# US 11,994,521 B2 · Methods for measuring inter- and/or intra-molecular interactions

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- Number: US 11,994,521 B2
- Document ID: US11994521B2
- Status: Granted
- Kind: B2
- Application number: 16/625,551
- Filed: 2018-06-22
- Published: 2020-09-03
- Granted: 2024-05-28
- Earliest claimed: 2017-06-23
- Assignees: Nanotemper Technologies GmbH
- Applicants: NanoTemper Technologies GmbH
- Inventors: Philipp Baaske; Stefan Duhr; Dennis Breitsprecher; Christian Osseforth; Axel Rohde; Amin Jean Gupta; Nuska Tschammer
- Web page: https://patentgrep.com/patent/US11994521B2

## CPC

- G01N 33/582: {with fluorescent label}
- G01N 33/54313: {the carrier being characterised by its particulate form}
- 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

A method for measuring interactions between labeled particles and ligands comprising the steps: a) providing a sample comprising labeled particles and ligands in a solution, wherein the labeled particles are dissolved or dispersed in the solution or are immobilized on a solid support; b) exciting fluorescently the labeled particles and detecting the fluorescence of the excited particles at a predetermined temperature; c) repeating steps (a) and (b) multiple times at different concentrations of the ligands in the solution; and d) determining the interaction between the labeled particles and the ligands based on the ligand concentration dependent change of the fluorescence of the labeled particles, wherein the labeled particles are labeled with one or more dyes.

## Claims

### Claim 1

A method for measuring interactions between labeled particles and ligands comprising the steps:
a) providing a sample comprising labeled particles and ligands in a solution, wherein the labeled particles are dissolved or dispersed in the solution or are immobilized on a solid support, wherein the labeled particles are present in the solution in a concentration from 10 picomolar to 1 micromolar;
b) exciting fluorescently the labeled particles and detecting the fluorescence of the excited particles at a first predetermined temperature;
c) heating or cooling the solution to a second predetermined temperature, wherein the heating or cooling is carried out using a heating or cooling source selected from the group consisting of a heating or cooling fluid, a heating or cooling gas, a heating element, a peltier element, electromagnetic radiation, and combinations thereof, wherein the second predetermined temperature is different from the first predetermined temperature, and wherein the difference between the second temperature and the first temperature is in the range from +/−0.1 Kelvin (K) to +/−90 K;
d) exciting fluorescently the labeled particles and detecting the fluorescence of the excited particles at the second predetermined temperature;
e) repeating (a) through (d) multiple times at different concentrations of the ligands in the solution thereby obtaining a first and second binding curve showing the ligand concentration dependent change of the fluorescence of the excited particles at the first and second predetermined temperature respectively; and
f) determining the interaction between the labeled particles and the ligands based on the ligand concentration dependent change of the fluorescence of the labeled particles, wherein the labeled particles are labeled with a dye selected from the group consisting of Chromeo P543, DY630, DY631, DY650, DyLight 655 B2, DyLight 655 B3, Cyanine 2, Cy3, Monosulfo Cy3, Cy5, Monosulfo Cy5 (version 1), Monosulfo Cy5 (version 2), Disulfo Cy5, Cy5.5, Z-Cy2, Z-Cy5, Monosulfo Z-Cy5, Alexa 647, DY547P1, DY647P1, TAMRA, TAMRA X, DY495, and Oregon Green 488;
and
wherein the particle is a protein.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the predetermined temperature is in the range of ˜20° C. to 115° C.

### Claim 3 (depends on 2)

The method according to claim 2, wherein the predetermined temperature is different from room temperature.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the predetermined temperature is controlled within +/−1 K, preferably within +/−0.5 K.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the first and second predetermined temperatures are in the range of ˜20° C. to 115° C.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the first and second predetermined temperatures are controlled within +/−1 K, preferably within +/−0.5 K.

### Claim 7 (depends on 1)

The method according to claim 1, wherein the concentration of the labeled particles in the solution is from 100 picomolar to 100 nanomolar.

### Claim 8 (depends on 1)

The method according to claim 1, wherein the concentration of the ligand is from 0.01 picomolar to 1 molar.

### Claim 9 (depends on 1)

The method according to claim 1, wherein said ligands are selected from the group consisting of ions, metals, compounds, drug fragments, carbohydrates, small molecules, drugs, prodrugs, lipids, proteins, peptides, peptoids, enzymes, nucleic acids, nanoparticles, liposomes, SUVs, GUVs, polymers, organic molecules, inorganic molecules, metal complexes, hormones, flavors, odorants, particles and (micro)beads.

### Claim 10 (depends on 1)

The method according to claim 1, wherein the particle and ligand are selected from the group consisting of the following combinations denoted as particle—ligand:
enzyme—lipid, receptor—hormone, receptor—chemokine, enzyme—inhibitor, receptor— neurotransmitter, receptor—cytokine, enzyme—ion, receptor—ion, receptor—amino acid, enzyme—cofactor, receptor—lipid, receptor—sterol, enzyme—fragment, receptor— peptide, receptor—fragment, enzyme—metabolite, receptor—receptor, receptor— glycolipid, enzyme—DNA, receptor—odorant, receptor—prodrug, enzyme—RNA, receptor—drug, enzyme—mono-/di- or polysaccharide, enzyme—fatty acid, enzyme vitamin, enzyme—prodrug, enzyme—drug, liposome—protein, transporter protein substrate, antibody—antigen, antibody—Fc receptor, chaperone—ATP, aptamer—ligand, chaperon—protein, structural protein—structural protein, signaling protein—signaling protein, signaling protein—small molecule, signaling protein—prodrug, signaling protein—drug, signaling protein—lipid, and structural protein-ions.

### Claim 11 (depends on 1)

The method according to claim 1, wherein the method of measuring interactions between labeled particles and ligands is a method of measuring the dissociation constant of the particles and the ligands.