# US 2014/0154724 A1 · Biosensing system with extended lifetime via cofactor recycling

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- Number: US 2014/0154724 A1
- Document ID: US20140154724A1
- Status: Published application
- Kind: A1
- Application number: 14/236,531
- Filed: 2012-08-02
- Published: 2014-06-05
- Earliest claimed: 2011-08-02
- Assignees: Colorado State University Research Foundation
- Inventors: Kenneth F. Reardon
- Web page: https://patentgrep.com/patent/US20140154724A1

## CPC

- C12Q 1/26: involving oxidoreductase
- C12N 9/0004: {Oxidoreductases (1.)}
- C12N 9/0069: {acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)}
- C12N 9/0071: {acting on paired donors with incorporation of molecular oxygen (1.14)}
- C12Q 1/32: involving dehydrogenase
- C12Y 102/01002: Formate dehydrogenase (1.2.1.2)
- C12Y 114/13: with NADH or NADPH as one donor, and incorporation of one atom of oxygen (1.14.13)
- G01N 21/6408: {with measurement of decay time, time resolved fluorescence}
- G01N 21/6486: {Measuring fluorescence of biological material, e.g. DNA, RNA, cells (G01N21/6428 takes precedence)}
- C12M 1/34: Measuring or testing with condition measuring or sensing means, e.g. colony counters
- 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
- C12Y 113/00: Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13)
- C12Y 114/00: Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
- G01N 2021/6432: {Quenching}
- G01N 2333/90241: acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
- G01N 2333/90245: acting on paired donors with incorporation of molecular oxygen (1.14)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS}

## 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

### Claim 1

A biosensing system that measures the concentration of an analyte comprising a biocomponent wherein said biocomponent comprises oxygenase enzymes and dehydrogenase enzymes; wherein said biocomponent comprises:
a plasmid or plasmids within a whole cell and said plasmid or plasmids comprise a nucleotide coding sequence of said biocomponent enzymes; or
a chromosome of a whole cell biocomponent and said chromosome comprises a nucleotide coding sequence of said biocomponent enzymes.

### Claim 2

(canceled)

### Claim 3

(canceled)

### Claim 4

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.

### Claim 5 (depends on 4)

The biosensing system of claim 4 wherein said transducer layer is an optical transducer that constitutively fluoresces and wherein the fluorescence is quenched by oxygen in solution.

### Claim 6 (depends on 4)

The biosensing system of claim 4 wherein said first biocomponent is selected from the group consisting of monooxygenases and dioxygenases.

### Claim 7 (depends on 4)

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

### Claim 8

(canceled)

### Claim 9 (depends on 4)

The biosensing system of claim 4 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.

### Claim 10

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.

### Claim 11 (depends on 10)

The method of claim 10 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.

### Claim 12 (depends on 10)

The method of claim 10 wherein said first biocomponent is toluene ortho-monooxygenase or a toluene ortho-monooxygenase variant; and
said second biocomponent is formate dehydrogenase and said electron donor is formate.

### Claim 13

(canceled)

### Claim 14

(canceled)

### Claim 15 (depends on 10)

The method of claim 10 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.

### Claim at position 16

16-24. (canceled)

### Claim 25 (depends on 4)

The biosensing system of claim 4, wherein
said analyte catalyzes the reaction of an 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.

### Claim 26 (depends on 25)

The biosensing system of claim 25 wherein said first biocomponent is toluene ortho-monooxygenase.

### Claim 27 (depends on 25)

The biosensing system of claim 25 wherein said first biocomponent is a toluene ortho-monooxygenase variant.

### Claim 28 (depends on 25)

The biosensing system of claim 25 wherein said second biocomponent is formate dehydrogenase and said electron donor is formate.

### Claim at position 21

29-38. (canceled)

### Claim 39 (depends on 4)

The biosensing system of claim 4, 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, 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 Km and Vmax of the reaction between said biocomponent and said analyte are selected such that the quotient between Da2 and 4β is from about 10 to about 1000.

### Claim 40 (depends on 39)

The biosensing system of claim 39 wherein said biocomponent layer comprises toluene ortho-monooxygenase, a toluene ortho-monooxygenase variant, or a toluene ortho-monooxygenase variant and formate dehydrogenase.

### Claim 41

(canceled)

### Claim 42

(canceled)

### Claim 43 (depends on 39)

The biosensing system of claim 39 wherein said biocomponent comprises a toluene ortho-monooxygenase variant, formate dehydrogenase and at least one enzyme selected from the group consisting of epoxide hydrolase, glutathione synthetase, glutathione S-transferase and gamma-glutamylcysteine sythetase.

### Claim 44

(canceled)

### Claim 45

(canceled)

### Claim 46 (depends on 39)

The biosensing system of claim 39 wherein said transducer layer comprises RuDPP.

### Claim 47 (depends on 39)

The biosensing system of claim 39 wherein said porous layer is track-etched polycarbonate.

### Claim 48

(canceled)

### Claim at position 32

49-56. (canceled)

### Claim 57 (depends on 4)

The biosensing system of claim 4 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.

### Claim at position 34

58-61. (canceled)