Devices for the ratiometric characterization of fluorescent particles

Abstract

Device for characterizing fluorescently labeled particles in solution by analyzing changes in fluorescence intensity resulting from spectral shifts and / or a broadening or narrowing of the spectrum or combinations thereof of the fluorescent labeling of the fluorescently labeled particles, the device comprising: a sample holder for holding a sample of fluorescently labeled particles in solution under a variety of conditions; Means for exciting the fluorescently labeled particles at a first wavelength; Means for detecting the fluorescence emission intensity of the fluorescently labeled particles in a second and third wavelength range using filter elements; Means for calculating a ratio between the fluorescence intensities detected in the second and third wavelength ranges, wherein the third wavelength range differs from the second wavelength range, wherein the calculated ratios are used to determine parameters selected from the group consisting of dissociation constants, mean effective concentrations (EC50), equilibrium constants, binding kinetics, enzyme reaction kinetics, thermodynamic parameters, unfolding or refolding kinetics, opening and closing reactions and combinations thereof; wherein the device is designed to successively excite fluorescently, detect fluorescence emissions and calculate the ratio for samples under different conditions; Means for characterizing the fluorescently labeled particles based on the calculated ratios obtained for the different conditions, the device is designed to detect the second and third wavelengths simultaneously, where the wavelengths of the second wavelength range are shorter and the wavelengths of the third wavelength range are longer than an absolute emission maximum of the fluorescence emission of the fluorescently labeled particles under a first condition of the various conditions.

Description

Field of invention

[0001] The present invention relates to devices and methods for characterizing fluorescently labeled particles in solution by analyzing changes in the fluorescence spectrum of the fluorescently labeled particles. In particular, a sample of fluorescently labeled particles is analyzed under different conditions / environments by fluorescence excitation and detection of the corresponding fluorescence emissions. The particles are characterized by analyzing the detected fluorescence emissions under these different conditions / environments. More precisely, the present invention relates to methods and devices for the ratiometric characterization of inter- and / or intramolecular interactions and / or conformational changes and / or the localization of the fluorescently labeled particles. background

[0002] The fluorescence spectrum of a fluorescent marker is sensitive to changes in the environment, such as changes in the chemical environment and temperature. Therefore, one and the same fluorescent marker can exhibit changes in its fluorescence spectrum with respect to intensity and / or spectral shifts and / or spectral shape.

[0003] Since this effect is well known, it is used to investigate interactions of intrinsically or extrinsically fluorescently labeled particles. In the prior art, changes in fluorescence intensity are primarily used to study binding reactions by determining dissociation constants (Kd). dto characterize (NPL1, NPL2). In addition to characterizing intermolecular interactions, the interactions to be determined can also include intramolecular interactions and / or modifications (conformational changes) and / or (changes in) localization of fluorescently labeled particles. For example, conformational changes of proteins and analyte concentrations are determined in the prior art based on methods involving a mechanism called Förster resonance energy transfer (FRET) (WO 2017 / 087912 A2). The basic mechanism of FRET involves a donor fluorophore in an excited electronic state, which can transfer its excitation energy to a nearby acceptor fluorophore via dipole-dipole coupling due to interaction-mediated (e.g., ligand-mediated) changes in the distance and / or angle between the two fluorophores.Therefore, FRET measurements require two or more fluorescent markers, i.e., at least one donor and at least one acceptor fluorophore. However, since two different fluorescent markers are used, each of which may have different sensitivities to environmental changes, FRET measurements can be corrupted by unwanted changes in the local environment of the fluorophores. Furthermore, the fluorescence emission of the acceptor fluorophore upon excitation by energy transfer usually yields a lower signal strength than the emission intensity of the acceptor fluorophore upon direct excitation. In general, FRET measurements require precise positioning of the donor and acceptor fluorophore(s) within a specific distance on the target molecule, e.g., by applying two site-specific labeling chemistries, which in turn may not be practical for every type of target molecule.In contrast, random labeling, e.g., with lysine-reactive or cysteine-reactive dyes, would lead to different FRET distances and thus to a failure of the FRET measurements. In summary, FRET measurements exhibit a lower signal-to-noise ratio compared to measurements involving only a single fluorescent marker, as in the present invention, and it is desirable that the fluorescently labeled particles to be analyzed are labeled with only a single type of fluorescent marker.

[0004] Depending on the interaction and the properties of the fluorescent marker itself, changes in the fluorescence spectra can be less than 1% and thus fall within the usual range of pipetting errors. Currently, it is difficult, if not impossible, to resolve changes smaller than 1% using state-of-the-art methods / devices.

[0005] Changes in the fluorescence spectrum of a fluorescent marker are typically detected using fluorescence spectrophotometers designed to record / measure over a large spectral range, thus failing to resolve small changes in fluorescence intensity. Furthermore, since commercially available fluorescent markers are currently designed to be more robust to environmental changes, there is a need to tailor highly sensitive fluorescent markers to increase the resolution of spectrophotometric measurements (NPL3, NPL4, NPL5).

[0006] Another way to improve the resolution of spectrophotometric measurements is by combining multiple measurements. In this case, the sample under investigation is excited at a first wavelength in a first measurement, and its emission is measured at a second wavelength. This is followed by a second measurement in which the excitation step is repeated at the first wavelength and its emission is measured at a third wavelength. The emission intensities obtained from the first and second measurements are then combined. This allows the pure information of interest to be determined independently of any artifacts that might arise, for example, from manual handling. However, due to the sequential execution of two measurements, this method is more cumbersome and time-consuming. Furthermore, the conditions during the first and second measurements can vary.Furthermore, bleaching may have occurred, which could lead to further distortion of the results. Using this approach, fluorescence changes of up to 4.5% were resolved in measurements at room temperature (NPL2).

[0007] In general, measurements with commercially available fluorescence spectrophotometer devices require large sample volumes (i.e., when using quartz cuvettes or microtiter plates) and consequently higher sample concentrations to adequately resolve changes in fluorescence intensity. Further methods known in the prior art that require smaller sample volumes (i.e., 10 µl) are discussed below.

[0008] For example, if the absolute intensity variation at room temperature is small compared to pipetting errors, pipetting errors can be eliminated by applying a characterization method based on the temperature-related intensity change (TRIC) (WO 2018 / 234557). By calculating F heiß / F kalt By determining the ratio of the fluorescence intensity based on the intensity measured at room temperature and the intensity measured at a second, usually higher, temperature, meaningful measurements of inter- and / or intramolecular interactions can still be obtained.

[0009] However, an increase in temperature, even by just a few degrees Celsius (°C), - not always tolerated by the particles being measured. Therefore, this method is not suitable for characterizing interactions with unstable samples, such as unstable proteins. - The fluorescence intensity can change equally for the fluorescently labeled particle and for the fluorescently labeled particle complexed with a ligand. In this case, “F heiß / F kalt “In both cases the same value and although an interaction takes place between the interacting partners, no bonding curve can be obtained. - Inhomogeneous samples, such as samples containing a certain proportion of aggregates, can lead to non-reproducible fluorescence profiles due to convection, which means that the noise in "F heiß / F kalt “can become very large. Thus, it is impossible to obtain binding curves for systems with small signal amplitudes.

[0010] Furthermore, when analyzing interactions of ternary complexes, such as complexes containing a labeled molecule A, another molecule B bound to A, and a third molecule C bound to B, changing the TRIC is often insufficient to resolve the interactions.

[0011] If the interaction changes rapidly with temperature and ligand binding causes changes in fluorescence intensity, the TRIC method may either lead to biphasic dose-response curves that cannot be analyzed using a sigmoidal 1:1 binding model, or the measured value of the dissociation constant may differ from the actual value (e.g., the measured binding affinity corresponds to the weaker binding at the higher temperature rather than at the lower temperature).

[0012] Another approach to investigating intra- and / or intermolecular interactions of fluorescent particles is nano-differential scanning fluorometry (WO 2017 / 055583). This method is based on measuring changes in the intrinsic fluorescence intensity of proteins containing tryptophan (Trp) and tyrosine (Tyr) residues. However, proteins typically contain several of these fluorescent aromatic amino acid residues. Since not all of them are usually involved in a binding reaction, excitation results in a high fluorescence background, which reduces the signal amplitude. Furthermore, Trp and Tyr residues are usually located in the hydrophobic core of a protein and may be only minimally affected by ligand binding. The autofluorescence of the ligands is often in the same range as the fluorescence of Trp and Tyr, thus interfering with the readout.

[0013] In contrast, labeling with an extrinsic fluorescent marker allows control over the binding of only one dye (i.e., one type of dye) to a target molecule, and only this dye is affected by ligand binding. Furthermore, the labeling chemistry can be tailored to place the dye at a location optimal for detecting changes in the chemical microenvironment (e.g., near the ligand binding site). Moreover, extrinsic dyes are typically located on the protein surface and are therefore ideally exposed to detect changes in the chemical microenvironment. The fluorescence range of the dye can be chosen to avoid interference from the autofluorescence of a ligand.Finally, extrinsic dyes are much brighter and measurements can be performed at a much lower concentration of the target molecule, thus reducing sample consumption and enabling the measurement of even picomolar affinities.

[0014] Therefore, there is a need for improved or alternative methods or improved or alternative devices for characterizing inter- and / or intramolecular interactions and / or modifications (conformational changes) and / or (changes) in the localization of fluorescently labeled particles. Brief description of the invention

[0015] The present invention provides novel methods and devices for characterizing inter- and / or intramolecular interactions and / or modifications (conformational changes) and / or (changes) localization of fluorescently labeled particles, as defined by the features of the independent claims. Further preferred embodiments of the present invention are defined in the dependent claims. In particular, the present invention solves the technical problem of economically resolving arbitrarily small changes in the fluorescence spectrum of labeled fluorescent particles, based preferably on exactly one fluorescent marker and independent of the properties (e.g., temperature stability, aggregation) and size of the fluorescently labeled particle.

[0016] The present invention relates to methods and devices for the ratiometric characterization of inter- and / or intramolecular interactions and / or conformational changes and / or the localization of fluorescently labeled particles. In particular, the methods of the present invention offer improved sensitivity in detecting changes in the fluorescence spectrum of fluorescent markers within small sample volumes of fluorescently labeled particles, which could not be resolved using previously known methods. Furthermore, the method of the present invention enables rapid measurements even of temperature-sensitive and / or unstable samples. In combination with a defined temperature perturbation, the method of the present invention enables measurements of thermodynamic and kinetic parameters of interactions.The methods of the present invention employ fluorescently labeled particles, preferably labeled with at least one fluorescent marker. The method of the present invention makes it possible to determine the localization of fluorescently labeled particles (e.g., when the fluorescently labeled particle is localized within lipid nanoparticles (LNPs) and / or within cells and / or within a buffer solution surrounding the LNPs and cells).

[0017] In a first aspect, the present invention relates to a method for characterizing fluorescently labeled particles in solution by analyzing changes in the fluorescence spectrum of the fluorescently labeled particles. The method of the first aspect comprises the following steps: a) Providing a sample of the fluorescently labeled particles in a solution under initial conditions, b) Excitation of the fluorescently labeled particles at a first wavelength, c) Measuring the fluorescence emission intensity of the fluorescently labeled particles at a second and a third wavelength, d) Calculating the ratio between the fluorescence intensities at the second and third wavelengths, wherein the third wavelength differs from the second wavelength. Furthermore, the following is preferred: e1) Repeat 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, where the second conditions differ from the first conditions. f) The fluorescently labeled particles can then be characterized based on the calculated ratios obtained for the different conditions, wherein the second and third wavelengths are preferably detected simultaneously and wherein the second wavelength is preferably shorter and the third wavelength is preferably longer than an emission maximum of the fluorescence emission of the fluorescently labeled particles under the first conditions.

[0018] In a second aspect, the present invention relates to a method for characterizing fluorescently labeled particles in solution by analyzing changes in the fluorescence spectrum of the fluorescently labeled particles in combination with a defined temperature disturbance / temperature change. The method of the second aspect comprises the following steps: a) Providing a sample of the fluorescently labeled particles in a solution under initial conditions, b) Excitation of the fluorescently labeled particles at a first wavelength, c) Measuring the fluorescence emission intensity of the fluorescently labeled particles at a second and a third wavelength, where the intensities are recorded during a defined temperature disturbance, d) Calculating the ratio between the fluorescence intensities at the second and third wavelengths, where the third wavelength differs from the second wavelength.

[0019] As with the first aspect, the following is also preferred: e1) Repeat 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, the second conditions differ from the first conditions, and f) Characterizing the fluorescently labeled particles based on the calculated ratios obtained for the different conditions, wherein the second and third wavelengths are preferably detected simultaneously and wherein the second wavelength is preferably shorter and the third wavelength is longer than an emission maximum of the fluorescence emission of the fluorescently labeled particles under the first conditions.

[0020] In a third aspect, the present invention relates to a method for characterizing the thermodynamic and / or kinetic parameters of fluorescently labeled particles in solution by analyzing changes in the fluorescence spectrum of the fluorescently labeled particles in combination with a defined temperature disturbance / temperature change.

[0021] In a fourth aspect, the present invention relates to a method for characterizing the localization of fluorescently labeled particles in solution by analyzing changes in the fluorescence spectrum of the fluorescently labeled particles with or without a defined temperature disturbance / temperature change.

[0022] Within the scope of the present invention, it is preferred that the fluorescence emission intensities at a second and a third wavelength of step c) are simultaneously detected, preferably within a short time interval of less than 1 s, more preferably less than 750 ms, more preferably less than 500 ms, more preferably less than 250 ms, more preferably less than 100 ms, more preferably less than 50 ms, more preferably less than 25 ms, more preferably less than 10 ms, more preferably less than 5 ms, and even more preferably less than 2.5 ms. Within this range, typical time intervals are 50 ms, 10 ms, and 1 ms.

[0023] Some special aspects of the invention can be summarized as follows: In some aspects, the procedures described herein include the steps 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) measuring the fluorescence emission intensity of the fluorescently labeled particles at a second and a third wavelength, d) calculating the ratio between the fluorescence intensities at the second and third wavelengths, the third wavelength being different from the second wavelength, e1) repeating steps b) to d) for the sample of fluorescently labeled particles under second conditions, or e2) repeating steps a) to d) for a second sample of fluorescently labeled particles under second conditions, the second conditions being different from the first conditions.f) Characterizing the fluorescently labeled particles based on the calculated ratios obtained for the different conditions, wherein the second and third wavelengths are detected simultaneously and wherein the second wavelength is shorter and the third wavelength is longer than an emission maximum of the fluorescence emission of the fluorescently labeled particles under the first conditions.

[0024] In some aspects of the present invention, the sample volume containing the fluorescently labeled particles is less than 100 µl, preferably between 1 µl and 25 µl, i.e. the sample containing the fluorescently labeled particles is provided in a volume of less than 100 µl, preferably between 1 µl and 25 µl.

[0025] In some aspects of the present invention, it is preferred that the sample containing the fluorescently labeled particles is provided in a volume between 1 µl and 25 µl.

[0026] In some aspects of the present invention, the sample containing the fluorescently labeled particles is provided in a capillary.

[0027] In some aspects of the present invention, the fluorescently labeled particles are labelled with an environmentally sensitive marker.

[0028] In some aspects of the present invention, the particles are selected from the group consisting of organic molecules, biomolecules, nanoparticles, microparticles, vesicles, biological cells or subcellular fragments, biological tissues, virus particles, viruses, cellular organelles, lipid nanoparticles (LNPs) and virus-like particles.

[0029] In some aspects of the present invention, the biomolecules are selected from the group consisting of amino acids, proteins, peptides, mono- and disaccharides, polysaccharides, lipids, glycolipids, fatty acids, sterols, vitamins, neurotransmitters, enzymes, nucleotides, metabolites, nucleic acids and combinations thereof.

[0030] In some aspects of the present invention, the concentration of the fluorescently labeled particles in the solution is between 10 pM and 10 µM, preferably 50 pM to 500 nM.

[0031] In some aspects of the present invention, the changes in the detected fluorescence intensity of the fluorescently labeled particles result from spectral shifts or broadenings of the spectrum or narrowings of the spectrum or combinations thereof.

[0032] In some aspects of the present invention, the fluorescence intensity of the fluorescently labeled particles changes due to mechanisms selected from the group consisting of conformational changes of the fluorescently labeled particles, relocalization of the fluorescently labeled particles, interactions between the fluorescently labeled particles and one or more ligands, and combinations thereof.

[0033] In some aspects of the present invention, the calculated ratios obtained in step f) are used to determine the localization of the fluorescently labeled particles or parameters selected from the group consisting of dissociation constants, mean effective concentrations (EC₁₀). 50), equilibrium constants, bonding kinetics, enzyme reaction kinetics, thermodynamic parameters, unfolding or refolding kinetics, opening and closing reactions and combinations thereof.

[0034] In some aspects of the present invention, the second conditions of step e) are modified by adding a ligand and / or different concentrations of the ligand, and the calculated ratios obtained in step f) are used to determine the dissociation constant of the fluorescently labeled particles and the ligand.

[0035] In some aspects of the present invention, the first and second conditions of the fluorescently labeled particles differ with respect to their temperature and / or chemical composition.

[0036] In some aspects of the present invention, the fluorescence emission intensity of the fluorescently labeled particles is recorded at a second and a third wavelength of step c) during a defined temperature disturbance.

[0037] This also provides an apparatus for characterizing fluorescently labeled particles in solution by analyzing changes in the fluorescence spectrum of the fluorescently labeled particles. In some aspects, the apparatus of the present invention is configured to carry out the methods described herein. In some aspects, the apparatus comprises a sample holder for holding a sample of fluorescently labeled particles in solution under a variety of conditions (i.e., several different conditions); means for exciting the fluorescently labeled particles at a first wavelength; and means for detecting the fluorescence emission intensity of the fluorescently labeled particles at a second and third wavelength.Means for calculating a ratio between the fluorescence intensities at the second and third wavelengths, wherein the third wavelength differs from the second wavelength, the device being designed to successively excite fluorescently, detect fluorescence emissions, and calculate the ratio for samples under different conditions; means for characterizing the fluorescently labeled particles based on the calculated ratios obtained for the different conditions, the device being designed to detect the second and third wavelengths simultaneously, and wherein the second wavelength is shorter and the third wavelength is longer than an emission maximum of the fluorescence emission of the fluorescently labeled particles under a first condition (of the different conditions).

[0038] In some aspects of the present invention, the excitation means is an excitation light source, preferably at least one light source from the group consisting of laser, laser fiber laser, diode laser, LED, HXP, halogen, LED array, HBO.

[0039] In some aspects of the present invention, the means for detection is a light detector, preferably at least one detector from the group consisting of PMT, siPM, APD, CCD or CMOS camera.

[0040] Also provided herein is a computer program comprising instructions which, when the program is executed by a computer, cause the computer used to perform the procedures described herein.

[0041] Also provided herein is a computer-readable data carrier containing instructions which, when executed on a computer, cause the computer used to perform the procedure described herein.

[0042] Also provided herein is the use of a device for characterizing fluorescently labeled particles in solution according to the methods described herein.

[0043] Also provided herein is the use of a capillary for characterizing fluorescently labeled particles in solution by analyzing the changes in the fluorescence spectrum of the fluorescently labeled particles, wherein a sample of the fluorescently labeled particles in solution is filled into the capillary and made available for analysis, according to the procedures described herein. Brief description of the drawings

[0044] Preferred embodiments of the present invention are described in more detail below with reference to the figures. Fig.Figure 1 (A) shows the excitation spectra of four different protein samples labeled with the same fluorescent dye. The samples were excited between 520 and 670 nm. Emission from the samples was recorded at 690 nm. Figure 2 (B) shows a magnified view of the excitation peaks from Figure 1 (A). Fig. Figure 2 (A) shows the emission spectra of four different protein samples labeled with the same fluorescent dye. The samples were excited at 605 nm. Emission from the samples was recorded between 620 and 750 nm. Figure 2 (B) shows a magnified view of the emission peaks from Figure 2 (A). Fig. Figure 3 shows possible effects of (A) proximity to ligands and (B) conformational changes of a labeled molecule on changes in the fluorescence spectrum of a fluorescent dye, including (C) hypsochromic (blue) or bathochromic (red) shifts and / or (D) broadening or narrowing of the spectrum. Fig.Figure 4 (A) shows the emission peaks of the fluorescently labeled protein streptavidin alone and in combination with its natural ligand biotin. Figure 4 (B) Enlarged view of the emission peaks of (A). Fig. Figure 5 (A) shows the emission peaks of the fluorescently labeled protein lysozyme alone and in combination with the inhibitor tri-N-acetyl-D-glucosamine (NAG3). Figure 5 (B) Enlarged view of the emission peaks of (A). Fig. Figure 6 (A) shows the ratio profiles of carbonic anhydrases in complex with furosemide, recorded using the dual emission configuration of the present invention. The change in the fluorescence ratio of carbonic anhydrase upon binding of furosemide is 0.5%. Figure 6 (B) shows the resulting dose-response curve of the binding interaction. Fig.Figure 7 (A) shows the 350 nm / 330 nm ratio of intrinsic tryptophan fluorescence of unlabeled lysozyme and various concentrations of NAG3 at increasing temperatures. To monitor the denaturation temperature, each sample was heated from 35 °C to 95 °C. Increasing concentrations of NAG3 lead to thermal stabilization, i.e., a thermal shift, of lysozyme. This shift cannot be used to determine the dissociation constant of this interaction. (B) Plotting the initial 350 nm / 330 nm ratio of intrinsic protein fluorescence at 35 °C against the concentration of NAG3 yields a sigmoidal dose-response curve and thus the dissociation constant (KD). d ) at this temperature. Fig.Figure 8 shows various embodiments according to the present invention. (A) Preferred embodiment with dual emission optics and the IR laser. (B) Embodiment with dual emission optics. (C) Embodiment with dual excitation optics and the IR laser. (D) Embodiment with dual excitation optics. (E) Embodiment with dual excitation and dual emission optics and the IR laser. (F) Embodiment with dual excitation and dual emission optics. Fig. Figure 9 shows exemplary filter configurations that can be used for the (A) dual excitation and (B) dual emission configuration for red (Cy5) and green (Cy3) fluorescent dyes, respectively. Fig.Figure 10 shows dose-response curves between Cy5-labeled DNA aptamer and AMP obtained by ratiometric characterization based on either (A) a measurement using a commercially available microtiter plate reader or (B and C) a measurement using the dual emission configuration according to the present invention. Fig. Figure 11 shows the fluorescence profiles of a Cy5-labeled DNA aptamer mixed with a dilution series of AMP. At time 0 s, an IR laser is switched on, and the response of the fluorescence intensity is measured over a period of 31 s. In the present dual-emission configuration, the emitted fluorescence profiles were recorded simultaneously (A) at a wavelength of 628 to 653 nm (“650 nm”) and (B) at a wavelength of 665 to 727 nm (“670 nm”). Fig. Figure 12 shows the analysis of the original fluorescence intensities of the measurement of Fig.9. Based on the original fluorescence intensities obtained for (A) 650 nm and (B) 670 nm, no sigmoidal dose-response curve and therefore no interaction affinities are obtained. Fig. Figure 13(A) shows the ratiometric analysis of the fluorescence intensity curves of the measurements of Fig. 11, which were obtained by pointwise division of the fluorescence profiles at 670 nm by the fluorescence profiles at 650 nm. The different phases, i.e., phase 1 to phase 3, of the measurement are highlighted. (B) By analyzing the ratio during phase 1, i.e., before the IR laser is switched on, a dose-response curve with a signal-to-noise ratio of over 300 is obtained. Fig. 14(A) shows a curve of K d against time for the ratiometric data of Fig.13A. “Vertical slices” taken at 200 ms intervals are analyzed to obtain a dose-response curve for each time interval. If the temperature change over time is known and the interaction equalizes faster than the temperature change, a relationship of K can be established. d (B) Performing a van't Hoff analysis allows the bond enthalpy (ΔH) and bond entropy (ΔS) of the interaction to be determined from the relationship of K d against the temperature. Fig.Figure 15 shows the fluorescence profiles of a Cy3-labeled DNA aptamer mixed with a dilution series of AMP. At time 0 s, an IR laser is switched on, and the response of the fluorescence intensity is measured over a period of 6 s. In the present dual excitation configuration, the emitted fluorescence profiles are recorded sequentially by a single detector, (A) with a first excitation using a blue LED at a wavelength of 475 to 495 nm and (B) with a subsequent excitation using a green LED at a wavelength of 550 to 575 nm. Fig. Figure 16(A) shows the ratiometric analysis of the initial fluorescence intensities of the measurement of Fig.15, which are obtained by pointwise division of the fluorescence profiles measured under excitation with the green LED by those measured under excitation with the blue LED. The different phases, i.e., phase 1 to phase 3, of the measurement are highlighted. (B) By analyzing the ratio during phase 1, i.e., before the IR laser is switched on, a dose-response curve with a signal-to-noise ratio of approximately 80 is obtained. (C) By analyzing the ratio during phase 3, i.e., after the IR laser has been switched on, a dose-response curve with an improved signal-to-noise ratio of over 130 is obtained. Fig.Figure 17 shows the dose-response curve of a 12-step dilution series of biotin mixed with fluorescently labeled streptavidin, obtained by ratiometric measurement using the dual-emission configuration. The dashed line is a 1:1 binding model fit. Since the target concentration is much higher than the dissociation constant (Kd), the dose-response curve is calculated using a 1:1 binding model. d ), a characteristic kink can be observed at the stoichiometric point. Fig. Figure 18 shows the dose-response curve of a 15-step dilution series of the small molecule acetazolamide mixed with fluorescently labeled bovine carbonic anhydrase II, obtained by ratiometric measurement using the dual emission configuration. The dashed line is a 1:1 binding model fit. The ratio changes by only about 0.7%. Fig.19(A) shows the dose-response curve of a 16-step dilution series of the unlabeled monoclonal antibody Herceptin (trastuzumab) mixed with (B) a preformed complex of biotinylated protein L and fluorescently labeled monovalent streptavidin, resulting in a ternary complex, obtained by ratiometric measurement using the dual emission configuration. Fig. Figure 20(A) shows a schematic representation of a complex of maltose, biotinylated maltose-binding protein, streptavidin, and fluorescently labeled biotinylated DNA. Figure 20(B) shows the dose-response curve between biotinylated maltose-binding protein, fluorescently labeled by mixing with unlabeled streptavidin and fluorescently labeled biotinylated DNA, and maltose, obtained by ratiometric measurement using the dual-emission configuration. Fig.Figure 21 shows the dose-response curve of a 14-step dilution series of angiotensin-converting enzyme 2 (ACE2) mixed with 20 nM Cov-19 spike protein labeled by the addition of 5 nM of the fluorescently labeled therapeutic antibody CR3022. Fig. Figure 22 shows four consecutive measurements of the fluorescence ratio of fluorescently labeled mitogen-activated protein kinase 14 (p38-α) over a period of approximately 20 minutes. The fluorescence ratio is not constant across the four measurements but appears to increase linearly, indicating that the protein is not stable at room temperature but gradually denatures. Fig. 23 shows K dTime-vs. curves for the interaction between (A) Cy5-labeled DNA aptamer for adenosine and the small molecule AMP. (B and C) DNA hybridization between two complementary 11-mer DNA strands, one strand being labeled with Cy5, measured at (B) 32 °C and (C) 22 °C. The rate at which the K d The -vs-time curve of the temperature perturbation of the IR laser indicates how fast the binding and dissociation kinetics of this interaction are. Fig. 24(A) shows normalized K d Time-vs. curves for DNA hybridization between 22 °C and 32 °C. The y-axis represents the magnitude of the increase in K. d during the measurement (all normalized to 1 for comparison). The x-axis shows the on-time of the IR laser. (B) shows a K d-vs.-time curve for a measurement of DNA hybridization with the dual emission configuration using an IR laser according to the present invention. The sample temperature was 22 °C and the temperature after IR laser heating was approximately 32 °C. The K d The value changes from approximately 10 nM to approximately 500 nM during the IR laser's on-time. Figure (C) shows the results of a van't Hoff analysis of the two K d Values ​​at the two different temperatures. (D and E) show the thermodynamic parameters of the interaction obtained from the classical van't Hoff analysis of the fluorescence ratio measurements at six different sample temperatures (22 °C, 24 °C, 26 °C, 28 °C, 30 °C, 32 °C), which yielded very similar thermodynamic parameters, but takes longer than the thermodynamic measurement with the IR laser. Fig.Figure 25 shows simulated Kd-versus-time curves for different dissociation rates. The legend indicates the different off rates used in the simulation. (A) shows that the dissociation rates between 10 s -1 and 0.001 s -1 with a typical measurement involving 20-second IR laser heating. (B) shows that even small differences between 0.036 s -1 and 0.154 s -1 can be resolved well. Fig.Figure 26 shows measurements of the slow binding kinetics using ratiometric fluorescence signals from a mix-and-measure approach of a fluorescently labeled nanobody rapidly mixed with six different concentrations of Cov-19 spike RBD. A 2 nM fluorescently labeled nanobody is rapidly mixed with six different concentrations of COV-19 spike RBD (20 nM–625 pM). Ratiometric fluorescence measurements are then performed every 90 s to monitor the slow binding kinetics. A global fitting model can be used to determine the kinetics of the nanobody. on , k off and K d provide the interaction. Fig.Figure 27(A) shows schematic representations of fluorescently labeled mRNA, a lipid nanoparticle (LNP), and a cell. Ratiometric measurements can be used to determine the localization of the fluorescently labeled mRNA molecules. Figure 27(B) shows that all fluorescently labeled mRNA molecules are localized within an LNP. Figure 27(C) shows that all fluorescently labeled mRNA molecules are localized in the buffer-containing chamber. Figure 27(D) shows that all fluorescently labeled mRNA molecules are localized in a cell. Figure 27(E) shows the uniform distribution of the labeled mRNA molecules in the LNP, the buffer chamber, and the cell. Fig.Figure 28 (A) shows ratiometric measurements of LNPs loaded with fluorescently labeled mRNA in different states. (B) Additional information about the state of the LNPs can be obtained by analyzing the fluorescence profiles at a single wavelength (here: 670 nm), revealing “bumpy” aggregation profiles, such as those obtained after a 20-minute centrifugation step, or “smooth” profiles, such as those obtained after 10 minutes of heating to 90°C. Fig.Figure 29(A) shows a schematic representation of a complex between a tetrameric streptavidin, two biotinylated fluorescently labeled linker molecules, and a biotinylated target molecule. Ratiometric measurements in the dual emission configuration can be used to determine (B) the stoichiometry of a complex between tetrameric streptavidin and biotinylated fluorescently labeled linker molecules and (C) the dose-response curve between the streptavidin-linker complex and a biotinylated target molecule. Detailed description of the invention

[0045] The present invention relates to methods and devices for the ratiometric characterization of inter- and / or intramolecular interactions and / or conformational changes and / or the localization of fluorescently labeled particles. In particular, the methods of the present invention offer improved sensitivity for detecting changes in the fluorescence spectrum of fluorescent markers within small sample volumes of fluorescently labeled particles, which could not be resolved using previously known methods (see, for example, Example 1). Furthermore, the method of the present invention is not necessarily based on temperature-induced changes in the fluorescence spectrum, thus enabling rapid measurements even of temperature-sensitive and / or unstable samples.In combination with a defined temperature disturbance, the method of the present invention enables measurements of thermodynamic and kinetic parameters of interactions.

[0046] The method of the present invention makes it possible to determine the localization of fluorescently labeled particles (i.e., when the fluorescently labeled particle is localized within lipid nanoparticles (LNPs) and / or within cells and / or within a buffer solution surrounding the LNPs and cells). The methods of the present invention employ fluorescently labeled particles that are labeled with only one fluorescent marker. "Labeled with only one fluorescent marker" here means "labeled with only one type of fluorescent marker." This can, in turn, be a label with only a single fluorescent unit (e.g., one Cy5 molecule) or with two or more fluorescent units of a single type (e.g., two or more Cy5 molecules).

[0047] Within the scope of the present invention, changes in the fluorescence spectrum of fluorescently labeled particles are measured and ratiometrically analyzed in order to characterize interactions, including binding affinities and the like, of the fluorescently labeled particles.

[0048] The steps of the method of the present invention comprise providing one or more samples of fluorescently labeled particles in solution. The one or more samples are preferably excited to fluorescent emission at a constant excitation wavelength. The emitted fluorescence is preferably recorded simultaneously at two different emission wavelengths, preferably at a predetermined constant temperature. The ratio of the fluorescence intensity at the two emission wavelengths can be determined. Thus, the two obtained measurements of the emitted fluorescence can be characterized in a ratiometric manner.

[0049] Since the acquisition of the two emission wavelengths is preferably carried out simultaneously, and thus identical disturbances or errors influence the measurements, ratiometric characterization leads to the extraction of pure information and thus improves the resolution of the measurement method.

[0050] The inventors of the present invention have found that changes in the fluorescence spectrum of a fluorescent marker bound to a particle such as a biomolecule can be used to determine, among other things, the conformational state (folded / unfolded state) and / or interaction parameters between a ligand and a biomolecule.

[0051] Within the scope of the present invention, the terms “detected” and “received” are used interchangeably and refer to the determination of the fluorescence signal of fluorescently labeled particles.

[0052] In a first aspect, the present invention relates to a method for characterizing fluorescently labeled particles in solution by analyzing changes in the fluorescence spectrum of the fluorescently labeled particles at, for example, a predetermined temperature.

[0053] The method of the first aspect of the invention comprises the following steps: a) Providing a sample of the fluorescently labeled particles in a solution under initial conditions, b) Excitation of the fluorescently labeled particles at a first wavelength, c) Measuring the fluorescence emission intensity of the fluorescently labeled particles at a second and a third wavelength, d) Calculating the ratio between the fluorescence intensities at the second and third wavelengths, where the third wavelength differs from the second wavelength, e1) Repeat 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, where the second conditions differ from the first conditions, f) Characterizing the fluorescently labeled particles based on the calculated ratios obtained for the different conditions, where the second and third wavelengths are detected simultaneously and where the second wavelength is shorter and the third wavelength is longer than an emission maximum of the fluorescence emission of the fluorescently labeled particles under the first conditions. Particles

[0054] According to the present invention, the term "particle" includes molecules, in particular organic molecules, biomolecules, nanoparticles, microparticles, and vesicles. The application of the present invention to biomolecules such as nucleic acids and proteins is particularly important. The term "particle" also includes biological cells (e.g., bacterial and eukaryotic cells) or subcellular fragments, biological tissues, virus particles, virus-like particles or viruses and cellular organelles, lipid nanoparticles (LNPs), and the like. Nanoparticles also include nanodiscs. A nanodisc is a synthetic model membrane system consisting of a lipid bilayer of phospholipids, wherein the hydrophobic rim is shielded by two amphipathic proteins.

[0055] Biomolecules are preferably selected from the group consisting of amino acids, proteins, peptides, mono- and disaccharides, polysaccharides, lipids, glycolipids, fatty acids, sterols, vitamins, neurotransmitters, enzymes, nucleotides, metabolites, nucleic acids, and combinations or complexes thereof. More preferably, the biomolecules are selected from the group consisting of proteins, peptides, enzymes, nucleic acids, and combinations or complexes thereof.

[0056] Preferably the particles (in the marked particles) are biomolecules, most preferably proteins or nucleic acids.

[0057] The proteins are selected from the group consisting of enzymes (e.g., carbonic anhydrase, beta-lactamase TEM1, or kinases such as MEK1 and p38), transporter proteins (e.g., MBP), inhibitory proteins (e.g., beta-lactamase inhibitor protein PLIP, anakinra), structural proteins, signaling proteins, ligand-binding proteins, chaperones (e.g., heat shock protein HSP90), antibodies (e.g., trastuzumab), membrane proteins, and receptors (e.g., interleukin-1 receptor).

[0058] Nucleic acids include DNA, RNA (e.g., mRNA, tRNA, rRNA, and the like), LNA, and PNA. Modified (e.g., chemically modified) nucleic acids can also be analyzed within the scope of the present invention. Locked nucleic acid (LNA), often referred to as inaccessible RNA, is a modified RNA nucleotide. The ribose unit of an LNA nucleotide is modified by an additional bridge connecting the 2' oxygen and the 4' carbon. Peptide nucleic acid (PNA) is an artificially synthesized polymer that resembles DNA or RNA. DNA and RNA have a deoxyribose or ribose sugar backbone, respectively, whereas the backbone of a PNA consists of a peptide, such as repeating N-(2-aminoethyl)glycine units linked by peptide bonds. The various purine and pyrimidine bases are linked to the main chain by a methylene bridge (-CH2-) and a carbonyl group (-(C=O)-).

[0059] In the context of the present invention, a nanoparticle is a particle with an average size of less than 100 nm. The term "average size" describes the average effective diameter, measured by dynamic light scattering using, for example, a Brookhaven 90Plus instrument or a Malvern Zetasizer Z90 particle size analyzer. Preferably, the size of the nanoparticles is in the range of 1 nm to 100 nm, more preferably 1 to 70 nm. The nanoparticles can be organic or inorganic. The nanoparticles can also be composite particles, such as an inorganic core with organic molecules bound to the surface.

[0060] A microparticle is a particle whose longest dimension is less than 1 mm, but typically more than 100 nm. Microparticles can be characterized using size determination techniques such as transmission electron microscopy (TEM), scanning electron microscopy (SEM), and quasi-elastic light scattering (QELS). Microparticles can also be found in the form of microspheres.

[0061] The microparticles can be, for example, coated or uncoated silicon dioxide / glass / biodegradable particles, polystyrene / coating / flow cytometry / PMMA / melamine / NIST particles, agarose particles, magnetic particles, coated or uncoated gold or silver particles or other metal particles, transition metal particles, biological materials, semiconductors, organic and inorganic particles, fluorescent polystyrene microspheres, non-fluorescent polystyrene microspheres, composite materials, liposomes, cells and the like.

[0062] Commercially available microparticles are available in a wide variety of materials, including ceramics, glass, polymers, and metals. Microparticles encountered in everyday life include pollen, sand, dust, flour, and powdered sugar. In biological systems, microparticles are small, membrane-bound vesicles that circulate in the blood and originate from cells in contact with the bloodstream, such as platelets and endothelial cells.

[0063] Microspheres are preferably manufactured solid plastic particles with a maximum dimension of less than 5 mm. Microspheres can also be uniform polymer particles, typically with a diameter of 0.5 to 500 µm.

[0064] The term "modified particle" or "modified sphere" refers in particular to spheres or particles that comprise or are linked to molecules, preferably biomolecules. This also includes coating such spheres or particles with these (bio)molecules.

[0065] Particles or spheres according to this invention can be modified in such a way that, for example, biomolecules, e.g., DNA, RNA, or proteins (in some embodiments specifically and / or covalently), can bind to the particles or spheres. Therefore, the analysis of the properties of spheres and / or particles, and in particular of molecules bound to or associated with such spheres or particles, is within the scope of protection of this invention. In particular, such molecules are biomolecules. Accordingly, the term "modified (micro)spheres / (nano- or micro)particles" refers in particular to spheres or particles that include additional molecules to be analyzed or characterized. Modified or unmodified microparticles / (nano- or micro)particles may be able to interact with other particles / molecules, such as biomolecules (e.g., DNA, RNA, or proteins), in solution.

[0066] The preferred concentration of the fluorescently labeled particles used in the present invention is preferably between 10 pM and 10 µM, and even more preferably between 50 pM and 500 nM.

[0067] In the methods of the present invention, it is preferred that the concentration of the fluorescently labeled particles in the solution is between 50 pM and 500 nM.

[0068] The methods of the present invention employ fluorescently labeled particles that are labeled with at least one fluorescent marker, preferably with exactly one fluorescent marker. Within the scope of the present invention, particles labeled with more than one fluorescent marker are labeled with only one type of fluorescent marker (i.e., only one type of marker per particle). Within the scope of the present invention, "labeled particles" refers to fluorescently labeled particles or other particles that can be detected by fluorescent agents, e.g., molecules / particles comprising an intrinsic fluorophore, or particles / molecules tagged with fusion proteins, or particles / molecules with attached extrinsic fluorophores.

[0069] In particular, the labeled particles are preferably particles that are bound to a marker, e.g. covalently (e.g. via NHA labeling, maleimide labeling and the like), reversibly bound to a marker via a high-affinity protein tag, e.g. HIS tag, AVI tag, SPOT tag, SNAP tag and the like, or bioconjugated via copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), voltage-activated azide-alkyne cycloaddition (SPAAC) and the like (also known as click chemistry) or similar processes.

[0070] Protein tags are peptide sequences that are genetically grafted onto a recombinant protein. These include poly(His) tags, polyanionic amino acids such as the FLAG tag, epitope tags such as the V5 tag, Myc tag, HA tag and the NE tag, as well as tags that allow specific enzymatic modification (such as biotinylation by biotin ligase) or chemical modification (such as reaction with FlAsH-EDT2 for fluorescence imaging). Fluorescence markers

[0071] Within the scope of the present invention, the terms “marker” and “dye” are used interchangeably and refer to a fluorophore / fluorochrome, i.e., a fluorescent chemical compound which re-emits light when excited.

[0072] In the present invention, useful markers are markers that are sensitive to environmental changes, i.e., the fluorescence spectrum of the dye changes when the environment changes, such as changes in the chemical microenvironment (ligand binding, conformational changes) and / or the macroenvironment (e.g., location in LNP versus location in cells) and / or temperature changes (e.g., heating or cooling).

[0073] In the context of the present invention, the fluorescent marker is advantageously bound to the particle in the vicinity of a location / position on the particle where a binding interaction is assumed to take place, e.g. the binding pocket of a protein or the like.

[0074] Fluorescence markers for use according to the present invention may be selected from the group consisting of intrinsic fluorescent markers, fusion proteins, extrinsic fluorescent markers or the like.

[0075] Intrinsic fluorescent markers include tryptophan residues, tyrosine residues, and phenylalanine residues. Fusion proteins can be selected from the group consisting of blue-emitting, cyan-emitting, green-emitting, yellow-emitting, and red-emitting fluorescent proteins, and the like. Reference is made here to FPbase, a prior art database that provides a comprehensive list of currently known fluorescent proteins (https: / / www.fpbase.org / table / ; Lambert, TJ (2019) FPbase: a community-editable fluorescent protein database. Nature Methods. 16, 277-278. doi: 10.1038 / s41592-019-0352-8).

[0076] In a preferred embodiment of the present invention, the fluorescent markers are extrinsic fluorescent markers.

[0077] Extrinsic fluorescent markers may include, but are not limited to, commercially available markers such as cyanine dyes, including Cy5, Cy3, Atto647, Atto647N, Alexa647, Dy647, and the like.

[0078] Preferred extrinsic fluorescent markers are environmentally sensitive dyes, such as those described in WO 2018 / 234557, which is incorporated herein by reference. Environmentally sensitive dyes are known in the prior art and are described, for example, in Klymchenko, AS (2017) (Solvatochromic and fluorogenic dyes as environment-sensitive probes: design and biological applications. Accounts of chemical research, 50(2), 366-375). In particular, WO 2018 / 234557 relates to fluorescent markers that are very sensitive to environmental changes, such as changes in chemical composition, temperature changes, and the like. According to a preferred embodiment, these dyes are selected from the group consisting of the dyes NanoTemper RED, GREEN, and BLUE (commercially available, e.g., as Protein Labeling Kits from NanoTemper Technologies GmbH, Munich, Germany).

[0079] Labeling with an extrinsic fluorescent marker allows control over ensuring that only one dye binds to a target molecule, and only that dye is affected by ligand binding. Furthermore, the labeling chemistry can be tailored to place the dye at a location optimal for detecting changes in the chemical environment (e.g., near the ligand binding site). In addition, extrinsic dyes are typically located on the protein surface and are therefore ideally exposed to detect changes in the chemical microenvironment. The fluorescence range of the dye can be selected to avoid interference from the autofluorescence of a ligand.Finally, extrinsic dyes are much brighter and measurements can be performed at a much lower concentration of the target molecule, thus reducing sample consumption and enabling the measurement of even picomolar affinities.

[0080] Changes in the fluorescence spectrum according to the present invention include changes in the fluorescence intensity of a fluorescent marker, but also include spectral shifts (see Fig. 3C) and / or a broadening or narrowing of their spectrum (see Fig.3D). According to the present invention, it is preferred to detect the fluorescence at different wavelengths or wavelength ranges, which can be achieved, for example, by using bandpass filters. The detected intensities at these different wavelengths / wavelength ranges and their respective ratios enable the detection of a spectral shift of the entire emission spectrum, a broadening and / or narrowing of the spectrum.

[0081] Within the scope of the present invention, the spectral shifts preferably include bathochrome (i.e., red) shifts and / or hypsochrome (i.e., blue) shifts.

[0082] Within the scope of the present invention, the magnitude of the spectral shifts is preferably at least 50 pm, more preferably at least 100 pm, and even more preferably at least 500 pm.

[0083] According to the present invention, the fluorescence intensity of the fluorescently labeled particles changes preferably due to mechanisms selected from the group consisting of conformational changes of the fluorescently labeled particles, relocalization of the fluorescently labeled particles, interactions between the fluorescently labeled particles and one or more ligands and combinations thereof, and the like. Sample chambers

[0084] The sample to be used in the present invention is preferably provided in a sample chamber, which is preferably selected from the group consisting of capillaries, microtiter plates, a microfluidic chip, a cuvette, a reaction tube, a pipette tip, microfluidics, droplets, native tissues, organelles, 3D-printed tissues, 3D-printed organelles, and a translucent container. The translucent container may be a glass container or a plastic container.

[0085] In a preferred embodiment of the present invention, the sample containing the fluorescently labeled particles is provided in a capillary.

[0086] In a further preferred embodiment of the present invention, the sample containing the fluorescently labeled particles is provided in a microtiter plate, for example a 96-well, a 384-well or a 1536-well plate.

[0087] Preferably, the capillary consists of glass and / or a polymer and / or at least one of the elements of borosilicate glass, borosilicate glass 3.3 (for example, DURAN glass), fused silica such as Suprasil, Infrasil, synthetic fused silica, soda-lime glass, Bk-7, ASTM Type 1 Class A glass, ASTM Type 1 Class B glass. The polymers may include PTFE, PMMA, Zeonor™ Zeonex™, Teflon AF, PC, PE, PET, PPS, PVDF, PFA, FEP and / or acrylic glass.

[0088] In particular, it is preferred that at least one region of the capillaries is transparent to light with a wavelength of 200 nm to 1000 nm, preferably from 250 nm to 900 nm. Particularly preferred, but not limited thereto, is the transparent region of the capillary also to light with one of the following wavelength ranges: from 940 nm to 1040 nm (preferably 980 nm ± 10 nm), from 1150 nm to 1210 nm, from 1280 nm to 1600 nm (preferably 1450 nm ± 20 nm and / or 1480 nm ± 20 nm and / or 1550 nm ± 20 nm), from 1900 nm to 2000 nm (preferably 1930 nm ± 20 nm). It is understood by those skilled in the art that the transparent region(s) may also extend over the entire capillary. In other words, the capillaries can be transparent and preferably consist entirely of one of the aforementioned materials.

[0089] Preferably, the capillaries used have an inner diameter of 0.1 mm to 0.8 mm, more preferably 0.2 mm to 0.6 mm, and more preferably 0.5 mm. The outer diameter of the preferred capillaries is preferably between 0.2 mm and 1.0 mm, more preferably 0.3 mm to 0.65 mm.

[0090] The geometry of the capillaries is not limited to one shape. Preferably, tubular capillaries with a round or oval cross-section are used. However, it is also possible to use capillaries with other cross-sections, for example, triangular, square, pentagonal, or polygonal. Preferably, a capillary comprises one of these specific cross-sections over its entire length. Furthermore, it is also preferred that the inner and / or outer dimension of the capillary is constant along its entire length. For example, it is preferred that a cylindrical (tubular) capillary has the same inner and outer diameter over its entire length. In other words, capillaries can be used that have a diameter and / or cross-section that is constant or variable along the length of the capillary.

[0091] In particular, the sample chambers used for the present invention exhibit low autofluorescence over a broad spectral range. The autofluorescence is preferably less than 20%, more preferably less than 10%, and even more preferably less than 5%.

[0092] It is advantageous to provide the sample probe within a chamber having a thickness of 1 µm to 20 mm, in particular 1 µm to 6 mm, in particular 1 µm to 500 µm, in particular 1 µm to 250 µm, in particular 1 µm to 100 µm, in particular 3 µm to 50 µm, in particular 5 µm to 30 µm, in the direction of the fluorescence excitation beam. A person skilled in the art understands that the term "chamber" also refers, for example, to a capillary, a microfluidic chip, or a microtiter plate. silicon surface

[0093] Preferred surfaces on which the sample chambers are placed are described, for example, in WO 2017 / 055583, which is incorporated herein by reference. In particular, WO 2017 / 055583 relates to a silicon surface on / over which the sample chambers (e.g., capillaries) of the present invention are preferably placed. Sample volume

[0094] Typically, the sample volume containing the fluorescently labeled particles is less than 500 µl, preferably less than 200 µl, more preferably less than 100 µl, and even more preferably between 1 µl and 25 µl. sample

[0095] The sample to be used in the process according to the present invention is typically a solution comprising fluorescently labeled particles and ligands. The labeled particles may be dissolved or dispersed in the solution.

[0096] The labeled particles can be immobilized on a solid support that is brought into contact with the ligand-containing solution. Preferably, the labeled particles are dissolved or dispersed in the solution, which is selected from the group consisting of organic solutions and / or aqueous solutions, preferably buffered aqueous solutions. The buffered aqueous solution is preferably adjusted to a pH of 2 to 10, more preferably 4 to 10, even more preferably 5 to 9, and most preferably 6 to 8.5, using a buffer. Fluorescence measurement (excitation / emission)

[0097] According to the present invention, preferred means for excitation, preferably for fluorescent excitation of the labeled particles / molecules, can be any suitable device selected from the group consisting of lasers, fiber lasers, diode lasers, light-emitting diodes (LEDs), halogen lamps, LED arrays, HBO lamps (HBO lamps are, for example, short-arc lamps in which the discharge arc burns in an atmosphere of mercury vapor under high pressure), HXP lamps (HXP lamps are, for example, short-arc lamps in which the discharge arc burns in an atmosphere of mercury vapor under very high pressure; for example, unlike HBO lamps, they are operated at a significantly higher pressure and use a halogen circuit. HXP lamps produce UV and visible light, including a considerable proportion of red light), and the like.

[0098] Preferably, the excitation light source in the context of the present invention enables highly focused excitation. Within the scope of the present invention, the excitation light source is preferably a laser, and even more preferably an LED.

[0099] It is clear to those skilled in the art that the term "fluorescence," as used herein, is not limited to "fluorescence" per se, but that the means, methods, and devices disclosed herein can also be used and employed using other means, in particular luminescence, such as phosphorescence. Accordingly, the term in step b) "excitation of the fluorescently labeled particles at a first wavelength" refers to the "excitation step" in the method identified above and may include the corresponding excitation of the luminescence; for example, the excitation is carried out at a shorter wavelength than the subsequent emission detection. Therefore, the term "detection of the fluorescence emission intensities of the fluorescently labeled particles at a second and a third wavelength" in the context of this invention means a step of detection of the emissions after excitation.Those skilled in the art are aware that, in connection with this invention, the "excitation" wavelengths and the "emission" wavelengths must be separated. Furthermore, those skilled in the art are aware that the detected third wavelength differs from the detected second wavelength in the context of the present invention. The two signals required for ratiometric analysis can be obtained either using a "dual excitation" configuration or a "dual emission" configuration.

[0100] Ratiometric analysis is typically based on the "dual emission" configuration, e.g., using the exemplary dual emission optics described in Fig.8B is provided. In particular, the one or more samples containing the fluorescently labeled particles are excited at a single constant wavelength and their emission spectrum is recorded at two different wavelengths (see Examples 1 and 2 in combination with Fig. 10 or 13 as well as examples 4 to 12 in combination with Fig. 17, Fig. 18, Fig. 19, Fig. 20, Fig. 21, Fig. 22, Fig. 23, Fig. 24, Fig. 25, Fig. 26, Fig. 27 to Fig.28). By using the “dual emission” configuration, the two emission signals can be detected at the same position and at the same time. Furthermore, many fluorescent markers (e.g., Cy5) exhibit a smaller second excitation peak that can be used for efficient excitation, while at the same time providing sufficient bandwidth at longer wavelengths to split the emission spectra into two parts ( Fig. 9A).

[0101] In a preferred embodiment of the present invention, the dual-emission configuration is used in combination with red fluorescent markers, such as Cy5, RFP, and the like. Since such markers have their excitation maximum at approximately 650 nm with a secondary excitation peak at approximately 600 nm and their emission maximum at approximately 660 nm, suitable excitation and emission wavelengths include excitation at approximately 570 nm and 615 nm, detection of the first emission between approximately 625 nm and 650 nm, and detection of the second emission between approximately 670 nm and 725 nm. Fig. 9A). Exemplary components that may be useful for the “dual emissions” configuration are provided in Table 1. Table 1 component Example Art Manufacturer name Part number wavelength area Excitation light source LED Cree LM1-UYL1-11-N1-00001 LM1-UYL1-11-N1-00001 591 nm Excitation filter Bandpass Semrock 593 / 46 BrightLine HC AOI 0°, diameter 25 mm, ring thickness 3.5 mm F39-593 (FF01-593 / 46-25) 570 ... 615 nm Light separating element Dichroic mirror custom-made by AHF Beam splitter T 622LPXR AOI 45°, Reflection 465-615 nm > 90%, Transmission 626-900 nm > 90%, Dimensions 25.5 × 36 × 1 mm F48-622(T622LPXR) 615...626 nm Second light separator Dichroic mirror Semrock Beam splitter T 660 LPXR AOI 45°, Reflectance 500-653 nm > 90%, Transmittance 661-800 nm > 90%, Dimensions 25.5 x 36 x 1 mm F48-660(T660LPXR) 653...661 nm Emission filter Bandpass Semrock 640 / 20 BrightLine HC AOI 0°, diameter 25 mm, ring thickness 3.5 mm F39-641 (FF01-640 / 20-25) 628...653 nm Second emission filter Bandpass Semrock 697 / 58 BrightLine HC AOI 0°, diameter 25 mm, substrate thickness 1.05 mm, ring thickness 3.5 mm F37-697 (FF01-697 / 58-25) 665...727 nm Detectors PMT Hamamatsu H10722-20 H 10722-20 230...920 nm

[0102] According to the present invention, the second wavelength is preferably detected at a shorter wavelength and the third wavelength at a longer wavelength as an emission maximum of the fluorescently labeled particles under the first condition. It is understood by those skilled in the art that an emission maximum in connection with the present invention can be a local emission maximum or an absolute emission maximum. Alternatively, detection can also be performed around a saddle point of the emission spectrum instead of an emission maximum.

[0103] Small changes (e.g. wavelength shifts) in the regions flanking the emission maximum have relatively large effects on changes in the fluorescence spectrum (e.g. in terms of intensities).

[0104] Thus, in a preferred embodiment of the present invention, the emission fluorescence intensities are detected in the immediate vicinity of an emission maximum, e.g. the second wavelength is detected at a wavelength at least 2.5 m shorter (e.g. 10 nm shorter) and the third wavelength at a wavelength at least 2.5 nm longer (e.g. 10 nm longer) than the emission maximum of the fluorescently labeled particles under first conditions.

[0105] According to the present invention, the preferred means for detecting the excited, fluorescently labeled particles, in particular for detecting the fluorescence, can be any suitable device selected from the group consisting of charge-coupled (CCD) cameras (2D or line-scan CCD), line-scan cameras, photomultiplier tubes (PMT), silicon photomultiplier tubes (siPM), avalanche photodiodes (APD), photodiode arrays (PDA), CMOS cameras (complementary metal-oxide semiconductor, CMOS) and the like. Alternative fluorescence measurement

[0106] In a further embodiment of the present invention, the ratiometric analysis is based on a “dual excitation” configuration, e.g. by using the in Fig.8D provided, exemplary dual excitation optics. In particular, the one or more samples containing the fluorescently labeled particles are excited at two different wavelengths and their emission spectrum is recorded at a single wavelength (see Example 3 in combination with Fig. 16) Using this configuration, the two excitation spectra cannot be acquired simultaneously, meaning they must be recorded later. This approach is more time-consuming, and the time delay between the two successive measurements could lead to significant differences between the measurements, for example, due to bleaching after the first excitation, sample aggregation, etc.

[0107] Some of the aforementioned problems could be solved by using a "stroboscopic" excitation approach, in which the two excitation light sources are switched on and off in less than 1 second or even faster, and the data are acquired alternately. However, the two different excitation light sources can lead to different bleaching rates of the sample, which can negatively affect the evaluation of the ratiometric signal in the case of longer acquisition times.

[0108] In a further embodiment of the present invention, the “dual excitation” configuration is used in combination with “green” fluorescent markers, such as Cy3, GFP, and the like. Since such markers have their excitation maximum at approximately 540 nm and their emission maximum at approximately 560 nm with a secondary emission peak at approximately 600 nm, suitable excitation and emission wavelengths include excitation between approximately 475 nm and 495 nm, detection of the first emission between approximately 550 nm and 575 nm, and detection of the second emission between approximately 590 nm and 680 nm. Fig. 9B). Exemplary components that may be useful for the “dual emissions” configuration are provided in Table 2. Table 2 Example component Art Manufacturer er name Part number wavelength area Excitation light source LED Cree LM1-EBL1-01-N2 LM1-EBL1-01-N2 480 nm Second excitation light source LED Cree LM1-EPG1-01-N2 LM1-EPGl-01-N2 540 nm Excitation filter Two-volume set Chroma FITC / CY3 ET-Dualband Excitation filter F59-023FITC / CY3 475...495 nm 550...575 nm Second light separator Dichroic mirror Chroma ET-Dualband bs FITC / CY3 F58-023FITC / CY3 500...540 nm 590...680 nm Emission filter Two-volume set Chroma FITC / CY3 ET dual-band blocking filter F57-023FITC / CY3 590...680 nm Detectors PMT Hamamatsu H10722-20 H 10722-20 230...920 nm

[0109] The excitation volume is generally the portion of the sample volume that is fluorescently excited by the excitation light source. The acquisition volume is the portion of the sample volume from which the emission spectrum is acquired. According to the present invention, the excitation volume and / or the acquisition volume preferably has a size of 2 mm × 2 mm × 5 mm or less, more preferably 1 mm × 1 mm × 5 mm or less, and even more preferably 0.5 mm × 0.5 mm × 5 mm or less.

[0110] However, the means for stimulating the fluorescently labeled particles and detecting the fluorescence of the stimulated particles are not limited, and any suitable means known to a person skilled in the art can be used. Ratiometric characterization

[0111] According to the present invention, the ratiometric analysis is preferably based on establishing the ratio between the fluorescence intensities detected at the second and third wavelengths by pointwise division. It is known to those skilled in the art that, within the scope of the present invention, the ratio of the fluorescence intensities can be obtained by dividing either the third wavelength by the second wavelength or vice versa (e.g., the second by the third wavelength).

[0112] Within the scope of the present invention, exemplary relative percentage changes of the ratio (i.e., the percentage change of the ratio after a spectral shift) are at least 0.5%, at least 1.1%, at least 5.5%, and at least 37.3%. Preferably, the relative percentage change is at least 3%. Characterization of interactions

[0113] Within the scope of the present invention, the interactions of fluorescently labeled particles include in particular biomolecules with, for example, other (bio)molecules, particles, spheres, as well as the stability of (bio)molecules, their conformation during folding and unfolding, or their chemical environment (such as their position within aqueous solutions, a lipid nanoparticle, or a cell).

[0114] Further equilibrium measurements to characterize interactions, measurements of bond kinetics and measurements of thermodynamic parameters.

[0115] According to the present invention, the calculated ratios are preferably used to determine the localization of the fluorescently labeled particles or parameters selected from the group consisting of dissociation constants, mean effective concentrations (EC1000). 50), equilibrium constants, binding kinetics, enzyme reaction kinetics, thermodynamic parameters, stability parameters (e.g., thermal denaturation of proteins, chemical denaturation of proteins, and the like), unfolding or refolding kinetics, opening and closing reactions and / or combinations thereof, and the like.

[0116] Within the scope of the present invention, it is known to those skilled in the art that the dissociation constant (K) d ) an interaction can be obtained from the fluorescence intensity ratio by fitting the data using the Langmuir equation, and the mean effective concentrations (EC₂) 50) can be determined from the obtained fluorescence intensity ratio by fitting it using the Hill equation (Ganellin, CR, Jefferis, R. and Roberts, SM (Eds.). (2013). Introduction to biological and small molecule drug research and development: theory and case studies. Academic Press., Chapter 1, pages 38 and 39).

[0117] According to the present invention, the first and second conditions of the fluorescently labeled particles can differ with respect to their chemical composition and / or temperature and / or localization within a chemical macroenvironment (e.g., the first conditions of the fluorescently labeled particles relate to the position of the particles within a first support, e.g., a vector, and the second conditions relate to the position of the particles within a second support, e.g., a recipient or in a buffer solution containing both supports).

[0118] The fluorescence spectrum of a fluorescent marker according to the present invention can also change when the fluorescently labeled particle / molecule is present in a complex with one or more other molecules, e.g., ligands (e.g., due to proximity to ligands (see Fig. 3B) and / or conformational changes upon binding of a ligand (see Fig. 3B).

[0119] Thus, in a preferred embodiment of the present invention, the second conditions can be changed by adding a ligand and / or different concentrations of the ligand, and the calculated ratios obtained are used to determine a dose-response curve and the dissociation constant (K). d ) to determine the fluorescently labeled particles and the ligand.

[0120] Within the scope of the present invention, the term “binding” of the ligand to the labeled particle preferably refers to a covalent bond or the bond through intermolecular forces such as ionic bonds, hydrogen bonds and van der Waals forces.

[0121] Ligand binding to a target biomolecule such as a protein can lead to a wide range of conformational changes, such as movement of the amino acid side chain, loop, or domain.The ligand that can be used according to the present invention can be selected from (but is not limited to) the group consisting of ions, metals, compounds, drug fragments (small chemical fragments that may bind only weakly to the biological target), carbohydrates, small molecules (organic compounds with a low molecular weight (< 900 Daltons); small molecules can support the regulation of a biological process and are usually on the order of 1 nm in size), drugs, prodrugs, lipids, proteins, peptides, peptoids, enzymes, nucleic acids, aptamers, nanoparticles, liposomes, unilamellar vesicles (including small unilamellar vesicles (SUVs) and unilamellar giant vesicles (GUVs)), polymers, organic molecules, inorganic molecules, metal complexes, hormones, flavorings, odorants, particles, and (micro)spheres.Preferably, the ligands are selected from the group consisting of ions, metals, compounds, drug fragments, carbohydrates, small molecules, active substances, prodrugs, lipids, proteins, peptides, peptoids, enzymes, nucleic acids, aptamers, hormones, flavorings and odorants.

[0122] The ligand concentration is preferably between 0.01 pM and 1 M, preferably between 1 pM and 100 mM, more preferably between 1 pM and 10 mM. Combination with temperature-dependent intensity change (TRIC) and microscale thermophoresis (MST)

[0123] Although the ratiometric analysis of fluorescently labeled particles according to the present invention is not necessarily based on temperature-induced changes in fluorescence intensity, the measurement of the fluorescence intensity can be carried out at a constant predetermined temperature or during a defined temperature disturbance.

[0124] In a second aspect, the present invention relates to a method for characterizing fluorescently labeled particles in solution by analyzing changes in the fluorescence spectrum of the fluorescently labeled particles in combination with a defined temperature disturbance.

[0125] The method of the second aspect of the invention comprises the following steps: a) Providing a sample of the fluorescently labeled particles in a solution under initial conditions, b) Excitation of the fluorescently labeled particles at a first wavelength, c) Measuring the fluorescence emission intensity of the fluorescently labeled particles at a second and a third wavelength, where the intensities are recorded during a defined temperature disturbance. d) Calculating the ratio between the fluorescence intensities at the second and third wavelengths, where the third wavelength differs from the second wavelength, e1) Repeat 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, where the second conditions differ from the first conditions, f) Characterizing the fluorescently labeled particles based on the calculated ratios obtained for the different conditions, wherein the second and third wavelengths are detected simultaneously and wherein the second wavelength is shorter and the third wavelength is longer than an emission maximum of the fluorescence emission of the fluorescently labeled particles under the first conditions.

[0126] In the preferred embodiment of the second aspect of the present invention, heating or cooling can be carried out using a temperature control element (i.e., a heating and / or cooling source) selected from the group consisting of heating and / or cooling of fluids or gases, heating elements (for example, a heating resistor or other Joule-based elements such as metallic heating elements, ceramic heating elements, polymer PTC heating elements, composite heating elements, semiconductor heating elements), or a thermoelectric element, for example, a Peltier element, or electromagnetic radiation (such as an LED, e.g., an IR LED, or a laser, e.g., an IR laser, or a microwave). The use of an IR laser enables rapid sample heating.

[0127] A Peltier element is preferred because it can be used to heat and / or cool the sample (e.g., to cool the sample below ambient temperature). In particular, it is possible to switch from heating to cooling by reversing the direction of the current through the Peltier element. A Peltier element is one of the few elements that can not only heat at room temperature but also actively cool below room temperature.

[0128] A laser, preferably a laser whose electromagnetic radiation is directly absorbed by the sample, is preferably used because the temperature can be changed quickly and directly in the sample without mechanical contact with the sample.

[0129] It is also preferred that the laser is a high-power laser in the range of 0.01 W to 10 W, preferably from 4 W to 6 W.

[0130] It is also preferred that the laser is a laser in the range of 1 mW to 1 W, preferably from 1 mW to 500 mW, more preferably from 1 mW to 250 mW.

[0131] Laser radiation is absorbed directly by the sample and converted into heat. For example, IR laser light with wavelengths of 980 nm ± 30 nm, 1480 nm ± 30 nm, 1550 nm ± 30 nm, and 1940 nm ± 30 nm is very well absorbed by water and heats up very quickly. This heating method is non-contact and can therefore be carried out quickly and without risk of contamination. The sample chamber only needs to be transparent to the laser light and does not require high thermal conductivity, unlike contact heating using a heating element.

[0132] With an IR laser, very small volumes (e.g., in the volume range of nanoliters) can be heated, the fluorescence of which is measured with a fluorescence optic (usually only 100 µm × 100 µm × 100 µm = 1 nl volume).

[0133] According to the present invention, the samples to be examined can also be subjected to linear temperature ramps by heating and / or cooling the temperature control element at defined constant rates, e.g., 1 °C / min or 1 K / min. Typically, the heating and / or cooling rates are between 0.1 K / min and 50 K / min using contact heating, for example, with Peltier elements.

[0134] In another embodiment, the samples can be heated with an IR laser (“optical heating”) at typical heating rates of 1 K / s to 100 K / s.

[0135] The method according to the second aspect of the present invention is preferably carried out in a temperature range of -20 °C to 160 °C, more preferably from 0 °C to 120 °C.

[0136] The preferred data acquisition time for measuring the initial ratio is between 1 s and 5 s, the preferred data acquisition time for the ratio obtained during the temperature disturbance is between 5 s and 20 s, however, the acquisition times can also be shorter, for example only 10 ms to 100 ms, or longer, for example minutes, hours or even days.

[0137] The ratio can also be analyzed at any later point in the temperature change. This can be useful if the amplitude is very small at room temperature but increases at higher temperatures (see Example 2 in combination with Fig. 13A, where the analysis in phase 3 leads to a larger amplitude than the analysis in phase 1.).

[0138] In a preferred embodiment of the second aspect of the present invention, the ratiometric analysis is based on the “dual emission” configuration in combination with a temperature perturbation, i.e., using the Fig. 8A provided exemplary dual emission optics and an IR laser.

[0139] In a further embodiment of the present invention, the ratiometric analysis is based on a “dual excitation” configuration in combination with a temperature perturbation, e.g. using the in Fig. 8C provided exemplary dual excitation optics and an IR laser.

[0140] In a further embodiment of the present invention, the ratiometric analysis is based on both the “dual excitation” and the “dual emission” configuration, i.e., using the configuration described in Fig. 8F provided exemplary “dual excitation / dual emission” optics.

[0141] In a further embodiment of the present invention, the ratiometric analysis is based on both the “dual excitation” and the “dual emission” configuration in combination with a temperature perturbation, e.g. using the in Fig. 8E provided, exemplary “dual excitation / dual emission” optics and an IR laser. Determination of thermodynamic and kinetic parameters by combining the ratiometric method with TRIC / MST

[0142] In a third aspect, the present invention relates to a method for characterizing the thermodynamic and / or kinetic parameters of fluorescently labeled particles in solution by analyzing changes in the fluorescence spectrum of the fluorescently labeled particles in combination with a defined temperature disturbance / temperature change.

[0143] Within the scope of the present invention, the thermodynamic parameters enthalpy, entropy, heat capacity (c) include P ) one, but are not limited to that.

[0144] Within the scope of the present invention, kinetic parameters include, but are not limited to, equilibrium constants, dissociation rates, association rates, enzymatic rates, folding and unfolding rates, release rates (e.g., payload release rates in the case of LNP), aggregation rates, and penetration rates (e.g., the rate at which a payload such as mRNA enters a cell).

[0145] According to the third aspect of the present invention, thermodynamic parameters of interactions can preferably be determined when ratiometric fluorescence data are collected from a single measurement using an IR laser. Since the sample temperature is known at each point in time during the measurement (determined during a calibration measurement in which the plate is heated in a controlled manner and the fluorescence of a reference marker is measured), a dissociation constant K can be determined. d can be obtained for any given time (see example 2 in combination with Fig. 14A).

[0146] In addition, according to the third aspect of the present invention, the thermodynamics of a reaction can also be determined by classical K dMeasurements can be performed at different, fully equilibrated sample temperatures, for example by successively setting the sample temperature to 22 °C, 24 °C, 26 °C, 28 °C, 30 °C and 32 °C and performing a binding affinity measurement for each temperature (see Example 10 in combination with Fig. 24).

[0147] Even if the accuracy of this approach does not always reach that of modern isothermal titration calorimetry (ITC), the measurement is approximately 100 times faster, and the significantly lower sample consumption can compensate for the lack of accuracy. For example, for certain molecules, the information on whether they bind entropically or enthalpially can be very valuable, especially in early stages of development.

[0148] According to the third aspect of the present invention, thermodynamic parameters of interactions can preferably be determined if the ratiometric fluorescence data are collected from a single measurement using an IR laser. Application: Monitoring the localization of fluorescently labeled mRNA by combining the ratiometric method with TRIC / MST.

[0149] In a fourth aspect, the present invention preferably relates to a method for characterizing the localization of fluorescently labeled particles in solution by analyzing changes in the fluorescence of the fluorescently labeled particles with or without a defined temperature disturbance / temperature change.

[0150] Within the scope of the present invention, the terms “location” and “localization” are used interchangeably and refer to the determination of the place / position of fluorescently labeled particles.

[0151] All gene and cell therapy procedures involving nucleic acids such as DNA and RNA, etc., have the inherent problem of determining the success of drug delivery (e.g., the delivery of the transported nucleic acid into the target cell by incorporation and release from carriers such as lipid nanoparticles (LNPs) dissolved in a buffer solution; see Fig. 27A).

[0152] Even before determining the success of the delivery, bioproduction (e.g., loading the delivery systems, including an unloaded, partially loaded, fully loaded and / or overloaded state of the LNP) is a crucial step that must be carefully assessed.

[0153] According to the fourth aspect of the present invention, carriers can be selected from the group consisting of metal nanoparticles and nanoconstructs, polymeric nanoparticles, lipid-based carrier systems (e.g., liposomes, other lipid-containing complexes), carbon-containing carrier systems, nanoemulsions, nanosuspensions, nanomicrons, dendrimers, milk-derived carriers, endosomes, viral vectors (e.g., adenoviruses, adeno-associated viruses (AAV), retroviruses), virus-like particles (VLPs), eukaryotic cells, prokaryotic cells, cellular fragments, and the like. In light of the foregoing, combinations of the carriers are also within the scope of protection of the present invention.

[0154] In a preferred embodiment of the fourth aspect of the present invention, the fluorescently labeled particle is a fluorescently labeled mRNA and the carrier is an LNP.

[0155] An LNP refers to any particle with a diameter of less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm. For example, an mRNA-filled LNP in a suitable buffer may have a hydrodynamic diameter between 65 nm and 85 nm. Alternatively, a nanoparticle may have a size in the range of 1–1000 nm, 1–500 nm, 1250 nm, 25–200 nm, 25–100 nm, 35–85 nm, or 25–60 nm.

[0156] LNPs can consist of cationic, anionic, or neutral lipids. Neutral lipids, such as the fusogenic phospholipid DOPE or the membrane component cholesterol, can be incorporated into LNPs as "helper lipids" to enhance transfection activity and nanoparticle stability. Limitations of cationic lipids include low efficacy due to their poor stability and rapid excretion, as well as the potential for generating inflammatory or anti-inflammatory responses.

[0157] LNPs can also be composed of hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids.

[0158] Any lipid or combination of lipids known in the prior art can be used to produce an LNP. Examples of lipids used to produce LNPs include: DOTMA, DOSPA, DOTAP, DMRIE, DC cholesterol, DOTAP cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG). Examples of cationic lipids include: 98N12-5, C12-200, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, and 7C1.

[0159] Examples of neutral lipids are: DPSC, DPPC, POPC, DOPE, and SM. Examples of PEG-modified lipids are PEG-DMG, PEG-CerC14, and PEG-CerC20.

[0160] Since the fluorescence spectrum of a fluorescent marker is very sensitive to environmental changes, such as changes in the chemical environment, and the spectrum changes when the environment changes, the position of the fluorescently labeled particles (e.g. mRNA) can be deduced from the ratiometric measurement according to the present invention.

[0161] As an example of a non-restrictive combination of dyes and a second and a third emission wavelength: if all fluorescently labeled mRNA molecules are properly localized in the LNPs, the ratio after characterization according to the present method is 2.1 (see Example 12 in combination with Fig. 27B). If all fluorescently labeled mRNA molecules are localized outside the LNP, the resulting ratio is 1.2 (see Example 12 in combination with Fig. 27C).

[0162] Since the chemical environment inside the cell differs significantly from that in an LNP or buffer solution, it is understood that, within the scope of the present invention, the ratio obtained will differ from the aforementioned values ​​(i.e., 1.2 and 2.1) when all fluorescently labeled mRNA molecules have been successfully delivered into the target cell (see Example 12 in combination with Fig. 27D). Furthermore, the fluorescence ratio obtained in intermediate states, e.g., when fluorescently labeled mRNA molecules are partially localized in the cell, the LNP, and the buffer solution, is a linear combination of the ratios obtained for the three states mentioned above.

[0163] According to this exemplary aspect of the present invention, the determination of the localization of fluorescently labeled particles (e.g., mRNA) is based on very small sample and detection volumes as well as short experimental procedures. In the same vein, currently used and prior art methods (such as field-flow fractionation and liquid chromatography) are based on other techniques and require not only more time but also a much larger sample volume, which is often limited and expensive. Kits for biotinylated molecules

[0164] In a fifth aspect, the present invention relates to a kit and the use of a kit for the characterization of fluorescently labeled particles, e.g. particles labelled with one or more biotin molecules (i.e., biotinylated particles), in solution according to the methods of the present invention.

[0165] The kit includes as its main components a defined stoichiometric ratio (i) of a (preferably tetrameric) biotin-binding protein comprising at least two (preferably four) binding sites for biotin, and (ii) a linking unit (hereinafter also referred to as a "linker"). The kit preferably further includes instructions for use, which explain the use of the kit in at least one of the methods of the present invention.

[0166] According to the fifth aspect of the present invention, the biotin-binding protein can be selected from the group consisting of streptavidin, avidin, and mutants thereof. Within the scope of the present invention, mutants include neutravidin, flavidin, divalent streptavidin, and the like.

[0167] The kit useful in the present invention can include a single vial comprising (i) the biotin-binding protein and (ii) a linker, preferably modified / labeled with a biotin molecule at one end and a fluorescent marker at the other end. In a preferred embodiment of the fifth aspect of the present invention, the biotin-binding protein is preferably a tetrameric protein, more preferably a tetrameric streptavidin.

[0168] Streptavidin is a homotetramer that exhibits an exceptionally high affinity for biotin (also known as vitamin B7). Due to the stability of the streptavidin-biotin complex to organic solvents, denaturing agents (e.g., guanidinium chloride), surfactants (e.g., SDS, Triton), proteolytic enzymes, and extreme temperatures and pH values, it is widely used in molecular biology and bionanotechnology.

[0169] In a further preferred embodiment of the fifth aspect of the present invention, the fluorescent marker is bound to a linker, e.g. as described in the prior art (NPL6).

[0170] According to the fifth aspect of the present invention, a linker is a polymer, preferably a nucleic acid, preferably a single-stranded nucleic acid, more preferably a single-stranded DNA, and even more preferably a single-stranded DNA oligomer (i.e., an oligonucleotide), preferably having a length of 6 to 24 nucleotides. For example, a 12-mer oligo-dT strand may be used.

[0171] However, the linker used in the present invention is not limited by the length (as long as the linker is long enough for the fluorophore to reach the target) and / or the type of linker (as long as the type of linker has the property that the length can be adjusted, e.g. DNA, aromatic rings enclosing aryl groups, and the like) and any suitable linker known to the skilled person can be used to indirectly bind a fluorescent marker to the particle to be characterized by the method of the present invention.

[0172] A linker for use according to the present invention contains a biotin molecule at one end and a fluorescent marker at the other end. In a preferred embodiment of the fifth aspect of the present invention, the linker is modified with biotin at its 3' end and with a fluorescent marker at its 5' end, or vice versa.

[0173] Each kit can contain enough material for multiple labeling reactions. Depending on the kit size and the amount of biomolecule used, enough material can be provided for approximately 500 to 3840 ratiometric single-point characterization experiments.

[0174] In a preferred embodiment of the fifth aspect of the present invention, the fluorescently labeled particle is a complex between tetrameric streptavidin molecules, biotinylated, fluorescently labeled linker molecules and biotinylated target molecules (see Fig. 29A). It is particularly preferred that the fluorescently labeled particle is a complex of a tetrameric streptavidin molecule, two biotinylated fluorescently labeled linker molecules and a biotinylated target molecule.

[0175] The stoichiometric ratio of tetrameric biotin-binding protein and modified linker is preferably adjusted such that, on average, two of the four binding sites on the tetrameric biotin-binding protein are occupied (e.g., by providing the vial with 2 nM streptavidin and 4 nM linker) and the remaining two binding sites are available for binding events with the biotinylated particles of interest.

[0176] Thus, in the fifth aspect of the present invention, it is further preferred that the tetrameric streptavidin and the linker are mixed in a 1:2 ratio such that, on average, one streptavidin molecule is labeled with two linker molecules, i.e., one streptavidin molecule carries two fluorescent markers. The remaining two binding sites of the streptavidin molecule can capture a biotinylated molecule (e.g., a protein). By mixing the labeled streptavidin and the biotinylated molecule in a 1:1 ratio, it can be achieved that, on average, only one biotinylated molecule binds to the streptavidin-linker complex. However, the stoichiometry can also be exploited to the advantage of biotinylated dimeric proteins (e.g., biotinylated interferon gene stimulator (STING), divalent streptavidin), wherein the functional dimer can be labeled with a streptavidin-linker complex.In the case of divalent biotin-binding proteins, the stoichiometric ratio of divalent biotin-binding protein and modified linker would be adjusted such that, on average, one of the two free binding sites on the divalent biotin-binding protein is occupied (e.g., by adding 2 nM streptavidin and 2 nM linker to the vial), and the remaining binding site is available for binding events with the biotinylated particles of interest.

[0177] The correct stoichiometry can be verified during the labeling process by measuring the spectral shift according to the present invention (e.g. using the dual emission configuration), wherein streptavidin is titrated against the linker.

[0178] While the free linker molecule (i.e., a 12-mer poly-T strand tagged with biotin at its 3' end and Cy5 at its 5' end) has a ratio less than 0.8 (see Fig. 29B, free linker) and streptavidin, which is labeled with only one linker molecule, has a ratio of approximately 1.05 (see Fig. 29B, a linker), a streptavidin labeled with two linker molecules exhibits a higher ratio, i.e., approximately 1.15, which corresponds to a peak in the biphasic dose-response curve resulting from the interaction of both fluorescent markers with the remaining two binding sites of the streptavidin molecule (see Fig.29B, ideal ratio). When the biotinylated molecule is added to the streptavidin-linker complex, the ratio decreases. Since all biotin binding sites retain their full activity, biotinylated molecules can be captured with extremely high affinity, resulting in a characteristic kink at the stoichiometry point in the dose-response curve (see Fig. 29C).

[0179] According to this exemplary aspect of the present invention, very low final concentrations of kit components and the biotinylated target molecule (e.g., 1 nM streptavidin, 2 nM linker, and 1 nM biotinylated molecule) are used. Thus, according to the fifth aspect of the present invention, the kit is suitable for measuring picomolar affinities based on a highly controllable and reproducible process (compared to prior art labeling kits, e.g., Protein His-Tag Labeling Kit RED-tris-NTA 2nd Generation (NanoTemper Technologies)) by using the methods of the present invention. In the same context, the currently used, prior art kits (such as the Protein His-Tag Labeling Kit RED-tris-NTA 2nd Generation (NanoTemper Technologies)) exhibit the following limitations:Generation) further limitations, including buffer limitations, slow label binding kinetics, incompatibility with already biotinylated molecules, and the labeling kits known in the prior art are costly and labor-intensive. General principles and illustrative aspects

[0180] The general principles of the present invention are explained in more detail below, based on illustrative examples or preferred embodiments of the present invention.

[0181] Since the fluorescence spectrum of a fluorescent marker depends strongly on the microenvironment in which it is located, one and the same marker can show very different fluorescence, for example depending on which molecule or particle it is bound to.

[0182] In particular, the microenvironment around the binding site of the fluorescent marker differs at each protein with respect to the amino acid residues that could quench the marker, collide with the marker, temporarily interact with the marker, or cause stacking.

[0183] Fig. Figure 1A shows the excitation spectra of four different proteins labeled with an identical fluorescent marker (Protein Labeling Kit RED-NHS 2nd Generation (NanoTemper Technologies)). Emission was recorded at a wavelength of 690 nm. Excitation varied between 520 nm and 670 nm. Although the four proteins are labeled with the same fluorescent marker, the wavelength of their maximum emission peak differs and lies in the range between approximately 659 nm and approximately 664 nm. Fig. 1B).

[0184] Out of Fig.Figure 2A shows that the same effect / phenomenon was observed when the four different proteins were excited at a constant wavelength (i.e., 605 nm) and the emission was recorded between 620 nm and 750 nm. Similar to what was explained above, the wavelengths of the maximum emission peak were in a range between approximately 659 nm and approximately 664 nm, even though the proteins were labeled with the same fluorescent marker ( Fig. 2B).

[0185] The microenvironment can also change depending on the position on the molecule / particle (e.g., which lysine residue of a protein the label is bound to, or whether a label is bound to the 3' or 5' end of a nucleic acid molecule), the conformation of the molecule / particle (e.g., folded or unfolded protein), or the length / composition of the linker between the molecule / particle and the label. The macroenvironment of the fluorescent label also affects its fluorescence spectrum. For example, the fluorescence spectrum of the label differs depending on the position of the particle / molecule (e.g., in an aqueous buffer solution, in an LNP, in a cell).

[0186] Furthermore, the fluorescence spectrum of the fluorescent marker can also change if the fluorescently labeled particle / molecule is present in a complex with one or more other molecules (i.e., ligands). For example, proximity to ligands ( Fig.3A) and / or conformational changes upon binding of a ligand ( Fig. 3B) to a shift ( Fig. 3C) and / or widening or narrowing ( Fig. 3D) of the excitation or emission fluorescence spectrum of the fluorescent marker. Using the ratiometric characterization method of the present invention, it is possible not only to detect changes in fluorescence intensity, but also changes in the complete absorption and emission spectra when the microenvironment of the fluorescent marker is altered.

[0187] In light of the foregoing, Fig. 4. The shift (i.e., 3 nm) of the wavelength of the maximum emission peak of streptavidin (200 nm) from approximately 664 nm to approximately 661 nm when it is in complex with its natural ligand biotin (2 µM) upon excitation with a wavelength of 605 nm. Along the same line, Fig.5. A slight shift (i.e., < 1 nm) of the wavelength of the maximum emission peak of lysozyme (100 nM) alone and in complex with the lysozyme inhibitor tri-N-acetyl-D-glucosamine (NAG3) (80 µM) was observed upon excitation at a wavelength of 585 nm. Shifts of 3 nm and approximately 500 pm correspond to relative changes in the fluorescence ratio of about 37.3% and 5.5%, respectively (Table 3). Table 3 Spectral shift Change in circumstances 3 nm 37,3 % 2 nm 23,6 % 1 nm 11,2 % 500 pm 5,5 % 100 pm 1,1 % 50 pm 0,5 %

[0188] Since changes in the excitation and / or emission spectrum of a fluorescent marker can be very small (e.g., only a few Å), it is not possible to measure / resolve them with the required precision using methods / devices known in the prior art. However, the methods and devices of the present invention allow even small changes in the fluorescence spectrum to be resolved. For example, wavelength shifts of only 50 pm, which lead to a change in the fluorescence ratio of 0.5% (Table 3), as in the case of the binding between carbonic anhydrase and furosemide ( Fig. 6A), can be easily measured / recorded and a sigmoidal dose-response curve can be obtained ( Fig. 6B).

[0189] The changes in the fluorescence spectrum are independent of the type of fluorescent marker; that is, they occur with both intrinsic and extrinsic fluorescent markers. However, the magnitude of the changes in the fluorescence spectrum may be greater for certain classes of markers. Fig. Figure 7A shows the use of measuring the fluorescence ratio of tryptophan fluorescence during a thermal melting ramp from 35 °C to 95 °C to characterize the denaturation and binding affinity between native lysozyme and its inhibitor tri-N-acetyl-D-glucosamine (NAG3). Very high concentrations of NAG3 lead to thermal stabilization, i.e., a thermal shift of lysozyme. However, this shift cannot be used to determine the dissociation constant (KD). d ) to obtain, however, a sigmoidal dose-response curve can be obtained, which the K dindicates (here: at 35 °C) when the initial ratio at 35 °C is plotted against the concentration of NAG3 ( Fig. 7B).

[0190] Fig. Figures 8A to 8F show various exemplary embodiments of measuring devices according to the present invention. In general, a device according to the present invention preferably comprises a sample holder for holding a sample of fluorescently labeled particles in solution under a variety of conditions. As mentioned above, the sample holder of the present invention can be a capillary, but is not limited to such a capillary. Other means for holding the sample can also be used, such as a microtiter plate or a chip.

[0191] Fig.Figures 8A to 8F show examples of arrangements of optical elements that help to direct the excitation light onto the sample and to detect the fluorescence emission(s) from the sample, the sample itself not being shown in the figures. Preferably, a sample container, e.g., a capillary, is located below the lens 1. The lens 1 is preferably an aspherical lens or a lens system with a plurality of lenses, which is also referred to below as the objective.

[0192] The apparatus of the present invention also includes at least one means for exciting the fluorescently labeled particles at a first wavelength. For example, a light source 8 can be provided to supply the excitation light. As discussed above, the present invention is not limited to a single excitation light source. As an alternative to using one light source, a second excitation light source 16 or even several additional light sources (not shown) can be provided (particularly for a “dual excitation” mode). The ratiometric analysis of the present invention can be obtained by using either a “dual excitation” configuration or a “dual emission” configuration. In the “dual emission” configuration, it is preferred to provide at least one light source. In the “dual excitation” configuration, it is preferred to provide two or even more light sources.However, a person skilled in the art further understands that a multitude of light sources may be provided for the “dual emission” configuration. In this case, however, it would suffice if one of these light sources were used for excitation.

[0193] The first excitation light source 8 is preferably at least one from the group consisting of a laser, fiber laser, diode laser, LED, HXP, halogen, LED array, HBO. The same applies to the second excitation light source 16.

[0194] Preferably, a first light-separating element 7, e.g., a dichroic mirror, is used to direct the excited light onto the sample and preferably to separate the fluorescence excitation light from the fluorescence emission light. Additional optical elements for directing the excitation light onto the sample can be provided, e.g., a lens system 9, for example, to determine the beam characteristics of the excitation light source (e.g., one, two, or more lenses). Furthermore, an excitation filter 10 can also be provided to filter the excited light, e.g., a bandpass / longpass filter. A person skilled in the art will understand which types of filters are preferable for the different light sources. Again, similar optical elements can be provided with respect to the second excitation light source 16. As, for example, in Fig. 8C, Fig. 8D, Fig. 8E and Fig.As shown in Figure 8F, a lens system 17 can be provided, e.g., to determine the beam characteristics of the second excitation light source 16. Furthermore, another light-separating element 18, e.g., a dichroic mirror, can be used, e.g., to combine the light from the two different excitation light sources 8 and 16.

[0195] The apparatus of the present invention also includes means for detecting the fluorescence emission intensity of the fluorescently labeled particles. In the “dual emission” configuration, it is preferred to provide a means for detecting two different wavelengths, and in the “dual excitation” configuration, it would be sufficient if the detection means were designed to detect only a single wavelength or a single wavelength range. According to the present invention, it is preferred to provide at least one light detector for each wavelength or wavelength range. In the “dual excitation” configuration, for example, it would be sufficient to provide a single light detector 14 (see, e.g., Figure 14). Fig. 8C and Fig. 8D). In the “dual emission” configuration, it is preferred to provide two separate light detectors 14 and 15, as shown in Fig. 8A, Fig. 8B, Fig. 8E and Fig.8F). However, a person skilled in the art further understands that two light detectors may be provided for the “dual excitation” configuration. In this case, however, it would be sufficient if one of these light detectors 14 and 15 is used for emission detection. The first and / or second light detector may be a light detector from the group consisting of PMT, siPM, APD, CCD or CMOS camera.

[0196] Again, additional optical elements, such as a light-separating element 11, can be provided to separate the emission light to the first and second light detectors 14 and 15. For example, shows Fig.8B describes a preferred configuration for a single-excitation and dual-emission configuration with an excitation light source 8 and two light detectors 14 and 15. The emission light to the individual detectors is separated by the light-separating element 11, e.g., a dichroic mirror. Additional filters 12 and 13 can be placed upstream of the two detectors 14 and 15 to define the different emission wavelengths, e.g., the second and third wavelengths, where the second wavelength is shorter and the third wavelength is longer than an emission maximum of the fluorescence emission of the fluorescently labeled particles under the first conditions. The emission filters 12 and 13 can be selected from any suitable type of filter element, e.g., a bandpass or longpass filter.

[0197] As in Fig. 8A, Fig. 8C and Fig.As shown in Figure 8E, a hot mirror 2 can also be provided, which is preferably used to direct IR light from an IR laser 3 onto the sample.

[0198] For example, the hot mirror 2 can offer high IR reflection and preferably a visible light transmittance of more than 80%. The IR light source 3 is preferably at least one IR laser, preferably with an emission wavelength of, for example, 1455 nm, 1480 nm, 1550 nm, and / or 980 nm. Furthermore, the power of the IR laser is preferably between 0.01 W and 10 W. To direct the IR light onto the sample, additional optical elements such as a laser fiber 4 (single-mode or multi-mode), a laser fiber coupler 5 (with or without a collimator), and / or a beam shaping module 6 for determining the laser beam diameter and focusing (e.g., a lens system with one, two, or more lenses) can be used (see [reference]). Fig. 8A, Fig. 8C and Fig.8E). However, the injection of IR light is only optional for additional temperature-dependent measurements or additional measurements.

[0199] The apparatus of the present invention also includes means for calculating a ratio between the fluorescence intensities at the second and the third wavelengths, wherein the third wavelength differs from the second wavelength. The means is preferably provided by a processor or a circuit with at least one processor. Examples Example 1

[0200] The following example illustrates the difference between using ratiometric characterization methods known in the prior art and using the ratiometric characterization method according to the present invention to obtain a dose-response curve between two molecules. A sample containing a fluorescently labeled DNA aptamer and adenosine monophosphate (AMP) was measured using a commercially available fluorescence spectrophotometer and the dual emission configuration of the present invention. Sample preparation

[0201] A 14-step 1:1 dilution series of unlabeled AMP was prepared. The DNA aptamer was fluorescently labeled with Cy5 and added in equal amounts to the AMP dilution series to obtain a final sample concentration of 20 nM. The highest AMP concentration corresponded to 5 mM. For measurements using a standard fluorescent microtiter plate reader (CLARIOstar, BMG Labtech), 95 µl of the sample was filled into a microtiter plate. For measurements using the dual-emission configuration of the present invention, 5 to 10 µl of the samples were filled into polymer-coated borosilicate glass capillaries (Monolith NT.115 Premium Capillaries, MO-K025, NanoTemper Technologies). measurement

[0202] For measurements using the microtiter plate reader, the sample was excited at a wavelength of 590 nm, and the emission was initially recorded at wavelengths from 628 nm to 652 nm. The sample was then excited again at a wavelength of 590 nm, and the emission was recorded at wavelengths from 665 nm to 725 nm. Three consecutive fluorescence intensity measurements of the dilution series were performed. For the ratiometric measurement according to the present invention, each sample was excited at a wavelength of 591 nm. The fluorescence profiles were measured simultaneously at a second wavelength between 628 nm and 653 nm and at a third wavelength between 665 nm and 727 nm. Ratiometric data analysis

[0203] To characterize the interaction with the fluorescence microtiter plate reader, the ratio between the fluorescence intensities per well was calculated manually using a commercially available calculation tool (Microsoft Excel). The fluorescence detected at the higher wavelength was divided by the fluorescence detected at the lower wavelength. The in Fig. The sigmoidal dose-response curve provided in section 10A begins at a ratio of approximately 0.95 and ends at a ratio of approximately 0.90. The mean value, which corresponds to the dissociation constant (K), d The μ corresponding to the interaction is approximately 20-30 µM. However, the signal-to-noise ratio (S / N) of the interaction is very low, and the variation between repetitions is very large.

[0204] When measuring the same samples according to the method of the present invention, the following shows Fig.In contrast, the dose-response curve provided in 10B shows an improved signal-to-noise ratio (S / N) and clearly demonstrates the K d the interaction with 39.3 µM. When using lower sample concentrations, i.e., 250 pM, in the ratiometric measurement according to the present invention, the signal-to-noise ratio (S / N) is still very good (19.8) and a K d The interaction can be easily determined ( Fig. 10C).

[0205] In summary, this example demonstrates that for the ratiometric characterization method according to the present invention, a sample approximately 1000 times thinner yields even better data compared to measurements with a plate reader. With commercially available microtiter plate readers, the noise is more than 10 times higher than the signal amplitude to be measured. Example 2

[0206] The following example describes the use of the ratiometric characterization method according to the present invention to obtain a dose-response curve between two molecules based on the dual emission configuration. Therefore, a sample containing fluorescently labeled DNA aptamer and AMP was excited at a first wavelength, and the emitted fluorescence intensity was measured at a second and a third wavelength. Sample preparation

[0207] A 12-step 1:1 dilution series of unlabeled AMP was prepared. The DNA aptamer was fluorescently labeled with Cy5 and added in equal amounts to the AMP dilution series to obtain a final sample concentration of 20 nM. The highest AMP concentration corresponded to 2 mM. The samples were filled into polymer-coated borosilicate glass capillaries (Monolith NT.115 Premium Capillaries, MO-K025, NanoTemper Technologies). measurement

[0208] Each sample was excited at a wavelength of 591 nm. At time 0 s, an IR laser was switched on. The response of the fluorescence intensity was measured simultaneously for 31 s. The fluorescence profiles recorded between 628 nm and 653 nm (“650 nm”) are shown in Fig. 11A provided. The fluorescence profiles recorded between 665 nm and 727 nm (“670 nm”) are in Fig. 11B provided. Since the initially recorded fluorescence intensities fluctuated considerably, a sigmoidal dose-response curve for affinity determination could not be obtained. Consequently, the initial fluorescence of neither of the two in Fig. Information about the binding can be extracted from the 12 provided emission wavelengths. Ratiometric data analysis

[0209] For ratiometric analysis (i.e., obtaining the ratios of the fluorescence profiles), a pointwise division of the fluorescence profiles at 670 nm and 650 nm was performed. The resulting ratio profiles can be either pre- ( Fig. 13A, Phase 1) or after switching on the IR laser ( Fig. 13A, Phase 2 or Phase 3). The data recorded before switching on the IR laser can be analyzed using ratiometric analysis ( Fig. 13A, Phase 1), a dose-response curve with a signal-to-noise ratio (S / N) of more than 300 was obtained, which is a K d between the two molecules at sample temperature, i.e. room temperature, yielded ( Fig. 13B).

[0210] By performing ratiometric analysis after switching on the IR laser ( Fig. 13A, Phase 2 or Phase 3) can K dValues ​​can be obtained at higher temperatures. This approach is particularly recommended if the amplitude is very small at room temperature and is expected to increase at higher temperatures. Determination of the thermodynamic parameters of the interaction

[0211] To a curve of the K d To obtain data against the time of the interaction, “vertical sections” were taken at a time interval of 200 ms of the ratio profiles, which were in Fig. 13A are provided, and the K d was determined from the dose-response curves for each of these sections ( Fig. 14A). Since the temperature change over time was known from a calibration measurement, i.e., a sample plate was heated in a controlled manner and the fluorescence of a reference dye was measured, and the interaction was equilibrated on a faster timescale than the occurring temperature change, a relationship of K could be established. dobtained against temperature. By performing a van't Hoff analysis: ln(Kd)=ΔH0RT−ΔS0R The bond enthalpy (ΔH) and the bond entropy (ΔS) were determined. Fig. 14B) of the interaction. For this interaction, it was found that AMP binds predominantly enthalpially (ΔH < 0). Example 3

[0212] The following example describes the use of the ratiometric characterization method according to the present invention to obtain a dose-response curve between two molecules based on the dual excitation configuration. Therefore, a sample containing fluorescently labeled DNA aptamer and AMP was excited at a first and a second wavelength, and the emitted fluorescence intensity was measured at a third wavelength. Sample preparation

[0213] A 16-step 1:1 dilution series of unlabeled AMP was prepared. The DNA aptamer was fluorescently labeled with Cy3 and added in equal amounts to the AMP dilution series to obtain a final sample concentration of 250 nM. The highest AMP concentration corresponded to 12.5 mM. The samples were filled into polymer-coated borosilicate glass capillaries (Monolith NT.115 Premium Capillaries, MO-K025, NanoTemper Technologies). measurement

[0214] Each sample was excited at a first wavelength of 480 nm (“blue” LED). At time 0 s, an IR laser was switched on. The response of the fluorescence intensity was recorded for 6 s from 590 nm to 680 nm. Subsequently, each sample was excited at a second wavelength of 540 nm (“green” LED). At time 0 s, an IR laser was switched on. The response of the fluorescence intensity was recorded for 6 s by a detector from 590 nm to 680 nm. The fluorescence profiles obtained by exciting the sample with the “blue” LED are shown in Fig. 15A provided. The fluorescence profiles obtained by exciting the sample with the "green" LED are shown in Fig. 15B provided. Ratiometric data analysis

[0215] For ratiometric analysis (i.e., obtaining the ratios of the fluorescence profiles), a pointwise division of the fluorescence profiles obtained by excitation with the green LED by those obtained by excitation with the blue LED was performed. The resulting ratio profiles can be either pre- ( Fig. 16A, Phase 1) or after switching on the IR laser ( Fig. 16A, Phase 2 or Phase 3). The data recorded before switching on the IR laser can be analyzed using ratiometric analysis ( Fig. 16A, Phase 1), a dose-response curve with a signal-to-noise ratio (S / N) of approximately 80 was obtained, which is a K d between the two molecules at sample temperature, i.e. room temperature, yielded ( Fig. 16B). By means of ratiometric analysis of the data recorded after switching on the IR laser ( Fig.16A, Phase 3), a dose-response curve with a signal-to-noise ratio (S / N) of more than 130 was obtained, which the K d between the two molecules resulted ( Fig. 16C). Compared to the analysis at room temperature, a better signal-to-noise ratio was obtained when the conditions were analyzed at higher temperatures. Example 4

[0216] The following example describes the use of the ratiometric characterization method according to the present invention to obtain a dose-response curve between two molecules based on the dual emission configuration. Therefore, a sample containing fluorescently labeled streptavidin and its naturally occurring ligand biotin was excited at a first wavelength, and the emitted fluorescence intensity was measured at a second and a third wavelength. Sample preparation

[0217] A 12-step 1:1 dilution series of unlabeled biotin was prepared. Streptavidin was fluorescently labeled using the RED-NHS 2nd Generation Protein Labeling Kit (NanoTemper Technologies) and added in equal amounts to the biotin dilution series to achieve a final sample concentration of 20 nM. The highest biotin concentration was 500 mM. Samples were filled into polymer-coated borosilicate glass capillaries (Monolith NT.115 Premium Capillaries, MO-K025, NanoTemper Technologies). measurement

[0218] Each sample was excited at a wavelength of 591 nm. The fluorescence profiles were simultaneously measured at a second wavelength between 628 nm and 653 nm (“650 nm”) and at a third wavelength between 665 nm and 727 nm (“670 nm”). Ratiometric data analysis

[0219] For the ratiometric analysis (i.e., obtaining the ratios of the fluorescence profiles), a pointwise division of "670 nm" by "650 nm" was performed. As can be seen from the in Fig. As shown in the dose-response curve on page 17, the ratio changed from approximately 2.2 to approximately 1.2 for unbound streptavidin and in complex with biotin, respectively. Since the target concentration is much higher than the Ki d , a characteristic kink at the stoichiometric point can be observed at a concentration of 80 nM biotin. Example 5

[0220] The following example describes the use of the ratiometric characterization method according to the present invention to obtain a dose-response curve between two molecules based on the dual emission configuration. Therefore, a sample containing fluorescently labeled bovine carbonic anhydrase II and acetazolamide was excited at a first wavelength, and the emitted fluorescence intensity was measured at a second and a third wavelength. Sample preparation

[0221] A 15-step 1:1 dilution series of unlabeled acetazolamide was prepared. Bovine carbonic anhydrase II was fluorescently labeled using the RED-NHS 2nd Generation Protein Labeling Kit (NanoTemper Technologies) and added in equal amounts to the acetazolamide dilution series to achieve a final sample concentration of 20 nM. The highest acetazolamide concentration was 2.5 µM. Samples were filled into polymer-coated borosilicate glass capillaries (Monolith NT.115 Premium Capillaries, MO-K025, NanoTemper Technologies). measurement

[0222] Each sample was excited at a wavelength of 591 nm. The fluorescence profiles were simultaneously measured at a second wavelength between 628 nm and 653 nm (“650 nm”) and at a third wavelength between 665 nm and 727 nm (“670 nm”). Ratiometric data analysis

[0223] For the ratiometric analysis (i.e., obtaining the ratios of the fluorescence profiles), a pointwise division of "670 nm" by "650 nm" was performed. In the Fig. In the dose-response curve provided, the ratio changed from approximately 0.944 to approximately 0.951, a change of approximately 0.7%. The resulting dose-response curve had a signal-to-noise ratio (S / N) of more than 30. This demonstrates that even small changes in the ratio can be measured using the ratiometric characterization method of the present invention. Example 6

[0224] The following example describes the use of the ratiometric characterization method according to the present invention to obtain a dose-response curve between three molecules based on the dual emission configuration. Therefore, a sample containing fluorescently labeled monovalent streptavidin, biotinylated protein L, and the antibody Herceptin was excited at a first wavelength, and the emitted fluorescence intensity was measured at a second and a third wavelength. Sample preparation

[0225] A 16-step 1:1 dilution series of unlabeled antibody Herceptin was prepared. Monovalent streptavidin was fluorescently labeled using the RED-NHS 2nd Generation Protein Labeling Kit (NanoTemper Technologies), and 20 nM of this was mixed with an equal volume of 4 nM of biotinylated protein L. This mixture was then added in equal amounts to the Herceptin dilution series to achieve a final sample concentration of 5 nM labeled monovalent streptavidin and 1 nM biotinylated protein L in the assay. The highest Herceptin concentration was 1 µM. Samples were filled into polymer-coated borosilicate glass capillaries (Monolith NT.115 Premium Capillaries, MO-K025, NanoTemper Technologies). measurement

[0226] Each sample was excited at a wavelength of 591 nm. The fluorescence profiles were simultaneously measured at a second wavelength between 628 nm and 653 nm (“650 nm”) and at a third wavelength between 665 nm and 727 nm (“670 nm”). Ratiometric data analysis

[0227] For ratiometric analysis (i.e., obtaining the ratios of the fluorescence profiles), a pointwise division of "670 nm" by "650 nm" was performed. The results in Fig. The dose-response curve provided in Figure 19A shows that the method according to the present invention enables measurements of ternary complexes where the labeling is carried out indirectly via a labeled third molecule ( Fig. 19B), such as labeled streptavidin for measuring the interaction between a biotinylated protein and a ligand. Example 7

[0228] The following example describes the use of the ratiometric characterization method according to the present invention to obtain a dose-response curve between a small molecule and a biotinylated protein (i.e., a biotin molecule is covalently bound to it) based on the dual emission configuration. For fluorescence labeling, the biotinylated protein was mixed with the protein streptavidin (SA) and a short nucleic acid modified at its 3' end with biotin and at its 5' end with the fluorophore Cy5 (bDNA). Sa is a homotetramer that exhibits an exceptionally high affinity for biotin (also known as vitamin B7). Due to the stability of the streptavidin-biotin complex towards organic solvents, denaturing agents (e.g., guanidinium chloride), and surfactants (e.g.,SDS, Triton), proteolytic enzymes and extreme temperatures and pH values, it is widely used in molecular biology and bio-nanotechnology.

[0229] In this example, a sample containing maltose-binding protein (MBP) was fluorescently labeled using this approach. MBP was mixed with the small molecule of maltose and excited at a first wavelength, and the emitted fluorescence intensity was measured at a second and a third wavelength. Sample preparation

[0230] Streptavidin was prepared at a stock concentration of 1 mg / ml (approximately 19 µM) and then diluted to 4 nM in phosphate-buffered saline. bDNA (a 12-mer oligodT sequence with a Cy5 molecule bound to its 5' end and a biotin molecule bound to its 3' end) was chemically synthesized and ordered from a DNA supplier. A 100 µM stock solution was prepared and subsequently diluted to a final concentration of 8 nM in double-distilled water (ddH₂O). SA and bDNA were then mixed in a 1:1 volume ratio to obtain a solution of 4 nM SA and 8 nM bDNA (1:2 stoichiometry). This step fluorescently labeled SA by binding the Cy5-labeled biotinylated bDNA.

[0231] Subsequently, 100 µl of 100 nM biotinylated MBP was mixed with 100 µl of the 4 nM SA, 8 nM bDNA-containing solution to obtain 200 µl of a 2 nM SA, 4 nM bDNA, 50 nM MBP-containing solution. Since SA is a tetrameric protein, on average two of its four biotin-binding sites are unoccupied and can bind the biotinylated MBP to generate a bDNA-SA-MBP complex, i.e., fluorescently labeled MBP. Fig. 20A).

[0232] A 16-step 1:1 dilution series of unlabeled maltose was then prepared. Fluorescently labeled MBP was added in equal amounts to the maltose dilution series to achieve a final target concentration of 1 nM SA, 2 nM bDNA, and 25 nM MBP. The highest maltose concentration corresponded to 500 µM. Samples were filled into polymer-coated borosilicate glass capillaries (Monolith NT.115 Premium Capillaries, MO-K025, NanoTemper Technologies). measurement

[0233] Each sample was excited at a wavelength of 591 nm. The fluorescence profiles were simultaneously measured at a second wavelength between 628 nm and 653 nm (“650 nm”) and at a third wavelength between 665 nm and 727 nm (“670 nm”). Each capillary was measured for a period of 3 seconds. Ratiometric data analysis

[0234] For ratiometric analysis (i.e., obtaining the ratios of the fluorescence profiles), a pointwise division of "670 nm" by "650 nm" was performed. The results in Fig. The dose-response curve provided in E. 20B shows that the method of the present invention enables measurements of quaternary complexes in which the labeling is carried out indirectly via an unlabeled third and a labeled fourth molecule, such as unlabeled SA and labeled biotinylated single-stranded DNA oligomers. Example 8

[0235] The following example describes the use of the ratiometric characterization method according to the present invention to obtain a dose-response curve between two molecules based on the dual emission configuration. Therefore, a sample containing fluorescently labeled therapeutic antibody CR3022, the spike protein Cov-19 (“SARS CoV-2”), and the protein angiotensin-converting enzyme 2 (ACE2) was excited at a first wavelength, and the emitted fluorescence intensity was measured at a second and a third wavelength. Sample preparation

[0236] A 14-step 1:1 dilution series of unlabeled ACE2 protein was prepared. The therapeutic antibody CR3022 was fluorescently labeled using the RED-NHS 2nd Generation Protein Labeling Kit (NanoTemper Technologies), and 10 nM of this labeled antibody was mixed with an equal volume of 80 nM of COVID-19 spike protein. This mixture was then added in equal amounts to the ACE2 dilution series to obtain a final sample concentration of 5 nM labeled CR3022 and 20 nM spike protein. The highest ACE2 concentration achieved was 250 nM. Samples were filled into polymer-coated borosilicate glass capillaries (Monolith NT.115 Premium Capillaries, MO-K025, NanoTemper Technologies). measurement

[0237] Each sample was excited at a wavelength of 591 nm. The fluorescence profiles were simultaneously measured at a second wavelength between 628 nm and 653 nm (“650 nm”) and at a third wavelength between 665 nm and 727 nm (“670 nm”). Ratiometric data analysis

[0238] For ratiometric analysis (i.e., obtaining the ratios of the fluorescence profiles), a pointwise division of "670 nm" by "650 nm" was performed. The results in Fig. The dose-response curve provided in Figure 21 shows that the method of the present invention enables measurements of ternary complexes in which the labeling is carried out indirectly via a labeled third molecule, such as a labeled antibody. Example 9

[0239] The following example describes the use of the ratiometric characterization method according to the present invention to characterize a conformational state of a protein based on its dual emission configuration. A sample containing fluorescently labeled protein was excited at a first wavelength, and the emitted fluorescence intensity was measured at a second and a third wavelength. Sample preparation

[0240] Mitogen-activated protein kinase 14 (p38-α) was fluorescently labeled using the RED-NHS 2nd Generation Protein Labeling Kit (NanoTemper Technologies). The labeled protein was then diluted to a concentration of 20 nM and filled into a polymer-coated borosilicate glass capillary (Monolith NT.115 Premium Capillaries, MO-K025, NanoTemper Technologies). measurement

[0241] The sample was excited at a wavelength of 591 nm. The fluorescence profile was simultaneously measured at a second wavelength between 628 nm and 653 nm (“650 nm”) and at a third wavelength between 665 nm and 727 nm (“670 nm”). The measurements were performed immediately after dilution (t = 0 min) and again after 3, 8, and 19 minutes in the capillary. Ratiometric data analysis

[0242] For ratiometric analysis (i.e., obtaining the ratios of the fluorescence profiles), a pointwise division of "670 nm" by "650 nm" was performed. When plotting the fluorescence ratio against the measurement start time, provided in Fig. In 22, it was shown that the ratio was not constant over the period of the four measurements, but increased linearly over time. This increase in the ratio indicates that the labeled protein is not stable at room temperature, but gradually denatures. Example 10

[0243] The following example describes the method of the present invention for measuring fast binding kinetics based on the dual emission configuration. A sample containing a fluorescently labeled DNA aptamer for adenosine and the small molecule AMP, and a sample containing two complementary 11-mer DNA strands, one of which was fluorescently labeled with Cy5, were excited at a first wavelength. The emitted fluorescence intensities of these samples were measured at a second and a third wavelength. Sample preparation

[0244] A 12-step 1:1 dilution series of unlabeled AMP was prepared for the aptamer. The DNA aptamer was fluorescently labeled with Cy5 and added in equal amounts to the AMP dilution series to obtain a final sample concentration of 20 nM. The highest AMP concentration corresponded to 2 mM. The samples were filled into polymer-coated borosilicate glass capillaries (Monolith NT.115 Premium Capillaries, MO-K025, NanoTemper Technologies).

[0245] For DNA hybridization, a 16-step 1:1 dilution series of the unlabeled 11-mer (sequence: 5' CCT GAA GTC C 3') was prepared. The complementary 11-mer (sequence: 5' GGA CTT CAG G 3') was fluorescently labeled with Cy5 at its 5' end and added to the dilution series in equal amounts to achieve a final sample concentration of 10 nM. The highest concentration of the unlabeled 11-mer corresponded to 100 µM. The samples were filled into polymer-coated borosilicate glass capillaries (Monolith NT.115 Premium Capillaries, MO-K025, NanoTemper Technologies). measurement

[0246] Each sample was excited at a wavelength of 591 nm. An IR laser was switched on at time 0 s. The fluorescence intensity response was measured simultaneously for 6 s (aptamer) and 21 s (DNA hybridization). Fluorescence profiles were measured at a second wavelength between 628 nm and 653 nm (“650 nm”) and at a third wavelength between 665 nm and 727 nm (“670 nm”).

[0247] It is evident from the above that information about the binding interaction (e.g., K) d ) can be derived from any “vertical” section over time together with the ratiometric curves when the method according to the present invention is used in combination with rapid heating with an IR laser. A new type of curve can be generated, which can be called the curve of K. dcan be described against time. This curve provides information not only about the thermodynamics but also about the bond kinetics of the interaction. In particular, if the equilibrium kinetics of an interaction is slower than the heating with the IR laser, the K d -versus-time curve shows a characteristic delay. Ratiometric data analysis

[0248] For ratiometric analysis (i.e., obtaining the ratios of the fluorescence profiles), a pointwise division of "670 nm" by "650 nm" was performed. The results in Fig. 23 provided K d Time-versus-time curves yield three different interactions with different dissociation constants K. d and binding kinetics. From the interaction between the Cy5-labeled DNA aptamer, a K d determined by approximately 30 µM and a k off of more than 10 seconds -1 was estimated ( Fig. 23A). For the interaction, i.e., the DNA hybridization between the two complementary 11-mer DNA strands, measured at 32 °C, a K d determined by approximately 500 nm and a k off of approximately 1 second -1 was estimated ( Fig. 23B). Measurements of the aforementioned complementary 11-mer DNA strands at 22 °C yielded a K d of approximately 5 nM and a k off of less than 0.01 s -1 ( Fig. 23C).

[0249] Detailed information on the K measurements d against the time between 22 °C and 32 °C for the DNA hybridization described above are in Fig. 24A provided. The y-axis shows the height of the rise of K. d during measurement. In interactions with slow kinetics, the K follows dThe -vs-time curve does not directly correlate with the temperature change, but shows a significant delay, with the delay increasing the slower the interaction kinetics. Analyzing this delay can therefore provide valuable information about the bonding kinetics of an interaction. Even if no exact values ​​for k are available. off and k on The ability to compare ligands and identify ligands that dissociate faster is already a huge advantage of this method, in addition to the fact that the results can be obtained.

[0250] Assuming that equilibrium was restored after 20 seconds of heating with an IR laser at the new, higher temperature, a van't Hoff analysis was further performed at the two different temperatures, i.e., 22 °C as the initial sample temperature and approximately 32 °C as the temperature after heating with the IR laser (determined from a calibration experiment as described above). The enthalpy and entropy of the interaction were obtained (see Fig. 24B and Fig. 24°C). The thermodynamic parameters obtained are similar to those obtained from a classical van't Hoff analysis by adjusting the sample plate temperature to several different temperatures (e.g., 22°C, 24°C, 26°C, 28°C, 30°C, 32°C) and measuring K d at each of these temperatures (see Fig. 24D and Fig. 24E).

[0251] In Fig. 25 are simulation data for K dTime-vs. curves for different dissociation rates are provided. The in Fig. 25A provided K d -versus-time curves show that dissociation rates between 10 s -1 and 0.001 s -1 by carrying out the method of the present invention in combination with 20-second heating with an IR laser, they can be resolved. The in Fig. 25B provided K d Time-versus-time curves show that even small differences between 0.036 s -1 and 0.154 s -1 can be resolved. Example 11

[0252] The following example describes the method of the present invention for measuring slow binding kinetics based on the dual emission configuration. A sample containing fluorescently labeled nanobodies and Cov-19 spike RBD protein was excited at a first wavelength, and the emitted fluorescence intensity was measured at a second and a third wavelength. Sample preparation

[0253] A nanobody targeting the COVID-19 spike protein was fluorescently labeled using the RED-NHS 2nd Generation Protein Labeling Kit (NanoTemper Technologies). 2 nM of the fluorescently labeled nanobody were rapidly mixed with six different concentrations of the COVID-19 spike RBD protein (20 nM to 625 pM). The samples were filled into polymer-coated borosilicate glass capillaries (Monolith NT.115 Premium Capillaries, MO-K025, NanoTemper Technologies). measurement

[0254] Each sample was excited at a wavelength of 591 nm. The fluorescence ratio was simultaneously measured at a second wavelength between 628 nm and 653 nm (“650 nm”) and at a third wavelength between 665 nm and 727 nm (“670 nm”). In this example, no IR laser was switched on because the samples were measured repeatedly and a temperature change could have affected the binding kinetics. Ratiometric data analysis

[0255] For ratiometric analysis (i.e., obtaining the ratios of the fluorescence profiles), a pointwise division of "670 nm" by "650 nm" was performed. The results in Fig.The ratio-versus-time curve provided in Figure 26 shows that the method according to the present invention enables the tracking of slow binding kinetics when such a "mix-and-measure" approach is used and the binding kinetics are slower than the time required for sample preparation and the start of the measurement. By applying a global fitting model, k on = 6.9 × 10 5 M -1 s -1 , k off = 2.9 × 10 -4 s -1 and K d = 415 pM of the interaction between the nanobody and the Cov-19 spike RBD protein was determined. Example 12

[0256] The following example describes the use of the ratiometric characterization method according to the present invention to localize fluorescently labeled particles based on the dual emission configuration ( Fig.27). Therefore, a sample containing fluorescently labeled mRNA was excited at a first wavelength and the emitted fluorescence intensity was measured at a second and a third wavelength. Sample preparation

[0257] mRNA was fluorescently labeled with the dye Atto647N and incorporated into lipid nanoparticles (LNPs). Duplicates of these mRNA-containing LNP preparations were subjected to various stresses: 0.25% of the detergent polysorbate 20 (Tween-20) was added, they were boiled at 90 °C for 10 min, vortexed for 1 min, or centrifuged at 14,000 rpm for 20 min. Untreated mRNA-containing LNPs served as controls. The samples were filled into polymer-coated borosilicate glass capillaries (Monolith NT.115 Premium Capillaries, MO-K025, NanoTemper Technologies). measurement

[0258] Each sample was excited at a wavelength of 591 nm. The fluorescence profiles were simultaneously measured at a second wavelength between 628 nm and 653 nm (“650 nm”) and at a third wavelength between 665 nm and 727 nm (“670 nm”).

[0259] For measurements solely to determine the absolute fluorescence ratio, each capillary was measured for 3 seconds. For measurements to determine the aggregation state of the LNP, measurements were performed using an IR laser (laser activation time 60 seconds).

[0260] Previously performed control measurements show that the ratiometric fluorescence signal of fluorescently labeled mRNA with Atto647N located within the LNP is approximately 2.1 in the dual emission configuration according to the invention. In contrast, when all fluorescently labeled mRNA molecules are located outside the LNP, the ratiometric fluorescence signal is approximately 1.2. Ratiometric data analysis

[0261] For ratiometric analysis (i.e., obtaining the ratios of the fluorescence profiles), a pointwise division of "670 nm" by "650 nm" was performed. In the untreated control, i.e., where all fluorescently labeled mRNA molecules are within the LNP, the ratiometric fluorescence signal is 2.1 ( Fig.28A, control). The addition of high concentrations of detergents ruptures / damages the lipid membrane of the LNP, and thus the fluorescently labeled mRNA molecules are no longer incorporated into the LNP ( Fig. 28A + Fig. 0, Fig. 25% Tween). In this case, the ratiometric fluorescence signal is 1.2. As can be seen from a ratio of approximately 2.1, the fluorescently labeled mRNA is still present in the vortexed or centrifuged LNP preparations ( Fig. 28A, Fig. 1 min vortexing, 20 min centrifugation). Analysis of the “bumpy” fluorescence profiles obtained after switching on the IR laser ( Fig. 28B), however, these treatments lead to aggregation of the LNP preparations. In contrast, the ratio of 1.2 obtained by boiling the LNP preparations at 90 °C for 10 minutes indicated that the fluorescently labeled mRNA was no longer incorporated into the LNP ( Fig. 28A, Fig. (10 min at 90 °C). This was also confirmed by analyzing the fluorescence profiles after switching on the IR laser. Since no uneven fluorescence profiles were observed after boiling, it was confirmed that the fluorescently labeled mRNA molecules had left the (possibly still aggregated) LNPs.

[0262] As can be seen from the foregoing, the ratiometric characterization method of the present invention enables the determination of the localization of fluorescently labeled mRNA. List of reference symbols: 1 lens (for example, an aspherical lens) or a lens system or an objective 2 hot mirrors, high IR reflection, visible light transmittance > 80% 3 IR lasers (e.g. 1455 nm, 1480 nm, 1550 nm, 980 nm, 0.01 W-10 W) or lasers for positioning 4 Laser fibers (single-mode or multi-mode) 5 laser fiber couplers without collimator 6 Beam shaping module for determining the laser beam diameter and focusing (e.g., a lens system comprising one, two, or more lenses) 7. First light-separating element (e.g. dichroic mirror) for separating fluorescence excitation and emission 8 First excitation light source (e.g. laser, fiber laser, diode laser, LED, HXP, halogen, LED array, HBO) 9. Lens system for determining the beam properties of the excitation light source (e.g., one, two, or more lenses) 10 excitation filters (e.g. bandpass / longpass) 11 Second light-separating element (e.g. dichroic mirror) for splitting the emission into a component with a lower and a higher wavelength 12 First emission filter (e.g. bandpass / longpass) 13 Second emission filter (e.g. bandpass / longpass) 14 First light detector (e.g. PMT, siPM, APD, CCD or CMOS camera) 15 Second light detector (e.g. PMT, siPM, APD, CCD or CMOS camera) 16 Second excitation light source (e.g. laser, fiber laser, diode laser, LED, HXP, halogen, LED array, HBO) 17 Lens system for determining the beam properties of the excitation light source 18 Third light-separating element (e.g. dichroic mirror) for combining two excitation light sources Cited non-patent literature [NPL1] Sindrewicz, P., Li, X., Yates, EA, Turnbull, JE, Lian, LY and Yu, LG (2019). Intrinsic tryptophan fluorescence spectroscopy reliably determines galectin-ligand interactions. Scientific reports, 9(1), 1-12. [NPL2] Mayer-Wrangowski, SC and Rauh, D. (2015). Monitoring ligand-induced conformational changes for the identification of estrogen receptor agonists and antagonists. Applied Chemistry International Edition, 54(14), 4379-4382. [NPL3] Chen, H. J., Chew, C. Y., Chang, E. H., Tu, Y. W., Wei, L. Y., Wu, B. H., ... und Tan, K. T. (2018). S-cis diene conformation: a new bathochromic shift strategy for near-infrared fluorescence switchable dye and the imaging applications. Journal of the American Chemical Society, 140(15), 5224-5234. [NPL4] Niu, W., Wei, Z., Jia, J., Shuang, S., Dong, C. und Yun, K. (2018). A ratiometric emission NIR-fluorescent probe for sensing and imaging pH changes in live cells. Dyes and Pigments, 152, 155-160. [NPL5] Pauli, J., Grabolle, M., Brehm, R., Spieles, M., Hamann, F. M., Wenzel, M., ... und Resch-Genger, U. (2011). Suitable labels for molecular imaging-influence of dye structure and hydrophilicity on the spectroscopic properties of IgG conjugates. Bioconjugate chemistry, 22(7), 1298-1308. [NPL6] Harroun, SG, Lauzon, D., Ebert, MC, Desrosiers, A., Wang, X. and Vallee-Belisle, A. (2022). Monitoring protein conformational changes using fluorescent nanoantennas. Nature methods, 19(1), 71-80.

[0263] All patent and non-patent documents cited herein are hereby incorporated by reference in their entirety. QUOTES INCLUDED IN THE DESCRIPTION

[0000] This list of documents cited by the applicant was automatically generated and is included solely for the reader's convenience. The list is not part of the German patent or utility model application. The DPMA accepts no liability for any errors or omissions. Cited patent literature

[0000] WO 2017 / 087912 A2

[0003] WO 2018 / 234557 [0008, 0078] WO 2017 / 055583 [0012, 0093] Cited non-patent literature

[0000] https: / / www.fpbase.org / table /

[0075] Lambert, TJ (2019) FPbase: a community-editable fluorescent protein database. Nature Methods. 16, 277-278. doi: 10.1038 / s41592-019-0352-8

[0075] Klymchenko, A. S. (2017) (Solvatochromic and fluorogenic dyes as environment-sensitive probes: design and biological applications. Accounts of chemical research, 50(2), 366-375

[0078] Sindrewicz, P., Li, X., Yates, E. A., Turnbull, J. E., Lian, L. Y. und Yu, L. G. (2019). Intrinsic tryptophan fluorescence spectroscopy reliably determines galectin-ligand interactions. Scientific reports, 9(1), 1-12

[0262] Mayer-Wrangowski, S. C. und Rauh, D. (2015). Monitoring ligand-induced conformational changes for the identification of estrogen receptor agonists and antagonists. Angewandte Chemie International Edition, 54(14), 4379-4382

[0262] Chen, H. J., Chew, C. Y., Chang, E. H., Tu, Y. W., Wei, L. Y., Wu, B. H., ... und Tan, K. T. (2018). S-cis diene conformation: a new bathochromic shift strategy for near-infrared fluorescence switchable dye and the imaging applications. Journal of the American Chemical Society, 140(15), 5224-5234

[0262] Niu, W., Wei, Z., Jia, J., Shuang, S., Dong, C. und Yun, K. (2018). A ratiometric emission NIR-fluorescent probe for sensing and imaging pH changes in live cells. Dyes and Pigments, 152, 155-160

[0262] Pauli, J., Grabolle, M., Brehm, R., Spieles, M., Hamann, F. M., Wenzel, M., ... und Resch-Genger, U. (2011). Suitable labels for molecular imaging-influence of dye structure and hydrophilicity on the spectroscopic properties of IgG conjugates. Bioconjugate chemistry, 22(7), 1298-1308

[0262] Harroun, S. G., Lauzon, D., Ebert, M. C., Desrosiers, A., Wang, X. und Vallee-Belisle, A. (2022). Monitoring protein conformational changes using fluorescent nanoantennas. Nature methods, 19(1), 71-80

[0262]

Claims

Apparatus for characterizing fluorescently labeled particles in solution by analyzing changes in fluorescence intensity resulting from spectral shifts and / or a broadening or narrowing of the spectrum or combinations thereof of the fluorescent labeling of the fluorescently labeled particles, the apparatus comprising: a sample holder for holding a sample of fluorescently labeled particles in solution under a variety of conditions; means for exciting the fluorescently labeled particles at a first wavelength; means for detecting the fluorescence emission intensity of the fluorescently labeled particles in a second and third wavelength range using filter elements;Means for calculating a ratio between the fluorescence intensities detected in the second and third wavelength ranges, wherein the third wavelength range differs from the second wavelength range, wherein the calculated ratios are used to determine parameters selected from the group consisting of dissociation constants, mean effective concentrations (EC50), equilibrium constants, binding kinetics, enzyme reaction kinetics, thermodynamic parameters, unfolding or refolding kinetics, opening and closing reactions and combinations thereof; wherein the apparatus is designed to successively excite fluorescently, detect fluorescence emissions and calculate the ratio for samples under different conditions;Means for characterizing the fluorescently labeled particles based on the calculated ratios obtained for the various conditions, wherein the device is designed to detect the second and third wavelengths simultaneously, wherein the wavelengths of the second wavelength range are shorter and the wavelengths of the third wavelength range are longer than an absolute emission maximum of the fluorescence emission of the fluorescently labeled particles under a first condition of the various conditions. Device according to claim 1, wherein the excitation means is an excitation light source, preferably at least one light source from the group consisting of laser, laser fiber laser, diode laser, LED, HXP, halogen, LED array, HBO. Device according to claim 1 or 2, wherein the means for detection is a light detector, preferably at least one detector from the group consisting of PMT, siPM, APD, CCD or CMOS camera. Device according to claim 1, wherein the sample holder is a capillary.

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