Enzyme Detection Technique

Abstract

A diagnostic test kit for detecting the presence or absence of an enzyme (e.g., leukocyte esterase) within a test sample is provided. The test kit comprises a substrate that is capable of being cleaved in the presence of the enzyme to release a nucleophilic aromatic compound. The kit also comprises a lateral flow device that comprises a chromatographic medium. The chromatographic medium defines a detection zone within which is contained a first reagent (e.g., diazonium ion) that is capable of reacting with the nucleophilic aromatic compound to form a second reagent (e.g., aromatic azo compound). The second reagent exhibits a color that is different than the color of the first reagent. The lateral flow device also includes an absorbent material located adjacent to the chromatographic medium, the absorbent material receiving the test sample after flowing through the chromatographic medium.

1 . A diagnostic test kit for detecting the presence or absence of an enzyme within a test sample, the test kit comprising:
 a substrate that is capable of being cleaved in the presence of the enzyme to release a nucleophilic aromatic compound; and   a lateral flow device that comprises:
 a chromatographic medium defining a detection zone within which is contained a first reagent, the first reagent being capable of reacting with the nucleophilic aromatic compound to form a second reagent, the second reagent exhibiting a color that is different than the color of the first reagent; and 
 an absorbent material that receives the test sample after flowing through the chromatographic medium. 
   
   
   
       2 . The diagnostic test kit of  claim 1 , wherein the enzyme is an esterase. 
   
   
       3 . The diagnostic test kit of  claim 2 , wherein the esterase is leukocyte esterase. 
   
   
       4 . The diagnostic test kit of  claim 2 , wherein the substrate is an aromatic ester. 
   
   
       5 . The diagnostic test kit of  claim 4 , wherein the substrate is an indoxyl ester, lactate ester, thiazole ester, pyrrole ester, thiophene ester, naphthyl ester, phenoxyl ester, or quinolinyl ester. 
   
   
       6 . The diagnostic test kit of  claim 1 , wherein the nucleophilic aromatic compound is a benzopyrrole. 
   
   
       7 . The diagnostic test kit of  claim 1 , wherein the first reagent is a diazonium ion. 
   
   
       8 . The diagnostic test kit of  claim 7 , wherein the diazonium ion is provided by a salt selected from the group consisting of diazonium chlorides, diazonium acid sulphates, diazonium alkyl sulphates, diazonium fluoborates, diazonium benzenesulphonates, and diazonium acid 1,5-naphthalenedisulphonates. 
   
   
       9 . The diagnostic test kit of  claim 7 , wherein the second reagent is an aromatic azo compound. 
   
   
       10 . The diagnostic test kit of  claim 1 , wherein the chromatographic medium is a porous membrane. 
   
   
       11 . The diagnostic test kit of  claim 1 , wherein the substrate is disposed on the lateral flow assay device. 
   
   
       12 . The diagnostic test kit of  claim 11 , wherein the lateral flow assay device further comprises a reagent zone within which is contained the substrate. 
   
   
       13 . The diagnostic test kit of  claim 12 , further comprising a sample application zone that is located upstream from the reagent zone. 
   
   
       14 . The diagnostic test kit of  claim 1 , wherein the lateral flow device comprises a quenching zone located upstream from the detection zone. 
   
   
       15 . The diagnostic test kit of  claim 14 , wherein the quenching zone contains an agent capable of reacting with a contaminant in the test sample. 
   
   
       16 . The diagnostic test kit of  claim 1 , wherein the lateral flow device comprises a control zone that is capable of signaling the presence of the test sample. 
   
   
       17 . The diagnostic test kit of  claim 16 , further comprising a control reagent that is capable being detected within the control zone. 
   
   
       18 . The diagnostic test kit of  claim 1 , wherein the test kit further comprises an accelerator for the substrate. 
   
   
       19 . The diagnostic test kit of  claim 18 , wherein the accelerator is contained within an accelerator zone located upstream from the detection zone. 
   
   
       20 . The diagnostic test kit of  claim 1 , wherein the test sample is urine. 
   
   
       21 . A diagnostic test kit for detecting the presence or absence of leukocyte esterase within urine, the test kit comprising:
 an aromatic ester substrate that is capable of being hydrolyzed in the presence of leukocyte esterase to release a nucleophilic aromatic compound; and   a lateral flow device that comprises:
 a porous membrane defining a detection zone within which is contained a diazonium ion, the diazonium ion being capable of reacting with the nucleophilic aromatic compound to form an aromatic azo compound, the aromatic azo compound exhibiting a color that is different than the color of the diazonium ion; and 
 an absorbent material that receives the urine after flowing through the porous membrane. 
   
   
   
       22 . The diagnostic test kit of  claim 21 , wherein the substrate is an indoxyl ester, lactate ester, thiazole ester, pyrrole ester, thiophene ester, naphthyl ester, phenoxyl ester, or quinolinyl ester. 
   
   
       23 . The diagnostic test kit of  claim 21 , wherein the nucleophilic aromatic compound is a benzopyrrole. 
   
   
       24 . The diagnostic test kit of  claim 21 , wherein the diazonium ion is provided by a salt selected from the group consisting of diazonium chlorides, diazonium acid sulphates, diazonium alkyl sulphates, diazonium fluoborates, diazonium benzenesulphonates, and diazonium acid 1,5-naphthalenedisulphonates. 
   
   
       25 . The diagnostic test kit of  claim 21 , wherein the substrate is disposed on the lateral flow assay device. 
   
   
       26 . The diagnostic test kit of  claim 25 , wherein the lateral flow assay device further comprises a reagent zone within which the substrate is disposed. 
   
   
       27 . The diagnostic test kit of  claim 26 , further comprising a sample application zone that is positioned upstream from the reagent zone. 
   
   
       28 . The diagnostic test kit of  claim 21 , wherein the lateral flow device comprises a quenching zone located upstream from the detection zone. 
   
   
       29 . The diagnostic test kit of  claim 21 , wherein the lateral flow device comprises a control zone that is capable of signaling the presence of the urine. 
   
   
       30 . The diagnostic test kit of  claim 21 , wherein the test kit further comprises an accelerator for the substrate, the accelerator being contained within an accelerator zone located upstream from the detection zone.