Biosensing system with extended lifetime via cofactor recycling

• US 9,499,853 B2
• Filed: 08/02/2012
• Issued: 11/22/2016
• Est. Priority Date: 08/02/2011
• Status: Active Grant

• Current Assignee: Colorado State University Research Foundation
• Original Assignee: Colorado State University Research Foundation
• Inventors: Reardon, Kenneth F.
• Primary Examiner(s): Humphrey, Louise W
• Assistant Examiner(s): Kosar, Aaron J
• Application Number:
  US14/236,531
• Publication Number:
  US 20140154724A1
• Time in Patent Office: 1,573 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12M 1/34 Measuring or testing with condition measuring or sensing means, e.g. colony counters
• CPC Class Codes: C12M 1/40 Apparatus specially designed for the use of free, immobilised, or carrier-bound enzymes, e.g. apparatus containing a fluidised bed of immobilised enzymes
• CPC Class Codes: C12N 9/0004 Oxidoreductases (1.)
• 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: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: C12Q 1/32 involving dehydrogenase
• CPC Class Codes: C12Y 102/01002 Formate dehydrogenase (1.2.1.2)
• 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: C12Y 114/13 with NADH or NADPH as one donor, and incorporation of one atom of oxygen (1.14.13)
• CPC Class Codes: G01N 2021/6432 Quenching
• CPC Class Codes: G01N 21/6408 with measurement of decay time, time resolved fluorescence
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 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)

Abstract

The present disclosure relates to biosensing systems and biosensing elements having increased storage capability and increased functional lifetimes through using compositions and methods for recycling cofactors.

Claims

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

a first biocomponent that catalyzes the reaction of said analyte and oxygen and uses a cofactor selected from the group consisting of NADH, NADPH, FADH, FADH₂, FMNH, FMNH₂, and

a second biocomponent that catalyzes the reaction of a cofactor selected from the group consisting of NAD⁺, NADP⁺, FADH, FMNH, FAD and FMN, and an electron donor, and

wherein said first biocomponent and said second biocomponent are immobilized within a matrix, and

wherein said matrix is in contact with a transducer layer, and

wherein said transducer layer is part of an optode.

2. The biosensing system of claim 1 wherein said transducer layer is an optical transducer that constitutively fluoresces and wherein the fluorescence is quenched during use by oxygen in an oxygen-containing solution.

3. The biosensing system of claim 1 wherein said first biocomponent is selected from the group consisting of monooxygenases and dioxygenases.

4. The biosensing system of claim 1 wherein said second biocomponent is formate dehydrogenase and said electron donor is formate.

5. The biosensing system of claim 1 further comprising a tip of a capillary tube, wherein said capillary tube contains formic acid or a salt of formate anion, such that formate is delivered through capillary action to said first and second biocomponent.

6. The biosensing system of claim 1, wherein

said analyte is a reactant in a reaction catalyzed by of an oxygenase enzyme, said oxygenase enzyme selected from the group consisting of Enzyme Commission numbers 1.13 and 1.14, and

wherein said oxygenase enzyme requires a reduced cofactor selected from the group consisting of NADH, NADPH, FADH, FADH₂, FMNH, and FMNH₂, and

wherein said first biocomponent comprises said oxygenase enzyme, and

wherein said second biocomponent comprises a dehydrogenase enzyme that catalyzes the reaction of an oxidized cofactor selected from the group consisting of NAD⁺, NADP⁺, FADH, FMNH, FAD and FMN and an electron donor, and

wherein a third biocomponent comprises an enzyme selected from the group consisting of epoxide hydrolase, glutathione synthetase, glutathione S-transferase and gamma-glutamylcysteine sythetase, and

wherein said first biocomponent catalyzes the reaction of said analyte and said cofactor selected from the group consisting of NADH, NADPH, FADH, FADH₂, FMNH, and FMNH₂ while consuming oxygen and producing oxidized cofactor and an epoxide product, and

wherein said oxidized cofactor is reduced by said second biocomponent and said electron donor, and

wherein said epoxide product is a reactant in the reaction catalyzed by said third biocomponent, and

wherein a transducer layer fluoresces photons, and

wherein oxygen quenches at least some of the fluorescent photons, and

wherein said photons 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 analyte in said solution.

7. The biosensing system of claim 6 wherein said first biocomponent is toluene ortho-monooxygenase.

8. The biosensing system of claim 6 wherein said first biocomponent is toluene ortho-monooxygenase-Green.

9. The biosensing system of claim 6 wherein said second biocomponent is formate dehydrogenase and said electron donor is formate.

10. The biosensing system of claim 1, wherein

said optode comprises a fiber optical cable having a first tip and a second tip, and

said first tip is covered by said transducer layer, and

said transducer layer is covered by a biocomponent layer comprising said first and second biocomponent within said matrix, and

said biocomponent layer is covered by a porous layer, and

said second tip is coupled to a photon-detection device, and

said photon-detection device is coupled to a signal processing system, and

said analyte concentration in said solution, the depth of the biocomponent layer, the depth of the porous layer, the diffusion coefficient of said porous layer, the K_m and V_max of the reaction between said biocomponent and said analyte are selected such that the quotient between Da^e and 4β

is from about 10 to about 1000.

11. The biosensing system of claim 10 wherein said biocomponent layer comprises toluene ortho-monooxygenase, toluene ortho-monooxygenase-Green, or toluene ortho-monooxygenase Green and formate dehydrogenase.

12. The biosensing system of claim 10 wherein said biocomponent comprises toluene ortho-monooxygenase-Green, formate dehydrogenase and at least one enzyme selected from the group consisting of epoxide hydrolase, glutathione synthetase, glutathione S-transferase and gamma-glutamylcysteine sythetase.

13. The biosensing system of claim 10 wherein said transducer layer comprises RuDPP.

14. The biosensing system of claim 10 wherein said porous layer is track-etched polycarbonate.

15. The biosensing system of claim 1 wherein said first biocomponent and said second biocomponent are within or on the surface of a whole cell biocomponent that is immobilized within a matrix, and

wherein said matrix is adhered to said transducer layer.

16. A method for measuring the concentration of an analyte in a solution using a biosensing system wherein

said biosensing system comprises biocomponents, and

wherein a first biocomponent of said biosensing system is an oxygenase enzyme that catalyzes a reaction that uses oxygen, said analyte and a reduced cofactor as reactants and produces oxidized cofactor, and

wherein a second biocomponent of said biosensing system is a dehydrogenase enzyme that catalyzes a reaction that uses said oxidized cofactor and an electron donor as reactants and produces said reduced cofactor, and

wherein said reduced cofactor is used in the reaction catalyzed by said first biocomponent, and

wherein the oxygen concentration in said solution is measured by said biosensing system, and

wherein the measured oxygen concentration in said solution is used to calculate the measurement of the concentration of said analyte in said solution.

17. The method of claim 16 wherein said biosensing system further comprises a transducer layer that fluoresces photons, and

wherein oxygen quenches the emission of at least some of the fluorescent photons, and

wherein said photons 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 analyte in said solution.

18. The method of claim 16 wherein said first biocomponent is toluene ortho-monooxygenase or toluene ortho-monooxygenase-Green;

and

said second biocomponent is formate dehydrogenase and said electron donor is formate.

19. The method of claim 16 wherein

said analyte is a reactant in the reaction catalyzed by an oxygenase enzyme, and

wherein said oxygenase enzyme requires a cofactor selected from the group consisting of NADH, NADPH, FADH, FADH₂, FMNH, FMNH₂, and

wherein said second biocomponent comprises a dehydrogenase enzyme that catalyzes the reaction of an oxidized cofactor selected from the group consisting of NAD⁺, NADP⁺, FADH, FMNH, FAD and FMN, and an electron donor, and

wherein a third biocomponent comprises an enzyme selected from the group consisting of epoxide hydrolase, glutathione synthetase, glutathione S-transferase and gamma-glutamylcysteine sythetase, and

wherein said first biocomponent catalyzes the reaction of said analyte and said cofactor selected from the group consisting of NADH, NADPH, FADH, FADH₂, FMNH, FMNH₂, while consuming oxygen and producing oxidized cofactor and an epoxide product, and

wherein said oxidized cofactor is reduced by said second biocomponent and said electron donor, and

wherein said epoxide product is a reactant in the reaction catalyzed by said third biocomponent, and

wherein a transducer layer fluoresces photons, and

wherein oxygen quenches the emission of at least some of the fluorescent photons, and

wherein said photons 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 analyte in said solution.