# US 2023/0053493 A1 · One step methods, kits, and systems for the measurement of concentrations of unbound bilirubin in biological fluids

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- Number: US 2023/0053493 A1
- Document ID: US20230053493A1
- Status: Published application
- Kind: A1
- Application number: 17/632,202
- Filed: 2020-08-27
- Published: 2023-02-23
- Earliest claimed: 2019-08-30
- Inventors: Alan Marc Kleinfeld; Andrew H. Huber; Hiroto Kameyama; Tung-Chih Sun; Divya Subramonian
- Web page: https://patentgrep.com/patent/US20230053493A1

## CPC

- G01N 33/72: involving blood pigments, e.g. haemoglobin, bilirubin {or other porphyrins; involving occult blood}
- G01N 33/582: {with fluorescent label}
- 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)

## 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 sensor for measuring free bilirubin in a sample, the sensor comprising:
a bilirubin responsive probe labeled with a first fluorophore; and
a non-responder probe labeled with a second fluorophore,
wherein the first and second fluorophores excite at the same wavelength, and
wherein the first and second fluorophore emit a fluorescence at different wavelengths.

### Claim 2 (depends on 1)

The sensor of claim 1, wherein the bilirubin responsive probe comprises a first intracellular lipid binding protein (iLBP), wherein the first iLBP has a peptide sequence comprising SEQ ID NO: 1, comprising:
Arg substituting for fourteen accessible lysines (KR14 as set forth in SEQ ID NO: 2);
a C terminal double His tag linker (C2XH11) having a sequence as set forth in SEQ ID NO: 3;
an N terminal addition of MGI; and
no more than 62 amino acid substitutions and additions including a single cysteine.

### Claim 3 (depends on 1)

The sensor of claim 1, wherein the bilirubin responsive probe comprise a sequence of any one of the probes as set forth in Table 1.

### Claim 4 (depends on 1)

The sensor of claim 1, wherein the non-responder probe comprises a second iLBP, wherein the second iLBP has a peptide sequence comprising SEQ ID NO: 1, comprising:
substitutions at positions 72, 73, 74, 126, and 131;
a substitution to Cys at any one of positions 27, 31, 33, 54, 73, 74, 76, or 98;
no more than three additional amino acid substitutions; and
a C terminal double His tag linker (C2XH11) having a sequence as set forth in SEQ ID NO: 3.

### Claim 5 (depends on 1)

The sensor of claim 1, wherein the non-responder probe comprises a sequence of any one of the probes as set forth in Table 2.

### Claim 6 (depends on 1)

The sensor of claim 1, wherein the first fluorophore and the second fluorophore are different fluorophores.

### Claim 7 (depends on 1)

The sensor of claim 1, wherein the bilirubin responsive probe comprises a single cysteine to which the first fluorophore is attached.

### Claim 8 (depends on 1)

The sensor of claim 1, wherein the non-responder probe comprises a single cysteine to which the second fluorophore is attached.

### Claim 9 (depends on 1)

The sensor of claim 1, wherein the first fluorophore is LICOR 700DX maleimide or LICOR 800CW maleimide attached to a cysteine substitution.

### Claim 10 (depends on 1)

The sensor of claim 1, wherein the bilirubin responsive probe is configured to bind to an unconjugated IX-α (Z,Z) isomer of bilirubin.

### Claim 11 (depends on 1)

The sensor of claim 1, wherein the bilirubin responsive probe is configured to minimally bind to conjugated bilirubin (below 4 mg/dl).

### Claim 12 (depends on 1)

The sensor of claim 1, wherein the bilirubin responsive probe is configured to not bind to Z,E or E,Z photoisomers of bilirubin, to lumirubin, to fatty acids, to any other naturally occurring blood components, and/or to neonatal drugs.

### Claim 13 (depends on 12)

The sensor of claim 12, wherein the neonatal drugs is not spironolactone.

### Claim 14 (depends on 1)

The sensor of claim 1, wherein the non-responder probe does not bind to an unconjugated IX-α (Z,Z) isomer of bilirubin or to conjugated bilirubin.

### Claim 15 (depends on 1)

The sensor of claim 1, wherein the non-responder probe is configured to not bind to Z,E or E,Z photoisomers of bilirubin, to lumirubin, to fatty acids, to any other naturally occurring blood components, and/or to neonatal drugs.

### Claim 16 (depends on 1)

The sensor of claim 1, wherein when the first fluorophore is LICOR 700DX maleimide, the second fluorophore is LICOR 800CW maleimide attached to a cysteine substitution, and when the first fluorophore is LICOR 800CW maleimide, the second fluorophore is LICOR 700DX maleimide.

### Claim 17 (depends on 1)

The sensor of claim 1, wherein the first fluorophore or the second fluorophore is attached to a cysteine substitution and wherein the cysteine substitution is at position 22, 24, 25, 26, 27, 29, 30, 33, 54, 74, 76, 97, or 98 of SEQ ID NO: 1.

### Claim 18 (depends on 1)

The sensor of claim 1, wherein an emission intensity of the first fluorophore or the second fluorophore is not affected by the absorbance of blood components selected from bilirubin and hemoglobin.

### Claim 19 (depends on 1)

The sensor of claim 1, wherein the bilirubin responsive probe or the non-responder probe further comprising at least one linker.

### Claim at position 20

20-24. (canceled)

### Claim 25 (depends on 1)

A solid substrate comprising the sensor of claim 1, wherein the bilirubin responsive probe and/or the non-responder probe is attached to the solid substrate.

### Claim 26 (depends on 25)

The solid substrate of claim 25, wherein the solid substrate is a Ni-polystyrene, Ni-latex, or Ni-agarose bead.

### Claim 27 (depends on 26)

The solid substrate of claim 26, wherein the Ni-polystyrene, Ni-latex, or Ni-agarose bead comprises iron.

### Claim 28 (depends on 25)

The solid substrate of claim 25, wherein the bilirubin responsive probe or the non-responder probe comprises substitutions 7R 16R 20R 29R 37R 46R 50R 88R 92R 94R 100R 125R 129R and 130R (KR14) as set forth in SEQ ID NO: 3.

### Claim 29 (depends on 25)

The solid substrate of claim 25, wherein the bilirubin responsive probe and/or the non-responder probe comprises a tag and the solid substrate comprises a receptor for the tag.

### Claim 30 (depends on 29)

The solid substrate of claim 29, wherein the tag comprises one or more of His-tag, biotin, Flag-epitope, c-myc epitope, HA-tag, glutathione-S-transferase (GST), maltose binding protein (MBP), a chitin binding domain (CBD), Thioredoxin, β-Galactosidase, VSV-Glycoprotein, calmodulin binding protein, a polystyrene (PS) hydrophobic tag, or a metal affinity tag.

### Claim 31 (depends on 29)

The solid substrate of claim 29, wherein the tag is a poly-histidine tag and the solid substrate comprises an immobilized metal chelate.

### Claim 32 (depends on 25)

The solid substrate of claim 25, wherein the first fluorophore is attached to a cysteine residue on the bilirubin response probe.

### Claim 33 (depends on 25)

The solid substrate of claim 25, wherein the second fluorophore is attached to a cysteine residue on the non-responder probe.

### Claim at position 30

34-58. (canceled)