Method and device for ratiometric characterization of fluorescent particles - Patents.com

Abstract

The present invention relates to a device and a method for characterization of fluorescently labeled particles in a solution by analyzing the variations in the fluorescence spectrum of the fluorescently labeled particles. In particular, a sample of fluorescently labeled particles is analyzed by fluorescence excitation and detection of the corresponding fluorescence emission under various conditions / environments. The particles are characterized by analyzing the detected fluorescence emission under these various conditions / environments. More specifically, the present invention relates to a method and a device for ratiometric characterization of the inter- and / or intra-molecular interactions and / or conformational modifications and / or localization of fluorescently labeled particles.

Description

[Technical field]

[0001] The present invention relates to a device and a method for characterization of fluorescently labeled particles in a solution by analyzing the variations in the fluorescence spectrum of the fluorescently labeled particles. In particular, a sample of fluorescently labeled particles is analyzed by fluorescence excitation and detection of the corresponding fluorescence emission under various conditions / environments. The particles are characterized by analyzing the detected fluorescence emission under these various conditions / environments. More specifically, the present invention relates to a method and a device for ratiometric characterization of the inter- and / or intra-molecular interactions and / or conformational modifications and / or localization of fluorescently labeled particles. [Background technology]

[0002] The fluorescence spectrum of a fluorescent label is sensitive to environmental changes such as changes in the chemical environment and temperature changes, and therefore the same fluorescent label may exhibit variations in its fluorescence spectrum in terms of intensity and / or spectral shift and / or spectral shape.

[0003] Because this effect is well known, it is used to study the interactions of intrinsically or extrinsically fluorescently labeled particles. In the art, the fluctuation in fluorescence intensity is referred to as the dissociation constant (K d) (NPL1, NPL2) are primarily used to characterize binding reactions. In addition to characterizing intermolecular interactions, the determined interactions may further include intramolecular interactions and / or modifications (conformational changes) and / or localization (changes) of fluorescently labeled particles. For example, conformational changes of proteins and analyte concentrations are determined in the art based on methods involving a mechanism called Förster resonance energy transfer (FRET) (WO2017 / 087912A2). 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 through dipole-dipole coupling due to an interaction-mediated (e.g., ligand-mediated) change in the distance and / or angle between the two fluorophores. Therefore, FRET measurements require two or more fluorescent labels, i.e., at least one donor and at least one acceptor fluorophore. However, because two different fluorescent labels are used, each of which may have different sensitivity to environmental changes, FRET measurements may be distorted by undesired changes in the local environment of the fluorophore. Furthermore, the fluorescence emission from the acceptor fluorophore when excited by energy transfer usually provides a lower signal strength than the measurement of the acceptor fluorophore emission intensity resulting from direct excitation. In general, FRET measurements require precise positioning of the donor and acceptor fluorophores within a defined distance on the target molecule, for example by applying two site-specific labeling chemicals, which in turn may not be feasible for all types of targets. In contrast, random labeling, for example with lysine- or cysteine-reactive dyes, may result in various FRET distances and thus failure of the FRET measurement.In summary, compared to measurements involving only a single fluorescent label as in the present invention, FRET measurements have a lower signal-to-noise ratio, and it is desirable that the fluorescently labeled particles being analyzed are labeled with only one type of fluorescent label.

[0004] Depending on the interactions and characteristics of the fluorescent labels themselves, the variations in the fluorescence spectra may be less than 1%, and therefore fall within the normal range of pipetting errors. Currently, it is difficult, if not impossible, to resolve variations less than 1% with the methods / devices available in the art.

[0005] Fluctuations in the fluorescence spectrum of fluorescent labels are usually detected by fluorescence spectrophotometers, which are designed to record / measure over a large spectral range and therefore cannot resolve small changes in fluorescence intensity. In addition, commercially available fluorescent labels are currently designed to be more robust to environmental changes, so ultrasensitive fluorescent labels need to be adapted to increase the resolution of spectrophotometric measurements (NPL3, NPL4, NPL5).

[0006] Another possibility to improve the resolution of spectrophotometric measurements can be achieved by combining several measurements. In this case, the sample to be investigated is excited at a first wavelength and its emission is measured at a second wavelength in a first measurement, followed by repeating the excitation step at the first wavelength and the measurement of its emission at a third wavelength in a second measurement. The detected emission intensities obtained from the first and second measurements are then combined. Pure information of interest can therefore be determined, independent of any artifacts introduced, for example due to manual handling. However, due to the subsequent execution of the two measurements, this method is more cumbersome and time-consuming. Also, the conditions during the first and second measurements may vary. Furthermore, bleaching may have occurred, which may further lead to a distortion of the results. By using this approach, fluorescence changes of up to 4.5% when measured at room temperature were resolved (NPL2).

[0007] Generally, measurements with commercially available fluorescence spectrophotometric devices require large sample volumes (i.e., by using quartz cuvettes or multi-well plates) and, consequently, higher sample concentrations in order to adequately resolve fluctuations in fluorescence intensity. Below, further methods known in the art are described that require smaller sample volumes (i.e., 10 μl).

[0008] For example, if the absolute intensity variation at room temperature is too small compared to the pipetting error, the pipetting error can be eliminated by applying a characterization method based on temperature-related intensity change (TRIC) (WO2018 / 234557). 熱 / F 冷 , i.e., by calculating the ratio of the fluorescence intensity based on the intensity measured at room temperature to the intensity measured at a second, typically higher temperature, a meaningful measure of the inter- and / or intra-molecular interactions can still be determined.

[0009] However, an increase in temperature, even of just a few degrees Celsius (°C): - The particles being measured are not necessarily tolerant. Therefore, the method is not suitable for characterizing interactions involving labile samples such as labile proteins.

[0010] - The fluorescence intensity may vary equally for fluorescently labeled particles and for fluorescently labeled particles complexed with a ligand. In this case, "F 熱 / F 冷 " has the same value in both cases and although the interaction between the interaction partners occurs, no binding curves can be obtained.

[0011] - Inhomogeneous samples, such as those containing certain small fractions of aggregates, may result in irreproducible fluorescence traces due to convection, which is the basis of the "F 熱 / F 冷This means that the noise in the signal amplitude can be substantially large. It is therefore not possible to obtain binding curves for systems with small signal amplitudes.

[0012] Furthermore, when analyzing interactions of ternary complexes, e.g., a complex containing a labeled molecule A, another molecule B that binds to A, and a third molecule C that binds to B, changes in TRIC are often not sufficient to disrupt the interaction.

[0013] If rapid interaction changes due to temperature and ligand binding cause fluorescence intensity changes, the TRIC method may result in biphasic dose-response curves that cannot be analyzed by a sigmoidal one-to-one binding model, or the measured values ​​of the dissociation constants may deviate from the actual values ​​(e.g., the measured binding affinity corresponds to weaker binding at higher temperatures but not at lower temperatures).

[0014] A different approach to investigate intra- and / or intermolecular interactions of fluorescent particles is nano differential scanning fluorimetry (WO2017 / 055583). This method is based on measuring the change in the intrinsic fluorescence intensity of proteins containing tryptophan (Trp) and tyrosine (Tyr) residues. However, proteins typically contain several of their fluorescent aromatic amino acid residues. Usually, not all of them are involved in the binding reaction, so upon excitation a high fluorescence background occurs that reduces the amplitude of the signal. Furthermore, Trp and Tyr residues are typically located in the hydrophobic core of the protein and may not be significantly affected by the binding of the ligand. Often, the autofluorescence of the ligand interferes with the readout, since it is in the same range as the fluorescence of Trp and Tyr.

[0015] In contrast, by labeling with exogenous fluorescent labels, it can be controlled that only one dye (i.e., one type of dye) binds to the target molecule and only that dye needs to be affected by ligand binding. Furthermore, the labeling chemistry can be adapted so that the dye can be placed in a location that is optimal for detecting changes in the chemical microenvironment (e.g., proximal to the ligand binding site). Furthermore, exogenous dyes are usually located on the protein surface and therefore have ideal exposure for sensing changes to the chemical microenvironment. The dye fluorescence range can be selected so that it does not interfere with the autofluorescence of the ligand. Finally, exogenous dyes are brighter and measurements can be performed at lower concentrations of the target molecule, thus reducing sample consumption and allowing even picomolar affinity measurements.

[0016] Thus, there is a need for improved or alternative methods or devices for characterizing the inter- and / or intra-molecular interactions and / or modifications (conformational changes) and / or (changes in) localization of fluorescently labeled particles. Summary of the Invention [Means for solving the problem]

[0017] The present invention provides novel methods and devices for the characterization of inter- and / or intra-molecular interactions and / or modifications (conformational changes) and / or (changes in) localization of fluorescently labeled particles, as defined by the features of the independent claims. Further preferred embodiments of the invention are defined in the independent claims. In particular, the present invention solves the technical problem of economically resolving any small fluctuations in the fluorescence spectrum of labeled fluorescent particles, preferably based on exactly one fluorescent label and independently of the characteristics (e.g. temperature stability, aggregate formation) and size of the fluorescently labeled particles.

[0018] The present invention relates to a method and device for ratiometric characterization of intermolecular and / or intramolecular interactions and / or conformational modification and / or localization of fluorescently labeled particles. In particular, the method of the present invention provides improved sensitivity in detecting variations in the fluorescence spectrum of fluorescent labels within small sample volumes of fluorescently labeled particles that could not be resolved by previously known methods. Furthermore, the method of the present invention allows even short-time measurements of temperature-sensitive and / or unstable samples. In combination with a defined temperature perturbation, the method of the present invention allows the measurement of thermodynamic and kinetic parameters of the interaction. In the method of the present invention, fluorescently labeled particles are used, preferably labeled with at least one fluorescent label. The method of the present invention allows the determination of the localization of fluorescently labeled particles (e.g., whether the fluorescently labeled particles are located within lipid nanoparticles (LNPs) and / or within cells and / or in the buffer solution surrounding the LNPs and cells).

[0019] In a first aspect, the present invention relates to a method for the characterization of fluorescently labeled particles in a solution by analyzing variations in the fluorescence spectrum of the fluorescently labeled particles. The method of the first aspect comprises the following steps: a) providing a sample of fluorescently labeled particles in a solution under first conditions; b) exciting the fluorescently labeled particles at a first wavelength; c) detecting the fluorescent emission intensity of the fluorescently labeled particles at a second and a third wavelength; d) calculating a ratio between said fluorescence intensities at a second and a third wavelength, the third wavelength being different from the second wavelength; Includes.

[0020] e1) repeating steps b) through d) on said sample of fluorescently labeled particles under second conditions; or e2) repeating steps a) to d) on a second sample of fluorescently labeled particles under second conditions, the second condition being different from the first condition. is even more preferred.

[0021] f) The fluorescently labeled particles may then be characterized based on the calculated ratios obtained for the different conditions; The second and third wavelengths are preferably detected simultaneously, the second wavelength preferably being shorter and the third wavelength preferably being longer than the maximum emission of the fluorescent emission of the fluorescently labeled particle under the first conditions.

[0022] In a second aspect, the present invention relates to a method for the characterization of fluorescently labeled particles in a solution by analyzing the variations in the fluorescence spectrum of the fluorescently labeled particles in combination with a defined temperature perturbation / change. The method of the second aspect comprises the following steps: a) providing a sample of fluorescently labeled particles in a solution under first conditions; b) exciting the fluorescently labeled particles at a first wavelength; c) detecting the fluorescent emission intensity of the fluorescently labeled particles at a second and a third wavelength, the intensity is detected during a defined temperature perturbation; and d) calculating a ratio between the fluorescence intensities at a second and a third wavelength, the third wavelength being different from the second wavelength.

[0023] As for the first aspect, and e1) repeating steps b) through d) on said sample of fluorescently labeled particles under second conditions; or e2) repeating steps a) to d) on a second sample of fluorescently labeled particles under second conditions, the second condition is different from the first condition; and f) characterizing the fluorescently labeled particles based on the calculated ratios obtained for the different conditions, a second and a third wavelength are preferably detected simultaneously, the second wavelength being preferably shorter than a maximum emission of the fluorescent emission of the fluorescently labeled particle under the first conditions, and the third wavelength being longer; is preferred.

[0024] In a third aspect, the present invention relates to a method for the characterization of thermodynamic and / or kinetic parameters of fluorescently labeled particles in solution by analyzing the variations in the fluorescence spectra of the fluorescently labeled particles in combination with defined temperature perturbations / changes.

[0025] In a fourth aspect, the present invention relates to a method for characterization of the localization of fluorescently labeled particles in a solution by analyzing the variations in the fluorescence spectra of the fluorescently labeled particles, in combination with or without defined temperature perturbations / changes.

[0026] In the context of the present invention, the fluorescence radiation intensities at the second and third wavelengths in step c) are preferably detected simultaneously, which means within a short time interval, preferably less than 1 second, more preferably less than 750 milliseconds, more preferably less than 500 milliseconds, more preferably less than 250 milliseconds, more preferably less than 100 milliseconds, more preferably less than 50 milliseconds, more preferably less than 25 milliseconds, more preferably less than 10 milliseconds, more preferably less than 5 milliseconds, even more preferably less than 2.5 milliseconds. Typical time intervals in this context are 50 milliseconds, 10 milliseconds and 1 millisecond.

[0027] Some particular aspects of the present invention can be summarized as follows: In some embodiments, the methods described herein include the steps of: a) providing a sample of fluorescently labeled particles in solution under first conditions; b) exciting the fluorescently labeled particles at a first wavelength; c) detecting the fluorescent emission intensities of the fluorescently labeled particles at a second and a third wavelength; d) calculating a ratio between the fluorescent intensities at the second and third wavelengths, wherein the third wavelength is different from the second wavelength; and e1) detecting the ratio between the fluorescent intensities at the second and third wavelengths, wherein the third wavelength is different from the second wavelength. 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; and f) characterizing the fluorescently labeled particles based on the calculated ratio obtained for the different conditions, wherein a second and a third wavelength are detected simultaneously, the second wavelength being shorter than a maximum emission of the fluorescent emission of the fluorescently labeled particles under the first conditions and the third wavelength being longer.

[0028] In some embodiments of the 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.

[0029] In some embodiments of the present invention, the sample containing the fluorescently labeled particles is preferably provided in a volume of between 1 μl and 25 μl. In some embodiments of the invention, a sample containing fluorescently labeled particles is provided in a capillary.

[0030] In some embodiments of the invention, the fluorescently labeled particles are labeled with an environmentally sensitive label. In some embodiments of the invention, the particle is selected from the group consisting of an organic molecule, a biomolecule, a nanoparticle, a microparticle, a vesicle, a biological cell or subcellular fragment, a biological tissue, a viral particle, a virus, an organelle, a lipid nanoparticle (LNP), and a virus-like particle.

[0031] In some aspects of the invention, the biomolecule is 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.

[0032] In some embodiments of the present invention, the concentration of the fluorescently labeled particles in the solution is from 10 pM to 10 μM, preferably from 50 pM to 500 nM. In some embodiments of the invention, the fluctuations in the detected fluorescence intensity of fluorescently labeled particles result from a spectral shift, or a spectral broadening or a spectral narrowing, or a combination thereof.

[0033] In some embodiments of the invention, the fluorescence intensity of the fluorescently labeled particle changes due to a mechanism selected from the group consisting of a conformational change of the fluorescently labeled particle, relocalization of the fluorescently labeled particle, an interaction between the fluorescently labeled particle and one or more ligands, and combinations thereof.

[0034] In some embodiments of the invention, the calculated ratio obtained in step f) is used to determine the localization of the fluorescently labeled particles, or the dissociation constant, 50% effective concentration (EC 50 ), equilibrium constants, binding kinetics, enzyme kinetics, thermodynamic parameters, unfolding or refolding kinetics, opening and closing reactions, and combinations thereof.

[0035] In some embodiments of the invention, the second conditions of step e) are varied by adding a ligand and / or different concentrations of the ligand, and the calculated ratio obtained in step f) is used to determine the dissociation constant of the fluorescently labeled particle and the ligand.

[0036] In some embodiments of the invention, the first and second conditions of the fluorescently labeled particles differ in their temperature and / or chemical composition. In some embodiments of the invention, the fluorescence emission intensities of the fluorescently labeled particles at the second and third wavelengths in step c) are detected during a defined temperature perturbation.

[0037] Also provided herein is a device for characterization of fluorescently labeled particles in solution by analyzing variations in the fluorescence spectra of the fluorescently labeled particles. In some embodiments, the device of the invention is adapted to perform the methods described herein. In some embodiments, the device includes a sample holder for holding a sample of fluorescently labeled particles in solution under a plurality of conditions (i.e., a number of different conditions); means for exciting the fluorescently labeled particles at a first wavelength; means for detecting the fluorescent emission intensity of the fluorescently labeled particles at a second and a third wavelength; means for calculating a ratio between the fluorescent intensities at the second and third wavelengths, wherein the third wavelength is different from the second wavelength; means configured for the device to sequentially fluorescently excite and detect the fluorescent emission and calculate the ratio for the sample in the different conditions; and means for characterizing the fluorescently labeled particles based on the calculated ratios obtained for the different conditions, wherein the device is configured to simultaneously detect the second and third wavelengths, wherein the second wavelength is shorter than the maximum emission of the fluorescent emission of the fluorescently labeled particles under the first condition (the different condition) and the third wavelength is longer.

[0038] In some embodiments of the invention, the means for excitation 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 embodiments of the invention, the means for detecting is a photodetector, preferably at least one detector from the group consisting of a 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 to carry out the methods described herein for use.

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

[0042] Also provided herein is the use of the device for characterization of fluorescently labeled particles in solution according to the methods described herein. Also provided herein is the use of a capillary for characterization of fluorescently labeled particles in solution by analyzing variations in the fluorescence spectra of the fluorescently labeled particles, wherein a sample of the fluorescently labeled particles in solution is filled into the capillary and provided for analysis by the methods described herein.

[0043] In the following, preferred embodiments of the invention are explained in detail with reference to the figures. [Brief description of the drawings]

[0044] [Figure 1]Figure 1A shows the excitation spectra of four different protein samples labeled with the same fluorescent dye. The samples were excited between 520 and 670 nm. The emission of the samples was recorded at 690 nm. Figure 1B is a zoomed-in view of the excitation peak in (A). [Diagram 2] Figure 2A shows the emission spectra of four different protein samples labeled with the same fluorescent dye. The samples were excited at 605 nm. The emission of the samples was recorded from 620 to 750 nm. Figure 2B is a zoom of the emission peak in (A). [Diagram 3] Figure 3A shows the potential effect of ligand proximity of a labeled molecule on the variation of a fluorescent dye's fluorescence spectrum. Figure 3B shows the potential effect of conformational changes of a labeled molecule on the variation of a fluorescent dye's fluorescence spectrum. Figure 3C shows a hypsochromic (blue) or bathochromic (red) shift of the spectrum. Figure 3D shows a broadening or narrowing of the spectrum. [Figure 4] Figure 4A shows the emission peaks of the fluorescently labeled protein streptavidin alone and in combination with its natural ligand, biotin, and Figure 4B shows a magnified view of the emission peaks in (A). [Diagram 5] Figure 5A shows the emission peaks of the fluorescently labeled protein lysozyme alone and in combination with the inhibitor tri-N-acetyl-D-glucosamine (NAG3), and Figure 5B shows a magnified view of the emission peaks in (A). [Figure 6] Figure 6A shows the ratio trace of carbonic anhydrase complexed with furosemide detected by using the dual emission configuration of the present invention. The change in the fluorescence ratio of carbonic anhydrase upon furosemide binding is 0.5%. Figure 6B shows the resulting dose response curve of the binding interaction. [Figure 7]FIG. 7A shows the ratio 350 nm / 330 nm of the intrinsic tryptophan fluorescence of unlabeled lysozyme and various concentrations of NAG3 during increasing temperature. To monitor the temperature of denaturation, each sample is heated from 35° C. to 95° C. Increasing the concentration of NAG3 leads to a thermostabilization of lysozyme, i.e., a thermal shift. This shift cannot be used to extract the dissociation constant of this interaction. FIG. 7B shows that by plotting the initial ratio 350 nm / 330 nm of the intrinsic protein fluorescence at 35° C. against the concentration of NAG3, a sigmoidal dose-response curve and therefore the dissociation constant (Kd) at said temperature is obtained. [Figure 8] FIG. 8A shows various embodiments according to the present invention, illustrating a preferred embodiment with dual emission optics and an IR laser. FIG. 8B shows various embodiments according to the present invention, illustrating an embodiment with dual emission optics. FIG. 8C shows various embodiments according to the present invention, illustrating an embodiment with dual excitation optics and an IR laser. FIG. 8D shows various embodiments according to the present invention, illustrating an embodiment with dual excitation optics. FIG. 8E shows various embodiments according to the present invention, illustrating an embodiment with dual excitation and dual emission optics and an IR laser. FIG. 8F shows various embodiments according to the present invention, illustrating an embodiment with dual excitation and dual emission optics. [Figure 9] 9A and 9B show exemplary filter arrangements applicable to a dual excitation configuration for a red (Cy5) fluorescent dye and a dual emission configuration for a green (Cy3) fluorescent dye, respectively. [Figure 10A] FIG. 10 shows dose-response curves between Cy5-labeled DNA aptamers and AMP obtained by ratiometric characterization based on either (A) measurements with a commercial microplate reader or (B and C) measurements with a dual-radiation configuration according to the present invention. [Figure 10B]FIG. 10 shows dose-response curves between Cy5-labeled DNA aptamers and AMP obtained by ratiometric characterization based on either (A) measurements with a commercial microplate reader or (B and C) measurements with a dual-radiation configuration according to the present invention. [Figure 10C] FIG. 10 shows dose-response curves between Cy5-labeled DNA aptamers and AMP obtained by ratiometric characterization based on either (A) measurements with a commercial microplate reader or (B and C) measurements with a dual-radiation configuration according to the present invention. [Figure 11] FIG. 11A shows a fluorescence trace of a Cy5-labeled DNA aptamer mixed with a serial dilution of an AMP. At time 0 seconds, the IR laser is switched on and the fluorescence intensity response is measured over a period of 31 seconds. In the current dual emission configuration, the emitted fluorescence trace was recorded at a wavelength of 628-653 nm ("650 nm"). FIG. 11B shows a fluorescence trace of a Cy5-labeled DNA aptamer mixed with a serial dilution of an AMP. At time 0 seconds, the IR laser is switched on and the fluorescence intensity response is measured over a period of 31 seconds. In the current dual emission configuration, the emitted fluorescence trace was recorded at a wavelength of 665-727 nm ("670 nm"). The emitted fluorescence trace was recorded simultaneously in (A) and (B). [Figure 12] Figure 12A shows an analysis of the initial fluorescence intensity of the measurement of Figure 9. Based on the initial fluorescence intensity obtained at 650 nm, no sigmoidal dose-response curve is obtained, and therefore no affinity of the interaction. Figure 12B shows an analysis of the initial fluorescence intensity of the measurement of Figure 9. Based on the initial fluorescence intensity obtained at 670 nm, no sigmoidal dose-response curve is obtained, and therefore no affinity of the interaction. [Figure 13]FIG. 13A shows a ratiometric analysis of the fluorescence intensity traces of the measurements of FIG. 11, obtained by point-wise dividing the fluorescence trace at 670 nm by the fluorescence trace at 650 nm. Three different phases of the measurement are highlighted, namely phases 1 to 3. FIG. 13B shows that by analyzing the ratios during phase 1, i.e. before turning on the IR laser, a dose-response curve with a signal-to-noise ratio of over 300 is obtained. [Figure 14] FIG. 14A shows the Kd time course curve for the ratiometric data of FIG. 13A. "Vertical slices" taken at 200 millisecond intervals are analyzed to obtain a dose-response curve for each of the time intervals. If the temperature change over time is known and the interaction equilibrates on a timescale faster than the temperature change occurs, a relationship of Kd to temperature can be obtained. FIG. 14B shows that by performing a Van't Hoff analysis, the binding enthalpy (ΔH) and binding entropy (ΔS) of an interaction can be determined from the relationship of Kd to temperature. [Figure 15] FIG. 15A shows the fluorescence traces of Cy3-labeled DNA aptamers mixed with serial dilutions of AMP. At time 0 seconds, the IR laser is switched on and the fluorescence intensity response is measured over a period of 6 seconds. In the current dual excitation configuration, the emitted fluorescence traces are then recorded by a single detector upon (A) a first excitation by a blue LED at wavelengths of 475-495 nm. FIG. 15B shows the fluorescence traces of Cy3-labeled DNA aptamers mixed with serial dilutions of AMP. At time 0 seconds, the IR laser is switched on and the fluorescence intensity response is measured over a period of 6 seconds. In the current dual excitation configuration, the emitted fluorescence traces are then recorded by a single detector upon (B) a second excitation by a green LED at wavelengths of 550-575 nm. [Figure 16A]FIG. 16A shows a ratiometric analysis of the initial fluorescence intensity of the measurement of FIG. 15, obtained by point-wise dividing the fluorescence trace measured upon excitation with the green LED by the fluorescence trace measured upon excitation with the blue LED. Three different phases of the measurement are highlighted, namely phases 1 to 3. FIG. 16B shows that by analyzing the ratio during phase 1, i.e. before turning on the IR laser, a dose-response curve with a signal-to-noise ratio of approximately 80 is obtained. FIG. 16C shows that by analyzing the ratio during phase 3, i.e. after turning on the IR laser, a dose-response curve with an improvement in the signal-to-noise ratio of more than 130 is obtained. [Figure 16B] FIG. 16A shows a ratiometric analysis of the initial fluorescence intensity of the measurement of FIG. 15, obtained by point-wise dividing the fluorescence trace measured upon excitation with the green LED by the fluorescence trace measured upon excitation with the blue LED. Three different phases of the measurement are highlighted, namely phases 1 to 3. FIG. 16B shows that by analyzing the ratio during phase 1, i.e. before turning on the IR laser, a dose-response curve with a signal-to-noise ratio of approximately 80 is obtained. FIG. 16C shows that by analyzing the ratio during phase 3, i.e. after turning on the IR laser, a dose-response curve with an improvement in the signal-to-noise ratio of more than 130 is obtained. [Figure 16C] FIG. 16A shows a ratiometric analysis of the initial fluorescence intensity of the measurement of FIG. 15, obtained by point-wise dividing the fluorescence trace measured upon excitation with the green LED by the fluorescence trace measured upon excitation with the blue LED. Three different phases of the measurement are highlighted, namely phases 1 to 3. FIG. 16B shows that by analyzing the ratio during phase 1, i.e. before turning on the IR laser, a dose-response curve with a signal-to-noise ratio of approximately 80 is obtained. FIG. 16C shows that by analyzing the ratio during phase 3, i.e. after turning on the IR laser, a dose-response curve with an improvement in the signal-to-noise ratio of more than 130 is obtained. [Figure 17]Figure 17 shows the dose-response curve of 12-point serial dilutions of biotin mixed with fluorescently labeled streptavidin obtained by ratiometric measurement in a dual radial configuration. The dashed line is a one-to-one binding model fit. Since the target concentration is much higher than the dissociation constant (Kd), a characteristic kink at the stoichiometric point can be observed. [Figure 18] Figure 18 shows dose-response curves of 15 serial dilutions of the small molecule acetazolamide mixed with fluorescently labeled bovine carbonic anhydrase II obtained by ratiometric measurements in a dual radial configuration. The dashed lines are 1:1 binding model fits. The ratios vary by approximately 0.7%. [Figure 19] Figure 19A shows a dose response curve of a 16-point serial dilution of unlabeled monoclonal antibody Herceptin (Trastuzumab) mixed with a preformed complex of biotinylated Protein L and fluorescently labeled monovalent streptavidin obtained by ratiometric measurement in a dual radial configuration. Figure 19B shows unlabeled monoclonal antibody Herceptin (Trastuzumab) mixed with a preformed complex of biotinylated Protein L and fluorescently labeled monovalent streptavidin, thus resulting in a ternary complex. [Figure 20] Figure 20A shows a schematic diagram of the complex of maltose, biotinylated maltose binding protein, streptavidin, and fluorescently labeled biotinylated DNA. Figure 20B shows a dose-response curve between maltose and biotinylated maltose binding protein, which is fluorescently labeled by mixing with unlabeled streptavidin and fluorescently labeled biotinylated DNA, obtained by ratiometric measurement in a dual radial configuration. [Figure 21] FIG. 21 shows a dose response curve of 14-point serial dilutions of angiotensin-converting enzyme 2 (ACE2) mixed with 20 nM Cov-19 spike protein labeled by adding 5 nM fluorescently labeled therapeutic antibody CR3022. [Figure 22]22 shows four subsequent 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 for the four measurements, but appears to increase linearly, indicating that the protein is not stable at room temperature, but gradually denatures. [Figure 23] FIG. 23A shows the Kd time course curve for the interaction between the Cy5-labeled DNA aptamer for adenosine and the small molecule AMP. FIG. 23B shows the DNA hybridization between two 11-mer complementary DNA strands, one of which is labeled with Cy5, measured at 32° C. How quickly the Kd time course curve can follow the temperature perturbation of the IR laser indicates how fast the binding and dissociation kinetics of the interaction are. FIG. 23C shows the DNA hybridization between two 11-mer complementary DNA strands, one of which is labeled with Cy5, measured at 22° C. How quickly the Kd time course curve can follow the temperature perturbation of the IR laser indicates how fast the binding and dissociation kinetics of the interaction are. [Figure 24A]FIG. 24A shows normalized Kd time course curves for DNA hybridization between 22° C. and 32° C. The y-axis shows the fold increase in Kd throughout the measurement (all normalized to 1 for comparison). The x-axis shows the IR laser on-time. FIG. 24B shows Kd time course curves for DNA hybridization measurements with a dual emission configuration with 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 Kd value varies from approximately 10 nM to approximately 500 nM during the IR laser on-time. FIG. 24C shows the results of a van't Hoff analysis of two Kd values ​​at two different temperatures. Figures 24D and E show the thermodynamic parameters of the interaction obtained from classical van't Hoff analysis of fluorescence ratio measurements at six different sample temperatures (22°C, 24°C, 26°C, 28°C, 30°C, 32°C), which yields very similar thermodynamic parameters but takes longer than the IR laser thermodynamic measurements. [Figure 24B] FIG. 24A shows normalized Kd time course curves for DNA hybridization between 22° C. and 32° C. The y-axis shows the fold increase in Kd throughout the measurement (all normalized to 1 for comparison). The x-axis shows the IR laser on-time. FIG. 24B shows Kd time course curves for DNA hybridization measurements with a dual emission configuration with 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 Kd value varies from approximately 10 nM to approximately 500 nM during the IR laser on-time. FIG. 24C shows the results of a van't Hoff analysis of two Kd values ​​at two different temperatures. Figures 24D and E show the thermodynamic parameters of the interaction obtained from classical van't Hoff analysis of fluorescence ratio measurements at six different sample temperatures (22°C, 24°C, 26°C, 28°C, 30°C, 32°C), which yields very similar thermodynamic parameters but takes longer than the IR laser thermodynamic measurements. [Figure 24C]FIG. 24A shows normalized Kd time course curves for DNA hybridization between 22° C. and 32° C. The y-axis shows the fold increase in Kd throughout the measurement (all normalized to 1 for comparison). The x-axis shows the IR laser on-time. FIG. 24B shows Kd time course curves for DNA hybridization measurements with a dual emission configuration with 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 Kd value varies from approximately 10 nM to approximately 500 nM during the IR laser on-time. FIG. 24C shows the results of a van't Hoff analysis of two Kd values ​​at two different temperatures. Figures 24D and E show the thermodynamic parameters of the interaction obtained from classical van't Hoff analysis of fluorescence ratio measurements at six different sample temperatures (22°C, 24°C, 26°C, 28°C, 30°C, 32°C), which yields very similar thermodynamic parameters but takes longer than the IR laser thermodynamic measurements. [Figure 24D] FIG. 24A shows normalized Kd time course curves for DNA hybridization between 22° C. and 32° C. The y-axis shows the fold increase in Kd throughout the measurement (all normalized to 1 for comparison). The x-axis shows the IR laser on-time. FIG. 24B shows Kd time course curves for DNA hybridization measurements with a dual emission configuration with 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 Kd value varies from approximately 10 nM to approximately 500 nM during the IR laser on-time. FIG. 24C shows the results of a van't Hoff analysis of two Kd values ​​at two different temperatures. Figures 24D and E show the thermodynamic parameters of the interaction obtained from classical van't Hoff analysis of fluorescence ratio measurements at six different sample temperatures (22°C, 24°C, 26°C, 28°C, 30°C, 32°C), which yields very similar thermodynamic parameters but takes longer than the IR laser thermodynamic measurements. [Figure 24E]FIG. 24A shows normalized Kd time course curves for DNA hybridization between 22° C. and 32° C. The y-axis shows the fold increase in Kd throughout the measurement (all normalized to 1 for comparison). The x-axis shows the IR laser on-time. FIG. 24B shows Kd time course curves for DNA hybridization measurements with a dual emission configuration with 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 Kd value varies from approximately 10 nM to approximately 500 nM during the IR laser on-time. FIG. 24C shows the results of a van't Hoff analysis of two Kd values ​​at two different temperatures. Figures 24D and E show the thermodynamic parameters of the interaction obtained from classical van't Hoff analysis of fluorescence ratio measurements at six different sample temperatures (22°C, 24°C, 26°C, 28°C, 30°C, 32°C), which yields very similar thermodynamic parameters but takes longer than the IR laser thermodynamic measurements. [Diagram 25] FIG. 25A shows simulated Kd time course curves for different dissociation rates. The legend indicates the various off rates used in the simulation. It shows that dissociation rates from 10 s-1 to 0.001 s-1 can be resolved by a typical measurement involving 20 seconds of IR laser heating. FIG. 25B shows simulated Kd time course curves for different dissociation rates. The legend indicates the various off rates used in the simulation. It shows that even small differences from 0.036 s-1 to 0.154 s-1 can be resolved well. [Figure 26]Figure 26 shows measurements of slow binding kinetics using ratiometric fluorescence signal for a mix-and-measure approach of fluorescently labeled nanobody rapidly mixed with six different concentrations of CoV-19 spike RBD. 2 nM of fluorescently labeled nanobody is rapidly mixed with six different concentrations of CoV-19 spike RBD (20 nM to 625 pM). Ratiometric fluorescence measurements are then taken every 90 seconds to follow the slow binding kinetics. An overall fitting model can yield the k, k and K of the interaction. [Figure 27] FIG. 27A shows a schematic diagram of fluorescently labeled mRNA, lipid nanoparticles (LNPs) and cells. Ratiometric measurements can be used to determine the localization of fluorescently labeled mRNA molecules. FIG. 27B shows that all fluorescently labeled mRNA molecules are located within the LNP. FIG. 27C shows that all fluorescently labeled mRNA molecules are located in the chamber containing the buffer. FIG. 27D shows that all fluorescently labeled mRNA molecules are located within the cell. FIG. 27E shows the even distribution of labeled mRNA molecules within the LNP, buffer chamber and cell. [Figure 28] Figure 28A shows ratiometric measurements of LNPs loaded with fluorescently labeled mRNA in various states. Figure 28B shows that additional information of the state of the LNPs can be obtained by analyzing the fluorescence traces at a single wavelength (here: 670 nm), which reveal a "bumpy" aggregate trace obtained after a 20 minute centrifugation step, or a "smooth" trace obtained after 10 minutes of heating to 90°C. [Figure 29A]Figure 29A shows a schematic diagram of a complex of tetrameric streptavidin, two biotinylated fluorescently labeled linker molecules, and a biotinylated target molecule. Ratiometric measurements in a dual radial configuration can be used to determine (B) the stoichiometry of the complex of tetrameric streptavidin and a biotinylated fluorescently labeled linker molecule, and (C) the dose-response curve between the streptavidin-linker complex and the biotinylated target molecule. [Figure 29B] Figure 29A shows a schematic diagram of a complex of tetrameric streptavidin, two biotinylated fluorescently labeled linker molecules, and a biotinylated target molecule. Ratiometric measurements in a dual radial configuration can be used to determine (B) the stoichiometry of the complex of tetrameric streptavidin and a biotinylated fluorescently labeled linker molecule, and (C) the dose-response curve between the streptavidin-linker complex and the biotinylated target molecule. [Figure 29C] Figure 29A shows a schematic diagram of a complex of tetrameric streptavidin, two biotinylated fluorescently labeled linker molecules, and a biotinylated target molecule. Ratiometric measurements in a dual radial configuration can be used to determine (B) the stoichiometry of the complex of tetrameric streptavidin and a biotinylated fluorescently labeled linker molecule, and (C) the dose-response curve between the streptavidin-linker complex and the biotinylated target molecule. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] The present invention provides methods and devices for ratiometric characterization of intermolecular and / or intramolecular interactions and / or conformational modification and / or localization of fluorescently labeled particles.In particular, the method of the present invention provides improved sensitivity in detecting variations in the fluorescence spectrum of fluorescent labels within small sample volumes of fluorescently labeled particles that could not be resolved by previously known methods (see, for example, Example 1).In addition, the method of the present invention does not necessarily rely on temperature-induced fluorescence spectrum changes, and therefore allows even short-term measurements of temperature-sensitive and / or unstable samples.In combination with a defined temperature perturbation, the method of the present invention allows the measurement of thermodynamic and kinetic parameters of the interaction.

[0046] The method of the present invention allows to determine the localization of fluorescently labeled particles (i.e., whether the fluorescently labeled particles are located within lipid nanoparticles (LNPs) and / or cells and / or in the buffer solution surrounding the LNPs and cells). In the method of the present invention, fluorescently labeled particles are used that are labeled with only one fluorescent label. "Labeled with only one fluorescent label" means "labeled with only one type of fluorescent label" in this specification. This, in turn, can be labeling with only one single fluorescent moiety (e.g., one Cy5 molecule) or with two or more fluorescent moieties of only one type (e.g., two or more Cy5 molecules).

[0047] In accordance with the present invention, the shift in the fluorescence spectrum of fluorescently labeled particles is measured and ratiometrically analyzed to characterize interactions, including binding affinities, of said fluorescently labeled particles.

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

[0049] Since the detection of the two radiation wavelengths is preferably performed simultaneously and therefore the same disturbance values ​​or errors affect the measured value, the ratiometric characterization results in the extraction of pure information and thus improves the resolution of the measurement method.

[0050] The inventors have discovered that variations in the fluorescence spectrum of a fluorescent label attached to a particle, such as a biomolecule, can be used to determine, inter alia, the conformational state (folded / unfolded state) and / or interaction parameters between the ligand and the biomolecule.

[0051] In the context of the present invention, the terms "detected" and "recorded" are used interchangeably and refer to the determination of the fluorescent signal of a fluorescently labeled particle. In a first aspect, the present invention relates to a method for the characterization of fluorescently labeled particles in solution, for example by analyzing the variations in the fluorescence spectrum of the fluorescently labeled particles at a given temperature.

[0052] The method of the first aspect of the present invention comprises the steps of: a) providing a sample of fluorescently labeled particles in a solution under first conditions; b) exciting the fluorescently labeled particles at a first wavelength; c) detecting the fluorescent emission intensity of the fluorescently labeled particles at a second and a third wavelength; d) calculating a ratio between the fluorescence intensities at a second and a third wavelength, the third wavelength being different from the second wavelength; e1) repeating steps b) through d) on said sample of fluorescently labeled particles under second conditions; or e2) repeating steps a) to d) on a second sample of fluorescently labeled particles under second conditions, the second condition is different from the first condition; and f) characterizing the fluorescently labeled particles based on the calculated ratios obtained for the different conditions, a second wavelength and a third wavelength are detected simultaneously, the second wavelength being shorter than a maximum emission of the fluorescent emission of the fluorescently labeled particle under the first condition, and the third wavelength being longer; Includes. particle 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 or eukaryotic cells) or subcellular fragments, biological tissues, virus particles, virus-like particles or viruses and organelles, lipid nanoparticles (LNPs), etc. Nanoparticles also include nanodiscs. Nanodiscs are synthetic model membrane systems consisting of a lipid bilayer of phospholipids with hydrophobic ends interrupted by two amphipathic proteins.

[0053] The biomolecule is 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 biomolecule is selected from the group consisting of proteins, peptides, enzymes, nucleic acids and combinations or complexes thereof.

[0054] Preferably, the particle (in the labeled particle) is a biomolecule, most preferably a protein or a nucleic acid. The protein is selected from the group consisting of enzymes (e.g., carbonic anhydrase, beta-lactamase TEM1, or kinases such as MEK1 and p38), transport proteins (e.g., MBP), inhibitory proteins (e.g., beta-lactamase inhibitory protein BLIP, anakinra), structural proteins, signal transduction proteins, ligand binding proteins, chaperones (e.g., heat shock protein HSP90), antibodies (e.g., trastuzumab), membrane proteins and receptors (e.g., interleukin 1 receptor).

[0055] Nucleic acids include DNA, RNA (e.g., mRNA, tRNA, rRNA, etc.), LNA and PNA. Modified (e.g., chemically modified) nucleic acids may also be analyzed in accordance with the present invention. Locked Nucleic Acids (LNA), often also referred to as inaccessible RNA, are modified RNA nucleotides. The ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2' oxygen and the 4' carbon. Peptide Nucleic Acids (PNAs) are artificial synthetic polymers similar to DNA or RNA. DNA and RNA have deoxyribose and ribose sugar backbones, respectively, while the backbone of PNA consists of peptides such as repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. The various purine and pyrimidine bases are linked by methylene bridges (-CH 2 -) and a carbonyl group (-(C=O)-).

[0056] In the context of the present invention, nanoparticles are particles with an average size of less than 100 nm. The term "average size" describes the average effective diameter measured by dynamic light scattering, for example using a Brookhaven Instruments 90Plus or a Malvern Zetasizer Z90 particle sizer. Preferably, the nanoparticle size is in the range of 1 nm to 100 nm, preferably 1 to 70 nm. Nanoparticles can be organic or inorganic particles. Nanoparticles may also exist as composite particles, for example an inorganic core with organic molecules attached to the surface.

[0057] Microparticles are microscopic particles with the longest dimension less than 1 mm, but usually greater than 100 nm. Sizing methods using transmission electron microscopy (TEM), scanning electron microscopy (SEM) and quasi-elastic light scattering (QELS) can be used to characterize microparticles. Microparticles may also exist in the form of microbeads.

[0058] The microparticles may be, for example, coated or uncoated silica / glass / biodegradable particles, polystyrene / coated / flow cytometry / PMMA / melamine / NIST particles, agarose particles, magnetic particles, coated or uncoated gold or silver particles or other metal particles, transition metal particles, biomaterials, semiconductors, organic and inorganic particles, fluorescent polystyrene microspheres, non-fluorescent polystyrene microspheres, composite materials, liposomes, cells, and the like.

[0059] Commercially available microparticles are available in a wide variety of materials, including ceramics, glasses, polymers, and metals. Microparticles encountered in daily life include pollen, sand, dust, flour, and powdered sugar. In biological systems, microparticles are small membrane-bound vesicles that circulate in the blood, derived from cells in contact with the bloodstream, such as platelets and endothelial cells.

[0060] Microbeads are solid plastic particles manufactured, preferably less than 5 mm in their largest dimension, and can be uniform polymer particles, typically between 0.5 and 500 μm in diameter.

[0061] The term "modified particles" or "modified beads" particularly relates to beads or particles that contain or are linked to molecules, preferably biomolecules. It also includes the coating of such beads or particles with these molecules (biomolecules).

[0062] The particles or beads according to the present invention may be modified, for example, so that biomolecules, such as DNA, RNA or proteins, may be able to bind (specifically bind and / or covalently bind in some embodiments) to the particles or beads. Therefore, the analysis of the characteristics of the beads and / or particles, and in particular of the molecules bound or linked to such beads or particles, is within the scope of the present invention. In particular, such molecules are biomolecules. Thus, the term "modified (micro)beads / (nano or micro)particles" particularly relates to beads or particles that contain additional molecules to be analyzed or characterized. Modified or unmodified microparticles / (nano or micro)particles may be able to interact with other particles / molecules in solution, such as biomolecules (e.g., DNA, RNA or proteins).

[0063] The preferred concentration of the fluorescently labeled particles used in the present invention is preferably 10 pM to 10 μM, even more preferably 50 pM to 500 nM. In the method of the present invention, the concentration of the fluorescently labeled particles in the solution is preferably 50 pM to 500 nM.

[0064] In the method of the present invention, a labeled particle is used, which is labeled with at least one fluorescent label, preferably exactly one fluorescent label. In the context of the present invention, a particle labeled with two or more fluorescent labels is labeled with only one type of fluorescent label (i.e., only a single type of label per particle). In the context of the present invention, "labeled particle" refers to a fluorescently labeled particle or other particle that can be detected by fluorescence means, such as a molecule / particle that contains an intrinsic fluorophore, or a particle / molecule that is tagged with a fusion protein, or a particle / molecule that is bound to an exogenous fluorophore.

[0065] In particular, the labeled particles are preferably particles that are bound to a label, e.g., covalently attached (e.g., by NHA labeling, maleimide labeling, etc.); reversibly attached to a label across a high affinity protein tag, e.g., a HIS tag, an AVI tag, a SPOT tag, a SNAP tag, etc.; or bioconjugated by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), strain-promoted azide-alkyne cycloaddition (SPAAC), etc. (also known as click chemistry) or the like.

[0066] Protein tags are peptide sequences genetically grafted onto recombinant proteins. These include poly(His) tags, polyanionic amino acids, epitope tags such as FLAG tags, V5 tags, Myc tags, HA tags and NE tags, tags that can allow specific enzymatic modification (e.g. biotinylation with biotin ligase) or chemical modification (e.g. reaction with FlAsH-EDT2 for fluorescent imaging). Fluorescent labeling In the context of the present invention, the terms "label" and "dye" are used interchangeably and refer to fluorophores / fluorescent dyes, ie fluorescent chemical compounds that re-emit light upon excitation.

[0067] Labels useful in the present invention are those that are sensitive to environmental changes, i.e., the fluorescence spectrum of the dye shifts upon environmental changes, e.g., changes in the chemical microenvironment (ligand binding, conformational changes) and / or macroenvironment (e.g., location within the LNP versus location within a cell), and / or temperature changes (e.g., heating or cooling).

[0068] In the context of the present invention, fluorescent labels are advantageously attached to particles in close proximity to locations / positions on said particles where binding interactions are predicted to occur, such as binding pockets of proteins.

[0069] Fluorescent labels for use with the present invention may be selected from the group consisting of intrinsic fluorescent labels, fusion proteins, exogenous fluorescent labels, and the like. Intrinsic fluorescent labels include tryptophan, tyrosine, and phenylalanine residues. The fusion protein may be selected from the group consisting of blue-emitting fluorescent protein, cyan-emitting fluorescent protein, green-emitting fluorescent protein, yellow-emitting fluorescent protein, and red-emitting fluorescent protein, etc. Reference is made to FPbase, a database well known in the art, which 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).

[0070] In a preferred embodiment of the invention, the fluorescent label is an exogenous fluorescent label. Extrinsic fluorescent labels may include, but are not limited to, commercially available labels such as cyanine dyes including Cy5, Cy3, Atto647, Atto647N, Alexa647 Dy647, and the like.

[0071] Preferred exogenous fluorescent labels are environmentally sensitive dyes, for example as described in WO2018 / 234557, which is incorporated herein by reference. Environmentally sensitive dyes are known in the 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, WO2018 / 234557 relates to fluorescent labels that are highly sensitive to environmental changes, such as changes in chemical composition, temperature changes, etc. According to a preferred embodiment, these dyes are selected from the group consisting of NanoTemper red, green and blue dyes (e.g., commercially available as protein labeling kits from NanoTemper Technologies GmbH, Munich, Germany).

[0072] By labeling with exogenous fluorescent labels, it can be controlled so that only one dye binds to the target molecule and only that dye needs to be affected by ligand binding. Furthermore, the labeling chemistry can be adapted so that the dye can be placed in a location that is optimal for detecting changes in the chemical environment (e.g., proximal to the ligand binding site). Furthermore, the exogenous dye is usually located on the protein surface and therefore has ideal exposure to sense changes to the chemical microenvironment. The dye fluorescence range can be selected so that it does not interfere with the autofluorescence of the ligand. Finally, the exogenous dye is brighter and measurements can be performed at lower concentrations of the target molecule, thus reducing sample consumption and allowing measurements of even picomolar affinities.

[0073] The variation in the fluorescence spectrum according to the present invention includes a change in the fluorescence intensity of the fluorescent labels, but also a spectral shift (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 band-pass filters. The intensities detected at these different wavelengths / wavelength ranges and their respective ratios allow the detection of a spectral shift, a spectral broadening and / or a narrowing of the total emission spectrum.

[0074] In the context of the present invention, the spectral shift preferably comprises a bathochromic (ie red) shift and / or a hypsochromic (ie blue) shift. In the context of the present invention, the magnitude of the spectral shift is preferably at least 50 pm, more preferably at least 100 pm, and even more preferably at least 500 pm.

[0075] According to the present invention, the fluorescence intensity of the fluorescently labeled particle is preferably changed due to a mechanism selected from the group consisting of a conformational change of the fluorescently labeled particle, relocalization of the fluorescently labeled particle, interaction of the fluorescently labeled particle with one or more ligands, or a combination thereof. Sample Chamber The sample used in the present invention is preferably provided in a sample chamber, preferably selected from the group consisting of capillaries, multi-well plates, microfluidic chips, cuvettes, reaction tubes, pipette tips, microfluidic technology, droplets, natural tissues, organelles, 3D printed tissues, 3D printed organelles and translucent containers. The translucent containers can be glass or plastic containers.

[0076] In a preferred embodiment of the invention, a sample containing fluorescently labeled particles is provided in a capillary. In another preferred embodiment of the invention, the sample containing the fluorescently labeled particles is provided in a multi-well plate, such as a 96-well, 384-well or 1536-well plate.

[0077] Preferably, the capillary is made from at least one of the following elements: glass and / or polymer, and / or borosilicate glass, borosilicate 3.3 glass (e.g. DURAN glass), quartz glass 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 polymer may include PTFE, PMMA, Zeonor™ Zeonex™, Teflon AF, PC, PE, PET, PPS, PVDF, PFA, FEP, and / or acrylic glass.

[0078] In particular, at least one range of the capillary is preferably transparent for light having a wavelength of 200 nm to 1000 nm, preferably 250 nm to 900 nm. It is also particularly preferred, but not limited to, that said range of the capillary is transparent for light having the following wavelength ranges: 940 nm to 1040 nm (preferably 980 nm + / - 10 nm), 1150 nm to 1210 nm, 1280 nm to 1600 nm (preferably 1450 nm + / - 20 nm and / or 1480 nm + / - 20 nm and / or 1550 nm + / - 20 nm), 1900 nm to 2000 nm (preferably 1930 nm + / - 20 nm). The skilled person will also understand that the transparent range may extend over the entire capillary. In other words, the capillary may be transparent, preferably integrally made from one of the materials listed above.

[0079] Preferably, the capillary used has an inner diameter of 0.1 mm to 0.8 mm, preferably 0.2 mm to 0.6 mm, more preferably 0.5 mm. The outer diameter of the preferred capillary is preferably 0.2 mm to 1.0 mm, preferably 0.3 mm to 0.65 mm.

[0080] The geometry of the capillary is not limited to a certain shape. Preferably, a tube-like capillary with a circular or elliptical cross section is used. However, it is also possible to use capillaries with various cross sections, for example, triangular, rectangular, pentagonal or polygonal. Preferably, the capillary includes one of the specific cross sections throughout the length of the capillary. Furthermore, it is further preferred that the inner and / or outer dimensions of the capillary are constant along the entire length of the capillary. For example, a cylindrical (tubular) capillary preferably includes the same inner diameter and the same outer diameter along the entire length of the capillary. In other words, capillaries with diameters and / or cross sections that are constant or not constant over the length of the capillary can be used.

[0081] In particular, the sample chambers used in the present invention exhibit low autofluorescence over a broad spectral range, preferably below 20%, more preferably below 10%, even more preferably below 5%.

[0082] It is advantageous to provide the sample probe in a chamber having a thickness in the direction of the fluorescence excitation beam 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. The skilled person will understand that the term chamber also relates to, for example, a capillary, a microfluidic chip or a multiwell plate. Silicon Surface Preferred surfaces on which the sample chamber is placed are described, for example, in WO2017 / 055583, which is incorporated herein by reference. In particular, WO2017 / 055583 relates to silicon surfaces on which the sample chamber (e.g., capillary) of the present invention is preferably placed. Sample volume 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, even more preferably between 1 μl and 25 μl. sample Typically, the sample used in the method of the present invention is a solution containing fluorescently labeled particles and a ligand. Herein, the labeled particles can be dissolved or dispersed in the solution.

[0083] The labeled particles may be immobilized on a solid support that is brought into contact with a solution containing the ligand. Preferably, the labeled particles are dissolved or dispersed in a solution selected from the group consisting of organic and / or aqueous solutions, in particular aqueous buffer solutions. The aqueous buffer solutions are preferably adjusted to a pH value of 2 to 10, more preferably 4 to 10, even more preferably 5 to 9, most preferably 6 to 8.5 using a buffer solution. Fluorescence measurement (excitation / emission) According to the present invention, a preferred means for exciting, preferably fluorescently exciting, the labeled particles / molecules can be any suitable device selected from the group consisting of laser, fiber laser, diode laser, light emitting diode (LED), halogen, LED array, HBO (HBO lamps are, e.g., short arc lamps where a discharge arc ignites in a gas of mercury vapor under high pressure), HXP (HXP lamps are, e.g., short arc lamps where a discharge arc ignites in a gas of mercury vapor under very high pressure, in contrast to, e.g., HBO lamps, which operate at substantially higher pressures and which use the halogen cycle. HXP lamps produce visible light with a significant portion of UV and red light), etc.

[0084] Preferably, in the context of the present invention, the excitation light source allows for highly focused excitation. In the context of the present invention, the excitation light source is preferably a laser, even more preferably an LED. The term "fluorescence" as used herein is not limited to "fluorescence" per se, but the means, methods and devices disclosed herein are and can be used by other means, particularly the use of luminescence, e.g. phosphorescence. Thus, the term "exciting the fluorescently labeled particles at a first wavelength" in step b) may include the corresponding excitation of luminescence, e.g. the excitation is performed at a shorter wavelength than the subsequent detection of the emission, in relation to the "excitation step" in the above-specified method. Therefore, the term "detecting the fluorescent emission intensity of the fluorescently labeled particles at a second and third wavelength" in relation to the present invention refers to the step of detection of said emission after excitation. The skilled artisan will recognize that in relation to the present invention, the "excitation" wavelength and the "emission" wavelength must be separated. In addition, the skilled artisan will recognize that in relation to the present invention, the third wavelength detected is different from the second wavelength detected. The two signals required for ratiometric analysis can be obtained by using either a "dual excitation" or a "dual emission" configuration.

[0085] Typically, ratiometric analysis is based on a "dual emission" configuration, for example by using an exemplary dual emission optical system provided in FIG. 8B. In particular, one or more samples containing fluorescently labeled particles are excited at a single constant wavelength, and their emission spectra are detected at two different wavelengths (see Examples 1 and 2 with FIGS. 10 and 13, respectively, and Examples 4-12 with FIGS. 17-28). By using the "dual emission" configuration, two emission signals can be detected at the same location and at the same time. Furthermore, many fluorescent labels (e.g., Cy5) have excitation peaks of fewer seconds, which can be used for efficient excitation while allowing sufficient bandwidth at larger wavelengths to split the emission spectrum in two (FIG. 9A).

[0086] In a preferred embodiment of the invention, the "dual emission" configuration is used in combination with "red" fluorescent labels, such as Cy5, RFP, etc. Such labels have their maximum excitation at approximately 650 nm, a secondary excitation peak at approximately 600 nm, and a maximum emission at approximately 660 nm, and well-suited excitation and emission wavelengths include excitation at approximately 570 nm to 615 nm, detection of the first emission at approximately 625 nm to 650 nm, and detection of the second emission at approximately 670 nm to 725 nm (FIG. 9A). Exemplary components that can be used for the "dual emission" configuration are shown in Table 1.

[0087] [Table 1]

[0088] According to the present invention, the second wavelength is preferably detected at a wavelength shorter than the maximum emission of the fluorescently labeled particle under the first condition, and the third wavelength is detected at a wavelength longer than the maximum emission of the fluorescently labeled particle under the first condition. Those skilled in the art will recognize that the maximum emission in the context of the present invention can be a local maximum emission or an absolute maximum emission. Alternatively, the detection can be near a saddle point of the emission spectrum, rather than the maximum emission.

[0089] Small changes (eg, wavelength shifts) in the region adjacent to the emission maximum have a relatively large effect (eg, in terms of intensity) on the variation of the fluorescence spectrum. Thus, in a preferred embodiment of the invention, the emitted fluorescence intensity is detected close to the maximum emission, e.g., the second wavelength is detected at a wavelength at least 2.5 nm shorter (e.g., 10 nm shorter) and the third wavelength is detected at a wavelength at least 2.5 nm longer (e.g., 10 nm longer) than the maximum emission of the fluorescently labeled particle under the first conditions.

[0090] According to the present invention, a preferred means for detecting excited fluorescently labeled particles, in particular for detecting fluorescence, can be any suitable device selected from the group consisting of a charge-coupled device (CCD) camera (2D or line-scan CCD), a line camera, a photomultiplier tube (PMT), a silicon photomultiplier tube (siPM), an avalanche photodiode (APD), a photodiode array (PDA), a complementary metal-oxide semiconductor (CMOS) camera, and the like. Alternative Fluorescence Measurements In another embodiment of the present invention, the ratiometric analysis is based on a "dual excitation" configuration, for example by using the exemplary dual excitation optical system provided in Figure 8D. In particular, one or more samples containing fluorescently labeled particles are excited at two different wavelengths and their emission spectra are detected at a single wavelength (see Example 3 with Figure 16). By using this configuration, the two excitation spectra cannot be detected simultaneously, which means that they need to be acquired sequentially. This approach is more time-consuming, and the time delay between two subsequent measurements may cause substantial differences between the measurements, for example due to bleaching events induced after the first excitation, sample aggregation, etc.

[0091] Some of the problems listed above can be solved by using a "stroboscopic" excitation technique, where two excitation light sources are switched on and off in less than one second or even faster, and data are collected alternately. However, two different excitation light sources may result in different bleaching rates of the sample, which can have a negative effect on the evaluation of the ratiometric signal when dealing with longer acquisition times.

[0092] In another embodiment of the invention, a "dual excitation" configuration is used in combination with "green" fluorescent labels, such as Cy3, GFP, etc. Since such labels have their maximum excitation at approximately 540 nm, maximum emission at approximately 560 nm, and secondary emission peak at approximately 600 nm, well-suited excitation and emission wavelengths include excitation at approximately 475 nm to 495 nm, detection of the first emission at approximately 550 nm to 575 nm, and detection of the second emission at approximately 590 nm to 680 nm (Figure 9B). Exemplary components that can be used for the "dual emission" configuration are shown in Table 2.

[0093] [Table 2]

[0094] The excitation volume is typically the portion of the sample volume that is fluorescently excited by the excitation light source. The detection volume is the portion of the sample volume from which the emission spectrum is detected. According to the present invention, the excitation volume and / or detection volume is preferably a volume with a size of 2 mm x 2 mm x 5 mm or less, more preferably 1 mm x 1 mm x 5 mm or less, even more preferably 0.5 mm x 0.5 mm x 5 mm or less.

[0095] However, the means for exciting the fluorescently labeled particles and for detecting the fluorescence of said excited particles are not limited and any suitable means known to those of skill in the art may be used. Ratiometric Characterization According to the present invention, the ratiometric analysis is preferably based on constructing the ratio between the fluorescence intensities detected at the second and third wavelengths by point-wise division. Those skilled in the art will recognize that in the context of the present invention, said ratio of fluorescence intensities can be obtained by dividing the third wavelength by the second wavelength or vice versa (e.g. dividing the second wavelength by the third wavelength).

[0096] In the context of the present invention, exemplary relative percent changes in the ratio (i.e., the percent change in the ratio after the spectral shift) are at least 0.5%, at least 1.1%, at least 5.5%, at least 37.3%. Preferably, the relative percent change herein is at least 3%. Characterizing the Interaction In the context of the present invention, the interactions of fluorescently labeled particles include, in particular, for example, biomolecules with further (bio)molecules, particles, beads, and their conformation for the stability, folding and unfolding of (bio)molecules, or their chemical environment (e.g., their location in aqueous solution, lipid nanoparticles or cells). Further interaction characterizations include equilibrium measurements, binding kinetic measurements, and measurements of thermodynamic parameters.

[0097] According to the present invention, the calculated ratio is preferably a function of the localization of the fluorescently labeled particles, or the dissociation constant, half maximal effective concentration (EC 50 ), equilibrium constants, binding kinetics, enzyme kinetics, thermodynamic parameters, stability parameters (e.g., thermal denaturation of a protein, chemical denaturation of a protein, etc.), unfolding or refolding kinetics, ring-opening and ring-closing reactions, and / or combinations thereof.

[0098] In the context of the present invention, the skilled artisan will determine the dissociation constant (K d The half effective concentration (EC) can be determined from the resulting fluorescence intensity ratio by fitting the data to the Langmuir equation. 50) can be determined from the resulting fluorescence intensity ratio by fitting it to 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).

[0099] According to the present invention, the first and second conditions of the fluorescently labeled particles may differ in their chemical composition and / or temperature and / or localization within a chemical macroenvironment (e.g., the first condition of the fluorescently labeled particles relates to the location of the particles within a first carrier, e.g., a vector, and the second condition relates to the location of the particles within a second carrier, e.g., a recipient or in a buffer solution containing both carriers).

[0100] Also, the fluorescence spectrum of fluorescent labels according to the present invention may change when the fluorescently labeled particle / molecule is present in a complex with one or more other molecules, such as ligands (e.g., through the proximity of the ligand (see Figure 3B) and / or conformational changes upon binding of the ligand (see Figure 3B)).

[0101] Thus, in a preferred embodiment of the invention, the second condition may be varied by adding ligand and / or different concentrations of ligand, and the resulting calculated ratios are used to plot the dose-response curves and dissociation constants (K) of the fluorescently labeled particles and ligand. d ) is used to determine

[0102] In the context of the present invention, the term "bonding" of a ligand to a labelled particle preferably refers to binding by covalent bonds or by intermolecular forces such as ionic bonds, hydrogen bonds and van der Waals forces.

[0103] Ligand binding to target biomolecules such as proteins can result in a wide range of conformational changes, such as the movement of amino acid side chains, loops or domains. Ligands that can be used according to the present invention can be selected from (but are not limited to) the group consisting of ions, metals, compounds, drug fragments (small chemical fragments that can only weakly bind to biological targets), carbohydrates, small molecules (organic compounds with low molecular weights (less than 900 Daltons); small molecules help regulate biological processes and can usually have sizes on the order of 1 nm), drugs, prodrugs, lipids, proteins, peptides, peptoids, enzymes, nucleic acids, aptamers, nanoparticles, liposomes, unilamellar vesicles (including small unilamellar vesicles (SUVs) and giant unilamellar vesicles (GUVs)), polymers, organic molecules, inorganic molecules, metal complexes, hormones, fragrances, odorants, particles and (micro)beads. Preferably, the ligand is selected from the group consisting of ions, metals, compounds, drug fragments, carbohydrates, small molecules, drugs, prodrugs, lipids, proteins, peptides, peptoids, enzymes, nucleic acids, aptamers, hormones, fragrances and odorants.

[0104] The concentration of the ligand is preferably 0.01 pM to 1 M, preferably 1 pM to 100 mM, and more preferably 1 pM to 10 mM. Combining Temperature Related Intensity Change (TRIC) and Microscale Thermophoresis (MST) Ratiometric analysis of fluorescently labeled particles according to the present invention does not necessarily rely on temperature induced fluorescence intensity changes, although fluorescence intensity measurements can be made at a constant, predefined temperature or during defined temperature perturbations.

[0105] In a second aspect, the present invention relates to a method for the characterization of fluorescently labeled particles in solution by analyzing the variations in the fluorescence spectra of the fluorescently labeled particles in combination with a defined temperature perturbation.

[0106] The method of the second aspect of the present invention comprises the steps of: a) providing a sample of fluorescently labeled particles in a solution under first conditions; b) exciting the fluorescently labeled particles at a first wavelength; c) detecting the fluorescent emission intensity of the fluorescently labeled particles at a second and a third wavelength, the intensity is detected during a defined temperature perturbation; d) calculating a ratio between the fluorescence intensities at a second and a third wavelength, the third wavelength being different from the second wavelength; e1) repeating steps b) through d) on said sample of fluorescently labeled particles under second conditions; or e2) repeating steps a) to d) on a second sample of fluorescently labeled particles under second conditions, the second condition is different from the first condition; and f) characterizing the fluorescently labeled particles based on the calculated ratios obtained for the different conditions, a second wavelength and a third wavelength are detected simultaneously, the second wavelength being shorter than a maximum emission of the fluorescent emission of the fluorescently labeled particle under the first condition, and the third wavelength being longer; Includes.

[0107] In the method of the preferred embodiment of the second aspect of the present invention, the heating or cooling can be performed using a tempering element (i.e., a heating and / or cooling source) selected from the group consisting of a heating and / or cooling liquid or gas, a heating element (e.g., a heating resistor or other element based on Joule heating such as a metal heating element, a ceramic heating element, a polymer PTC heating element, a composite heating element, a semiconductor heating element) or a thermoelectric element, e.g., a Peltier element, or electromagnetic radiation (e.g., an LED, e.g., an IR-LED, or a laser, e.g., an IR laser, or a microwave). The use of an IR laser allows for fast sample heating.

[0108] Peltier elements are preferably used because they 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 current through the Peltier element. Peltier elements are one of the few elements that can be heated but also actively cooled below room temperature.

[0109] Lasers, preferably lasers whose electromagnetic radiation is directly absorbed by the sample, are preferably used since the temperature can be changed rapidly and directly in the sample, without mechanical contact to the sample.

[0110] In addition, it is preferable that the laser is a high-output laser in the range of 0.01W to 10W, preferably 4W to 6W. Moreover, it is preferable that the laser is within a range of 1 mW to 1 W, preferably 1 mW to 500 mW, and more preferably 1 mW to 250 mW.

[0111] The laser radiation is directly absorbed by the sample and converted to heat, for example IR laser light with wavelengths of 980nm+ / -30nm, 1480nm+ / -30nm, 1550nm+ / -30nm, 1940nm+ / -30nm is very well absorbed by water and heats up very quickly. This heating method can be non-contact and therefore fast and without the risk of contamination. The sample chamber must only be transparent for the laser light, but does not require good thermal conductivity in contrast to contact heating by means of heating elements.

[0112] IR lasers can heat even very small volumes (eg, in the nanoliter volume range) whose fluorescence is measured by fluorescence optics (typically just 100 μm×100 μm×100 μm=1 nl volume).

[0113] According to the invention, the sample to be investigated may also be subjected to a linear temperature gradient by heating and / or cooling a tempering element at a defined constant rate, e.g. 1° C. / min or 1 K / min. Typically, the heating and / or cooling rate is between 0.1 K / min and 50 K / min, e.g. using contact heating by a Peltier element.

[0114] In another embodiment, the sample can be heated by an IR laser ("optical heating") with typical heating rates of 1 K / s to 100 K / s. The process according to the second aspect of the present invention is preferably carried out within a temperature range of -20°C to 160°C, more preferably 0°C to 120°C.

[0115] Preferred data acquisition times for measurements of the initial ratio are 1 to 5 seconds, and for ratios obtained during temperature perturbations are 5 to 20 seconds, although acquisition times may also be shorter, e.g., only 10 to 100 milliseconds, or longer, e.g., minutes, hours or even days.

[0116] The ratio can also be analyzed at any later time after the temperature change, which can be beneficial if the amplitude is very small at room temperature but increases at higher temperatures (see Example 2 with FIG. 13A, where analysis in phase 3 results in a larger amplitude than analysis in phase 1).

[0117] In a preferred embodiment of the second aspect of the present invention, the ratiometric analysis is based on a "dual emission" configuration in combination with temperature perturbation, i.e., by using the exemplary dual emission optics and IR laser shown in FIG. 8A.

[0118] In another embodiment of the invention, ratiometric analysis is based on a "dual excitation" configuration in combination with temperature perturbation, for example, by using the exemplary dual excitation optics and IR laser shown in FIG. 8C.

[0119] In another embodiment of the present invention, ratiometric analysis is based on both "dual excitation" and "dual emission" configurations, i.e., by using an exemplary "dual excitation / dual emission" optical system shown in FIG. 8F.

[0120] In another embodiment of the invention, ratiometric analysis is based on both "dual excitation" and "dual emission" configurations in combination with temperature perturbation, for example, by using the exemplary "dual excitation / dual emission" optical system and IR laser shown in FIG. 8E. Determination of thermodynamic and kinetic parameters by combining ratiometric methods with TRIC / MST. In a third aspect, the present invention relates to a method for the characterization of thermodynamic and / or kinetic parameters of fluorescently labeled particles in solution by analyzing the variations in the fluorescence spectra of the fluorescently labeled particles in combination with defined temperature perturbations / changes.

[0121] In the context of the present invention, thermodynamic parameters include, but are not limited to, enthalpy, entropy, and heat capacity (Cp). In the context of the present invention, kinetic parameters include, but are not limited to, equilibrium constants, dissociation rates, association rates, enzymatic reaction rates, folding and unfolding rates, release rates (e.g., in the case of LNPs, the rate of payload release), aggregation rates, entry rates (e.g., the rate at which a payload such as mRNA enters a cell).

[0122] According to the third aspect of the invention, preferably the thermodynamic parameters of the interaction can be determined when collecting ratiometric fluorescence data with an IR laser from a single measurement. Since the sample temperature at each time point of the measurement is known (determined in a calibration measurement where the tray is heated in a controlled manner and the fluorescence of the reference label is measured), the dissociation constant K d can be obtained for each time point (see Example 2 in conjunction with FIG. 14A).

[0123] According to a third aspect of the invention, the thermodynamics of the reaction are also determined based on classical K d It is added that this can be determined by performing measurements, for example, by sequentially setting the sample temperature at 22°C, 24°C, 26°C, 28°C, 30°C and 32°C and performing binding affinity measurements for each temperature (see Example 10 in conjunction with Figure 24).

[0124] Even if the accuracy of this technique does not necessarily reach that of state-of-the-art isothermal titration calorimetry (ITC) measurements, the measurements are approximately 100 times faster and the very low sample consumption may outweigh the lack of accuracy. For example, for a particular molecule, information on entropy or enthalpy binders may be very valuable, especially during early development stages.

[0125] According to the third aspect of the invention, preferably the thermodynamic parameters of the interaction can be determined when collecting ratiometric fluorescence data with an IR laser from a single measurement. Application: Ratiometric methods combined with TRIC / MST to monitor the localization of fluorescently labeled mRNA. In a fourth aspect, the present invention relates to a method for the characterization of the localization of fluorescently labeled particles in a solution by analyzing the variations in the fluorescence of the fluorescently labeled particles, preferably in combination with or without a defined temperature perturbation / change.

[0126] In the context of the present invention, the terms "location" and "localization" are used interchangeably and refer to the determination of the place / position of a fluorescently labeled particle. All gene and cell therapy approaches involving nucleic acids, such as DNA and RNA, have inherent problems that determine the success of drug delivery (e.g., delivery of the carried nucleic acid to target cells by incorporation and release from carriers such as lipid nanoparticles (LNPs) dissolved in a buffer solution; see FIG. 27A).

[0127] Even before determining successful delivery, bioproduction (e.g., loading of the delivery system, including unloaded, partially loaded, fully loaded and / or overloaded states of LNPs) is a crucial step, and this step needs to be thoroughly evaluated.

[0128] According to the fourth aspect of the present invention, the carrier may be selected from the group consisting of metal nanoparticles and nanoconstructs, polymeric nanoparticles, lipid-based carrier systems (e.g., liposomes, other lipid-containing complexes), carbonaceous carriers, nanoemulsions, nanosuspensions, nanomicelles, dendrimers, milk-derived carriers, endosomes, viral vectors (e.g., adenoviruses, adeno-associated viruses (AAV), retroviruses), virus-like particles (VLPs), eukaryotic cells, prokaryotic cells, cell fragments, etc. In view of the above, combinations of said carriers are also within the scope of the present invention.

[0129] In a preferred embodiment of the fourth aspect of the invention, the fluorescently labeled particle is a fluorescently labeled mRNA and the carrier is a LNP. LNP refers to any particle with a diameter less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm. For example, in an appropriate buffer, an LNP filled with mRNA may have a hydrodynamic diameter of 65 nm to 85 nm. Alternatively, the nanoparticles may range in size from 1 to 1000 nm, 1 to 500 nm, 1 to 250 nm, 25 to 200 nm, 25 to 100 nm, 35 to 85 nm, or 25 to 60 nm.

[0130] LNP can be made of cationic, anionic or neutral lipid.Neutral lipid, such as membrane fusogenic phospholipid DOPE or membrane component cholesterol, can be included in LNP as "helper lipid" to improve transfection activity and nanoparticle stability.Limitations of cationic lipid include poor stability and low effectiveness for rapid clearance, as well as generation of inflammatory or anti-inflammatory response.

[0131] Additionally, LNPs can be composed of hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids. Any lipid or lipid combination known in the art can be used to produce LNPs. Examples of lipids used to produce LNPs are: DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE and GL67A-DOPE-DMPE-polyethylene glycol (PEG). Examples of cationic lipids are: 98N12-5, C12-200, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1 and 7C1.

[0132] Examples of neutral lipids are: DPSC, DPPC, POPC, DOPE and SM. Examples of PEG-modified lipids are PEG-DMG, PEG-CerC14 and PEG-CerC20.

[0133] Since the fluorescence spectrum of fluorescent labels is highly sensitive to environmental changes, e.g., changes in the chemical environment, and the spectrum fluctuates upon environmental changes, the location of fluorescently labeled particles (e.g., mRNA) can be inferred from ratiometric measurements according to the present invention.

[0134] As an example of a non-limiting combination of dyes with second and third emission wavelengths: if all fluorescently labeled mRNA molecules are properly located within the LNP, the ratio after characterization by this method corresponds to 2.1 (see Example 12 in conjunction with FIG. 27B). If all fluorescently labeled mRNA molecules are located outside the LNP, the resulting ratio corresponds to 1.2 (see Example 12 in conjunction with FIG. 27C).

[0135] Since the chemical environment inside a cell is very different from that in a LNP or a buffer solution, those skilled in the art will recognize that in the present invention, when all fluorescently labeled mRNA molecules are successfully delivered to a target cell, the ratio obtained will be different from the values ​​listed above (i.e., 1.2 and 2.1) (see Example 12 with FIG. 27D). Furthermore, in intermediate states, such as when the fluorescently labeled mRNA molecules are partially located in a cell, a LNP, and a buffer solution, the fluorescence ratio obtained is a linear combination of the ratios obtained for the three above-listed states.

[0136] According to this exemplary embodiment 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 and short experimental procedures.In the same context, currently applied methods known in the art (e.g., field flow fractionation and liquid chromatography) are based on various methods and require not only more time but also larger sample volumes, which are often limited and expensive. Kits for biotinylated molecules In a fifth aspect, the present invention relates to a kit and the use of the kit for the characterization of fluorescently labeled particles in solution, such as particles labeled with one or more biotin molecules (i.e. biotinylated particles), according to the method of the present invention.

[0137] The kit comprises as main components a defined stoichiometric ratio of (i) a (preferably tetrameric) biotin-binding protein comprising at least two (preferably four) binding sites for biotin, and (ii) a linking moiety (also referred to herein as a "linker"). The kit preferably further comprises an instruction manual describing the use of the kit in at least one of the methods of the invention.

[0138] According to a fifth aspect of the present invention, the biotin-binding protein may be selected from the group consisting of streptavidin, avidin and variants thereof. In the context of the present invention, variants include neutravidin, flavidin, bivalent streptavidin, etc.

[0139] A kit useful in the present invention may comprise a single vial containing (i) a biotin-binding protein and (ii) a linker, preferably modified / labeled with a biotin molecule at one end and a fluorescent label at the other end. It is a preferred embodiment of the fifth aspect of the present invention that the biotin-binding protein is preferably a tetrameric protein, even more preferably tetrameric streptavidin.

[0140] Streptavidin is a homotetramer with very high affinity for biotin (also known as vitamin B7). It is widely used in molecular biology and bionanotechnology due to the resistance of the streptavidin-biotin complex to organic solvents, denaturants (e.g., guanidinium chloride), detergents (e.g., SDS, Triton), proteolytic enzymes, and extremes of temperature and pH.

[0141] It is a further preferred embodiment of the fifth aspect of the invention that a fluorescent label is attached to a linker, for example as described in the art (NPL6). According to a fifth aspect of the present invention, the linker is a polymer, preferably a nucleic acid, preferably a single-stranded nucleic acid, more preferably a single-stranded DNA, 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.

[0142] However, the linkers used in the context of the present invention are not limited by their length (as long as the linker is long enough to enable the fluorophore to reach the target) and / or type (as long as the type of linker has the property that its length can be adjusted, e.g., DNA, aromatic rings containing aryl groups, etc.), and any suitable linker known to those of skill in the art can be used to indirectly attach a fluorescent label to a particle as characterized by the methods of the present invention.

[0143] Linkers for use according to the invention contain a biotin molecule at one end and a fluorescent label at the other end. In a preferred embodiment of the fifth aspect of the invention, the linker is modified with a biotin molecule at its 3' end and a fluorescent label at its 5' end, or vice versa.

[0144] Each kit may contain enough material for multiple labeling reactions. Depending on the size of the kit and the amount of biomolecule used, enough material may be provided for approximately 500 up to 3840 single-point ratiometric characterization experiments.

[0145] In a preferred embodiment of the fifth aspect of the present invention, the fluorescently labeled particle is a complex of a tetrameric streptavidin molecule, a biotinylated fluorescently labeled linker molecule, and a biotinylated target molecule (see FIG. 29A). It is particularly preferred that the fluorescently labeled particle is a complex of one tetrameric streptavidin molecule, two biotinylated fluorescently labeled linker molecules, and one biotinylated target molecule.

[0146] The stoichiometric ratio of tetrameric biotin-binding protein to modified linker is preferably adjusted so that, on average, two of the four binding sites on the tetrameric biotin-binding protein are occupied (e.g., by providing a vial with 2 nM streptavidin and 4 nM linker), leaving the remaining two binding sites available for a binding event with the biotinylated particle of interest.

[0147] Therefore, in the fifth embodiment of the present invention, it is further preferred that the tetrameric streptavidin and the linker are mixed in a ratio of 1:2, so that on average, one streptavidin molecule is labeled with two linker molecules, i.e., one streptavidin molecule has two fluorescent labels. 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 ratio of 1:1, it can be achieved that on average, only one biotinylated molecule is bound to the streptavidin-linker complex. However, stoichiometry can also be advantageously utilized for biotinylated dimeric proteins (e.g., biotinylated stimulator of interferon genes (STING), bivalent streptavidin), where a functional dimer can be labeled with one streptavidin-linker complex. In the case of a bivalent biotin-binding protein, the ratio of bivalent biotin-binding protein to modified linker can be adjusted such that, on average, one of the two free binding sites on the bivalent biotin-binding protein is occupied (e.g., by providing a vial with 2 nM streptavidin and 2 nM linker) and the remaining binding site is available for a binding event with the biotinylated particle of interest.

[0148] The exact stoichiometry can be confirmed during the labeling process by spectral shift measurements according to the invention (eg, by using a dual emission configuration) in which streptavidin is titrated against the linker.

[0149] A free linker molecule (i.e., a 12-mer poly-T strand labeled with biotin at its 3' end and Cy5 at its 5' end) has a ratio less than 0.8 (see FIG. 29B, free linker), streptavidin labeled with only one linker molecule has a ratio of approximately 1.05 (see FIG. 29B, one linker), while streptavidin labeled with two linker molecules has a higher ratio, i.e., approximately 1.15, corresponding to a peak in the biphasic dose-response curve resulting from the interaction of both fluorescent labels with the remaining two binding sites of the streptavidin molecule (see FIG. 29B, ideal ratio). When a 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 very high affinity, resulting in a characteristic kink at the stoichiometric point in the dose-response curve (see Figure 29C).

[0150] According to this exemplary embodiment of the present invention, very low final concentrations of kit components and biotinylated target molecule (e.g. 1 nM streptavidin, 2 nM linker and 1 nM biotinylated molecule) are used. Therefore, the kit according to the fifth aspect of the present invention is suitable for measuring picomolar affinity based on a highly controllable and reproducible labeling process (compared to the labeling kits known in the art, such as Protein His-tag labeling kit RED-Tris-NTA 2nd generation (NanoTemper Technologies)) by using the method of the present invention. In the same context, currently applied kits known in the art (e.g. Protein His-tag labeling kit RED-Tris-NTA 2nd generation) have further limitations, including buffer limitations, slow labeling binding kinetics, incompatibility with already biotinylated molecules, and in addition, the labeling kits known in the art are costly and labor-intensive. General Principles and Illustrative Aspects In the following, the general principles of the invention are explained in more detail and based on exemplary examples or preferred embodiments of the invention.

[0151] The fluorescence spectrum of a fluorescent label strongly depends on the microenvironment in which it is located, so the same label can exhibit very different fluorescence, for example, depending on the molecule or particle to which it is bound. In particular, the microenvironment on each protein around the binding site of the fluorescent label differs in terms of amino acid residues that can quench the label, collide with the label, transiently interact with the label, or cause stacking.

[0152] FIG. 1A shows the excitation spectra of four different proteins labeled with the same fluorescent label (Protein Labeling Kit RED-NHS 2nd Generation (NanoTemper Technologies)). Emission was detected at a wavelength of 690 nm. Excitation varied from 520 nm to 670 nm. Although labeled with the same fluorescent label, the maximum emission peak wavelengths of the four proteins are different, ranging from approximately 659 nm to approximately 664 nm (FIG. 1B).

[0153] From Figure 2A, it is evident that the same effect / phenomenon was detected when exciting four different proteins at a constant wavelength (i.e., 605 nm) and recording the emission from 620 nm to 750 nm. As above, the proteins were labeled with the same fluorescent labels, but the maximum emission peak wavelengths ranged from approximately 659 nm to approximately 664 nm (Figure 2B).

[0154] The microenvironment may further vary based on the location on the molecule / particle (e.g., the label is attached to a lysine residue of a protein, or the label is attached 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 a fluorescent label also has an effect on its fluorescence spectrum. For example, the fluorescence spectrum of the label differs depending on the location of the particle / molecule (e.g., in an aqueous buffer solution, in an LNP, inside a cell).

[0155] In addition, the fluorescence spectrum of the fluorescent label may also change when the fluorescently labeled particle / molecule is present as a complex with one or more other molecules (i.e., ligands). For example, ligand proximity (FIG. 3A) and / or conformational change upon ligand binding (FIG. 3B) may result in a shift (FIG. 3C) and / or broadening or narrowing (FIG. 3D) of the excitation or emission fluorescence spectrum of the fluorescent label. By using the ratiometric characterization method of the present invention, it is possible to detect not only changes in fluorescence intensity, but also changes in the complete absorption and emission spectrum upon perturbation of the microenvironment of the fluorescent label.

[0156] In view of the above, Figure 4 shows a shift (i.e., 3 nm) in the maximum emission peak wavelength of streptavidin (200 nM) when present in complex with the natural ligand biotin (2 μM) from approximately 664 nm to approximately 661 nm upon excitation at a wavelength of 605 nm. In the same row, Figure 5 shows a slight shift (i.e., less than 1 nm) in the maximum emission peak wavelength of lysozyme (100 nM) alone and in complex with the lysozyme inhibitor tri-N-acetyl-D-glucosamine (NAG3) (80 μM) upon excitation at a wavelength of 585 nm. The 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).

[0157] [Table 3]

[0158] Fluctuations in the excitation and / or emission spectrum of fluorescent labels can be so small (e.g., only a few Å) that it is not possible to measure / resolve them with the required accuracy by methods / devices known in the art. However, by the methods and devices of the present invention, even small changes in the fluorescence spectrum can be resolved. For example, in the case of the binding between carbonic anhydrase and furosemide (Figure 6A), a wavelength shift of only 50 pm, which results in a 0.5% fluorescence ratio change (Table 3), can be easily measured / detected, and a sigmoidal dose-response curve can be obtained (Figure 6B).

[0159] The variations in the fluorescence spectra are independent of the nature of the fluorescent label, i.e., they appear equally in endogenous and exogenous fluorescent labels. However, the degree of variation in the fluorescence spectra can be increased for certain classes of labels. Figure 7A shows the use of fluorescence ratio measurements of tryptophan fluorescence during a thermal melting gradient from 35 °C to 95 °C for characterization of the denaturation and binding affinity between native lysozyme and its inhibitor tri-N-acetyl-D-glucosamine (NAG3). Very high concentrations of NAG3 result in thermostabilization, i.e., a thermal shift of lysozyme. This shift can be used to determine the dissociation constant (K d ) cannot be obtained, but K d A sigmoidal dose-response curve revealing (here: at 35° C.) can be obtained when plotting the initial ratio at 35° C. against the concentration of NAG3 (FIG. 7B).

[0160] 8A-8F show various exemplary embodiments of a measurement device according to the present invention. In general, the device according to the present invention preferably includes a sample holder for holding a sample of fluorescently labeled particles in solution under multiple conditions. As mentioned above, the sample holder of the present invention can be a capillary, but is not limited to such a capillary. Also, other means for holding a sample can be used, such as a multi-well or a chip.

[0161] 8A-8F show examples of arrangements of optical elements to help direct light to a sample for excitation and to detect fluorescent emission from the sample, where the sample itself is not shown in the figures. Preferably, a sample container, e.g. a capillary, is located below lens 1. Said lens 1 is preferably an aspheric lens or a lens system with multiple lenses, also referred to below as objective lens.

[0162] The device 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 for providing excitation light may be provided. As explained above, the present invention is not limited to a single excitation light source. Alternatively, the use of one light source may be provided with a second excitation light source 16 or even additional light sources (not shown) (particularly for the "dual excitation" mode). The ratiometric analysis of the present invention may be obtained by using either a "dual excitation" arrangement or a "dual emission" arrangement. For the "dual emission" arrangement, it is preferred to provide at least one light source. For the "dual excitation" arrangement, it is preferred to provide two or even more light sources. However, the skilled person will further understand that multiple light sources may be provided for the "dual emission" arrangement. However, in this case, it may be sufficient if one of these light sources is used for excitation.

[0163] The first excitation light source 8 is preferably at least one of the group consisting of a laser, a fiber laser, a diode laser, an LED, an HXP, a halogen, an LED array, an HBO. The same applies to the second excitation light source 16.

[0164] Preferably, a first light separation element 7, e.g., a dichroic mirror, is used to direct the excited light to the sample and preferably to separate the fluorescence excitation light from the fluorescence emission light. Additional optical elements, e.g., a lens system 9, for directing the excitation light to the sample may be provided, e.g., to determine the beam quality of the excitation light source (e.g., one, two or more lenses). In addition, an excitation filter 10 may also be provided to filter the excited light, e.g., bandpass / longpass. Those skilled in the art will understand which type of filter is preferred for different light sources. Again, similar optical elements may be provided for the second excitation light source 16. For example, a lens system 17 may be provided to determine the beam quality of the second excitation light source 16, as shown, e.g., in Figs. 8C, 8D, 8E and 8F. Furthermore, a further light separation element 18, e.g., a dichroic mirror, may be used, e.g., to combine the light from the two different excitation light sources 8 and 16.

[0165] The device of the present invention also includes a means for detecting the fluorescence emission intensity of the fluorescently labeled particles. For the "dual emission" configuration, it is preferable to provide a means for detecting two different wavelengths, and for the "dual excitation" configuration, it may be sufficient if the means for detecting are configured to detect only a single wavelength or a single wavelength range. According to the present invention, it is preferable to provide at least one photodetector for each wavelength or wavelength range. For example, for the "dual excitation" configuration, it may be sufficient to provide a single photodetector 14 (see, for example, Figures 8C and 8D). For the "dual emission" configuration, it is preferable to provide two separate photodetectors 14 and 15, as shown in Figures 8A, 8B, 8E and 8F. However, the skilled person will further understand that two photodetectors may be provided for the "dual excitation" configuration. However, in this case, it may be sufficient if only one of these photodetectors 14 and 15 is used for emission detection. The first and / or second photodetector may be a photodetector from the group consisting of PMT, siPM, APD, CCD or CMOS camera.

[0166] Here too, additional optical elements, such as a light separation element 11, may be provided to separate the emission light to the first and second photodetectors 14 and 15. For example, FIG. 8B shows a preferred arrangement for a "single excitation", "dual emission" arrangement, with an excitation light source 8 and two photodetectors 14 and 15. The emission light to the individual detectors is separated by a light separation element 11, such as a dichroic mirror. Additional filters 12 and 13 upstream of the two detectors 14 and 15 may be provided to define different emission wavelengths, such as a second and a third wavelength, where the second wavelength is shorter than the maximum emission of the fluorescence emission of the fluorescently labeled particles under the first conditions and the third wavelength is longer. The emission filters 12 and 13 may be selected from any suitable type of filter element, such as a bandpass or longpass filter.

[0167] As shown in Figures 8A, 8C and 8E, a hot mirror 2 may additionally be provided, which is preferably used to direct IR light from the IR laser 3 to the sample. For example, the hot mirror 2 may provide high IR reflection and preferably visible light transmission >80%. The IR light source 3 is preferably at least one IR laser, preferably with emission wavelengths of, for example, 1455 nm, 1480 nm, 1550 nm and / or 980 nm. Furthermore, the power of the IR laser is preferably preferably 0.01 W to 10 W. To direct the IR light to the sample, further optical elements may be used, such as a laser fiber 4 (single mode or multimode), a laser fiber coupler 5 (with or without a collimator), and / or a beam shaping module 6 (for example a lens system including one, two or more lenses), for example to determine the laser beam diameter and focus (see Figures 8A, 8C and 8E). However, the input of IR light is merely optional for additional temperature-dependent measurements or additional measurements.

[0168] The device of the invention also comprises means for calculating a ratio between said fluorescence intensities at a second and a third wavelength, said third wavelength being different from said second wavelength, said means being preferably provided by a processor or a circuit comprising at least one processor. EXAMPLES

[0169] Example 1 The following example illustrates the difference between using the ratiometric characterization method known in the art compared to using the ratiometric characterization method according to the present invention to obtain dose-response curves between two molecules.Therefore, a sample containing fluorescently labeled DNA aptamer and adenosine monophosphate (AMP) was measured by a commercially available fluorescence spectrophotometer and by the dual emission configuration of the present invention. Sample preparation Fourteen 1:1 serial dilutions of unlabeled AMP were prepared. DNA aptamers were fluorescently labeled with Cy5 and added in equal amounts to the AMP serial dilutions to obtain a final sample concentration of 20 nM. The highest concentration of AMP corresponded to 5 mM. For measurements with a standard fluorescence microplate reader (CLARIOstar, BMG Labtech), 95 μl of sample was loaded into a microwell plate. For measurements with the dual radial configuration of the present invention, 5-10 μl of sample was loaded into polymer-coated borosilicate glass capillaries (Monolith NT.115 Premium Capillaries, MO-K025, NanoTemper Technologies). measurement For the microplate reader measurements, the samples were excited at a wavelength of 590 nm and the emission was first detected at wavelengths between 628 nm and 652 nm. Then the samples were excited again at a wavelength of 590 nm and the emission was detected at wavelengths between 665 nm and 725 nm. The following three fluorescence intensity measurements of the serial dilutions were performed. For the ratiometric measurements according to the invention, each sample was excited at a wavelength of 591 nm. The fluorescence traces were simultaneously measured at a second wavelength between 628 nm and 653 nm and a third wavelength between 665 nm and 727 nm. Ratiometric Data Analysis To characterize the interactions with a fluorescence microplate reader, the ratio between the fluorescence intensities per well was calculated manually by 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 sigmoidal dose-response curve provided in FIG. 10A starts at a ratio value of approximately 0.95 and ends at a ratio value of approximately 0.90. The dissociation constant (K d ) is located at approximately 20-30 μM. However, the signal-to-noise ratio (S / N) of the interaction is very low and the deviation between replicates is very large.

[0170] In contrast, by measuring the same sample with the method of the present invention, the dose-response curve provided in FIG. 10B shows an improved signal-to-noise ratio (S / N) and a K of interaction of 39.3 μM. d When using a lower sample concentration, i.e., 250 pM, the signal-to-noise ratio (S / N) for the ratiometric measurement according to the invention is still very good (19.8), and the K d can be easily determined (FIG. 10C).

[0171] In summary, this example demonstrates that the ratiometric characterization method according to the invention gives better data, even with approximately 1000 times fewer samples, compared to measurements with a plate reader, for which the noise is more than 10 times higher than the signal amplitude that needs to be measured.

[0172] Example 2 The following example illustrates the use of the ratiometric characterization method according to the present invention to obtain a dose-response curve between two molecules based on a dual emission configuration. Thus, a sample containing a fluorescently labeled DNA aptamer and an AMP was excited at a first wavelength and the emitted fluorescence intensity was measured at a second and a third wavelength. Sample preparation Twelve 1:1 serial dilutions of unlabeled AMP were prepared. DNA aptamers were fluorescently labeled with Cy5 and added in equal amounts to the AMP serial dilutions to obtain a final sample concentration of 20 nM. The highest concentration of AMP corresponded to 2 mM. Samples were then loaded into polymer-coated borosilicate glass capillaries (Monolith NT.115 Premium Capillaries, MO-K025, NanoTemper Technologies). measurement Each sample was excited at a wavelength of 591 nm. At time 0 seconds, the IR laser was switched on. Fluorescence intensity responses were measured simultaneously for 31 seconds. Fluorescence traces recorded at 628 nm to 653 nm ("650 nm") are shown in FIG. 11A. Fluorescence traces recorded at 665 nm to 727 nm ("670 nm") are shown in FIG. 11B. Because the initially recorded fluorescence intensities showed large fluctuations, a sigmoidal dose-response curve for affinity determination could not be obtained. As a result, no binding information could be extracted from the initial fluorescence at either of the two emission wavelengths shown in FIG. 12. Ratiometric Data Analysis For ratiometric analysis (i.e., obtaining the ratio of the fluorescence traces), a point-by-point division of the fluorescence traces at 670 nm and 650 nm was performed. The obtained ratio traces could be analyzed either before (Figure 13A, phase 1) or after (Figure 13A, phase 2 or phase 3) turning on the IR laser. By ratiometric analysis of the data recorded before turning on the IR laser (Figure 13A, phase 1), a dose-response curve with a signal-to-noise ratio (S / N) higher than 300 was obtained, indicating a K between both molecules at the sample temperature, i.e., room temperature. d was produced (Figure 13B).

[0173] By performing ratiometric analysis after turning on the IR laser (Figure 13A, phase 2 or phase 3), the K at higher temperatures d This approach is especially recommended when the amplitude at room temperature is very small and is expected to increase at higher temperatures. Determination of thermodynamic parameters of the interaction K for interaction d To obtain the time course curves, we took "vertical slices" at 200 ms time intervals of the ratio trace shown in FIG. 13A and calculated the K of the dose-response curve for each of these slices. dwas determined (FIG. 14A). The temperature change over time was known from a calibration measurement, i.e., the sample tray was heated in a controlled manner and the fluorescence of the reference dye was measured, and the interaction was allowed to equilibrate on a faster timescale, after which temperature changes occurred, so the K d By performing van't Hoff analysis, we obtain the following relationship:

[0174]

number

[0175] The binding enthalpy (ΔH) and binding entropy (ΔS) of the interaction (FIG. 14B) were determined. For this interaction, AMP was found to bind predominantly enthalpically (ΔH<0).

[0176] Example 3 The following example illustrates the use of the ratiometric characterization method according to the present invention to obtain a dose-response curve between two molecules based on a dual excitation configuration. Thus, a sample containing a fluorescently labeled DNA aptamer and an AMP was excited at a first and a second wavelength, and the emitted fluorescence intensity was measured at a third wavelength. Sample preparation Sixteen 1:1 serial dilutions of unlabeled AMP were prepared. DNA aptamers were fluorescently labeled with Cy3 and added in equal amounts to the AMP serial dilutions to obtain a final sample concentration of 250 nM. The highest concentration of AMP corresponded to 12.5 mM. Samples were then loaded into polymer-coated borosilicate glass capillaries (Monolith NT.115 Premium Capillaries, MO-K025, NanoTemper Technologies). measurement Each sample was excited at a first wavelength of 480 nm ("blue" LED). At time 0 seconds, the IR laser was switched on. The fluorescence intensity response was detected from 590 nm to 680 nm for 6 seconds. In the following, each sample was excited at a second wavelength of 540 nm ("green" LED). At time 0 seconds, the IR laser was switched on. The fluorescence intensity response was recorded by the detector from 590 nm to 680 nm for 6 seconds. The fluorescence trace obtained by exciting the sample with the "blue" LED is shown in FIG. 15A. The fluorescence trace obtained by exciting the sample with the "green" LED is shown in FIG. 15B. Ratiometric Data Analysis For ratiometric analysis (i.e. obtaining the ratio of the fluorescence traces), a point-by-point division of the fluorescence trace obtained by excitation with the green LED by the fluorescence trace obtained by excitation with the blue LED was performed. The obtained ratio traces could be analyzed either before (Fig. 16A, phase 1) or after (Fig. 16A, phase 2 or phase 3) turning on the IR laser. By ratiometric analysis of the data recorded before turning on the IR laser (Fig. 16A, phase 1), a dose-response curve with a signal-to-noise ratio (S / N) of approximately 80 was obtained, which indicates that the K between both molecules at the sample temperature, i.e. room temperature, d (Figure 16B). Ratiometric analysis of the data recorded after turning on the IR laser (Figure 16A, phase 3) yielded a dose-response curve with a signal-to-noise ratio (S / N) of over 130, revealing a K d (FIG. 16C). An improved signal-to-noise ratio was obtained when this ratio was analyzed at higher temperatures compared to analysis at room temperature.

[0177] Example 4 The following example illustrates the use of the ratiometric characterization method according to the invention to obtain a dose-response curve between two molecules based on a dual emission configuration. Thus, 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 Twelve 1:1 serial dilutions of unlabeled biotin were prepared. Streptavidin was fluorescently labeled with the Protein Labeling Kit RED-NHS 2nd Generation (NanoTemper Technologies) and added in equal amounts to the biotin serial dilutions to obtain a final sample concentration of 20 nM. The highest concentration of biotin corresponded to 500 nM. The samples were then loaded into polymer-coated borosilicate glass capillaries (Monolith NT.115 Premium Capillaries, MO-K025, NanoTemper Technologies). measurement Each sample was excited at a wavelength of 591 nm. Fluorescence traces were measured simultaneously at a second wavelength from 628 nm to 653 nm ("650 nm"), and a third wavelength from 665 nm to 727 nm ("670 nm"). Ratiometric Data Analysis For ratiometric analysis (i.e., obtaining the ratio of the fluorescence traces), a point-by-point division of "670 nm" by "650 nm" was performed. As is evident from the dose-response curves shown in Figure 17, the ratio changed from a value of approximately 2.2 to approximately 1.2 when streptavidin was unbound or complexed with biotin, respectively. The target concentration was K d Since the concentration of biotin is much higher than that of 80 nM, a characteristic kink in the stoichiometry point can be observed at a concentration of 80 nM biotin.

[0178] Example 5 The following example illustrates the use of the ratiometric characterization method according to the present invention to obtain a dose-response curve between two molecules based on a dual emission configuration. Thus, 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 Fifteen 1:1 serial dilutions of unlabeled acetazolamide were prepared. Bovine carbonic anhydrase II was fluorescently labeled with the protein labeling kit RED-NHS 2nd generation (NanoTemper Technologies) and added in equal amounts to the acetazolamide serial dilutions to obtain a final sample concentration of 20 nM. The highest concentration of acetazolamide corresponded to 2.5 μM. The samples were then loaded into polymer-coated borosilicate glass capillaries (Monolith NT.115 Premium Capillaries, MO-K025, NanoTemper Technologies). measurement Each sample was excited at a wavelength of 591 nm. Fluorescence traces were measured simultaneously at a second wavelength from 628 nm to 653 nm ("650 nm"), and a third wavelength from 665 nm to 727 nm ("670 nm"). Ratiometric Data Analysis For ratiometric analysis (i.e. obtaining the ratio of the fluorescent traces), a point-wise division of "670 nm" by "650 nm" was performed. In the dose-response curve shown in Figure 18, the ratio changed from approximately 0.944 to a value of approximately 0.951, and therefore approximately about 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 by the ratiometric characterization method of the present invention.

[0179] Example 6 The following example illustrates the use of the ratiometric characterization method according to the invention to obtain dose-response curves between three molecules based on a dual emission configuration. Thus, 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 Sixteen 1:1 serial dilutions of unlabeled antibody Herceptin were prepared. Monovalent streptavidin was fluorescently labeled with the protein labeling kit RED-NHS 2nd generation (NanoTemper Technologies) and 20 nM of it was mixed with an equal volume of 4 nM biotinylated protein L. The mixture was then added in equal volume to the Herceptin serial dilutions to obtain a final sample concentration of 5 nM labeled monovalent streptavidin and 1 nM biotinylated protein L in the assay. The highest concentration of Herceptin corresponded to 1 μM. The samples were then loaded into polymer-coated borosilicate glass capillaries (Monolith NT.115 Premium Capillaries, MO-K025, NanoTemper Technologies). measurement Each sample was excited at a wavelength of 591 nm. Fluorescence traces were measured simultaneously at a second wavelength from 628 nm to 653 nm ("650 nm"), and a third wavelength from 665 nm to 727 nm ("670 nm"). Ratiometric Data Analysis For ratiometric analysis (i.e. obtaining the ratio of the fluorescence traces), a point-wise division of "670 nm" by "650 nm" was performed. The dose-response curves shown in Figure 19A reveal that the method of the invention allows the measurement of ternary complexes, where the labeling is indirectly performed via a labeled third molecule (Figure 19B), e.g., labeled streptavidin, for the measurement of the interaction of a biotinylated protein with a ligand.

[0180] Example 7 The following example illustrates 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 a dual-radiation configuration. For fluorescent labeling, the biotinylated protein was mixed with the protein streptavidin (SA) and a small nucleic acid (bDNA) modified with biotin at the 3' end and the fluorophore Cy5 at the 5' end. SA is a homotetramer with an unusually high affinity for biotin (also known as vitamin B7). It is widely used in molecular biology and bionanotechnology due to the resistance of the streptavidin-biotin complex to organic solvents, denaturants (e.g., guanidinium chloride), detergents (e.g., SDS, Triton), proteolytic enzymes, and extremes of temperature and pH.

[0181] In the examples, a sample containing maltose binding protein (MBP) was fluorescently labeled by this procedure following: MBP was then mixed with the small molecule maltose, excited at a first wavelength, and the emitted fluorescence intensity was measured at a second and third wavelength. Sample preparation 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 oligo-dT sequence with a Cy5 molecule attached to the 5' end and a biotin molecule attached to the 3' end) was chemically synthesized and aligned by a DNA commercial supplier. A stock solution of 100 μM was prepared and then diluted with double distilled water (ddH2O). 2 0) to a final concentration of 8 nM. SA and bDNA were then mixed in a 1:1 volume ratio to obtain a 4 nM SA, 8 nM bDNA solution (1:2 stoichiometry). Through this step, SA was fluorescently labeled by binding to Cy5-labeled biotinylated bDNA.

[0182] Next, 100 μl of 100 nM biotinylated MBP was mixed with 100 μl of 4 nM SA, 8 nM bDNA solution to obtain 200 μl of 2 nM SA, 4 nM bDNA, 50 nM MBP solution. Because SA is a tetrameric protein, on average, two of its four binding sites for biotin are unoccupied and can bind to biotinylated MBP, creating the bDNA-SA-MBP complex, i.e., fluorescently labeled MBP (FIG. 20A).

[0183] Next, 16 1:1 serial dilutions of unlabeled maltose were prepared. Fluorescently labeled MBP was added in equal amounts to the maltose serial dilutions to obtain final target concentrations of 1 nM SA, 2 nM bDNA, and 25 nM MBP. The highest concentration of maltose corresponded to 500 μM. Samples were then loaded into polymer-coated borosilicate glass capillaries (Monolith NT.115 Premium Capillaries, MO-K025, NanoTemper Technologies). measurement Each sample was excited at a wavelength of 591 nm. Fluorescence traces were measured simultaneously at a second wavelength of 628 nm to 653 nm ("650 nm"), and a third wavelength of 665 nm to 727 nm ("670 nm"). Each capillary was measured for a period of 3 seconds. Ratiometric Data Analysis For ratiometric analysis (i.e., obtaining the ratio of the fluorescence traces), a point-wise division of "670 nm" by "650 nm" was performed. The dose-response curves shown in Figure 20B reveal that the method of the invention allows the measurement of a quaternary complex, where labeling is indirect via an unlabeled third molecule and a labeled fourth molecule, e.g., unlabeled SA and a labeled biotinylated single-stranded DNA oligomer.

[0184] Example 8 The following example illustrates the use of the ratiometric characterization method according to the invention to obtain a dose-response curve between two molecules based on a dual emission configuration. Thus, a sample containing fluorescently labeled therapeutic antibody CR3022, Cov-19 ("SARS CoV-2") spike protein, and 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 Fourteen 1:1 serial dilutions of unlabeled protein ACE2 were prepared. The therapeutic antibody CR3022 was fluorescently labeled with the protein labeling kit RED-NHS 2nd generation (NanoTemper Technologies) and 10 nM of it was mixed with an equal volume of 80 nM Cov-19 spike protein. The mixture was then added in equal volume to the ACE2 serial dilutions to obtain a final sample concentration of 5 nM labeled CR3022 and 20 nM spike protein. The highest concentration of ACE2 corresponded to 250 nM. The samples were then loaded into polymer-coated borosilicate glass capillaries (Monolith NT.115 Premium Capillaries, MO-K025, NanoTemper Technologies). measurement Each sample was excited at a wavelength of 591 nm. Fluorescence traces were measured simultaneously at a second wavelength from 628 nm to 653 nm ("650 nm"), and a third wavelength from 665 nm to 727 nm ("670 nm"). Ratiometric Data Analysis For ratiometric analysis (i.e. obtaining the ratio of the fluorescent traces), a point-wise division of "670 nm" by "650 nm" was performed. The dose-response curves shown in Figure 21 reveal that the method of the invention allows the measurement of a ternary complex, where labeling is indirectly achieved via a labeled third molecule, e.g. a labeled antibody.

[0185] Example 9 The following example illustrates the use of a ratiometric characterization method according to the invention to characterize the conformational state of a protein, based on a dual emission configuration, whereby a sample containing a fluorescently labeled protein is excited at a first wavelength and the emitted fluorescence intensity is measured at a second and a third wavelength. Sample preparation Mitogen-activated protein kinase 14 (p38-α) was fluorescently labeled with the protein labeling kit RED-NHS 2nd generation (NanoTemper Technologies). The labeled protein was then diluted to a concentration of 20 nM and loaded into polymer-coated borosilicate glass capillaries (Monolith NT.115 Premium Capillaries, MO-K025, NanoTemper Technologies). measurement The samples were excited at a wavelength of 591 nm. Fluorescence traces were measured simultaneously at a second wavelength of 628 nm to 653 nm ("650 nm"), and a third wavelength of 665 nm to 727 nm ("670 nm"). Measurements were taken immediately after dilution (t=0 min) and again after 3, 8 and 19 min in the capillary. Ratiometric Data Analysis For ratiometric analysis (i.e., obtaining the ratio of the fluorescence traces), a point-wise division of "670 nm" by "650 nm" was performed. Plotting the fluorescence ratio over the start of the measurements, as shown in Figure 22, reveals that the ratio is not constant over the period of the four measurements, but increases linearly over time. This increase in ratio indicates that the labeled protein is not stable at room temperature, but gradually denatures.

[0186] Example 10 The following example describes the method of the present invention for measuring short-term binding kinetics based on dual emission configuration.Therefore, a sample containing a fluorescently labeled DNA aptamer for adenosine and small molecule AMP, and a sample containing two 11-mer complementary DNA strands, one of which is fluorescently labeled by Cy5, are excited at a first wavelength.The emitted fluorescence intensity of said sample is measured at a second and third wavelength. Sample preparation Twelve 1:1 serial dilutions of the aptamer with unlabeled AMP were prepared. The DNA aptamer was fluorescently labeled with Cy5 and added in equal amounts to the AMP serial dilutions to obtain a final sample concentration of 20 nM. The highest concentration of AMP corresponded to 2 mM. The samples were then loaded into polymer-coated borosilicate glass capillaries (Monolith NT.115 Premium Capillaries, MO-K025, NanoTemper Technologies).

[0187] For DNA hybridization, 16 one-to-one serial dilutions of an unlabeled 11-mer (sequence: 5'CCT GAA GTC C3') were prepared. A complementary 11-mer (sequence: 5'GGA CTT CAG G3') was fluorescently labeled at its 5' end with Cy5 and added in equal amounts to the serial dilutions to obtain a final sample concentration of 10 nM. The highest concentration of unlabeled 11-mer corresponded to 100 μM. Samples were then loaded into polymer-coated borosilicate glass capillaries (Monolith NT.115 Premium Capillaries, MO-K025, NanoTemper Technologies). measurement Each sample was excited at a wavelength of 591 nm. At time 0 seconds, the IR laser was switched on. Fluorescence intensity responses were measured simultaneously for 6 seconds (aptamer) and 21 seconds (DNA hybridization), respectively. Fluorescence traces were measured at a second wavelength of 628 nm to 653 nm ("650 nm"), and at a third wavelength of 665 nm to 727 nm ("670 nm").

[0188] From the above, when the method of the present invention is used in combination with short-term IR laser heating, information about the binding interactions (e.g., K d It is clear that K can originate from any "vertical" slice in time along the ratiometric trace. d A new kind of curve can be generated that can be described as a time course curve. From this curve, information can be determined not only about the thermodynamics, but also about the binding kinetics of the interaction. In particular, when the equilibrium kinetics of the interaction is slower than the heating by the IR laser, the K d The time course curve shows a characteristic lag. Ratiometric Data Analysis For ratiometric analysis (i.e., obtaining the ratio of the fluorescence traces), a point-wise division of "670 nm" by "650 nm" was performed. d The time course curves are plotted for different dissociation constants K d and reveal three different interactions with binding kinetics. For the interaction between the Cy5-labeled DNA aptamer and AMP, a K of approximately 30 μM was obtained. d Determine and set the time for 10s. -1 Super K off was estimated (Figure 23A). The interaction between two 11-mer complementary DNA strands, i.e., DNA hybridization, measured at 32°C, was found to have a K of approximately 500 nM. d Determine approximately 1s -1 k off Measurements of the two 11-mer complementary DNA strands listed above at 22°C gave a K of approximately 5 nM (Figure 23B). d and 0.01s -1 k smaller off (Figure 23C).

[0189] K of 22°C to 32°C for DNA hybridization as described above d Detailed information about the time course measurements is shown in FIG. 24A. The y-axis shows the K d For interactions with slower kinetics, Kd The time course curve does not immediately follow the temperature change, but rather shows a separate lag, which leads to a larger lag and slower interaction kinetics. Therefore, analyzing this lag can provide valuable information about the binding kinetics of the interaction. off and k on Even when exact values ​​of β are not available, the ability to compare ligands and identify those that dissociate faster is already a great advantage of the method.

[0190] Additionally, assuming that equilibrium is restored at the new, higher temperature after 20 seconds of IR laser heating, van't Hoff analysis, enthalpy and entropy of interaction at two different temperatures, i.e., 22°C as the initial sample temperature and approximately 32°C as the temperature after IR laser heating (determined from the calibration experiment described above), were obtained (see Figures 24B and 24C). The sample tray temperature was adjusted to a number of different temperatures (e.g., 22°C, 24°C, 26°C, 28°C, 30°C, 32°C) and the K at each of them was calculated. d By measuring the ΔΨ σ, the thermodynamic parameters obtained are similar to those obtained from classical van't Hoff analysis (see Figures 24D and 24E).

[0191] K for different dissociation rates d The simulated data for the time course curves are shown in FIG. 25. The simulated K d The time course curve is -1 ~0.001s -1 It is clear that the dissociation rate of K can be resolved by carrying out the method of the present invention in combination with 20 seconds of IR laser heating. d The time course is 0.036s -1 ~0.154s -1 It is clear that even small differences in

[0192] Example 11 The following example describes a method of the invention for measuring slow binding kinetics based on a dual emission configuration, whereby a sample containing a fluorescently labeled nanobody and a 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 Nanobodies against Cov-19 spike protein were fluorescently labeled with the Protein Labeling Kit RED-NHS 2nd Generation (NanoTemper Technologies). 2 nM of fluorescently labeled nanobody was rapidly mixed with six different concentrations of Cov-19 spike RBD protein ranging from (20 nM to 625 pM). Samples were then loaded into polymer-coated borosilicate glass capillaries (Monolith NT.115 Premium Capillaries, MO-K025, NanoTemper Technologies). measurement Each sample was excited at a wavelength of 591 nm. Fluorescence traces were measured simultaneously at a second wavelength of 628 nm to 653 nm ("650 nm"), and a third wavelength of 665 nm to 727 nm ("670 nm"). In this example, samples were measured in duplicate, and the IR laser was not turned on, since temperature changes may have affected the binding kinetics. Ratiometric Data Analysis For ratiometric analysis (i.e. obtaining the ratio of the fluorescence traces), a point-wise division of "670 nm" by "650 nm" was performed. The ratio time course curves shown in Figure 26 reveal that the method according to the invention is capable of following slow binding kinetics when using such a "mix-and-measure" approach, and that the binding kinetics is slower than the time required to prepare the sample and start the measurement. By applying a global fitting model, the k of the interaction between the nanobody and the Cov-19 spike RBD protein was calculated. on =6.9×10 5 M -1 s -1 , k off =2.9×10-4 s -1 and K. d = 415 pM was determined.

[0193] Example 12 The following example illustrates the use of a ratiometric characterization method according to the present invention to locate fluorescently labeled particles, based on a dual emission configuration (Figure 27). Thus, 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 The mRNA was fluorescently labeled with Atto647N fluorescent dye and incorporated into lipid nanoparticles (LNPs). These duplicated mRNA-containing LNP preparations were exposed to different types of stress, namely, adding 0.25% detergent polysorbate 20 (Tween-20), boiling at 90°C for 10 min, vortexing for 1 min, or centrifugation at 14,000 rpm for 20 min. Untreated mRNA-containing LNPs were used as controls. The samples were then loaded into polymer-coated borosilicate glass capillaries (Monolith NT.115 Premium Capillaries, MO-K025, NanoTemper Technologies). measurement Each sample was excited at a wavelength of 591 nm. Fluorescence traces were measured simultaneously at a second wavelength from 628 nm to 653 nm ("650 nm"), and a third wavelength from 665 nm to 727 nm ("670 nm").

[0194] For measurements determining only the absolute fluorescence ratio, each capillary was measured for a period of 3 seconds. For measurements determining the aggregation state of the LNPs, measurements were performed with an IR laser (60 seconds laser on time).

[0195] From the previously performed control measurements shown, it is clear that the ratiometric fluorescence signal of Atto647N fluorescently labeled mRNA located within the LNP in the dual emission configuration of the present invention is approximately equal to 2.1 In contrast, when all fluorescently labeled mRNA molecules are located outside the LNP, the ratiometric fluorescence signal is approximately equal to 1.2. Ratiometric Data Analysis For ratiometric analysis (i.e. obtaining the ratio of the fluorescence traces), a point-wise division of "670 nm" by "650 nm" was performed. For the untreated control, i.e. all fluorescently labeled mRNA molecules are located within the LNP, the ratiometric fluorescence signal is equal to 2.1 (Figure 28A, control). By adding high concentrations of detergent, the lipid membrane of the LNP is ruptured / damaged, and therefore the fluorescently labeled mRNA molecules are no longer incorporated within the LNP (Figure 28A, +0.25% Tween). In this case, the ratiometric fluorescence signal is equal to 1.2. As is evident from the ratio of approximately 2.1, the fluorescently labeled mRNA is still located within the vortexed or centrifuged LNP preparation (Figure 28A, vortex 1 min, centrifuge 20 min). However, the treatment leads to aggregation of the LNP preparation, as can be seen by analyzing the "bumpy" fluorescence trace obtained after switching on the IR laser (Figure 28B). In contrast, a ratio of 1.2 was obtained when the LNP preparation was boiled at 90° C. for 10 min, indicating that the fluorescently labeled mRNA was no longer incorporated into the LNPs ( FIG. 28A , 90° C. for 10 min). This was further confirmed by analyzing the fluorescent trace after turning on the IR laser. A fluorescent trace without bumps was observed after boiling, confirming that the fluorescently labeled mRNA molecules had left the LNPs (potentially still aggregated LNPs).

[0196] As is evident from the above, the ratiometric characterization method of the present invention makes it possible to determine the localization of fluorescently labeled mRNA. Citation table: 1: Lens (e.g., aspheric lens) or lens system, or objective lens 2: Hot mirror, high IR reflection, visible light transmission >80% 3: IR laser (e.g., 1455nm, 1480nm, 1550nm, 980nm, 0.01W~10W) or laser for positioning 4: Laser fiber (single mode or multimode) 5: Laser fiber coupler w / o collimator 6: A beam shaping module (e.g., a lens system containing one, two or more lenses) for determining the laser beam diameter and focusing. 7: First light separation element (e.g., a dichroic mirror) for separating the fluorescence excitation from the emission 8: First excitation light source (e.g., laser, fiber laser, diode laser, LED, HXP, halogen, LED array, HBO) 9: A lens system (e.g., one, two or more lenses) for determining the beam characteristics of the excitation light source. 10: Excitation filter (e.g. bandpass / longpass) 11: A second light separation element (e.g., a dichroic mirror) for separating the radiation in lower and higher wavelength components 12: First emission filter (e.g. bandpass / longpass) 13: Second radial filter (e.g. bandpass / longpass) 14: First photodetector (e.g., PMT, siPM, APD, CCD or CMOS camera) 15: Second photodetector (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 characteristics of the excitation light source 18: A third light separation element (e.g., a dichroic mirror) for combining two different excitation light sources Cited non-patent literature

[0197] [Table 4]

[0198] All patent and non-patent literature cited herein is hereby incorporated by reference in its entirety.

Claims

1. A method for characterizing fluorescently labeled particles in a solution by analyzing changes in fluorescence intensity, spectral shifts, and / or broadening or narrowing of the spectrum of the fluorescent label of the fluorescently labeled particles, comprising: a) providing a sample of fluorescently labeled particles in a solution under a first condition; b) exciting the fluorescently labeled particles at a first wavelength; c) detecting the fluorescence emission intensity of the fluorescently labeled particles at second and third wavelengths; d) calculating the ratio between the fluorescence intensities at the second and third wavelengths; wherein the third wavelength is different from the second wavelength; e1) repeating steps b) to d) for the sample of fluorescently labeled particles under a second condition; or e2) repeating steps a) to d) for a second sample of fluorescently labeled particles under a second condition; wherein the second condition is different from the first condition; f) characterizing the fluorescently labeled particles based on the calculated ratios obtained for different conditions. The method includes: The second and third wavelengths are detected simultaneously and are close to the maximum emission of the fluorescence emission of the fluorescently labeled particles under the first condition; The second wavelength is detected at a wavelength that is at least 2.5 nm shorter than the maximum emission of the fluorescence emission of the fluorescently labeled particles under the first condition, and the third wavelength is detected at a wavelength that is at least 2.5 nm longer than the maximum emission of the fluorescence emission of the fluorescently labeled particles under the first condition; The spectral shift is in the range of 50 pm to 3 nm; The particles are covalently attached to the label, reversibly bound to the label across a high-affinity protein tag, or bioconjugated by click chemistry; 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, organelles, lipid nanoparticles (LNP), and virus-like particles; The above method.

2. The method according to claim 1, wherein the volume of the sample containing the fluorescently labeled particles is less than 100 μl, preferably 1 μl to 25 μl.

3. The method according to claim 1, wherein the sample containing the fluorescently labeled particles is provided in a capillary.

4. The method according to claim 1, wherein the fluorescently labeled particles are labeled with an environmentally sensitive label.

5. The method according to claim 1, wherein the fluorescently labeled particles are labeled with an exogenous fluorescent label that is a cyanine dye, preferably Cy5.

6. The method according to claim 1, wherein the biomolecule is selected from the group consisting of amino acids, proteins, peptides, monosaccharides and disaccharides, polysaccharides, lipids, glycolipids, fatty acids, sterols, vitamins, neurotransmitters, enzymes, nucleotides, metabolites, nucleic acids, and combinations thereof.

7. The method according to claim 1, wherein the concentration of the fluorescently labeled particles in the solution is from 10 pM to 10 μM, preferably from 50 pM to 500 nM.

8. The method according to claim 1, wherein the change in the detected fluorescence intensity of the fluorescently labeled particles results from a spectral shift, or broadening or narrowing of the spectrum, or a combination thereof.

9. The method according to claim 1, wherein the fluorescence intensity of the fluorescently labeled particles changes due to a conformational change of the fluorescently labeled particles, relocalization of the fluorescently labeled particles, interaction between the fluorescently labeled particles and one or more ligands, and combinations thereof.

10. The calculated ratio obtained in step f) is for determining a parameter selected from the group consisting of the localization of the fluorescently labeled particles, or the dissociation constant, 50% effective concentration (EC ５０ ), equilibrium constant, binding kinetics, enzyme reaction kinetics, thermodynamic parameters, unfolding or refolding kinetics, opening and closing reactions, and combinations thereof, the method according to claim 1.

11. The method according to claim 1, wherein the second condition in step e) is varied by adding a ligand and / or different concentrations of the ligand, and the calculated ratio obtained in step f) is used to determine the dissociation constant of the fluorescently labeled particles and the ligand.

12. The method according to claim 1, wherein the first and second conditions of the fluorescently labeled particles differ in terms of their temperature and / or chemical composition.

13. The method according to claim 1, wherein the fluorescence emission intensity of the fluorescently labeled particles at the second and third wavelengths in step c) is detected during a defined temperature perturbation.

14. A device for characterizing fluorescently labeled particles in a solution by analyzing a change in the fluorescence intensity of the fluorescent label of the fluorescently labeled particles, and a spectral shift, and / or broadening or narrowing of the spectrum, adapted to perform the method according to any one of claims 1 to 13, comprising: a sample holder for holding samples of fluorescently labeled particles in a solution under a plurality of conditions; means for exciting fluorescently labeled particles at a first wavelength; means for detecting the fluorescence emission intensity of the fluorescently labeled particles at second and third wavelengths; means for calculating a ratio between the fluorescence intensities at the second and third wavelengths, the third wavelength being different from the second wavelength; comprising, wherein the device is configured to continuously excite fluorescence, detect fluorescence emission, and calculate ratios for samples under different conditions, comprising means for characterizing the fluorescently labeled particles based on the calculated ratios obtained for different conditions, the device being configured to simultaneously detect the second and third wavelengths, the second wavelength being detected at a wavelength at least 2.5 nm shorter than the maximum emission of the fluorescence emission of the fluorescently labeled particles under a first condition of different conditions, and the third wavelength being detected at a wavelength at least 2.5 nm longer than the maximum emission of the fluorescence emission of the fluorescently labeled particles under the first condition of different conditions, the spectral shift being in the range of 50 pm to 3 nm, the particles being covalently attached to the label, reversibly bound to the label across a high affinity protein tag, or bioconjugated by click chemistry, the particles being selected from the group consisting of organic molecules, biomolecules, nanoparticles, microparticles, vesicles, biological cells or subcellular fragments, biological tissues, virus particles, viruses, organelles, lipid nanoparticles (LNP) and virus-like particles, the device as described above.

15. The device according to claim 14, wherein the means for excitation is at least one light source selected from the group consisting of an excitation light source, preferably a laser, a fiber laser, a diode laser, an LED, an HXP, a halogen, an LED array, an HBO.

16. The device according to claim 14, wherein the means for detection is at least one detector selected from the group consisting of a photodetector, preferably a PMT, a siPM, an APD, a CCD or a CMOS camera.

17. A computer program comprising instructions for causing a computer to perform the method according to any one of claims 1 to 13 when the program is executed by the computer.

18. A computer-readable data carrier comprising instructions for causing a computer to perform the method according to any one of claims 1 to 13 when the program is executed by the computer.

19. Use of a device according to claim 14 for characterizing fluorescently labeled particles in solution by the method according to any one of claims 1 to 13.

20. Use of a capillary for characterizing fluorescently labeled particles in solution by analyzing changes in fluorescence intensity, spectral shifts, and / or broadening or narrowing of the spectrum of the fluorescent label of the fluorescently labeled particles, wherein a sample of the fluorescently labeled particles in solution is filled into the capillary and provided, in particular, for analysis by the method according to any one of claims 1 to 13.

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