Biosensing system with extended lifetime via cofactor recycling

1. A method for measuring the concentration of an analyte in a solution, the method comprising:

immersing a biosensing element comprising a first biocomponent, a second biocomponent, a third biocomponent, and a transducer layer in said solution, wherein:

said analyte is a reactant in a reaction catalyzed by an oxygenase enzyme that requires a cofactor selected from the group consisting of NADH, NADPH, FADH, FADH 2 , FMNH, FMNH 2 ; said first biocomponent comprises said oxygenase enzyme;

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

said first biocomponent catalyzes the reaction of said analyte and said cofactor, while consuming oxygen and producing oxidized cofactor and an epoxide product;

said oxidized cofactor is reduced by said second biocomponent and said electron donor; and

wherein said epoxide product is a reactant in a reaction catalyzed by said third biocomponent, causing said transducer layer to fluoresce photons, and

determining the concentration of said analyte in said solution, wherein:

oxygen quenches at least some of the fluorescent photons;

remaining unquenched photons enter into a fiber optic cable and are transmitted to a photomultiplier; and

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.

2. The method of claim 1 wherein said first biocomponent is toluene ortho-monooxygenase.

3. The method of claim 1 wherein said first biocomponent is a toluene ortho-monooxygenase green.

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

5. The method of claim 1 wherein said third biocomponent is selected from the group consisting of epoxide hydrolase, glutathione synthetase, glutathione S-transferase and gamma-glutamylcysteine synthetase.

6. A method of manufacturing a biosensing system having a linear response to the concentration of an analyte in a bulk solution, the method comprising:

covering a first tip of an optical fiber of an optode with a luminescent reagent layer;

covering said luminescent reagent layer with a biocomponent layer having a preselected thickness;

covering said biocomponent layer with a porous layer having a preselected thickness and a preselected diffusion coefficient;

coupling a second tip of said optical fiber to a photon-detection device, and

coupling said photon-detection device to a signal processing system, wherein

said preselected thickness of said biocomponent layer, said preselected thickness of said porous layer, and

said preselected diffusion coefficient of said porous layer, are selected such that Da is greater than the value of 1−β and a quotient between Da 2 and 4β is from about 10 to at least 1000.

7. The method of claim 6 wherein said biocomponent layer comprises toluene ortho-monooxygenase.

8. The method of claim 6 wherein said biocomponent layer comprises a toluene ortho-monooxygenase green.

9. The method of claim 6 wherein said biocomponent layer comprises a toluene ortho-monooxygenase green and formate dehydrogenase.

10. The method of claim 6 wherein said biocomponent layer comprises a 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.

11. The method of claim 6 wherein said transducer layer comprises RuDPP.

12. The method of claim 6 wherein said porous layer is track-etched polycarbonate.

13. The method of claim 6 wherein said porous layer is a polymer.

14. The method of claim 6 wherein said biocomponent layer comprises toluene ortho-monooxygenase and formate dehydrogenase.

15. The method of claim 6 wherein said biocomponent layer comprises toluene ortho-monooxygenase, formate dehydrogenase and at least one enzyme selected from the group consisting of epoxide hydrolase, glutathione synthetase, glutathione S-transferase and gamma-glutamylcysteine sythetase.

16. A method for measuring the concentration of an analyte in a solution, the method comprising:

immersing a biosensing element comprising a first biocomponent, a second biocomponent, and a transducer layer in said solution, wherein:

said analyte is a reactant in a reaction catalyzed by an oxygenase enzyme that requires a cofactor selected from the group consisting of NADH and NADPH;

said first biocomponent comprises said oxygenase enzyme;

said second biocomponent comprises a dehydrogenase enzyme that catalyzes a reaction of an oxidized cofactor selected from the group consisting of NAD + and NADP + and an electron donor;

said first biocomponent catalyzes the reaction of said analyte and said cofactor, while consuming oxygen and producing oxidized cofactor; and

said oxidized cofactor is reduced by said second biocomponent and said electron donor, causing said transducer layer to fluoresce photons, and

determining the concentration of said analyte in said solution, wherein:

oxygen quenches at least some of the fluorescent photons;

remaining unquenched photons enter into a fiber optic cable and are transmitted to a photomultiplier; and

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.

17. The method of claim 16 wherein said first biocomponent is toluene ortho-monooxygenase.

18. The method of claim 16 wherein said first biocomponent is a toluene ortho-monooxygenase green.

19. The method of claim 16 wherein said second biocomponent is formate dehydrogenase and said electron donor is formate.