Enzymatic Biosensors With Enhanced Activity Retention For Detection Of Organic Compounds

Abstract

Enzymatic biosensors and methods of producing distal tips for biosensor transducers for use in detecting one or more analytes selected from organic compounds susceptible to dehalogenation, organic compounds susceptible to oxygenation and organophosphate compounds susceptible to hydrolysis are disclosed herein, as well as biosensor arrays, methods of detecting and quantifying analytes within a mixture, and devices and methods for delivering reagents to enzymes disposed within the distal tip of a biosensor.

What is claimed is: 
     
         1 . An enzymatic biosensing system for measuring the concentration of an analyte, the biosensing system comprising:
 one or more biosensors comprising at least one optical transducer capable of responding to oxygen concentration positioned adjacent to at least one biocomponent, the at least one optical transducer and the at least one biocomponent each disposed in a distal portion of the one or more biosensors; and   an optoelectronic instrumentation system functionally coupled to the one or more biosensors.   
     
     
         2 . The enzymatic biosensing system of  claim 1 , wherein the at least one biocomponent comprises at least one oxygenase from EC number 1.13 or EC number 1.14 for carrying out oxidation or oxygenation of the analyte. 
     
     
         3 . The enzymatic biosensing system of  claim 2 , wherein the at least one oxygenase is selected from the group consisting of toluene o-monooxygenase (TOM), soluble methane monooxygenase (sMMO), toluene xylene-o-monooxygenase (ToMO), toluene p-monooxygenase (T4MO), toluene-m-monooxygenase (T3MO), phenol hydroxylase (PH) and TOM-Green. 
     
     
         4 . The enzymatic biosensing system of  claim 1 , wherein the at least one biocomponent is immobilized via entrapment within a hydrogel or a polymer matrix. 
     
     
         5 . The enzymatic biosensing system of  claim 4 , wherein the at least one biocomponent is immobilized within the hydrogel or the polymer matrix using one or more of a cross-linking agent and a stabilizing agent. 
     
     
         6 . The enzymatic biosensing system of  claim 5 , wherein the cross-linking agent or the stabilizing agent comprise one or more of glutaraldehyde, polyethyleneimine, hexamethylenediamine and formaldehyde. 
     
     
         7 . The enzymatic biosensing system of  claim 4 , wherein the hydrogel comprises one or more of algal polysaccharides, agar, agarose, alginate, K-carrageenan, gelatin, collagen, pectin, poly(carbamoyl) sulfonate, locust bean gum and gellan. 
     
     
         8 . The enzymatic biosensing system of  claim 4 , wherein the polymer matrix comprises one or more of polyacrylamide, polystyrene, polymethacrylate, polyvinylalcohol and polyurethane. 
     
     
         9 . The enzymatic biosensing system of  claim 1 , wherein the at least one biocomponent is layered atop a transducer layer comprising an oxygen-sensitive fluorophore. 
     
     
         10 . The enzymatic biosensing system of  claim 9 , wherein the oxygen-sensitive fluorophore comprises a ruthenium or platinum complex. 
     
     
         11 . The enzymatic biosensing system of  claim 1 , wherein the analyte is an organic compound, and wherein the organic compound is susceptible to dehalogenation, oxygenation or hydrolysis. 
     
     
         12 . The enzymatic biosensing system of  claim 11 , wherein the organic compound analyte is one or more halogenated ethenes. 
     
     
         13 . The enzymatic biosensing system of  claim 12 , wherein the one or more halogenated ethenes is one or more of tetrachloroethene (PCE), trichloroethene (TCE), dichloroethene isomers and vinyl chloride (VC). 
     
     
         14 . The enzymatic biosensing system of  claim 1 , wherein the biocomponent comprises cells or purified enzymes. 
     
     
         15 . The enzymatic biosensing system of  claim 1 , wherein the optoelectronic instrumentation system comprises an excitation source, a fiber optic system, a photodetector and signal processing circuitry. 
     
     
         16 . The enzymatic biosensing system of  claim 15 , wherein the fiber optic system carries excitation light to the optical transducer and carries an optical signal from the optical transducer to the photodetector. 
     
     
         17 . The enzymatic biosensing system of  claim 16 , wherein the photodetector detects the optical signal from the optical transducer. 
     
     
         18 . The enzymatic biosensing system of  claim 16 , wherein the signal processing circuitry generates a value from the optical signal detected by the photodetector that corresponds to the analyte concentration. 
     
     
         19 . The enzymatic biosensing system of  claim 1 , wherein the one or more biosensors form a biosensor array capable of detecting multiple analytes. 
     
     
         20 . The enzymatic biosensing system of  claim 1 , further comprising a device for delivering one or more reagents to the at least one biocomponent or the at least one optical transducer.