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EP-4320425-B1 - METHOD, APPARATUS AND SYSTEM FOR CHARACTERIZING TRANSIENT INTERACTIONS BETWEEN BIOMOLECULES

EP4320425B1EP 4320425 B1EP4320425 B1EP 4320425B1EP-4320425-B1

Inventors

  • CUI, JIAN
  • RIDDERBEEK, Korneel

Dates

Publication Date
20260506
Application Date
20220407

Claims (15)

  1. A method for characterizing transient interactions between biomolecules (1, 2) comprising following steps: - providing a plurality of plasmonic nanoparticles (3) which are configured to allow first biomolecules (1) to adhere thereto, - providing a mixture comprising the nanoparticles (3), first biomolecules (1) and second biomolecules (2) to allow the first biomolecules (1) to adhere to the nanoparticles (3) and to allow the second biomolecules (2) to transiently interact with the first biomolecules (1) adherent to the nanoparticles (3), - irradiating the mixture with first electromagnetic radiation (4), - detecting second electromagnetic radiation (5), which is scattered by the mixture while irradiating the mixture with the first electromagnetic radiation (4), in a time-resolved and spectrally-resolved manner so as to obtain intensity signals representing changes in the spectrum of the detected second electromagnetic radiation (5), and - determining at least one interaction parameter , such as k on , k off or k D , characterizing transient interactions of the second biomolecules (2) with the first biomolecules (1) based on the intensity signals, characterized in that the step of detecting the second electromagnetic radiation comprises - splitting the second electromagnetic radiation into a first partial beam and a second partial beam, providing one or more different path length differences (δ) between the first partial beam and the second partial beam, superimposing the first partial beam and the second partial beam for each of the path length differences (δ) so as to obtain a first interference beam and a second interference beam for each of the path length differences (δ), and separately detecting the first interference beam and the second interference beam by means of two separate detectors at different times and for each of the path length differences (δ) so as to obtain two series of intensity signals representing intensities of the first interference beam and second interference beam, respectively, at the different times and for the different path length differences (δ), or - applying the second electromagnetic radiation to at least one spectral splitting element (19; 14, 19a, 19b), in particular a dichroic optical element (19), so as to obtain at least one first partial beam of electromagnetic radiation having a first spectrum and at least one second partial beam of electromagnetic radiation having a second spectrum which is different from the first spectrum, and separately detecting the first partial beam and the second partial beam by means of two separate detectors at different times so as to obtain two series of intensity signals representing intensities of the first partial beam and second partial beam, respectively, at the different times and for the first and second spectrum.
  2. The method according to claim 1 further comprising determining at least one time auto-correlation function (intensity-time auto-correlation function) characterizing a time auto-correlation of a combination of the two series of intensity signals, wherein at least one diffusion parameter characterizing a diffusion of the nanoparticles in the mixture is determined based on the at least one time auto-correlation function.
  3. The method according to claim 1 or 2 further comprising determining at least one time cross-correlation function (intensity-time cross-correlation function) characterizing a time cross-correlation between the two series of intensity signals, wherein the at least one interaction parameter is determined based on the at least one time cross-correlation function and optionally also on the at least one time auto-correlation function.
  4. The method according to claim 2 or 3, wherein the at least one time auto-correlation function and/or the at least one time cross-correlation function is determined for the different path length differences (δ) between the first partial beam and the second partial beam or for the first and second spectrum, respectively.
  5. The method according to any one of the claims 2 to 4, wherein the time auto-correlation function (intensity-time auto-correlation function) is used to correct the time cross-correlation function (intensity-time cross-correlation function) to remove effects of nanoparticle diffusion on the time cross-correlation function.
  6. The method according to claim 5, wherein the at least one interaction parameter is determined based, in particular based only, on the corrected intensity cross-correlation function.
  7. The method according to any one of the claims 3 to 6 further comprising Fourier transforming the time cross-correlation functions, which are determined for the different path length differences, with respect to the dimension of the path length differences so as to obtain spectral correlation functions (spectral-time correlation function) at different times and for different wavelength changes, wherein the at least one interaction parameter is determined based on the spectral correlation functions.
  8. The method according to claim 7 further comprising determining a temporal behavior of the spectral correlation functions, wherein the at least one interaction parameter is determined based on the temporal behavior of the spectral correlation functions and/or intensity correlation functions.
  9. The method according to any one of the preceding claims, wherein determining the at least one interaction parameter , such as k on , k off or k D , includes determining at least one parameter characterizing reversible interactions between the first and second biomolecules (1, 2).
  10. The method according to any one of the preceding claims, wherein determining the at least one interaction parameter , such as k on , k off or k D , includes determining at least one of the following: - a first transition rate , k on , characterizing a time rate at which transitions occur between an unbound state, in which the second biomolecules (2) are not bound to and/or do not interact with first biomolecules (1), to a bound state, in which the second biomolecules (2) are bound to and/or interact with the first biomolecules (1), and/or - a second transition rate , k off , characterizing a time rate at which transitions occur between a bound state, in which the second biomolecules (2) are bound to and/or interact with the first biomolecules (1), to an unbound state, in which the second biomolecules (2) are not bound to and/or do not interact with the first biomolecules (1), and/or - a dissociation constant , k D , characterizing a tendency of the first and second biomolecules (1, 2) to reversibly dissociate between a bound state, in which the second biomolecules (2) are bound to and/or interact with the first biomolecules (1), and an unbound state, in which the second biomolecules (2) are not bound to and/or do not interact with the first biomolecules (1).
  11. The method according to any one of the preceding claims, wherein the mixture comprising the nanoparticles (3), first biomolecules (1) and second biomolecules (2) is a solution and/or dispersion, wherein the nanoparticles (3), first biomolecules (1) and second biomolecules (2) are dissolved in a solvent or dispersed in a dispersion agent, respectively.
  12. An apparatus for characterizing transient interactions between biomolecules (1,2) comprising: - a receptacle (13a) configured to receive a mixture comprising first biomolecules (1), second biomolecules (2) and a plurality of plasmonic nanoparticles (3) which are configured to allow at least a part of the first biomolecules (1) to adhere thereto, wherein at least a part of the second biomolecules (2) are allowed to transiently interact with first biomolecules (1) adherent to the nanoparticles (3), - an irradiation unit (10) configured to irradiate the mixture with first electromagnetic radiation (4), - a detection unit (7a, 7b, 14 - 17, 19, 19a, 19b) configured to detect second electromagnetic radiation (5), which is scattered by the mixture while being irradiated with the first electromagnetic radiation (4), in a time-resolved and spectrally-resolved manner so as to obtain intensity signals representing changes in the spectrum of the detected second electromagnetic radiation (5), and - a processing unit (18) configured to determine at least one interaction parameter , such as k on , k off or k D , characterizing transient interactions of the second biomolecules (2) with the first biomolecules (1) based on the intensity signals, characterized in that the detection unit comprises: - a beam splitter (14) configured to split the second electromagnetic radiation (5) into a first partial beam (5a) and a second partial beam (5b), a path length difference generating unit (15a, 15b, 16) configured to provide one or more different path length differences between the first partial beam (5a, 5a') and the second partial beam (5b, 5b'), wherein the first partial beam (5a') and the second partial beam (5b') are allowed to superimpose for each of the path length differences so as to obtain a first interference beam (6a) and a second interference beam (6b) for each of the path length differences, and two separate detectors (7a, 7b) which are configured to separately detect the first interference beam (6a) and the second interference beam (6b) at different times and for each of the path length differences so as to obtain two series of intensity signals representing intensities of the first interference beam (6a) and second interference beam (6b), respectively, at the different times and for the different path length differences, or - at least one spectral splitting element (19; 14, 19a, 19b), in particular a dichroic optical element (19), configured to reflect and/or transmit the second electromagnetic radiation (5) so as to obtain at least one first partial beam (8a) of electromagnetic radiation having a first spectrum and at least one second partial beam (8b) of electromagnetic radiation having a second spectrum which is different from the first spectrum, and two separate detectors (7a, 7b) which are configured to separately detect the first partial beam (8a) and the second partial beam (8b) at different times so as to obtain two series of intensity signals representing intensities of the first partial beam (8a) and second partial beam (8b), respectively, at the different times and for the first and second spectrum.
  13. The apparatus according to claim 12, wherein the first electromagnetic radiation (4) is configured to cause and/or excite a surface plasmon resonance of the nanoparticles (3), in particular one or more wavelengths or energies of the first electromagnetic radiation (4) match one or more wavelengths or energies, respectively, of the surface plasmon resonance.
  14. The apparatus according to claim 12 or 13, wherein the at least one spectral splitting element (19; 14, 19a, 19b), in particular the dichroic optical element (19), is configured to reflect and/or transmit the second electromagnetic radiation (5) such that one or more wavelengths or frequencies of the first spectrum and/or of the second spectrum match one or more wavelengths or energies, respectively, of the surface plasmon resonance.
  15. A system for characterizing transient interactions between biomolecules (1, 2), the system comprising the apparatus according to any one of the claims 12 to 14 and - a plurality of plasmonic nanoparticles (3) which are configured to allow first biomolecules (1) to adhere thereto and/or - a mixture comprising a plurality of plasmonic nanoparticles (3), which are configured to allow first biomolecules (1) to adhere thereto, first biomolecules (1) and second biomolecules (2).

Description

The present invention relates to a method, apparatus and system for characterizing transient interactions, in particular rapid transient interactions, between biomolecules. Biomolecular interactions form the foundation of nearly all cellular processes. However, many of these interactions occur on timescales that cannot be accessed using conventional methods, such as surface plasmon resonance (SPR). Nuclear magnetic resonance (NMR) can access the high temporal and spatial resolution needed to resolve these interactions, but NMR is generally limited to small biomolecules. Fluorescent dyes can be used via Förster resonance energy transfer (FRET) to potentially determine these interaction rates, however fluorescent methods are limited by emitter photobleaching and the need to specifically label biomolecules and remove unlabeled dyes. The patent application WO 2016/075229 A1 discloses a biosensor detecting binding and unbinding events based on the shift of the plasmonic resonance of gold nanoparticles. Plasmon shift between bound and unbound states result in changes of detected signal intensity. The present invention is based on the problem of providing an improved method, apparatus and system for characterizing transient interactions, in particular rapid transient interactions, between biomolecules. The problem is solved by a method and apparatus according to the independent claims and a system comprising such an apparatus. Aspects of the invention are based on the approach of using plasmonic nanoparticles as local sensors for sensing fast transient interactions between biomolecules. Plasmonic nanoparticles are particles made out of a, preferably metallic, material with dimensions preferably smaller than 1 µm (< 10-6 m). The high density of electrons in these particles can resonate with light incident on the particles generating so-called localized surface plasmon resonances (LSPRs). At this resonance, the electromagnetic waves are confined tightly around the particle, making the resonance very sensitive to changes in the local dielectric environment. Due to this effect, the optical response changes as a function of a changing environment, so that plasmonic nanoparticles act as local (refractive index) sensors, wherein interacting molecules, for example proteins, around the nanoparticles change the optical output of the nanoparticles. For further details regarding the use of plasmonic nanoparticles as local refractive index sensors it is referred to Maier, S. A., Plasmonics: fundamentals and applications, Springer Science & Business Media (2007). This local sensitivity of plasmonic nanoparticles implies that the optical output of the nanoparticle reflects the dynamics of events occurring around the particles. Therefore, the scattering signal of plasmonic nanoparticles can be used to study and characterize biomolecular interactions. Using the scattering of such particles yields orders of magnitude more signal than fluorescent emitters, because they are not limited to by "slow" absorption-emission photophysics and by photobleaching of dyes. In a preferred application, a biomolecule of interest A is immobilized on the nanoparticle surface, then biomolecule B is added to the mixture, light is shone on the sample, and the resulting scattering light is collected. Within the meaning of present disclosure, the term "transient interaction(s)" and its variations, such as "transiently interact" or "interact transiently", preferably relate to any non-stable and/or temporary and/or non-permanent and/or reversible interaction between biomolecules (being preferably in a thermal equilibrium), in particular in distinction to a non-transient and/or stable and/or permanent and/or irreversible interaction, in particular binding, of biomolecules. For example, a transient interaction between biomolecule A and biomolecule B means that A and B interact, e.g. bind and/or associate and/or are in a bound state, for a usually very short time, before reversibly dissociating to an unbound state and so on. In contrast to this, a non-transient interaction between A and B means that, once A and B are in a bound state or an according chemical reaction is completed, A and B remain in the bound state. Basically, the method, apparatus and system according to present disclosure can be used in any kind of biological, medical and/or industrial application, for example as an analytical tool in the field of protein biology or for drug screening. Drug screening is the process by which potential drugs are identified and optimized before selection of a candidate drug to progress to clinical trials. It can involve screening large libraries of chemicals for a particular biological activity in high-throughput screening assays. In the invention, plasmonic nanoparticles and biomolecules to be investigated are contained in a mixture, which is preferably in a thermal equilibrium, in particular a solution and/or dispersion, to allow first biomolecules to adhere t