Diagnostic methods for the detection and quantification of blood-related diseases

Abstract

A diagnostic method suitable for detection and quantification of blood-related diseases or conditions. The methods utilize biomarkers, such as hemoglobin and hemozoin as catalysts in an atom transfer radical polymerization (ATRP) reaction performed above a lower critical solution temperature (LCST) of a polymer which allows the polymerization to be tracked by rate of turbidity formation. The rate of turbidity formation is correlated to the concentration of the biomarker, making the tests useful quantitative techniques which can be utilized as point-of-care tests in the field.

What is claimed is: 
     
         1 . A diagnostic test kit for carrying out a diagnostic method for the detection of a biomarker in a biological sample, comprising:
 a first container comprising a plurality of atom transfer radical polymerization (ATRP) initiators;   a second container comprising a plurality of radically ATRP polymerizable monomers that are able to form a polymer exhibiting a lower critical solution temperature (LCST) at or above which the polymer precipitates, and a reducing agent; and   a device for determining a concentration of a biomarker in a biological sample.   
     
     
         2 . The test kit according to  claim 1 , further including a light source, a detector, and a temperature control device. 
     
     
         3 . The test kit according to  claim 2 , wherein the device for determining the concentration of the biomarker is a chart or a processor; and wherein the initiators, the monomers and the reducing agent are stable for at least two months at 50° C. 
     
     
         4 . The test kit according to  claim 1 , wherein the second container is a cuvette, glass capillary, microwell plate, micro reactor or a test strip. 
     
     
         5 . The test kit according to  claim 1 , wherein the plurality of atom transfer radically polymerizable monomers are N-isopropyl acrylamide monomers, wherein the polymerization reaction polymerizes the N-isopropyl acrylamide monomers to form the polymer poly(N-isopropyl acrylamide). 
     
     
         6 . The test kit according to  claim 5 , wherein the biomarker is a heme-containing molecule, and wherein the heme-containing molecule is one or more of hemoglobin, myoglobin, peroxidase, catalase, hemozoin, hemin, and hematin and dimers or oligomers thereof. 
     
     
         7 . The test kit according to  claim 1 , wherein the radically ATRP polymerizable monomers are N-isopropylacrylamide (NIPAM), N-n-propylacrylamide, N-cyclopropylacrylamide, N,N-diethylacrylamide, N—(N′-isobutylcarbamido)propyl methacrylamide, N—(N′-ethylcarbamido)propyl methacrylamide, N-(1-hydroxymethyl)propylmethacrylamide, [N-(2,2-dimethyl-1,3-dioxolane)methyl]acrylamide, N-(2-methoxy-1,3-dioxan-5-yl) methacrylamide, N-(2,2-di-methyl-1,3-dioxan-5-yl) methacrylamide, N-(2,2-di-methyl-1,3-dioxan-5-yl) acrylamide, N-vinylisobutyramide, N-vinyl-n-butyramide, N-acryloyl-N′-propylpiperazine, N-vinylcaprolactam, N-vinylpyrrolidone, N-(2-methacryloyloxyethyl) pyrrolidone, N-ethylpyrrolidine methacrylate, N-acryloylpyrrolidine, dimethylaminoethyl methacrylate, 2-(2-methoxyethoxy)ethyl methacrylate, 2-[2-(2-methoxyethoxy)ethoxy]ethyl methacrylate, poly(ethylene glycol) acrylate, poly(ethylene glycol) methacrylate, oligo(ethylene glycol) acrylate, oligo(ethylene glycol) methacrylate, endo,exo-bicyclo[2.2.1]hept-5-ene-2,3-dicarboxylic acid bis[2-[2-(2-ethoxyethoxy)ethoxy]ethyl] ester, 4-vinylbenzyl methoxytetrakis(oxyethylene)ether, bis((ethoxyethoxy)ethoxy)phosphazene, bis(2,3-bis(2-methoxyethoxy)propanoxy) phosphazene], methyl vinyl ether, 2-(2-ethoxy)ethoxyethyl vinyl ether, 2-methoxyethyl vinyl ether, 2-methoxyethyl vinyl ether, ethoxyethyl glycidal ether, N-acryloyl-I-proline methyl ester, N-acryloyl-L-aline N′-methylamide, 2-hydroxypropylacrylate, or N,N′-methylenebisacrylamide, or a combination thereof. 
     
     
         8 . The test kit according to  claim 5 , wherein the radically ATRP polymerizable monomers are N-isopropylacrylamide (NIPAM), N-n-propylacrylamide, N-cyclopropylacrylamide, N,N-diethylacrylamide, N—(N′-isobutylcarbamido)propyl methacrylamide, N—(N′-ethylcarbamido)propyl methacrylamide, N-(1-hydroxymethyl)propylmethacrylamide, [N-(2,2-dimethyl-1,3-dioxolane)methyl]acrylamide, N-(2-methoxy-1,3-dioxan-5-yl) methacrylamide, N-(2,2-di-methyl-1,3-dioxan-5-yl) methacrylamide, N-(2,2-di-methyl-1,3-dioxan-5-yl) acrylamide, N-vinylisobutyramide, N-vinyl-n-butyramide, N-acryloyl-N′-propylpiperazine, N-vinylcaprolactam, N-vinylpyrrolidone, N-(2-methacryloyloxyethyl) pyrrolidone, N-ethylpyrrolidine methacrylate, N-acryloylpyrrolidine, dimethylaminoethyl methacrylate, 2-(2-methoxyethoxy)ethyl methacrylate, 2-[2-(2-methoxyethoxy)ethoxy]ethyl methacrylate, poly(ethylene glycol) acrylate, poly(ethylene glycol) methacrylate, oligo(ethylene glycol) acrylate, oligo(ethylene glycol) methacrylate, endo,exo-bicyclo[2.2.1]hept-5-ene-2,3-dicarboxylic acid bis[2-[2-(2-ethoxyethoxy)ethoxy]ethyl] ester, 4-vinylbenzyl methoxytetrakis(oxyethylene)ether, bis((ethoxyethoxy)ethoxy)phosphazene, bis(2,3-bis(2-methoxyethoxy)propanoxy) phosphazene], methyl vinyl ether, 2-(2-ethoxy)ethoxyethyl vinyl ether, 2-methoxyethyl vinyl ether, 2-methoxyethyl vinyl ether, ethoxyethyl glycidal ether, N-acryloyl-1-proline methyl ester, N-acryloyl-L-aline N′-methylamide, 2-hydroxypropylacrylate, or N,N′-methylenebisacrylamide, or a combination thereof. 
     
     
         9 . The test kit according to  claim 1 , wherein the reducing agent is one or more of ascorbic acid, sodium ascorbate, formic acid, oxalic acid, galic acid, tartaric acid, quinic acid, malic acid, malonic acid, citric acid, fumaric acid, acetic acid, caffeic acid, lactic acid and sodium dithionite. 
     
     
         10 . The test kit according to  claim 7 , wherein the reducing agent is one or more of ascorbic acid, sodium ascorbate, formic acid, oxalic acid, galic acid, tartaric acid, quinic acid, malic acid, malonic acid, citric acid, fumaric acid, acetic acid, caffeic acid, lactic acid and sodium dithionite. 
     
     
         11 . The test kit according to  claim 8 , wherein the reducing agent is one or more of ascorbic acid, sodium ascorbate, formic acid, oxalic acid, galic acid, tartaric acid, quinic acid, malic acid, malonic acid, citric acid, fumaric acid, acetic acid, caffeic acid, lactic acid and sodium dithionite. 
     
     
         12 . The test kit according to  claim 2 , wherein the second container is a cuvette, glass capillary, microwell plate, micro reactor or a test strip. 
     
     
         13 . The test kit according to  claim 6 , wherein the device for determining the concentration of the biomarker is a chart or a processor; and wherein the initiators, the monomers and the reducing agent are stable for at least two months at 50° C. 
     
     
         14 . The test kit according to  claim 13 , wherein the second container is a cuvette, glass capillary, microwell plate, micro reactor or a test strip. 
     
     
         15 . The test kit according to  claim 7 , wherein the device for determining the concentration of the biomarker is a chart or a processor; and wherein the initiators, the monomers and the reducing agent are stable for at least two months at 50° C. and wherein the second container is a cuvette, glass capillary, microwell plate, micro reactor or a test strip. 
     
     
         16 . The test kit according to  claim 8 , wherein the device for determining the concentration of the biomarker is a chart or a processor; and wherein the initiators, the monomers and the reducing agent are stable for at least two months at 50° C. and wherein the second container is a cuvette, glass capillary, microwell plate, micro reactor or a test strip. 
     
     
         17 . The test kit according to  claim 2 , wherein the reducing agent is one or more of ascorbic acid, sodium ascorbate, formic acid, oxalic acid, galic acid, tartaric acid, quinic acid, malic acid, malonic acid, citric acid, fumaric acid, acetic acid, caffeic acid, lactic acid and sodium dithionite. 
     
     
         18 . The test kit according to  claim 11 , further including a light source, a detector, and a temperature control device.