# US 11,029,320 B2 · Development and use of fluorescent probes of unbound bilirubin

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- Number: US 11,029,320 B2
- Document ID: US11029320B2
- Status: Granted
- Kind: B2
- Application number: 16/366,143
- Filed: 2019-03-27
- Published: 2019-07-11
- Granted: 2021-06-08
- Earliest claimed: 2011-08-26
- Assignees: Alan Marc Kleinfeld
- Applicants: Alan Marc Kleinfeld
- Inventors: Alan Marc Kleinfeld; Andrew Henry Huber; James Patrick Kampf; Thomas Kwan; Baolong Zhu
- Web page: https://patentgrep.com/patent/US11029320B2

## CPC

- G01N 33/728: {Bilirubin; including biliverdin}
- C07K 14/47: from mammals
- 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 33/533: with fluorescent label
- G01N 33/582: {with fluorescent label}
- G01N 33/92: involving lipids, e.g. cholesterol {, lipoproteins, or their receptors (steroid hormones G01N33/743)}
- G01N 2800/52: Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
- Y10T 436/146666: Bile pigment
- 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)
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## Abstract

Identification and use of proteins fluorescently labeled and that undergo a change in fluorescence index upon binding bilirubin are described. Probes are disclosed which are labeled at a cysteine or lysine residue and also probes labeled at both cysteine and lysine with two different fluorophores. These probes are useful for determination of unbound bilirubin levels in a fluid sample.

## Claims

### Claim 1

A method of calibrating bilirubin probes to determine binding affinity of the bilirubin probes to bilirubin, the method comprising:
mixing a bilirubin probe with an aqueous sample of a known concentration of bilirubin (Bt), wherein the bilirubin probe comprises a first fluorophore and a second fluorophore;
measuring a change in fluorescence of the bilirubin probe in response to binding to bilirubin probe to bilirubin; and
determining binding affinity of the bilirubin probes to bilirubin based on the measured change in fluorescence by fitting the measured change in fluorescence with the following equations 2 or 3:
I
λ
⁢
⁢
em
I
0
=
1
-
(
K
d
+
B
t
+
P
T
)
-
(
K
d
+
B
t
+
P
T
)
2
-
4
⁢
B
T
⁢
P
T
2
⁢
P
T
(
2
)
R
=
-
(
P
T
2
+
(
2
⁢
K
d
-
2
⁢
B
T
)
⁢
P
T
+
K
d
2
+
2
⁢
B
T
⁢
K
d
+
B
T
2
)
⁢
(
rR
0
2
-
R
0
)
+
(
P
T
+
K
d
-
B
T
)
⁢
rR
0
2
+
(
-
P
T
⁢
K
d
+
B
T
)
⁢
R
0
2
⁢
(
B
T
⁢
r
2
⁢
R
0
2
-
(
P
T
+
K
d
+
B
T
)
⁢
rR
0
+
P
T
)
(
3
)
wherein Iλem is the fluorescence intensity of the probe in the sample with blank subtracted, Io is the intensity of the probe in the absence of bilirubin, PT is the total bilirubin probe concentration, BT is the total bilirubin concentration, R is the measured fluorescence ratio (Iλ1/Iλ2), wherein Iλ1 is a fluorescence intensity from the first fluorophore at wavelength λ1 and Iλ2 is a fluorescence intensity from the second fluorophore at wavelength λ2, Ro is the ratio in the absence of bilirubin, r is the Iλ2/Iλ1 ratio of the probe in the absence of the second fluorophore, and Kd is the dissociation constant.

### Claim 2 (depends on 1)

The method of claim 1, wherein the sample is mixed with a carrier macromolecule for the bilirubin selected from the group consisting of albumin, lipid binding proteins, lipid vesicles and cyclodextrin.

### Claim 3 (depends on 1)

The method of claim 1, wherein the bilirubin probe is attached to a solid support.

### Claim 4 (depends on 1)

The method of claim 1, wherein concentrations of free bilirubin (Bf) are determined using disposable microfluidics devices that optionally allow measurements of undiluted blood samples.

### Claim 5 (depends on 1)

The method of claim 1, wherein the sample is from a human, an animal or a plant.

### Claim 6 (depends on 1)

The method of claim 1, wherein the sample is selected from the group consisting of whole blood, blood plasma, blood serum, urine, CSF, saliva, gastric juices, interstitial fluid and lymph.

### Claim 7 (depends on 1)

The method of claim 1, wherein the sample is from patients receiving intravenous infusion of oil emulsions.

### Claim 8 (depends on 1)

The method of claim 1, wherein the sample is from patients that are receiving drugs that displace bilirubin from albumin and/or from patients that may be producing, from the infused oil emulsion 1, disease, or stress, molecules that displace bilirubin from albumin.

### Claim 9 (depends on 1)

The method of claim 1, wherein the sample is from patients that are undergoing phototherapy, transfusion, or other therapies that reduce bilirubin levels.