DEVICES AND METHODS FOR THE RATIOMETRIC CHARACTERIZATION OF FLUORESTENT PARTICLES

Abstract

Method for characterizing fluorescently labeled particles in solution by analyzing changes in fluorescence intensity due to spectral shifts or broadening or narrowing of the spectrum, or combinations thereof, of the fluorescent label of the fluorescently labeled particles, comprising the steps of: a) providing a sample of the fluorescently labeled particles in a solution under first conditions; b) exciting the fluorescently labeled particles at a first wavelength; c) detecting the fluorescence emission intensity of the fluorescently labeled particles in second and third wavelength ranges; d) calculating the ratio between the fluorescence intensities detected in the second and third wavelength ranges, wherein the third wavelength range differs from the second wavelength range; e) repeating steps b) to d) for the sample of fluorescently labeled particles under second conditions.or e2) Repeat steps a) to d) for a second sample of the fluorescently labeled particles under second conditions, wherein the second conditions differ from the first conditions, f) Characterize the fluorescently labeled particles based on the ratios calculated for the different conditions, wherein the calculated ratios obtained in step f) are used to determine parameters selected from the group consisting of dissociation constants, half-maximal effective concentrations (EC50), equilibrium constants, binding kinetics, enzymatic reaction kinetics, thermodynamic parameters, unfolding or refolding kinetics, opening and closing reactions, and combinations thereof, wherein the second and third wavelength ranges are detected simultaneously,wherein the wavelengths in the second wavelength range are shorter and the wavelengths in the third wavelength range are longer than an absolute emission maximum of the fluorescence emission of the fluorescently labeled particles under the first conditions, wherein the concentration of the fluorescently labeled particles in the solution is 50 pM to 500 nM, wherein the particles are covalently bound to the label, reversibly bound to the label via a high-affinity protein tag, or bioconjugated via click chemistry, wherein the particles are selected from the group consisting of organic molecules, biomolecules, nanoparticles, microparticles, vesicles, biological cells or subcellular fragments, biological tissues, viral particles, viruses, cellular organelles, lipid nanoparticles (LNPs), and virus-like particles, and wherein the fluorescently labeled particles are labeled with an extrinsic fluorescent label.