Enzymatic biosensors with enhanced activity retention for detection of organic compounds

• US 9,493,805 B2
• Filed: 04/09/2008
• Issued: 11/15/2016
• Est. Priority Date: 06/01/2001
• Status: Active Grant

• Current Assignee: Colorado State University Research Foundation, Texas A&M University System
• Original Assignee: Colorado State University Research Foundation
• Inventors: Wood, Thomas K., Reardon, Kenneth F., Dandy, David S., Fritzsche, Michael
• Primary Examiner(s): Shen, Bin
• Application Number:
  US12/100,308
• Publication Number:
  US 20090026092A1
• Time in Patent Office: 3,142 Days
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/002 Electrode membranes
• CPC Class Codes: C12Q 1/005 involving specific analytes or enzymes including groups of enzymes, e.g. oxydases; C12Q1/004 takes precedence
• CPC Class Codes: G01N 2333/90241 acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
• CPC Class Codes: G01N 2333/90245 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: G01N 2333/914 Hydrolases (3)
• CPC Class Codes: G01N 2333/988 Lyases (4.), e.g. aldolases, heparinase, enolases, fumarase

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.

Claims

1. A biosensor for use in measuring a concentration of an organic compound analyte, said biosensor comprising:

a distal portion;

an optical transducer disposed in the distal portion of said biosensor capable of measuring oxygen concentration in situ;

a biocomponent disposed in the distal portion of said biosensor, said biocomponent immobilized within a hydrogel or polymer matrix, and said biocomponent comprising one or more oxygenases from EC family 1.13 and EC family 1.14 for carrying out an oxidation of the organic compound analyte.

2. The biosensor of claim 1, wherein the one or more oxygenases are 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.

3. The biosensor of claim 1, wherein the organic compound analyte is one or more halogenated ethene.

4. The biosensor of claim 3, wherein the one or more halogenated ethene is a chlorinated ethene selected from the group consisting of tetrachloroethene (PCE), trichloroethene (TCE), dichloroethene isomers and vinyl chloride (VC).

5. The biosensor of claim 1, further comprising a device for delivering one or more reagents.

6. The distal biosensor of claim 5, wherein the reagent is selected from oxygen and hydrogen peroxide.

7. The biosensor of claim 1 wherein the biocomponent is a microorganism containing the one or more oxygenases selected from the group consisting of Burkholderia cepacia G4, Pseudomonas stutzeri OX1, P. mendocina KR1, M. trichosporium OB3b, M capsulatus Bath, P. stutzeri OX1, and R. pickettii PK01.

8. The biosensor of claim 7 wherein the one or more oxygenases include a monooxygenase.

9. The biosensor of claim 8 wherein the monooxygenase is encoded by a polynucleotide comprising one or more molecular modifications obtainable using one or more methods selected from the group consisting of site-directed mutagenesis, DNA shuffling and saturation mutagenesis.

10. The biosensor of claim 7 wherein the one or more oxygenases include a dioxygenase.

11. The biosensor of claim 7, wherein the microorganism comprises at least one plasmid comprising one or more polynucleotides encoding for the one or more oxygenases.

12. The biosensor of claim 11 wherein one or more nucleic acids in the at least one plasmid is mutated using one or more methods selected from the group consisting of site-directed mutagenesis, DNA shuffling and saturation mutagenesis.

13. The biosensor of claim 7 wherein said microorganism is selected from the group consisting of live cells and dead cells.

14. The biosensor of claim 1 wherein the biocomponent is layered atop a transducer layer of an oxygen-sensitive fluorophore.

15. The biosensor of claim 1 wherein the biocomponent is lyophilized.

16. The biosensor of claim 1, wherein said biocomponent further comprises one or more of a gel-hardening agent and a stabilizing agent.

17. The biosensor of claim 16, wherein the one or more of a gel-hardening agent and a stabilizing agent comprises one or more of glutaraldehyde, polyethyleneimine, hexamethylenediamine and formaldehyde.

18. The biosensor of claim 1, 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.

19. The biosensor of claim 1, wherein the polymer matrix comprises one or more of polyacrylamide, polystyrene, polymethacrylate, polyvinylalcohol and polyurethane.

20. The biosensor of claim 1, wherein said biocomponent is encapsulated in a microporous semipermeable membrane.

21. The biosensor of claim 20, wherein the membrane comprises one or more of cellulose acetate, polycarbonate, collagen, acrylate copolymers, poly(ethylene glycol), polytetrafluoroethylene (PTFE), agarose, and alginate-polylysine-alginate microcapsules formed of alginate and polylysine.