Molecular diagnostic assay device and method of use

Abstract

Lateral flow devices and methods of use for a molecular diagnostic assay are provided. The method is suitable for detection or monitoring of targets, including biological, chemical, and material targets that exist in very low concentrations in biological samples. The methods and devices of the present application are amenable to power source-free point of care testing.

1 . A method of detecting an analyte in a test sample, the method comprising;
 i) providing a lateral flow assay device that comprises a chromatographic medium that includes:
 (a) a sample loading zone located upstream of a detection zone; 
 (b) a reporting carrier zone located between the sample loading zone and a detection zone, wherein said reporting carrier zone comprises a reporting carrier capable of forming a complex with the analyte said reporting carrier comprising a carrier and one or more proficient enzyme cassettes; and 
 (c) a detection zone, wherein the detection zone comprises a capture component for the analyte and an indicator; 
   ii) Contacting the sample application zone with the test sample, wherein the test sample travels through the reporting carrier zone along the chromatographic medium from the sample loading zone to the detection zone and beyond the detection zone;   iii) adding a substrate to the detection zone wherein the substrate undergoes a reaction in the presence of proficient enzyme analyte containing reporting carrier; and   iv) generating a response of the indicator within the detection zone that corresponds to the presence or absence of the analyte in the test sample.   
     
     
         2 . The method of  claim 1 , wherein the lateral flow device further comprises a control zone down stream of the detection zone and the control zone comprises a capture component for the reporting carrier and an indicator;
 adding a substrate to the control zone wherein the substrate undergoes a reaction in the presence of proficient enzyme containing reporting carrier; and   generating a response of the indicator within the control zone that corresponds to the presence or absence of the reporting carrier.   
     
     
         3 . The method of  claim 1  further comprising maintaining a reaction temperature maintained at about 4° C. to about 95° C., or higher while still maintaining enzyme activity. 
     
     
         4 . The method of  claim 1 , wherein the analyte is selected from the group consisting of a protein; a small molecule; or a nucleic acid. 
     
     
         5 - 6 . (canceled) 
     
     
         7 . The method of  claim 1 , wherein the proficient enzyme conjugate is selected from the group consisting of an antibody; a nucleic acid or urease, phosphocholine phosphatase, betagalactosidase, xylose reductase, shikimate dehydrogenase, malate dehydrogenase, carboxylesterase, neopullulanase, subtilisin, 4-phytase, acetylcholinesterase, laccase, bacterial leucyl aminopeptidase, tripeptidyl-peptidase I, coagulation factor VIIa, trypsin, beta-fructofuranosidase. 
     
     
         8 - 10 . (canceled) 
     
     
         11 . The method of  claim 7 , wherein the antibody interacts non-covalently with a proficient enzyme to form the proficient enzyme conjugate; or wherein the nucleic acid is covalently attached to a proficient enzyme to form the proficient enzyme conjugate; or wherein the antibody is covalently attached to a proficient enzyme to form the proficient enzyme conjugate. 
     
     
         12 - 13 . (canceled) 
     
     
         14 . The method of  claim 1 , further providing one or more inactive pro-enzymes in the detection zone. 
     
     
         15 . The method of  claim 1 , wherein the activity of the proficient enzyme conjugate is detected by monitoring the effect of the proficient enzyme assisted reaction. 
     
     
         16 . The method of  claim 15 , wherein the effect of the proficient enzyme assisted reaction is proton release. 
     
     
         17 . The method of  claim 16 , wherein the proton release produces a pH change and wherein the pH change is determined using a pH sensitive potassium I-hydroxy-4-[1-(2-hydroxyethylsulphonyl)phenylazo]-naphthalene-2-sulphonate indicator polymer that is immobilized on the membrane; and wherein the colorimetric change is due to silver ion reduction. 
     
     
         18 . (canceled) 
     
     
         19 . The method of  claim 17 , wherein the pH change is determined using a pH sensitive cellulose acetate coupled dye. 
     
     
         20 . The method of  claim 1 , wherein the activity of the proficient enzyme conjugate is detected from the group consisting of colorimetric change; fluorescence emission; or electrochemical methods. 
     
     
         21 - 23 . (canceled) 
     
     
         24 . The method of  claim 1 , wherein the activity of the proficient enzyme conjugate is detected by precipitation of a soluble component and wherein the soluble component is the protein BSA or a pH sensitive polymer selected from the group consisting of methyl acrylic acid, methyl methacrylate, methacrylic acid 2-(dimethylamino) ethyl ester, and N-hydroxymethyl acrylamide. 
     
     
         25 - 27 . (canceled) 
     
     
         28 . The method of  claim 1  further comprising adding a pre-reporter carrier to the test sample prior to contacting the sample application zone with the test sample. 
     
     
         29 . The method of  claim 1  further comprising an absorptive pad at the distal end of the chromatographic medium. 
     
     
         30 . The method of  claim 1 , wherein the substrate is allyl hexanoate and proficient enzyme is carboxy lesterase. 
     
     
         31 . A lateral flow assay device for detecting the presence of an analyte within a test sample, the lateral flow assay device comprising:
 a chromatographic medium that includes:
 a sample loading zone located upstream of a detection zone; 
 a reporting carrier zone located between the sample loading zone and a detection zone, wherein said reporting carrier zone comprises a reporting carrier capable of forming a complex with the analyte said reporting carrier comprising a carrier and one or more proficient enzyme cassettes; and 
 a detection zone, wherein the detection zone comprises a capture component for the analyte and an indicator, wherein the indicator detects a reaction of a substrate in the presence of a proficient enzyme thereby detecting the presence of the analyte. 
   
     
     
         32 . The lateral flow assay device of  claim 31  further comprising a control zone, wherein the control zone comprises a capture component for the reporting carrier and an indicator for detecting a reaction of a substrate in the presence of a proficient enzyme selected from the group consisting of an antibody; a nucleic acid; urease, phosphocholine phosphatase, betagalactosidase, xylose reductase, shikimate dehydrogenase, malate dehydrogenase, carboxylesterase, neopullulanase, subtilisin, 4-phytase, acetylcholinesterase, laccase, bacterial leucyl aminopeptidase, tripeptidyl-peptidase I, coagulation factor VIIa, trypsin, beta-fructofuranosidase; wherein the product of the enzyme reporting carrier complex and substrate is detected thereby detecting the presence of the reporting carrier. 
     
     
         33 - 35 . (canceled) 
     
     
         36 . The lateral flow assay device of  claim 31  wherein the antibody interacts non-covalently with a proficient enzyme to form the proficient enzyme conjugate. 
     
     
         37 . The lateral flow assay device of  claim 31  wherein the nucleic acid is covalently attached to a proficient enzyme to form the proficient enzyme conjugate. 
     
     
         38 . The lateral flow assay device of  claim 31  further providing one or more inactive pro-enzymes in the detection zone. 
     
     
         39 . The lateral flow assay device of  claim 31 , wherein the activity of the proficient enzyme conjugate is detected by monitoring the effect of the proficient enzyme assisted reaction. 
     
     
         40 . The lateral flow assay device of  claim 31 , wherein the indicator is selected from the group consisting of a pH sensitive indicator; Potassium I-hydroxy-4-[1-(2-hydroxyethylsulphonyl) phenylazo]-naphthalene-2-sulphonate; or a pH sensitive cellulose acetate coupled dye. 
     
     
         41 - 42 . (canceled) 
     
     
         43 . The lateral flow assay device of  claim 31 , wherein the reporting carrier zone comprises a conjugate pad. 
     
     
         44 . The lateral flow assay device of  claim 31 , wherein the sample loading zone comprises a sample loading pad. 
     
     
         45 . The lateral flow assay device of  claim 31 , further comprising a rigid or flexible backing material. 
     
     
         46 . The lateral flow assay device of  claim 31 , wherein the antibody is associated with the proficient enzyme by noncovalent interactions; or wherein the antibody or nucleic acid is covalently attached to the proficient enzyme. 
     
     
         47 . (canceled) 
     
     
         48 . The lateral flow assay device of  claim 31 , further comprising a source of one or more inactive pro-enzymes. 
     
     
         49 . The lateral flow assay device of  claim 31 , wherein the detection zone detects a product of the proficient enzyme by detection of a pH change; wherein the detection zone detects the product of the proficient enzyme by colorimetric change; wherein the detection zone detects the product of the proficient enzyme by fluorescence emission; or wherein the detection zone detects the product of the proficient enzyme by electrochemical methods. 
     
     
         50 . The lateral flow assay device of  claim 31 , wherein the pH change is determined using a pH sensitive hydrogel. 
     
     
         51 - 53 . (canceled) 
     
     
         54 . The lateral flow assay device of  claim 49 , wherein the colorimetric change is due to silver ion reduction. 
     
     
         55 . The lateral flow assay device of  claim 31 , the detection zone detects the product of the proficient enzyme by precipitation of a soluble component. 
     
     
         56 . The lateral flow assay device of  claim 31 , wherein the soluble component is a protein BSA or pH sensitive polymer selected from the group consisting of methyl acrylic acid, methyl methacrylate, methacrylic acid 2-(dimethylamino) ethyl ester, and N-hydroxymethyl acrylamide. 
     
     
         57 - 58 . (canceled) 
     
     
         59 . The lateral flow assay device of  claim 31  further comprising an absorptive pad at the distal end of the chromatographic medium. 
     
     
         60 . The lateral flow assay device of  claim 31 , wherein the substrate is allyl hexanoate and proficient enzyme is carboxylesterase. 
     
     
         61 . A lateral flow assay kit for detecting the presence of an analyte within a test sample, the lateral flow assay device comprising:
 a porous membrane comprising:
 a sample loading zone; 
 a reporting carrier zone down stream of the loading zone, wherein said reporting carrier zone comprises a reporting carrier capable of forming a complex with the analyte; 
 a detection zone down stream of the reporting carrier zone, wherein the detection zone comprises a capture component and an indicator; and 
 a substrate for the proficient enzyme; wherein the substrate is applied to the detection zone after the test sample has been allowed to flow through the lateral flow device and the product of the enzyme and substrate is detected. 
   
     
     
         62 . The lateral flow assay kit of  claim 61  further comprising a control zone, wherein the control zone comprises a capture component for the reporting carrier and an indicator for detecting a reaction of a substrate in the presence of a proficient enzyme; wherein the product of the enzyme reporting carrier complex and substrate is detected thereby detecting the presence of the reporting carrier. 
     
     
         63 . The lateral flow assay kit of  claim 61  wherein the proficient enzyme conjugate is selected from the group consisting of an antibody; a nucleic acid; or urease, phosphocholine phosphatase, betagalactosidase, xylose reductase, shikimate dehydrogenase, malate dehydrogenase, carboxylesterase, neopullulanase, subtilisin, 4-phytase, acetylcholinesterase, laccase, bacterial leucyl aminopeptidase, tripeptidyl-peptidase I, coagulation factor VIIa, trypsin, beta-fructofuranosidase. 
     
     
         64 - 65 . (canceled) 
     
     
         66 . The lateral flow assay kit of  claim 61  wherein the antibody interacts non-covalently with a proficient enzyme to form the proficient enzyme conjugate; or wherein the nucleic acid is covalently attached to a proficient enzyme to form the proficient enzyme conjugate. 
     
     
         67 . (canceled) 
     
     
         68 . The lateral flow assay kit of  claim 61  further providing one or more inactive pro-enzymes the detection zone. 
     
     
         69 . The lateral flow assay kit of  claim 61 , wherein the activity of the proficient enzyme conjugate is detected by monitoring the effect of the proficient enzyme assisted reaction. 
     
     
         70 . The lateral flow assay kit of  claim 61 , wherein the indicator is selected from the group consisting of a pH sensitive indicator; Potassium I-hydroxy-4-[1-(2-hydroxyethylsulphonyl)phenylazo]-naphthalene-2-sulphonate; or a pH sensitive cellulose acetate coupled dye. 
     
     
         71 - 72 . (canceled) 
     
     
         73 . The lateral flow assay kit of  claim 61 , wherein the reporting carrier zone comprises a conjugate pad. 
     
     
         74 . The lateral flow assay kit of  claim 61 , wherein the sample loading zone comprises a sample loading pad. 
     
     
         75 . The lateral flow assay kit of  claim 61 , further comprising a rigid or flexible backing material. 
     
     
         76 . The lateral flow assay kit of  claim 61 , wherein the antibody is associated with the proficient enzyme by noncovalent interactions; or wherein the antibody or nucleic acid is covalently attached to the proficient enzyme. 
     
     
         77 . (canceled) 
     
     
         78 . The lateral flow assay kit of  claim 61 , further comprising a source of one or more inactive pro-enzymes. 
     
     
         79 . The lateral flow assay kit of  claim 61 , wherein the detection zone detects a product of the proficient enzyme by detection of a pH change and wherein the pH change is determined using a pH sensitive hydrogel. 
     
     
         80 . (canceled) 
     
     
         81 . The lateral flow assay kit of  claim 61 , wherein the detection zone detects the product of the proficient enzyme by colorimetric change via a change due to silver ion reduction; wherein the detection zone detects the product of the proficient enzyme by fluorescence emission; and wherein the detection zone detects the product of the proficient enzyme by electrochemical methods. 
     
     
         82 - 84 . (canceled) 
     
     
         85 . The lateral flow assay device of  claim 61 , the detection zone detects the product of the proficient enzyme by precipitation of a soluble component; wherein the soluble component is a protein BSA or pH sensitive polymer and wherein the pH sensitive polymer is selected from the group consisting of methyl acrylic acid, methyl methacrylate, methacrylic acid 2-(dimethylamino) ethyl ester, and N-hydroxymethyl acrylamide. 
     
     
         86 . (canceled) 
     
     
         87 . (canceled) 
     
     
         88 . (canceled) 
     
     
         89 . The lateral flow assay device of  claim 60  further comprising an absorptive pad at the distal end of the chromatographic medium. 
     
     
         90 . The lateral flow assay device of  claim 60 , wherein the substrate is allyl hexanoate and proficient enzyme is carboxylesterase.