# US 11,906,524 B2 · Urea biosensors and uses thereof

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- Number: US 11,906,524 B2
- Document ID: US11906524B2
- Status: Granted
- Kind: B2
- Application number: 15/776,697
- Filed: 2016-11-19
- Published: 2020-10-08
- Granted: 2024-02-20
- Earliest claimed: 2015-11-20
- Assignees: Duke University
- Applicants: Duke University
- Inventors: Homme W. Hellinga; Malin J. Allert
- Web page: https://patentgrep.com/patent/US11906524B2

## CPC

- G01N 33/62: involving urea
- C07K 14/00: Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K 14/195: from bacteria
- C12Q 1/58: involving urea or urease
- 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)}
- G01N 33/542: with steric inhibition or signal modification, e.g. fluorescent quenching
- C07K 1/306: {by crystallization}
- C07K 2299/00: Coordinates from 3D structures of peptides, e.g. proteins or enzymes
- C07K 2319/20: containing a tag with affinity for a non-protein ligand
- G01N 2021/6439: {with indicators, stains, dyes, tags, labels, marks}
- G01N 2333/195: from bacteria
- G01N 2333/47: Assays involving proteins of known structure or function as defined in the subgroups
- 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)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- 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

## Abstract

The present subject matter provides urea biosensors as well as compositions, devices, and methods comprising such biosensors.

## Claims

### Claim 1

A biosensor for urea, comprising a urea-binding protein and a reporter group, wherein said urea-binding protein is at least 90% identical to SEQ ID NO: 18 and comprises primary complementary surface amino acids (PCS), wherein said PCS comprises residues selected from the group consisting of 92S, 111Y, 114Q, 157Y, 211N and 238S, and wherein said reporter group comprises a fluorophore and is functional to transduce a detectable signal when said biosensor binds to urea.

### Claim 2 (depends on 1)

The biosensor of claim 1, wherein the reporter group is selected from the group consisting of:
6-Acryloyl-2-Dimethylaminonaphthalene,
6-bromoacetyl-2-dimetylaminonaphtalene, and
5-(4-((5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)carbamoyl)phenyl)-2,3,3,7,7,8-hexamethyl-2,3,7,8-tetrahydro-1H-pyrano[3,2-f:5,6-f′]diindol-9ium-10,12-disfulfonate.

### Claim 3 (depends on 1)

The biosensor of claim 1, wherein the reporter group is covalently attached to the urea-binding protein.

### Claim 4 (depends on 1)

The biosensor of claim 1, wherein the reporter group is conjugated to a cysteine of the urea-binding protein.

### Claim 5 (depends on 1)

A method of detecting the presence or concentration of a urea in a sample, the method comprising:
(a) contacting the biosensor of claim 1 with the sample;
(b) measuring a signal from the biosensor; and
(c) comparing the signal to a control value, wherein a difference in signal indicates the presence of urea in the sample.

### Claim 6 (depends on 1)

A method for monitoring the level of urea in a subject, comprising
(a) administering a biosensor according to claim 1 or a device comprising a biosensor according to claim 1 to the subject, wherein after administration the biosensor is in contact with a bodily fluid or surface of the subject, and
(b) detecting (i) a signal produced by a reporter group of the biosensor continuously or repeatedly at intervals less than about 30 minutes apart, and/or (ii) whether a signal is produced by a reporter group of the biosensor continuously or repeatedly at intervals less than about 30 minutes apart.

### Claim 7 (depends on 2)

The biosensor of claim 2, wherein said reporter group is attached to a cysteine of said urea-binding protein.

### Claim 8 (depends on 2)

The biosensor of claim 2, wherein the urea-binding protein comprises the amino acid sequence of SEQ ID NO: 32, and wherein said reporter group is attached to a cysteine of said urea-binding protein.

### Claim 9 (depends on 2)

The biosensor of claim 2, wherein the urea-binding protein comprises the amino acid sequence of SEQ ID NO: 98, and wherein said reporter group is attached to a cysteine of said urea-binding protein.

### Claim 10 (depends on 1)

The biosensor of claim 1, wherein the PCS further comprises one of 159 V or 159Y.

### Claim 11 (depends on 1)

The biosensor of claim 1, wherein the PCS further comprises one of 113V or 113I or 113L.

### Claim 12 (depends on 1)

The biosensor of claim 1, wherein the urea binding protein further comprises one or more mutations selected from the group consisting of T26X, M27X, S30X, E43X, S65X, T69X, W90X, T91X, S92X, A93X, R95X, Y111X, V113X, Q114X, Y115X, E116X, Y157X, V158X, F159X, L186X, N211X, S238X, E241X, K276X, K280X, D288X, and E329X, wherein X is any amino acid.

### Claim 13 (depends on 11)

The biosensor of claim 11, wherein X is cysteine.