Biosensing system with extended lifetime via cofactor recycling
First Claim
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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, FADH2, FMNH, FMNH2;
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, anddetermining 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.
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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.
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Citations
19 Claims
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1. A method for measuring the concentration of an analyte in a solution, the method comprising:
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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, FADH2, FMNH, FMNH2;
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. - View Dependent Claims (2, 3, 4, 5)
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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:
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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 Da2 and 4β
is from about 10 to at least 1000.- View Dependent Claims (7, 8, 9, 10, 11, 12, 13, 14, 15)
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16. A method for measuring the concentration of an analyte in a solution, the method comprising:
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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. - View Dependent Claims (17, 18, 19)
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Specification