Oxygenase-Based Biosensing Systems For Measurement Of Halogenated Alkene Concentrations

• US 20140234882A1
• Filed: 10/01/2012
• Published: 08/21/2014
• Est. Priority Date: 04/09/2007
• Status: Active Grant

• Current Assignee: Colorado State University Research Foundation
• Original Assignee: Colorado State University Research Foundation
• Inventors: Reardon, Kenneth F., Heinze, Brian C.
• Granted Patent: US 9,796,998 B2
• Time in Patent Office: Days
• US Class Current: 435/15
• CPC Class Codes: C12N 9/0069 acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
• CPC Class Codes: C12N 9/0071 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: C12N 9/1088 Glutathione transferase (2.5.1.18)
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: C12Q 1/34 involving hydrolase
• CPC Class Codes: C12Q 1/48 involving transferase
• CPC Class Codes: C12Y 113/00 Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13)
• CPC Class Codes: C12Y 114/00 Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
• CPC Class Codes: G01N 2021/7753 Reagent layer on photoelectrical transducer
• CPC Class Codes: G01N 2021/7786 Fluorescence
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/7703 using reagent-clad optical fibres or optical waveguides using measurement of total internal reflection or attenuated total reflection G01N21/552; optical fibres or waveguides per se G02B

Abstract

A biosensing system that measures the concentration of halogenated alkenes is disclosed.

Claims

1. A biosensing system that measures the concentration of a halogenated alkene in a solution, said biosensing system comprising

a first biocomponent that catalyzes the reaction of said halogenated alkene, and

a second biocomponent that catalyzes the reaction of a halogenated alkene epoxide, and

a transducer layer that luminesces, and

wherein said transducer layer is part of an optode.

6. The biosensing system of claim 1

wherein said first biocomponent catalyzes the reaction of said halogenated alkene and oxygen and said halogenated alkene epoxide is created by the reaction of said first biocomponent with said halogenated alkene,

wherein said transducer layer luminescence is altered by oxygen and/or hydrogen ions in said solution, and

wherein photons from the luminescence of said transducer layer enter into a fiber optic cable and are transmitted to a photomultiplier, and

wherein said photomultiplier produces an output signal that is coupled to an algorithm that transforms the signal generated by said photomultiplier into an output correlated to the concentration of said halogenated alkene in the solution.

7. The bio sensing system of claim 6 wherein said halogenated alkene is selected from the group consisting of tetrachloroethene, trichloroethene, dichloroethene, and monochloroethene.

8. The bio sensing system of claim 6 wherein

said first biocomponent is selected from the group consisting of toluene ortho-monooxygenase, toluene ortho-monooxygenase-Green, and toluene ortho-monooxygenase variant;

said second biocomponent is selected from the group consisting of epoxide hydrolase, glutathione synthetase, glutathione S-transferase and gamma-glutamylcysteine synthetase; and

said transducer layer comprises a luminescent reagent selected from the group consisting of RuDPP and fluorescein.

11. The biosensing system of claim 1

wherein said first biocomponent and said second biocomponent comprise cells, and

wherein said cells contain enzymes selected from the group consisting of oxygenases, monooxygenases, dioxygenases, toluene dioxygenase, toluene ortho-monooxygenase, toluene ortho-monooxygenase-Green, epoxide hydrolase, glutathione synthetase, glutathione S-transferase and gamma-glutamylcysteine synthetase and

wherein said cells are immobilized within a matrix, and

wherein said matrix is in contact with said transducer layer.

12. The bio sensing system of claim 11 wherein said cells are alive.

13. The bio sensing system of claim 11 wherein said cells are dead.

14. The bio sensing element of claim 11 wherein the nucleotide coding sequence of said biocomponent enzymes are on a plasmid or plasmids within a whole cell biocomponent or on a chromosome of a whole cell biocomponent.

16. The bio sensing element of claim 11 wherein said optode is selected from the group consisting of an oxygen optode and a pH optode.

18. The biosensing system of claim 1, wherein said halogenated alkene is trichloroethene,

wherein said first biocomponent catalyzes the reaction of trichloroethene and oxygen and said second biocomponent catalyzes the reaction of trichloroethene epoxide, and

wherein said transducer layer luminescence is altered by oxygen in said solution, and

wherein photons from the luminescence of said transducer layer enter into a fiber optic cable and are transmitted to a photomultiplier, and

wherein said photomultiplier produces an output signal that is coupled to an algorithm that transforms the signal generated by said photomultiplier into an output correlated to the concentration of trichloroethene in the solution.

44. The biosensing system of claim 1, wherein

said first biocomponent catalyzes the reaction of a halogenated alkene and oxygen and said second biocomponent catalyzes the reaction of a halogenated alkene epoxide created by the reaction of said first biocomponent with said halogenated alkene, and

wherein said transducer layer comprises compounds and chemical complexes containing ruthenium, and

wherein said transducer layer luminescence is altered by oxygen in said solution, and

wherein photons from the luminescence of said transducer layer enter into a fiber optic cable and are transmitted to a photomultiplier, and

wherein said photomultiplier produces an output signal that is coupled to an algorithm that transforms the signal generated by said photomultiplier into an output correlated to the concentration of said halogenated alkene in the solution.

58. The biosensing system of claim 1, wherein said first and second biocomponents comprise a purified cell-free enzyme.

2. A method for measuring the concentration of a halogenated alkene in a solution whereby

a first biocomponent catalyzes the reaction of said halogenated alkene and oxygen, and

whereby a second biocomponent catalyzes the reaction of a halogenated alkene epoxide produced by said first biocomponent, and

whereby a transducer layer luminesces, and

whereby said transducer layer luminescence is altered by oxygen and/or hydrogen ions in said solution, and

whereby photons from the luminescence of said transducer layer enter into a fiber optic cable and are transmitted to a photomultiplier, and

whereby said photomultiplier produces an output signal that is coupled to an algorithm that transforms the signal generated by said photomultiplier into an output correlated to the concentration of said halogenated alkene in said solution.

3. The method of claim 2 wherein:

said first biocomponent is selected from the group consisting of toluene ortho-monooxygenase, toluene ortho-monooxygenase-Green, toluene ortho-monooxygenase variant, and toluene dioxygenase;

said second biocomponent is selected from the group consisting of epoxide hydrolase, glutathione synthetase, glutathione S-transferase and gamma-glutamylcysteine synthetase; and

said transducer layer comprises a luminescent reagent selected from the group consisting of RuDPP and fluorescein.

22. The method of claim 2, wherein said halogenated alkene is trichloroethene wherein

said first biocomponent selected from the group consisting of toluene ortho-monooxygenase, toluene ortho-monooxygenase-Green and, toluene ortho-monooxygenase variant, and catalyzes the reaction of trichloroethene and produces trichloroethene epoxide, and

wherein said trichloroethene epoxide is a substrate for a reaction catalyzed by said second biocomponent selected from the group consisting of epoxide hydrolase, glutathione synthetase, glutathione S-transferase and gamma-glutamylcysteine synthetase, and

wherein said transducer layer luminescence is altered by oxygen in said solution, and

wherein said photomultiplier produces an output signal that is coupled to an algorithm that transforms the signal generated by said photomultiplier into an output correlated to the concentration of said trichloroethene in said solution.

4-5. -5. (canceled)

9-10. -10. (canceled)

15. (canceled)

17. (canceled)

19-21. -21. (canceled)

23-43. -43. (canceled)

45. (canceled)

46. A biosensing system that measures the concentration of a halogenated alkene in a solution, said biosensing system comprising

a biocomponent that catalyzes the reaction of said halogenated alkene, and

a transducer layer that luminesces, and

wherein said transducer layer is part of an optode.

54. The biosensing system of claim 46, wherein said biocomponent is toluene ortho-monooxygenase, a toluene ortho-monooxygenase variant, or toluene dioxygenase.

55. The biosensing system of claim 46, wherein said biocomponent comprises a purified cell-free enzyme.

56. The biosensing system of claim 46, wherein said halogenated alkene is trichloroethene.

57. The biosensing system of claim 46, wherein said transducer layer comprises a luminescent reagent that is RuDPP or fluorescein.

47-53. -53. (canceled)