One-step rapid assay for the detection of inhibitors of enzymes
Inventors
Vallejo, JR., Yli Remo • Milunic, David • Yin, Ray • Street, Renee • Chen, De
Assignees
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Publication Number
US-9850522-B2
Publication Date
2017-12-26
Expiration Date
Abstract
A device and method for the rapid on-site detection of inhibitors of enzymes, such as acetylcholinesterase, is described wherein the device contains all reagents added to a sample pad containing dried releasable enzyme creating a reaction mixture wherein inhibitor deactivates the enzyme, while said reaction mixture travels via a longitudinal membrane to a distal porous pad containing a substrate for the enzyme. The reaction of the enzyme and the substrate results in a product that can generate a measurable signal such as color, fluorescence or luminescence to serve as a reporter. Signal that is generated at this reaction zone is inversely proportional to inhibitor concentration in the test sample. A device containing two such strips, one for a test sample, the other for a negative control fluid as an onboard comparator is described. 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.
Core Innovation
A one-step, on-site longitudinal test-strip device enables flow of fluid from one end to the other using two pads of porous matrices on either end of the test strip. A porous fluid transfer membrane is positioned between the pads along the length of the test strip, in liquid communication with each pad. The upstream pad is a sample pad comprising releasably dried enzyme, and the downstream pad is a substrate pad comprising an immobilized substrate of the enzyme. The substrate comprises a reporter which produces a detectable signal in the device on reaction with the enzyme, detectable visually or with a portable device.
In the device, an inhibitor reacts with the enzyme to form an inactivated enzyme that does not react with the immobilized substrate. As a result, the reporter generation is inhibited, producing a detectable inhibitor response in the device. The device does not comprise an antibody and/or an antigen as a capture site, relying instead on the enzyme-substrate-reporter chemistry and the longitudinal flow structure.
The disclosed approach is described in the context of detecting enzyme inhibitors, including avoidance of oxidizing/quenching steps by using a dual-enzyme strategy and/or an integrated oxidizer pretreatment pad. The document describes enzyme and reporter pairings, including acetylcholinesterase (AChE) and carboxylesterase (CE) strategies and fluorogenic reporter substrates such as N-methylindoxyl acetate and indoxyl acetate. Example inhibitor contexts include carbamate and organophosphate/organothiophosphate targets, with detection signals reported visually or by portable reader measurement, including defined ratio-based decision thresholds for hue/ratiometric outputs.
Claims Coverage
The independent claim set identified in the provided material consists of one independent device claim. The main inventive features cover the longitudinal test-strip structure with a sample pad containing releasably dried enzyme, a downstream substrate pad with an immobilized substrate/reporter, inhibitor-mediated enzyme inactivation producing a detectable visual/portable signal, and the absence of antibody/antigen capture sites.
Longitudinal inhibitor-detection test-strip with releasably dried enzyme and immobilized reporter substrate
A device for detecting an inhibitor of an enzyme comprising a longitudinal test strip enabling flow of fluid from one end to the other, with two pads of porous matrices at either end and a fluid transfer membrane between the pads in liquid communication with each pad, wherein an upstream sample pad comprises releasably dried enzyme and a downstream substrate pad comprises an immobilized substrate of the enzyme, the substrate comprising a reporter that produces a signal detectable visually or with a portable device, and wherein the inhibitor reacts with the enzyme to form an inactivated enzyme that does not react with the substrate; the device does not comprise an antibody and/or an antigen as a capture site.
Overall claim coverage (from the provided material) centers on an inhibitor-responsive longitudinal test strip that links a releasably dried enzyme upstream with an immobilized substrate/reporter downstream via a fluid transfer membrane, producing a visual or portable-device detectable signal that depends on whether the inhibitor inactivates the enzyme; antibody/antigen capture sites are excluded.
Stated Advantages
Enables inhibitor detection in the longitudinal test-strip device with a signal detectable visually or with a portable device.
Inhibitor reaction forms an inactivated enzyme that does not react with the substrate, providing an inhibitor-dependent reporter response.
Does not comprise an antibody and/or an antigen as a capture site.
Describes avoidance of oxidizing/quenching steps for organophosphate detection using a dual-enzyme strategy and/or an integrated oxidizer pretreatment pad.
Documented Applications
On-site, one-step testing using a portable device or visual readout for detecting enzyme inhibitors.
Detection context includes enzyme inhibitors such as organophosphates, organothiophosphates, organophosphonates, carbamates, and metals/heavy or toxic metals.
Example inhibitor discrimination is documented using oxamyl (carbamate) versus fenamiphos (organothiophosphate) with different enzyme strategies and reporter signal outputs including ratio-based hue/ratiometric thresholds.
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