Rapid Tests for the Detection of Inhibitors of Enzymes and Human Exposure to the Same

Abstract

A device and method for the rapid on-site detection of inhibitors of enzymes, such as, acetylcholinesterase is described where the device contains 2 reaction zones containing a reporter enzyme substrate. One reaction zone is for the test sample while the other is for an onboard negative control. Sample and control fluids are preincubated with the enzyme in separate reaction containers, then an aliquot of each reaction mixture is added to designated reaction zones on the test device. A purpose-built reader or an illuminating device, such as, containing an incandescent light source, a diode, a UV light source or any other illumination source that is suitable for the reporter or mere visualization is used to determine the level of reporter.

We claim: 
     
         1 . A test system for detecting enzyme inhibitors comprising:
 a) a device comprising two reaction zones, each reaction zone comprising a porous matrix comprising an immobilized reporter substrate of said enzyme, wherein said substrate is a reporter for activity of said enzyme; and   b) separate from the device, two reaction containers comprising said enzyme, and optionally,   c) separate from the device, a reader that detects said reporter substrate.   
     
     
         2 . The test system of  claim 1 , wherein said enzyme comprises an esterase. 
     
     
         3 . The test system of  claim 1 , wherein said enzyme comprises acetylcholinesterase, butylcholinesterase or carboxylesterase. 
     
     
         4 . The test system of  claim 1 , wherein said substrate comprises an N-methylindoxyl acetate (NMI). 
     
     
         5 . The test system of  claim 1 , wherein said inhibitor comprises an organophosphate, an organothiophosphate, an organophosphonate or a carbamate. 
     
     
         6 . The test system of  claim 1 , wherein said inhibitor comprises a pesticide, a toxicant or a nerve agent. 
     
     
         7 . The test system of  claim 1 , where said reaction containers comprise a liquid sample. 
     
     
         8 . The test system of  claim 7 , wherein said sample comprises a water for human consumption, a source water from natural and man-made bodies of water, an ocean, a wash of an edible product or a wipe of a surface. 
     
     
         9 . The test system of  claim 1 , wherein said reaction containers are free of an oxidizing agent. 
     
     
         10 . The test system of  claim 1 , wherein said reaction containers comprise a reagent that accelerates inhibitor/enzyme reaction kinetics. 
     
     
         11 . A method for detecting an inhibitor of an enzyme comprising:
 a) adding an aliquot of a sample to a reaction container comprising said enzyme, adding a control sample to a reaction containing comprising said enzyme and incubating said reaction containers;   b) adding an aliquot from each reaction container to a unique reaction zone comprising a porous matrix comprising a substrate of said enzyme of a device, said substrate comprises a reporter, and   c) following incubating said device, exposing said reactions zones to a device that detects said reporter to obtain a value comprising the amount of reporter in a reaction zone or visually comparing the reaction zones, wherein an inhibitor is correlated with a lower amount of reporter.   
     
     
         12 . The method of  claim 11 , wherein incubating said sample or control with said enzyme is in the absence of an oxidizing agent. 
     
     
         13 . The method of  claim 11 , wherein said device obtained value is compared to a statistically determined ratio value cutoff determining a positive result from a negative result, said ratio is the measured value of reporter comprising the reaction zone comprising said sample divided by the measured value of reporter comprising the reaction zone comprising said control sample. 
     
     
         14 . The method of  claim 11 , wherein said device that detects said reporter comprises an illuminating device. 
     
     
         15 . The method of  claim 11 , wherein said reaction containers of a) comprise a reagent to accelerate reaction kinetics of the inhibitor and the enzyme. 
     
     
         16 . The method of  claim 11  comprising exposing said containers of step a), said device of step b), or both to an elevated temperature. 
     
     
         17 . A method for detecting exposure of an enzyme to an inhibitor comprising:
 a) adding an aliquot of a sample and of a control sample each to a unique reaction zone, comprising a porous matrix comprising a substrate of said enzyme, of a device, said substrate comprising a reporter,   b) following incubating said device, exposing said reaction zones to a device that detects said reporter or visualizing said reaction zones, and   d) obtaining a value comprising the amount of reporter comprising a reaction zone or visually discerning a difference in the amount of reporter in said reaction zones,   wherein an inhibitor is correlated with a lower amount of reporter.   
     
     
         18 . The method of  claim 17 , wherein said sample, said control sample or both comprise a biological fluid of an individual. 
     
     
         19 . The method of  claim 17 , wherein said control sample comprises a known negative sample or a known positive sample. 
     
     
         20 . The method of  claim 17 , wherein said control sample comprises a biological fluid from said individual prior to exposure to an inhibitor.