covalent linking to copolymer of water soluble polycarboxylic carrier polymers with second carrier hydrophilic protein and hydrophilic uncharged polymer;   d) Water soluble hydrogen donor conjugates prepared by covalent linking to co-polymer of water soluble poly carboxylic carrier polymer with hydrophilic protein, hydrophilic uncharged polymer and bio-affinity partner; or   e) Water soluble hydrogen donor conjugates prepared by covalent linking to carrier protein conjugated with brunched polycarboxylic oligomers and carboxylic pendant polymers.   
     
     
         2 . The composition of  claim 1 , wherein hydrogen donors for covalent coupling are selected from the group consisting of amine-containing compounds comprising substituted anilines, quinolines, naphthols, and phenols, which produce dyes of different colors in a reaction with oxidized coupler with an absorbance maximum in a range of 380-650 nm. 
     
     
         3 . The composition of  claim 1 , wherein the hydrogen donors are 8-[4-amino-1-methylbutyl]-6-methoxy quinoline, N,N-Dimethyl-1,3-phenylenediamine, N-Phenylethylenediamine, or N-(1-Naphtyl)ethylenediamine. 
     
     
         4 . The composition of  claim 1 , wherein the polycarboxylic polymers and branched oligomers are selected from the group consisting of a fixed number of carboxyl groups, such as ethylenediamine tetraacetic acid, ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid, branched pendant carboxylic polymers, and linear polymers such as polyglutamic acid, polyaspartic acid, polyacrylic acid, polymaleic acid, polymethacrylic acid, polyacrylamide/polyacrylic acid copolymer, polyvinyl-maleic acid copolymer, polyethylene maleic acid copolymers, and polyacrylic-polymaleic acid copolymer at any combinations and molecular weights. 
     
     
         5 . The composition of  claim 1 , wherein the hydrophilic uncharged polymers are polyethylene glycols of various molecular weights (1-20 kDa) covalently linked with carboxylic groups of both carriers, polycarboxylic polymer and protein or with amino groups of a carrier protein. 
     
     
         6 . The composition of  claim 1 , wherein the protein carrier for conjugation with polymers and hydrogen donors is bovine serum albumin additionally hydrophilized by attachment of polyethylene glycols through amino or carboxyl groups of the protein. 
     
     
         7 . The composition of  claim 1 , wherein the bio-affinity partners are components of high-affinity binding pairs, biotin, digoxigenin, antigens, peptides, haptens, or other analyte capture reagents participating in analyte capture directly or indirectly. 
     
     
         8 . The composition of  claim 1 , wherein the water soluble hydrogen donor polymer conjugates are covalently immobilized on carboxylate or aminated particles. 
     
     
         9 . The composition of  claim 1 , which are covalently immobilized on particles together with hydrophilic polymers and proteins. 
     
     
         10 . The composition of  claim 1 , wherein the water soluble hydrogen donor conjugates are immobilized on particles through non-covalent passive adsorption. 
     
     
         11 . A method for producing colored reaction products comprising the step of: contacting at least one hydrogen donor polymer conjugate or particle immobilized hydrogen donor with an oxidizable coupling reagent in the presence of peroxide and peroxidase or a peroxidatively active substance. 
     
     
         12 . The method of  claim 11 , wherein the hydrogen donor polymer conjugate or particle immobilized hydrogen donor reagents are mixed with soluble or particle-bound analyte capture reagents and applied to a membrane or solid support for making diagnostic zones through mechanism of passive adsorption or trapping into pores. 
     
     
         13 . A method for application of a water-soluble and particle-bound hydrogen donor conjugate of  claim 1  in diagnostic tests wherein a variety of hydrogen donor conjugates are used for generation of multicolor result on diagnostic membranes containing multiple diagnostic and control zones. 
     
     
         14 . The method of  claim 11 , wherein the peroxide oxidizable coupling reagent is 3-Methyl-2-benzothiazolinone hydrazone or a derivative thereof. 
     
     
         15 . The method of  claim 11 , wherein the peroxide compound is sodium perborate, hydrogen peroxide or urea hydrogen peroxide. 
     
     
         16 . The method of  claim 11 , wherein the peroxide is provided by the conversion of a substrate of an oxidative enzyme. 
     
     
         17 . The method of  claim 11 , wherein the peroxidase, exemplary horseradish or another plant peroxidase, is a detector label in the assay for quantitative or qualitative analysis of a test analyte. 
     
     
         18 . The method of  claim 11 , wherein formation of the colored reaction product indicates the presence of a analyte with peroxidative activity. 
     
     
         19 . A method for application of a water-soluble and particle-bound hydrogen donor conjugate of  claim 1  in membrane-based diagnostic tests where membranes are used as a media for lateral flow chromatographic separation, vertical filtration, passive diffusion, dip-stick, bead filtration and slide microarray assays.