# US 8,465,981 B2 · Polypeptides, systems, and methods useful for detecting glucose

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- Number: US 8,465,981 B2
- Document ID: US8465981B2
- Status: Granted
- Kind: B2
- Application number: 12/672,403
- Filed: 2008-08-06
- Published: 2011-05-19
- Granted: 2013-06-18
- Earliest claimed: 2007-08-06
- Assignees: University of Kentucky Research Foundation
- Applicants: Sylvia Daunert; Kendrick Turner; Smita Joel; Laura Rowe
- Inventors: Sylvia Daunert; Kendrick Turner; Smita Joel; Laura Rowe
- Web page: https://patentgrep.com/patent/US8465981B2

## CPC

- A61B 5/14532: {for measuring glucose, e.g. by tissue impedance measurement}
- A61B 5/1459: invasive, e.g. introduced into the body by a catheter
- A61B 5/412: {Detecting or monitoring sepsis}
- Y10T 436/13: Tracers or tags
- Y10T 436/144444: Glucose
- A61B: DIAGNOSIS; SURGERY; IDENTIFICATION (usefulness limited to only animals A61D)
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## Abstract

The presently-disclosed subject matter provides biosensors for detecting molecules of interest. The biosensors include a polypeptide capable of selectively-binding glucose, wherein the polypeptide molecule is selected from: an unnatural analogue of wild type glucose binding protein; a fragment of wild type glucose binding protein; and an unnatural analogue fragment of wild type glucose binding protein.

## Claims

### Claim 1

A biosensor for detecting a molecule of interest, comprising:
an isolated polypeptide molecule capable of selectively-binding glucose, wherein the polypeptide molecule is selected from: an unnatural analogue of the amino acid sequence of SEQ ID NO: 2; and an unnatural analogue fragment of the amino acid sequence of SEQ ID NO: 2; and
a label associated with the polypeptide molecule;
wherein binding of glucose to the polypeptide molecule causes the label to generate a signal, such that the glucose can be detected.

### Claim 2 (depends on 1)

The biosensor of claim 1, wherein the polypeptide molecule is an unnatural analogue fragment of the amino acid sequence of SEQ ID NO: 2.

### Claim 3 (depends on 1)

The biosensor of claim 1, wherein the polypeptide molecule comprises the amino acid sequence of SEQ ID NO: 2, wherein one or more natural amino acids are replaced with one or more unnatural amino acids.

### Claim 4 (depends on 1)

The biosensor of claim 1, wherein one or more natural amino acids of the unnatural analogue or unnatural analogue fragment are replaced with one or more unnatural amino acids as set forth in Table A.

### Claim 5 (depends on 4)

The biosensor of claim 4, wherein one or more natural leucines are replaced with 5,5,5-trifluoroleucine.

### Claim 6 (depends on 4)

The biosensor of claim 4, wherein one or more natural tryptophans are replaced with 5-fluorotryptophan.

### Claim 7 (depends on 4)

The biosensor of claim 4, wherein one or more natural leucines are replaced with 5,5,5-trifluoro-DL-leucine.

### Claim 8 (depends on 4)

The biosensor of claim 4, wherein one or more natural tryptophans are replaced with 5-fluoro-L-tryptophan.

### Claim 9 (depends on 1)

The biosensor of claim 1, wherein the polypeptide molecule comprises the amino acid sequence of SEQ ID NO: 2, wherein one or more natural leucines are replaced with 5,5,5-trifluoroleucine.

### Claim 10 (depends on 1)

The biosensor of claim 1, wherein the polypeptide molecule comprises the amino acid sequence of SEQ ID NO: 2, wherein one or more natural tryptophans are replaced with 5-fluorotryptophan.

### Claim 11 (depends on 1)

The biosensor of claim 1, wherein the polypeptide molecule is stable over a wider range of temperatures relative to wild type GBP (wtGBP).

### Claim 12 (depends on 1)

The biosensor of claim 1, wherein the polypeptide molecule is stable at temperatures between about 55° C. and about 75° C.

### Claim 13 (depends on 12)

The biosensor of claim 12, wherein the polypeptide molecule is an unnatural analogue or an unnatural analogue fragment, wherein one or more natural leucines are replaced with unnatural leucines.

### Claim 14 (depends on 13)

The biosensor of claim 13, wherein the unnatural leucines are 5,5,5-trifluoroleucine.

### Claim 15 (depends on 1)

The biosensor of claim 1, wherein the label is a fluorophore.

### Claim 16 (depends on 15)

The biosensor of claim 15, wherein the polypeptide molecule comprises: an unnatural analogue of the amino acid sequence of SEQ ID NO: 2; or an unnatural analogue fragment of the amino acid sequence of SEQ ID NO: 2;
wherein the label is attached to an amino acid site of the polypeptide molecule selected from Asp 44, Val 68, Ala 71, Phe 90, Lys 92, Glu 93, Pro 94, Lys 97, Gly 109, Thr 110, Asp 111, Gly 151, Cys 152, Pro 153, Ala 155, Asp 184, Asp 212, Leu 255, Ala 258, Asn 260, Ala 262, Arg 292, Val 293, and Pro 294, wherein SEQ ID NO: 2 comprises amino acids numbered 14-296 of wild type GBP (wtGBP).

### Claim 17 (depends on 16)

The biosensor of claim 16, wherein label is the fluorophore N-[2-(1-maleimidyl(ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC).

### Claim 18 (depends on 16)

The biosensor of claim 16, wherein binding of glucose to the polypeptide molecule causes the label to generate a detectably altered fluorescence intensity of the fluorophore such that the glucose can be detected.

### Claim 19 (depends on 18)

The biosensor of claim 18, wherein the fluorescence intensity can be correlated to the concentration of the glucose in a sample.

### Claim 20 (depends on 1)

A method for detecting a glucose, comprising:
contacting the biosensor of claim 1 with a sample of interest;
detecting the signal; and
collecting and displaying the signal with a detection and data collection device, to thereby detect the glucose.

### Claim 21 (depends on 20)

The method of claim 20, wherein the glucose is continuously detected.

### Claim 22

An isolated polypeptide molecule capable of selectively-binding glucose, wherein the polypeptide molecule is selected from an unnatural analogue of the amino acid sequence of SEQ ID NO: 2; and an unnatural analogue fragment of the amino acid sequence of SEQ ID NO: 2.