US 11,994,521 B2
Methods for measuring inter- and/or intra-molecular interactions
Philipp Baaske, Munich (DE); Stefan Duhr, Munich (DE); Dennis Breitsprecher, Munich (DE); Christian Osseforth, Munich (DE); Axel Rohde, Munich (DE); Amin Jean Gupta, Munich (DE); and Nuska Tschammer, Munich (DE)
Assigned to Nanotemper Technologies GMBH, Munich (DE)
Appl. No. 16/625,551
Filed by NanoTemper Technologies GmbH, Munich (DE)
PCT Filed Jun. 22, 2018, PCT No. PCT/EP2018/066804
§ 371(c)(1), (2) Date Dec. 20, 2019,
PCT Pub. No. WO2018/234557, PCT Pub. Date Dec. 27, 2018.
Claims priority of application No. 17177746 (EP), filed on Jun. 23, 2017; and application No. 17177747 (EP), filed on Jun. 23, 2017.
Prior Publication US 2020/0278351 A1, Sep. 3, 2020
Int. Cl. G01N 33/58 (2006.01); G01N 33/543 (2006.01)
CPC G01N 33/582 (2013.01) [G01N 33/54313 (2013.01)] 11 Claims
 
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.