Methods and systems for tracking single molecules in cell-free systems

Abstract

This disclosure relates to single-molecule tracking methods, including high-throughput methods, for analyzing molecules in cell-free samples. The disclosure further provides a system for carrying out such methods.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 523,356, filed on 26 June 2023, and to U.S. Provisional Patent Application No. 63 / 613,704, filed on 21 December 2023, the contents of which are incorporated herein by reference in whole.

[0002] The subject matter described herein relates to methods for analyzing single molecules in cell-free systems. [Background technology]

[0003] The movement of molecules within cell-free systems is greatly influenced by their interactions with their environment. This environment includes, but is not limited to, the solution in which the molecule is distributed, other molecules present in the solution, and even physical conditions. Therefore, monitoring the movement of a target molecule provides biophysical information that underlies the interaction and influence of the environment on the target molecule. Single-molecule tracking (SMT) is one method of capturing the movement of a single molecule, for example, as a reporter of the conformation, interactions, and / or identity of a target molecule. In SMT, molecules such as fluorescently labeled molecules are imaged over time with high spatiotemporal resolution, and their movement is analyzed at the single-molecule level. However, currently, the application of SMT techniques to cell-free systems is limited in terms of scale, sensitivity, and success. [Overview of the project]

[0004] This disclosure relates to single-molecule methods, including, for example, high-throughput methods for analyzing target molecules in cell-free samples. This disclosure further provides systems for carrying out such methods.

[0005] In the first aspect, the disclosure relates to a method (e.g., a high-throughput method) for identifying interactions between a test molecule and a target molecule. The method includes (a) contacting a cell-free sample containing multiple target molecules with multiple test molecules; (b) tracking the multiple target molecules over time to obtain measurements of multiple spatiotemporal trajectories and / or rotational motions; (c) analyzing the measurements of the multiple spatiotemporal trajectories and / or rotational motions to determine the movement of the target molecules in the presence of the test molecules; and (d) comparing the movement of the target molecules obtained in (c) with the movement of a reference target molecule, where the movement of the reference target molecule is the movement of the target molecules in the absence of the test molecules, and the interaction between the target molecule and the test molecule is indicated by a change in the movement of the target molecule compared to the movement of the reference target molecule. In certain embodiments, the interaction between the target molecule and the test molecule is indicated by a decrease in the movement of the target molecule compared to the movement of the reference target molecule. In certain embodiments, the interaction between the target molecule and the test molecule is indicated by an increase in the movement of the target molecule compared to the movement of the reference target molecule. In certain embodiments, the duration and / or reversibility of the change in the target molecule's motion compared to the change in the reference target molecule's motion indicates the interaction between the target molecule and the test molecule.

[0006] In certain embodiments aimed at identifying interactions between test molecules and target molecules, the determination of molecular motion can be quantified by analysis of spatiotemporal trajectories. Motion characterized in this way may include, but is not limited to, measurements of diffusion coefficients. For example, the measurement may be a measurement of a diffusion coefficient maximum likelihood estimator, defined as an estimate of the maximum likelihood diffusion coefficient for multiple spatiotemporal trajectories under a single-state diffusion model with a constant position estimation error. In certain embodiments, spatiotemporal trajectories can be used to estimate the proportion of target molecules in multiple dynamic diffusion states (e.g., different diffusion states) using machine learning methods such as variational Bayesian inference. Such methods may include, but are not limited to, state arrays. In certain embodiments, molecular motion may be measured through analysis of the products of a link generation algorithm. Motion characterized in this way may include, but is not limited to, mean posterior diffusion coefficients, the mean of the posterior probability distribution of coefficients from a stochastic linking algorithm. Motion characterized in this way may include, but is not limited to, geometric mean posterior diffusion coefficients, the mean of the log-scale posterior probability distribution of coefficients from a stochastic linking algorithm. In certain embodiments, molecular motion can be measured through model-dependent analysis of multiple spatiotemporal trajectories. In certain embodiments, the determination of molecular motion can be quantified by calculating a jump length distribution. For example, for any given set of protein displacements between a given time point and a subsequent time point, a histogram of the probabilities of each displacement length ("jump length") can be constructed. The quantiles of this distribution can be used to describe the molecular motion. In certain embodiments, the quantile used is the median of the jump length distribution. In certain embodiments, the quantile used is the third quartile of the jump length distribution. In certain embodiments, molecular motion may be quantified by measuring mean squared displacement, defined by the mean of the squares of all displacements within the spatiotemporal trajectories averaged over multiple spatiotemporal trajectories. Motion characterized in this way may also include, but is not limited to, measurements of spatiotemporal trajectory lengths or distributions of spatiotemporal trajectory lengths.Motion characterized in this way may also include, but is not limited to, a measurement of the mean radius of rotation defined by the root mean square distance of all coordinates in the spacetime trajectory from the center of mass of the set of points contained in the spacetime trajectory, averaged over multiple spacetime trajectories. Motion characterized in this way may also include, but is not limited to, a measurement of the mean joint angle defined by the angle formed by three continuous spatial coordinates, averaged over multiple spacetime trajectories.

[0007] In certain embodiments, the determination of molecular motion can be quantified by analyzing measurements of rotational motion. Motion characterized in this way may include, but is not limited to, anisotropic decay time.

[0008] In certain embodiments, the motion can be characterized by determining the number and wavelength of the conjugated fluorescent labels. In certain embodiments, the motion can be characterized by determining the polarization of the conjugated fluorescent labels.

[0009] In certain embodiments, the determination of molecular interactions can be quantified by analyzing measurements of the spatial range of detection. In certain embodiments, such characterized interactions may include, but are not limited to, measurements of oligomerization. In certain embodiments, the oligomerization process may involve, but are not limited to, analyzing one or more detection channels to analyze the number and heterogeneity of monomers forming the oligomer.

[0010] In certain embodiments directed to identifying an interaction between a test molecule and a target molecule, for example, where movement is determined using a change in the diffusion coefficient relative to the diffusion coefficient of a reference target molecule, the change in the diffusion coefficient compared to the diffusion coefficient of the reference target molecule is at least about 0.001%, at least about 0.005%, at least 0.01%, at least about 0.5%, at least about 1%, at least about 1.5%, at least about 2% or at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10%.

[0011] In certain embodiments directed to identifying an interaction between a test molecule and a target molecule, a change in the movement of the target molecule compared to the movement of the reference target molecule indicates a direct interaction between the target molecule and the test molecule. In certain embodiments, a change in the movement of the target molecule compared to the movement of the reference target molecule indicates an indirect interaction between the target molecule and the test molecule. In certain embodiments, an interaction between the target molecule and the test molecule causes a conformational change in the target molecule. In certain embodiments, an interaction between the target molecule and the test molecule increases the mass of the target molecule. In certain embodiments, an interaction between the target molecule and the test molecule decreases the mass of the target molecule. In certain embodiments, an interaction between the target molecule and the test molecule causes a change in the temperature stability of the target molecule. In certain embodiments, an interaction between the target molecule and the test molecule causes oligomerization. In certain embodiments, a plurality of target molecules are conjugated to a plurality of fluorescent labels. In certain embodiments, the interaction between the target molecule and the test molecule is reversible. In certain embodiments, the interaction between the target molecule and the test molecule is irreversible.

[0012] In another embodiment, the Disclosure provides a method (e.g., a high-throughput method) for determining the morphology of a target molecule. This method includes (a) tracking multiple target molecules in a cell-free sample over time to provide measurements of multiple spatiotemporal trajectories and / or rotational motions; (b) analyzing the measurements of the multiple spatiotemporal trajectories and / or rotational motions to determine the motion of the target molecule; and (c) comparing the motion of the target molecule obtained in (b) with the motion of a reference target molecule, where the motion of the reference target molecule is the motion of one morphology of the target molecule, and (i) a change in the motion of the target molecule compared to the motion of the reference target molecule indicates that the target molecule is in a different morphology from the reference target molecule, or (ii) no change in the motion of the target molecule compared to the motion of the reference target molecule indicates that the target molecule is in the same morphology as the reference target molecule. In certain embodiments, the duration and / or reversibility of the change in the motion of the target molecule compared to the duration and / or reversibility of the change in the motion of the reference target molecule indicates that the target molecule is in a different morphology from the reference target molecule. In certain embodiments, the different morphologies of the target molecule may have the same chemical composition but different conformations. In certain embodiments, one form of the target molecule may be a product of alternative splicing. In certain embodiments, one form of the target molecule may be a post-translational modified form of the target molecule. In certain embodiments, one form of the target molecule may be the wild-type form of the target molecule. In certain embodiments, one form of the target molecule may be a mutant form of the target molecule. In certain embodiments, one form of the target molecule may be a homolog, ortholog, or paralog of the target molecule.

[0013] In certain embodiments, changes in the motion associated with determination of the conformation of a target molecule are changes in (a) the diffusion coefficient obtained from the maximum likelihood estimator, (b) the geometric mean posterior diffusion coefficient, (c) the median of the jump length distribution, (d) the third quartile of the jump length distribution, (e) the median of the radius of gyration, (f) the mean posterior diffusion coefficient, (g) the mean squared displacement, (h) the median binding angle, (i) the spatio-temporal trajectory length, (j) the anisotropy decay time, (k) the spatial extent of detection, (l) the number and wavelengths of conjugated fluorescent labels, (m) the occupancy in various diffusion states obtained from the state array, (n) the polarization of the conjugated fluorescent labels, or (o) the inferred state occupancy.

[0014] In certain embodiments directed to determining the conformation of a target molecule, for example, if the motion is determined using a change in the diffusion coefficient relative to the diffusion coefficient of a reference target molecule, the change in the diffusion coefficient is at least about 0.001%, at least about 0.005%, at least about 0.01%, at least about 0.05%, at least about 0.1%, at least about 0.5%, at least about 1%, at least about 1.5%, at least about 2% or at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9% or at least about 10%.

[0015] In a further embodiment, the disclosure provides a method (e.g., a high-throughput method) for identifying a test molecule that can distinguish between at least two target molecules. In certain embodiments, the method includes (a) contacting a cell-free sample containing a plurality of first target molecules with a plurality of test molecules; (b) tracking the plurality of first target molecules over time to obtain measurements of a plurality of spatiotemporal trajectories and / or rotational motions; (c) analyzing the measurements of the plurality of spatiotemporal trajectories and / or rotational motions to determine the movement of the first target molecules in the presence of the test molecules; (d) contacting a cell-free sample containing a plurality of second target molecules with a plurality of test molecules; (e) tracking the plurality of second target molecules over time to obtain measurements of a plurality of spatiotemporal trajectories and / or rotational motions; (f) analyzing the measurements of the plurality of spatiotemporal trajectories and / or rotational motions to determine the movement of the second target molecules in the presence of the test molecules; and (g) comparing the movements of the target molecules obtained in (c) and (f), demonstrating that the test molecules can distinguish between the two target molecules by changes in the movements of the first and second target molecules.

[0016] In certain embodiments, changes in motion related to the identification of test molecules that can distinguish between at least two target molecules include changes in (a) the diffusion coefficient obtained from the maximum likelihood estimator, (b) the geometric mean posterior diffusion coefficient, (c) the median of the jump length distribution, (d) the third quartile of the jump length distribution, (e) the median of the turning radius, (f) the mean posterior diffusion coefficient, (g) the mean square displacement, (h) the median of the bond angle, (i) the spatiotemporal trajectory length, or (j) the anisotropic decay time, (k) the spatial range of detection, (l) the number and wavelength of conjugated fluorescent labels, (m) the occupation in various diffusion states obtained from the state array, (n) the polarization of the conjugated fluorescent labels, or (o) the state occupation by inference.

[0017] In certain embodiments, changes in the movement of the first and second target molecules indicate that the test molecule can distinguish between the two target molecules. In certain embodiments, the two target molecules are unrelated. In certain embodiments, the two target molecules are related. In certain embodiments where the two target molecules are related, the first and second target molecules are the same or different forms of the target molecule. In certain embodiments where the two target molecules are related, one form of the target molecule is the wild-type form of the target molecule. In certain embodiments where the two target molecules are related, one form of the target molecule is a variant form of the target molecule. In certain embodiments, one form of the target molecule is a post-translational modified form of the target molecule. In certain embodiments where the two target molecules are related, the first and second target molecules are homologs, orthologues, or paralogs. In certain embodiments where the two target molecules are related, the first and second target molecules are different species of the target molecule. In certain embodiments involving two target molecules, the first and second target molecules are different splicing isoforms of the target molecule. In certain embodiments involving two target molecules, the first and second target molecules are the same or different conformations of the target molecule. In certain embodiments, the distinction between the first and second target molecules is achieved by differences in their fluorescent labeling. In certain embodiments, more than two target molecules are analyzed, for example, a third target molecule is analyzed. For example, though not intended to be limiting, this method may further include contacting a cell-free sample containing multiple third target molecules with multiple test molecules, tracking the multiple third target molecules over time to provide measurements of a third spatiotemporal trajectory and / or rotational motion, analyzing the measurements of the third spatiotemporal trajectory and / or rotational motion to determine the movement of the third target molecules in the presence of the test molecules, and comparing the movement of the third target molecules with the movement obtained in (c) and (f), demonstrating that the test molecules can distinguish between the three target molecules by changes in the movement of the third target molecules relative to the first and second target molecules.

[0018] In a further embodiment, the Disclosure provides a method (e.g., a high-throughput method) for identifying test molecules that bind to a target molecule and induce a conformational change in the target molecule. In a particular embodiment, the method includes (a) contacting a cell-free sample containing a plurality of target molecules with a plurality of test molecules, each sample being contacted with a different test molecule; (b) tracking the plurality of target molecules in each sample over time to provide measurements of a plurality of spatiotemporal trajectories and / or rotational motions; (c) analyzing the measurements of the plurality of spatiotemporal trajectories and / or rotational motions to determine the motion of the target molecule in the presence of the test molecule; and (d) comparing the motion of the target molecule obtained in step (c) with the motion of a reference target molecule, where the motion of the reference target molecule is the motion of the target molecule in the presence of a test molecule that does not induce a conformational change in the target molecule, and the change in the motion of the target molecule compared with the motion of the reference target molecule indicates an interaction between the target molecule and the test molecule that induces a conformational change in the target molecule. In certain embodiments, the duration and / or reversibility of the change in the motion of the target molecule compared to the duration and / or reversibility of the change in the motion of the reference target molecule indicates an interaction between the target molecule and one or more test molecules that induce a conformational change in the target molecule. In certain embodiments, the conformational change in the target molecule is related to a change in the temperature stability of the target molecule.

[0019] In certain embodiments, changes in motion related to the identification of a test molecule that binds to a target molecule and causes a conformational change in the target molecule include changes in (a) the diffusion coefficient obtained from the maximum likelihood estimator, (b) the geometric mean posterior diffusion coefficient, (c) the median of the jump length distribution, (d) the third quartile of the jump length distribution, (e) the median of the turning radius, (f) the mean posterior diffusion coefficient, (g) the mean square displacement, (h) the median of the binding angle, (i) the spatiotemporal trajectory length, (j) the anisotropic decay time, (k) the spatial range of detection, (l) the number and wavelength of conjugated fluorescent labels, (m) occupation in various diffusion states obtained from the state array, (n) the polarization of the conjugated fluorescent labels, or (o) the state occupation by inference.

[0020] In certain embodiments relating to identifying a test molecule that binds to a target molecule and causes a conformational change in the target molecule, for example, if the movement is determined using a change in the diffusion coefficient relative to the diffusion coefficient of the reference target molecule, the change in the diffusion coefficient relative to the diffusion coefficient of the reference target molecule is at least about 0.001%, at least about 0.005%, at least about 0.01%, at least about 0.05%, at least about 0.1%, at least about 0.5%, at least about 1%, at least about 1.5%, at least about 2%, or at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10%.

[0021] In another aspect, the Disclosure provides a method (e.g., a high-throughput method) for determining the dose response of a target molecule to a test molecule. This method includes (a) contacting a plurality of cell-free samples containing a plurality of target molecules with a test molecule, wherein the plurality of cell-free samples are contacted with a range of test molecule doses; (b) tracking the plurality of target molecules over time in the presence of the range of test molecule doses to provide measurements of a plurality of spatiotemporal trajectories and / or rotational motions; (c) analyzing measurements of the plurality of spatiotemporal trajectories and / or rotational motions of the target molecules in the presence of the range of test molecule doses to determine the motion of the target molecules at each dose of the test molecule; and (d) comparing the motion of the target molecules obtained in step (c) at various test molecule doses to determine the dose response of the target molecule to the test molecule.

[0022] In certain embodiments, changes in motion related to determining the dose response of the target molecule to the test molecule include changes in (a) the diffusion coefficient obtained from the maximum likelihood estimator, (b) the geometric mean posterior diffusion coefficient, (c) the median of the jump length distribution, (d) the third quartile of the jump length distribution, (e) the median of the turning radius, (f) the mean posterior diffusion coefficient, (g) the mean square displacement, (h) the median of the bond angle, (i) the spatiotemporal trajectory length, or (j) the anisotropic decay time, (k) the spatial range of detection, (l) the number and wavelength of conjugated fluorescent labels, (m) occupation in various diffusion states obtained from the state array, (n) the polarization of the conjugated fluorescent labels, or (o) the state occupation by inference.

[0023] In certain embodiments relating to determining the dose-response of a target molecule to a test molecule, for example, if the movement is determined using the change in the diffusion coefficient of the target molecule relative to the diffusion coefficient of the target molecule at each test molecule dose, the change in the diffusion coefficient of the target molecule relative to the diffusion coefficient of the target molecule at each test molecule dose is at least about 0.001%, at least about 0.005%, at least about 0.01%, at least about 0.05%, at least about 0.1%, at least about 0.5%, at least about 1%, at least about 1.5%, at least about 2%, or at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10%.

[0024] In a further embodiment, the disclosure provides a method (e.g., a high-throughput method) for determining the difference in dose response between two target molecules to a test molecule. The method comprises: (a) contacting a test molecule with a plurality of cell-free samples containing a plurality of first target molecules, wherein the plurality of cell-free samples are contacted with a range of test molecule doses; (b) tracking the plurality of target molecules over time in the presence of the range of test molecule doses to provide measurements of a plurality of spatiotemporal trajectories and / or rotational motions; (c) analyzing measurements of the plurality of spatiotemporal trajectories and / or rotational motions of the target molecules in the presence of the range of test molecule doses to determine the movement of the first target molecules at each dose of the test molecule; (d) comparing the movement of the target molecules obtained in (c) at various test molecule doses to determine the dose response of the target molecules to the test molecule; (e) repeating steps (a) to (d) with a second target molecule to determine the dose response of the second target molecule to the test molecule; and (f) comparing the dose response of the first target molecule with the dose response of the second target molecule to determine the difference in the responses of the first and second target molecules to the test molecule. In certain embodiments, the distinction between the first target molecule and the second target molecule is achieved by differences in their fluorescent labeling. In certain embodiments, more than two target molecules are analyzed, for example, three or more target molecules are analyzed.

[0025] In certain embodiments, changes in motion related to determining the difference in dose response between two target molecules to a test molecule include changes in (a) the diffusion coefficient obtained from the maximum likelihood estimator, (b) the geometric mean posterior diffusion coefficient, (c) the median of the jump length distribution, (d) the third quartile of the jump length distribution, (e) the median of the turning radius, (f) the mean posterior diffusion coefficient, (g) the mean square displacement, (h) the median of the bond angle, (i) the spatiotemporal trajectory length, or (j) the anisotropic decay time, (k) the spatial range of detection, (l) the number and wavelength of conjugated fluorescent labels, (m) occupation in various diffusion states obtained from the state array, (n) the polarization of the conjugated fluorescent labels, or (o) the state occupation by inference.

[0026] In certain embodiments relating to determining the difference in dose response to a test molecule by two target molecules, the first and second target molecules are not related. In certain embodiments, the first and second target molecules are related. In certain embodiments relating the first and second target molecules, the first and second target molecules are the same or different forms of the target molecule. In certain embodiments relating the first and second target molecules, one form of the target molecule is the wild-type form of the target molecule. In certain embodiments relating the first and second target molecules, one form of the target molecule is a variant form of the target molecule. In certain embodiments relating the first and second target molecules, one form of the target molecule is a post-translational modified form of the target molecule. In certain embodiments relating the first and second target molecules, the first and second target molecules are homologs, orthologues, or paralogs. In certain embodiments relating the first and second target molecules, the first and second target molecules are different species of the target molecule. In certain embodiments involving a first target molecule and a second target molecule, the first and second target molecules are different splicing isoforms of the target molecule. In certain embodiments involving a first and second target molecule, the first and second target molecules are the same or different conformations of the target molecule. In certain embodiments, the distinction between the first and second target molecules is achieved by differences in their fluorescent labeling. In certain embodiments, more than two target molecules are analyzed.

[0027] In certain embodiments aimed at determining the difference in dose-response between two target molecules and a test molecule, the target molecule is an organic molecule less than approximately 1 kDa. In certain embodiments, the target molecule is selected from the group consisting of peptides, protein domains, proteins, nucleic acid polymers, carbohydrates, and lipids. In certain embodiments, the target molecule is selected from the group consisting of glycoproteins, glycolipids, oligosaccharides, polysaccharides, and lipid micelles. In certain embodiments, the target molecule is an entity of multiple components (e.g., at least two, at least three, at least four, or at least five components) non-covalently bonded by interactions (including, but not limited to, hydrophobic, electrostatic, ionic, hydrogen, and van der Waals forces). In certain embodiments, the target molecule is an entity of multiple components covalently bonded together. In certain embodiments, the target molecule is a multi-subunit protein or protein complex composed of multiple subunits. In certain embodiments, the target molecule is a protein composed of one or more polypeptides and one or more ligands. In certain embodiments, the target molecule is an exosome composed of proteins, lipids, and other biological molecules. In certain embodiments, the target molecule is a virus, viroid, phage, or other biological particle. In certain embodiments, the protein is selected from the group consisting of antibodies, receptors, and enzymes. In certain embodiments, the protein contains a disordered domain. In certain embodiments, the protein does not contain a structured domain. In certain embodiments, the peptide is a ligand. In certain embodiments, the target molecule is a nanomaterial. In certain embodiments, the target molecule is a synthetic polymer. In certain embodiments, the target molecule is labeled. In certain embodiments, the target molecule is fluorescently labeled. In certain embodiments, the target molecule is labeled with an organic fluorescent dye or inorganic fluorescent particles. In certain embodiments, the target molecule is labeled with a fluorescent protein. In certain embodiments, the target molecule is labeled with a synthetic nanomaterial or polymer conjugate. In certain embodiments, the target molecule is purified. In certain embodiments, the target molecule is a component of a mixture.In certain embodiments, the target molecule is a component of a mixture consisting of bacterial extracts, cell extracts, tissue extracts, plant extracts, or animal extracts. In certain embodiments, the bacterial extracts, cell extracts, tissue extracts, plant extracts, or animal extracts are lysates. In certain embodiments, the target molecule is a component of a mixture consisting of serum, blood, and other biological samples. In certain embodiments, the target molecule is a component of a mixture containing a buffer and glycerol.

[0028] In a further embodiment, the Disclosure provides a high-throughput method (e.g., a high-throughput method) for identifying interactions between a test molecule and a target protein containing disordered domains. This method includes (a) contacting a cell-free sample containing multiple target proteins with multiple test molecules; (b) tracking the multiple target proteins over time to obtain multiple spatiotemporal trajectory and / or rotational motion measurements; (c) analyzing the multiple spatiotemporal trajectory and / or rotational motion measurements to determine the movement of the target proteins in the presence of the test molecules; and (d) comparing the movement of the target proteins obtained in (c) with the movement of a reference target protein, where the movement of the reference target protein is the movement of the target protein in the absence of the test molecules, wherein the change in the movement of the target protein containing disordered domains compared with the movement of the reference target protein indicates an interaction between the target protein containing disordered domains and the test molecule. In certain embodiments, the duration and / or reversibility of the change in the movement of the target protein containing disordered domains compared with the duration and / or reversibility of the change in the movement of the reference target protein indicates an interaction between the target molecule and the test molecule. In certain embodiments, the interaction between the target molecule and the test molecule is reversible. In certain embodiments, the interaction between the target molecule and the test molecule is irreversible.

[0029] In certain embodiments, changes in motion related to the identification of interactions between a test molecule and a target protein containing disordered domains are changes in (a) the diffusion coefficient obtained from the maximum likelihood estimator, (b) the geometric mean posterior diffusion coefficient, (c) the median of the jump length distribution, (d) the third quartile of the jump length distribution, (e) the median of the turning radius, (f) the mean posterior diffusion coefficient, (g) the mean square displacement, (h) the median of the bond angle, (i) the spatiotemporal trajectory length, or (j) the anisotropic decay time, (k) the spatial range of detection, (l) the number and wavelength of conjugated fluorescent labels, (m) occupation in various diffusion states obtained from the state array, (n) the polarization of the conjugated fluorescent labels, or (o) the state occupation by inference.

[0030] In certain embodiments aimed at identifying interactions between a test molecule and a target protein containing disordered domains, for example, if the movement is determined using a change in the diffusion coefficient relative to the diffusion coefficient of a reference target molecule, the change in the diffusion coefficient of the target protein containing disordered domains compared to the diffusion coefficient of the reference target protein is at least about 0.001%, at least about 0.005%, at least about 0.01%, at least about 0.05%, at least about 0.1%, at least about 0.5%, at least about 1%, at least about 1.5%, at least about 2%, or at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10%. In certain embodiments, the change in the diffusion coefficient of the target protein containing disordered domains compared to the diffusion coefficient of a reference target protein indicates a direct interaction between the target protein containing disordered domains and the test molecule. In certain embodiments, a change in the diffusion coefficient of the target protein compared to the diffusion coefficient of the reference target protein indicates an indirect interaction between the target protein containing disordered domains and the test molecule. In certain embodiments, a change in the diffusion coefficient of the target protein containing disordered domains compared to the diffusion coefficient of the reference target protein indicates a conformational change of the target protein. In certain embodiments, a conformational change in the target protein results in a change in the temperature stability of the target protein. In certain embodiments, a change in the diffusion coefficient of the target protein containing disordered domains compared to the diffusion coefficient of the reference target protein indicates an increase in the mass of the target protein. In certain embodiments, a change in the diffusion coefficient of the target protein containing disordered domains compared to the diffusion coefficient of the reference target protein indicates a decrease in the mass of the target protein. In certain embodiments, a change in the diffusion coefficient of the target protein containing disordered domains compared to the diffusion coefficient of the reference target protein indicates oligomerization. In certain embodiments, multiple target proteins are conjugated to multiple fluorescent labels. In certain embodiments, the interaction between the target protein and the test molecule is reversible.In certain embodiments, the interaction between the target protein and the test molecule is irreversible.

[0031] In certain embodiments aimed at identifying interactions between a test molecule and a target protein containing a disordered domain, the target protein containing the disordered domain is selected from the group consisting of antibodies, receptors, structural proteins, and enzymes. In certain embodiments, the target protein containing the disordered domain does not contain a structural domain. In certain embodiments, the target protein containing the disordered domain is a fragment of a native full-length protein. In certain embodiments, the target protein containing the disordered domain is labeled. In certain embodiments, the target protein containing the disordered domain is fluorescently labeled. In certain embodiments, the test molecule is fluorescently labeled. In certain embodiments, the target protein containing the disordered domain is purified.

[0032] In certain embodiments aimed at identifying interactions between a test molecule and a target protein containing a disordered domain, the test molecule is an organic molecule less than approximately 1 kDa. In certain embodiments, the test molecule is selected from the group consisting of peptides, proteins, nucleic acids, carbohydrates, and lipids. In certain embodiments, the protein is selected from the group consisting of antibodies, receptors, and enzymes. In certain embodiments, the protein contains a disordered domain. In certain embodiments, the protein does not contain a structured domain. In certain embodiments, the peptide is a ligand. In certain embodiments, the test molecule is a nanomaterial. In certain embodiments, the test molecule is labeled. In certain embodiments, the test molecule is fluorescently labeled. In certain embodiments, the test molecule is labeled with a fluorescent protein. In certain embodiments, the test molecule is labeled by a synthetic nanomaterial or polymer conjugate. In certain embodiments, the test molecule is purified. In certain embodiments, the test molecule is a component of a mixture. In certain embodiments, the test molecule is a component of a mixture consisting of bacterial extracts, cell extracts, tissue extracts, plant extracts, or animal extracts. In certain embodiments, the bacterial extracts, cell extracts, tissue extracts, plant extracts, or animal extracts are lysates. In certain embodiments, the test molecule is a component of a mixture consisting of serum, blood, and other biological samples. In certain embodiments, the target molecule is a component of a mixture containing a buffer and glycerol.

[0033] In certain embodiments aimed at identifying interactions between a test molecule and a target protein containing disordered domains, the interaction between the target protein containing disordered domains and the test molecule results in a conformational change in the target molecule. In certain embodiments, the conformational change in the target molecule results in a change in the temperature stability of the target protein. In certain embodiments, the interaction between the target protein containing disordered domains and the test molecule results in an increase in the mass of the target protein. In certain embodiments, the interaction between the target protein containing disordered domains and the test molecule results in a decrease in the mass of the target protein. In certain embodiments, the interaction between the target protein containing disordered domains and the test molecule results in oligomerization.

[0034] In another embodiment, the Disclosure provides a method (e.g., a high-throughput method) for analyzing a target molecule in a test solution. In a particular embodiment, the method includes (a) tracking a plurality of target molecules in a test solution over time to provide measurements of a plurality of spatiotemporal trajectories and / or rotational motions; (b) analyzing the measurements of the plurality of spatiotemporal trajectories and / or rotational motions to determine the motion of the target molecule in the test solution; and (c) comparing the motion of the target molecule obtained in (b) with the motion of a reference target molecule, the motion of the reference target molecule being the motion of the target molecule in a reference solution, where (i) a change in the motion of the target molecule compared to the motion of the reference target molecule indicates that the target molecule is affected by the test solution, or (ii) no change in the motion of the target molecule compared to the motion of the reference target molecule indicates that the target molecule is not affected by the test solution. In a particular embodiment, the duration and / or reversibility of the change in the motion of the target molecule compared to the duration and / or reversibility of the change in the motion of the reference target molecule indicates that the target molecule is affected by the test solution.

[0035] In certain embodiments, changes in motion related to the analysis of target molecules in a test solution include changes in (a) the diffusion coefficient obtained from the maximum likelihood estimator, (b) the geometric mean posterior diffusion coefficient, (c) the median of the jump length distribution, (d) the third quartile of the jump length distribution, (e) the median of the turning radius, (f) the mean posterior diffusion coefficient, (g) the mean square displacement, (h) the median of the bond angle, (i) the spatiotemporal trajectory length, or (j) the anisotropic decay time, (k) the spatial range of detection, (l) the number and wavelength of conjugated fluorescent labels, (m) the occupation in various diffusion states obtained from the state array, (n) the polarization of the conjugated fluorescent labels, or (o) the state occupation by inference.

[0036] In certain embodiments, which are intended to analyze target molecules in a test solution, the test solution contains a chaotropic agent. In certain embodiments, the chaotropic agent is urea. In certain embodiments, the test solution contains a viscous agent. In certain embodiments, the viscous agent is glycerol. In certain embodiments, the test solution contains a gradient. In certain embodiments, the gradient is a temperature gradient, a chemical gradient, or a combination thereof. In certain embodiments, the test solution contains at least two phases.

[0037] In certain embodiments, which aim to analyze target molecules in a test solution, the target molecule is an organic molecule with a magnitude of less than 1 kDa. In certain embodiments, the target molecule is selected from the group consisting of peptides, proteins, nucleic acids, carbohydrates, and lipids. In certain embodiments, the protein is selected from the group consisting of antibodies, receptors, and enzymes. In certain embodiments, the protein contains disordered domains. In certain embodiments, the protein does not contain structured domains. In certain embodiments, the peptide is a ligand. In certain embodiments, the target molecule is a nanomaterial. In certain embodiments, the target molecule is a synthetic polymer. In certain embodiments, the target molecule is labeled. In certain embodiments, the target molecule is fluorescently labeled. In certain embodiments, the target molecule is labeled with a fluorescent protein. In certain embodiments, the target molecule is labeled by a conjugate of synthetic nanomaterials or polymers. In certain embodiments, the target molecule is purified. In certain embodiments, the interaction between the target protein and the test solution causes a conformational change in the target molecule.

[0038] In certain embodiments relating to the analysis of a target molecule in a test solution, the sample used in connection with any of the methods described herein includes a solution. For example, if the interaction is determined between the target molecule (e.g., a target protein containing disordered domains) and the test molecule, then changes in the interaction between the target molecule and the solution in the presence of the test molecule can be identified. However, in certain embodiments, the presence of the test molecule is not required, and the interaction between the target molecule and the solution can be determined by changes in the movement of the target molecule in various solutions. In certain embodiments, the solution includes a pH buffer, salt, chaotropic agent, crowding agent, carrier, viscous agent, detergent, reducing agent, or a combination thereof. In certain embodiments, the viscous agent includes glycerol, sucrose, polyethylene glycol, dextran, Ficol, polyvinyl alcohol, and / or polyvinylpyrrolidone. In certain embodiments, the solution contains at least about 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% glycerol, or a range of glycerol constrained to such glycerol range. In certain embodiments, the chaotropic agent includes urea, guanidinium, alcohol, or ketone, etc. In certain embodiments, the crowding agent includes one or more of polyethylene glycol (PEG) polymers, Ficol, dextran polymers, or proteins (e.g., ovalbumin or hemoglobin). In certain embodiments, the carrier is a protein or a protein mixture. For example, but not limited to, the carrier protein or protein mixture prevents nonspecific interactions between the target molecule and / or test molecule (including other molecules) and the surface. Certain non-limiting examples of carriers include bovine serum albumin (BSA), lysozyme, casein, nonfat milk powder, and other biological extracts. In certain embodiments, the solution includes a temperature gradient, a chemical gradient, at least two liquid phases, or a combination thereof. In certain embodiments, the sample has a maximum volume of about 1 mL. In certain embodiments, the sample has a volume in the range of about 0.1 μL to about 100 μL.In certain embodiments, the sample has a volume of approximately 1 μL, approximately 5 μL, approximately 10 μL, approximately 20 μL, approximately 30 μL, approximately 40 μL, approximately 50 μL, approximately 60 μL, approximately 70 μL, approximately 80 μL, approximately 90 μL, or approximately 100 μL.

[0039] In another embodiment, the Disclosure provides a method (e.g., a high-throughput method) for analyzing a test solution containing a target molecule. In a particular embodiment, the method includes (a) tracking a plurality of target molecules in a test solution over time to provide measurements of a plurality of spatiotemporal trajectories and / or rotational motions; (b) analyzing the measurements of the plurality of spatiotemporal trajectories and / or rotational motions to determine the motion of the target molecule in the test solution; and (c) comparing the motion of the target molecule obtained in (b) with the motion of a reference target molecule, where the motion of the reference target molecule is the motion of the target molecule in a reference solution, and the comparison in step (c) is used to determine the properties of the test solution.

[0040] In a particular embodiment intended for the analysis of a test solution, the motion of the target molecule is calculated as follows: (a) diffusion coefficient obtained from the maximum likelihood estimator, (b) geometric mean posterior diffusion coefficient, (c) median of the jump length distribution, (d) third quartile of the jump length distribution, (e) median of the turning radius, (f) mean posterior diffusion coefficient, (g) mean square displacement, (h) median bond angle, (i) spatiotemporal trajectory length, (j) anisotropic decay time, (k) spatial range of detection, (l) number and wavelength of conjugated fluorescent labels, (m) occupation in various diffusion states obtained from a state array, (n) polarization of the conjugated fluorescent labels, or (o) state occupation by inference.

[0041] In certain embodiments, which are intended to analyze a test solution, the test solution contains a chaotropic agent. In certain embodiments, the chaotropic agent is urea. In certain embodiments, the test solution contains a viscous agent. In certain embodiments, the viscous agent is glycerol. In certain embodiments, the test solution contains a gradient. In certain embodiments, the gradient is a temperature gradient, a chemical gradient, or a combination thereof. In certain embodiments, the test solution contains at least two phases. In certain embodiments, the test solution is a biological sample taken from a subject.

[0042] In certain embodiments intended for the analysis of a test solution, the target molecule is an organic molecule with a size of less than 1 kDa. In certain embodiments, the target molecule is selected from the group consisting of peptides, proteins, nucleic acids, carbohydrates, and lipids. In certain embodiments, the protein is selected from the group consisting of antibodies, receptors, and enzymes. In certain embodiments, the protein contains disordered domains. In certain embodiments, the protein does not contain structured domains. In certain embodiments, the peptide is a ligand. In certain embodiments, the target molecule is a nanomaterial. In certain embodiments, the target molecule is a synthetic polymer. In certain embodiments, the target molecule is labeled. In certain embodiments, the target molecule is fluorescently labeled. In certain embodiments, the target molecule is labeled with a fluorescent protein. In certain embodiments, the target molecule is labeled by a conjugate of synthetic nanomaterials or polymers. In certain embodiments, the target molecule is purified. In certain embodiments, the interaction between the target protein and the test solution causes a conformational change in the target molecule.

[0043] In certain embodiments, which involve analyzing a test solution, the determined properties of the test solution are pH, ionic concentration, organic molecule concentration, or viscoelastic properties. In some embodiments, the viscoelastic property is viscosity. In certain embodiments, the properties of the test solution are used to diagnose a disease in the subject. In certain embodiments, the disease is cancer.

[0044] In certain embodiments intended to analyze a test solution, the sample used in connection with any of the methods described herein includes a solution. For example, if the interaction is determined between a target molecule (e.g., a target protein containing disordered domains) and the test molecule, then changes in the interaction between the target molecule and the solution in the presence of the test molecule can be identified. However, in certain embodiments, the presence of the test molecule is not required, and the interaction between the target molecule and the solution can be determined by changes in the movement of the target molecule in various solutions. In certain embodiments, the solution includes a pH buffer, salt, chaotropic agent, crowding agent, carrier, viscous agent, detergent, reducing agent, or a combination thereof. In certain embodiments, the viscous agent includes glycerol, sucrose, polyethylene glycol, dextran, Ficol, polyvinyl alcohol, and / or polyvinylpyrrolidone. In certain embodiments, the solution contains at least about 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% glycerol, or a range of glycerol constrained to such glycerol range. In certain embodiments, the chaotropic agent includes urea, guanidinium, alcohol, or ketone, etc. In certain embodiments, the crowding agent includes one or more of polyethylene glycol (PEG) polymers, Ficol, dextran polymers, or proteins (e.g., ovalbumin or hemoglobin). In certain embodiments, the carrier is a protein or a protein mixture. For example, but not limited to, the carrier protein or protein mixture prevents nonspecific interactions between the target molecule and / or test molecule (including other molecules) and the surface. Non-limiting examples of carriers include bovine serum albumin (BSA), lysozyme, casein, nonfat milk powder, and other biological extracts. In certain embodiments, the solution includes a temperature gradient, a chemical gradient, at least two liquid phases, or a combination thereof. In certain embodiments, the sample has a maximum volume of about 1 mL. In certain embodiments, the sample has a volume in the range of about 0.1 μL to about 100 μL.In certain embodiments, the sample has a volume of approximately 1 μL, approximately 5 μL, approximately 10 μL, approximately 20 μL, approximately 30 μL, approximately 40 μL, approximately 50 μL, approximately 60 μL, approximately 70 μL, approximately 80 μL, approximately 90 μL, or approximately 100 μL.

[0045] In certain embodiments, the change in motion determined by the disclosed method is calculated as a change in the polarization of the conjugated fluorescent label. In certain embodiments, the method for determining the change in the polarization of the conjugated fluorescent label may include (a) time-correlated single-photon counting (TCSPC), (b) step-scan pump-probe techniques, (c) ultra-long-lived fluorescent dyes and / or (d) advanced optical techniques. In certain embodiments, the target molecule and / or test molecule are labeled with an ultra-long-lived fluorescent dye.

[0046] In another aspect, the disclosure further provides a system for carrying out the disclosed method.

[0047] This patent or application document includes at least one drawing drawn in color. Copies of this patent or patent application publication, including the color drawing(s), will be provided by the Ministry upon request and payment of the required fees. [Brief explanation of the drawing]

[0048] [Figure 1A] This specification compares the diffusion of two very similar proteins, the Halo tag and the Halo protein 1 fusion, using the methods disclosed herein. Figures of the protein databank (PBD) structures of the exemplary proteins are shown. In the figure of the Halo tag on the left, the 51 amino acid residue tails are represented by a coil. [Figure 1B] This section compares the diffusion of two very similar proteins, HaloTag and Haloprotein 1 fusion, using the methods disclosed herein. A table is shown summarizing the theoretical and measured diffusion coefficients (measured at 10 pM) of each protein under 10% or 30% glycerol, as determined by the exemplary methods of this disclosure. [Figure 1C] This section shows a comparison of the diffusion of two very similar proteins, HaloTag and HaloProtein1 fusion, using the methods disclosed herein. The graph shows the diffusion coefficients (mean - / +98% confidence interval) of HaloTag and HaloProtein1 measured over the number of fields of view (FOV) used in the calculation. [Figure 2] The analysis of protein-protein interactions (PPIs) using SMT is shown. A shows the diffusion coefficient of Halo Protein 1 (40 pM) measured in 30% glycerol containing various concentrations of Protein 2, as determined by the exemplary method of this disclosure. B shows the diffusion coefficient of Halo Protein 1 (40 pM) measured in the presence of 30 nM of Protein 2 and increasing concentrations of unlabeled Protein 1 peptide (as a competitor), as determined by the exemplary method of this disclosure. [Figure 3A] An example of distinguishing conformational changes using SMT is shown. The diffusion coefficients of saltase-tagged protein 3 (closed conformation) and protein 3 mutant (open conformation) in 30% glycerol, as determined by the exemplary method of this disclosure, are shown. [Figure 3B] An example of distinguishing conformational changes using SMT is shown. The diffusion coefficients of protein 3 mutants in response to the addition of two small molecule ligands (molecule M1 and molecule M2) that can convert protein 3 mutants to closed conformations, as determined by the exemplary method of this disclosure, are shown. [Figure 3C] This example demonstrates the use of SMT to distinguish conformational changes. It shows state array profiles of protein 3 and protein 3 mutants treated with different concentrations of molecule M1 or MX. [Figure 3D] An example of distinguishing conformational changes using SMT is shown. Quantification of the upper left and lower right panels is shown in Figure 3C. [Figure 3E] This example demonstrates the use of SMT to distinguish conformational changes. It shows the state array profile of protein 3 in the presence of 16 μM molecule M1 and molecule MX in dose titration, as well as its quantification. [Figure 4A]Examples of buffer modification for analyzing changes caused by ligand binding to relatively rigid protein structures are shown. Exemplary structures are shown that do not contain ligand and that include an overlay containing the ligand ("Molecular M3") of protein 4. [Figure 4B] Examples of buffer modification for analyzing changes caused by ligand binding to relatively rigid protein structures are shown. The diffusion coefficients of saltase-tagged protein 4 in 30% glycerol in the presence of DMSO (vector control) without ligand or in the presence of 3 μM molecule M3, as determined by the exemplary methods of this disclosure, are shown. [Figure 4C] Examples of buffer modification for analyzing changes caused by ligand binding to relatively rigid protein structures are shown. Changes in the diffusion coefficient of protein 4, determined by the exemplary methods of this disclosure, are shown in the presence of various chaotropic agents (urea, guanidinium, acetonitrile, and acetone), including DMSO (ligand-free), or in the presence of molecular M3 ligand (3 μM). [Figure 4D] Examples of buffer modification for analyzing changes caused by ligand binding to relatively rigid protein structures are shown. Changes in the diffusion coefficient of protein 4 in the presence of various chaotropic agents (acetone, acetonitrile, urea, guanidinium, and DMSO) with or without molecule M3 (3 μM), as determined by the exemplary methods of this disclosure, are shown. Here, a high concentration of DMSO (different from the small amounts used as vector controls for chemical treatment elsewhere in this specification) was used as the chaotropic agent. [Figure 4E] This document presents an example of buffer modification for analyzing changes caused by ligand binding to relatively rigid protein structures. It shows the changes in the diffusion coefficient of protein 4 in DMSO (ligand-free) in the presence of various concentrations of chaotropic agents (urea, guanidinium, and acetone) or in the presence of ligand molecule M3 (3 μM), as determined by the exemplary method of this disclosure. The data are presented as box plots. [Figure 4F-1] Examples of buffer modification for analyzing changes caused by ligand binding to relatively rigid protein structures are shown. Dose titrations of molecule M3 and its analogues (M3 analogue 1 to M3 analogue 6) to protein 4 in the presence of 6M urea are shown. This table shows the Tm change measured by differential scanning fluorescence (DSF) assay and the IC50 determined by fluorescence polarization (FP) assay. The apparent EC50 determined by SMT is plotted against the potency of DSF (bottom left) and FP (bottom right). The EC50 and IC50 of protein 4 tool compound 2 were estimated based on available data. [Figure 4F-2] Examples of buffer modification for analyzing changes caused by ligand binding to relatively rigid protein structures are shown. Dose titrations of molecule M3 and its analogues (M3 analogue 1 to M3 analogue 6) to protein 4 in the presence of 6M urea are shown. This table shows the Tm change measured by differential scanning fluorescence (DSF) assay and the IC50 determined by fluorescence polarization (FP) assay. The apparent EC50 determined by SMT is plotted against the potency of DSF (bottom left) and FP (bottom right). The EC50 and IC50 of protein 4 tool compound 2 were estimated based on available data. [Figure 5] The diffusion coefficient of an inherently disordered region (IDR) found within protein 5, as determined by the exemplary method of this disclosure, and its response to the addition of a covalent binder molecule M4 are shown. [Figure 6A] The exemplary SMT method described herein highlights that it enables the measurement of protein diffusion in solution. Representative sequential images of SMT (top) and superimposed track (bottom) for JF549-labeled free Halo-tagged protein in 30% glycerol at a 192×192 pixel ROI at 200 Hz are shown. Scale bar represents 2 mm. [Figure 6B] The exemplary SMT method described in this disclosure highlights that it enables the measurement of protein diffusion in solution. The average diffusion coefficient is shown as a function of medium viscosity, compared to the theoretical Stokes-Einstein equations. [Figure 6C] The exemplary SMT method described herein highlights that it enables the measurement of protein diffusion in solution. The diffusion of Halo Protein 1 measured in dose responses for both monomer (monovalent / MV) Protein 2 and dimer (divalent / BV) Protein 2 is shown in pairs (n=8 wells). [Figure 6D] The exemplary SMT method described herein highlights that it enables the measurement of protein diffusion in solution. The diffusion coefficients of the protein 1 / protein 2 complex measured over the entire dose response of unlabeled protein 1 and molecule B1 are shown (n=32 FOV from two different plates). Error bars represent standard deviation. [Modes for carrying out the invention]

[0049] The subject matter disclosed herein relates to methods (e.g., high-throughput methods) for analyzing single molecules in cell-free systems. In certain embodiments, the methods described herein can be used for a variety of biophysical analytical applications, including, but not limited to, drug discovery activities, such as screening compound libraries and elucidating structure-activity relationships (SARs). In certain embodiments, the methods described herein can be used to characterize both known and novel pathway contributions to larger molecular assemblies, including targets such as protein signaling interaction networks. The subject matter disclosed herein further provides systems for carrying out such methods.

[0050] The subject matter of this disclosure will be described with reference to the drawings. The drawings are not drawn to scale and are provided solely to illustrate the embodiments disclosed herein. It should be understood that a number of specific details, relationships, and methods are provided in order to allow for a more complete understanding of the subject matter disclosed herein. Not intended to be limiting, but for clarity of disclosure, embodiments for carrying out this invention are divided into the following subsections. 1. Definition; 2. Molecule; 3. SMT method for biophysical analysis; 4. SMT system for biophysical analysis; 5. Examples; and 6. Exemplary Embodiments

[0051] 1.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. In case of any conflict, the definitions in this document shall prevail. Preferred methods and materials are described below, but similar or equivalent methods and materials described herein may also be used in carrying out or testing the subject matter of this disclosure. All publications, patent applications, patents, and other references referenced herein are incorporated herein by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative and not intended to limit the scope of the disclosure.

[0052] As used herein, the term “cell-free system” refers to a system that enables the biophysical analysis of molecules outside the context of intact whole cells. A sample containing such a cell-free system is not prevented from containing intact whole cells or cellular debris. Instead, the focus of the analysis of the cell-free system lies outside of intact whole cells. For example, though not intended to be limiting, a cell-free system may still contain a cell extract containing intact whole cells or cellular fragments, but the focus of the analysis of the cell-free system resides within the cell extract, not within the intact whole cells. In certain embodiments, the cell-free system does not contain a cell extract, but instead contains a solution in which the target molecules and / or test molecules described herein can be placed. In certain embodiments, the cell extract is a lysate, examples of which include bacterial cell extracts, fungal cell extracts, animal tissue or cell extracts, or plant tissue or cell extracts.

[0053] The terms “comprise,” “include,” “having,” “has,” “can,” “contain,” and their variations, when used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of further actions or structures. Unless otherwise explicitly stated by context, the singular forms “a,” “an,” and “the” include multiple referents. This disclosure also intends other examples of “comprising,” “consisting of,” and “consisting essentially of,” presented herein, whether expressly stated or not.

[0054] In the enumeration of numerical ranges described herein, each intervening number within a range is explicitly intended to have the same precision. For example, in the range 6–9, the numbers 7 and 8 are intended in addition to 6 and 9, and in the range 6.0–7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly intended.

[0055] As used herein, the terms “about” or “approximately” mean within an acceptable range of error for a particular value as determined by those skilled in the art, which depends in part on the limits of the method of measuring or determining the value, i.e., the measuring system. For example, “about” can mean a standard deviation of 3 or less or 3 or more, according to convention in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within one order of magnitude of the value, preferably up to five times, and more preferably up to two times.

[0056] In this specification, the term “antibody” is used in its broadest sense and encompasses a wide range of antibody structures, as long as they exhibit the desired antigen-binding activity, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies, and antibody fragments. Non-exclusive examples of antibody fragments include Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments.

[0057] As used herein, the term “chaotropic agent” refers to a substance that disrupts the structure of macromolecules such as proteins and nucleic acids (e.g., DNA and RNA), and / or denatures macromolecules. In certain embodiments, chaotropic agents interfere with intermolecular and / or intramolecular interactions mediated by non-covalent forces, such as hydrogen bonds, van der Waals forces, and hydrophobic effects.

[0058] As used herein, the term “contact” a sample (e.g., a cell-free sample) means exposing the sample to a molecule (e.g., a test molecule). Contact can be achieved using any preferred method. For example, “contact” can be achieved, for the purposes of this text, by adding a molecule (e.g., a test molecule) to a sample (e.g., a vessel holding the sample).

[0059] As used herein, the term “fluorescent molecule” refers to any molecule that emits a fluorescent signal. In certain embodiments, fluorescent emission occurs in response to exposure to light of a specific wavelength. An example of a naturally occurring fluorescent protein is green fluorescent protein (GFP). However, in certain embodiments, the test molecule or target molecule can be adapted to emit a fluorescent signal via the introduction of an encoded fluorescent tag, for example, by fusing a protein sequence to a target protein to emit fluorescence. In certain embodiments, the test molecule or target molecule can be adapted to emit a fluorescent signal via the binding of a fluorescent ligand. Non-limiting examples of such encoded fluorescent tags include Halo tags, SNAP tags, CLIP tags, TMP tags, and SunTags. Additionally or alternatively, the test molecule or target molecule can be adapted to emit a fluorescent signal via coupling with a fluorescent dye molecule (e.g., an amine or sulfhydryl reactive dye). Additionally or alternatively, the test molecule or target molecule can be adapted to emit a fluorescent signal via coupling with a fluorescent artificial material (e.g., polystyrene beads, quantum dots, and / or nanodiamonds).

[0060] As defined herein, "movement" of a molecule refers to a change in the position, orientation, conformation, and / or intermolecular bonds of the molecule over time.

[0061] In certain embodiments, molecular motion can be quantified by analyzing changes in spatial coordinates over a continuous time period, for example, by analyzing spatiotemporal trajectories. Motion characterized in this way may include, but is not limited to, measurements of diffusion coefficients. For example, for a given set of protein displacements between a single-state diffusion model with a constant position estimation error, the measurement may be a diffusion coefficient maximum likelihood estimator, defined as an estimate of the maximum likelihood diffusion coefficient for multiple spatiotemporal trajectories. In certain embodiments, molecular motion may be measured through analysis of the products of a link generation algorithm. Motion characterized in this way may include, but is not limited to, mean posterior diffusion coefficients, or the mean of the posterior probability distribution of coefficients from a stochastic linking algorithm. Motion characterized in this way may include, but is not limited to, geometric mean posterior diffusion coefficients, or the mean of the log-scale posterior probability distribution of coefficients from a stochastic linking algorithm. Motion characterized in this way may include, but is not limited to, measurements of jump length distributions. For example, for a given set of protein displacements between a given time point and a subsequent time point, a histogram of the probabilities of each displacement length ("jump length") can be constructed. The quantiles of this distribution can be used to describe the motion of molecules. In certain embodiments, the quantile used is the median of the jump length distribution. In certain embodiments, the quantile used is the third quartile of the jump length distribution. Motion characterized in this way may include, but is not limited to, a measurement of mean square displacement, defined by the mean of the squares of all displacements in a spacetime trajectory, averaged over multiple spacetime trajectories. Motion characterized in this way may also include, but is not limited to, a measurement of spacetime trajectory length or the distribution of spacetime trajectory lengths. Motion characterized in this way may also include, but is not limited to, a measurement of mean radius of rotation, defined by the mean square distance of all coordinates in a spacetime trajectory from the center of mass of the set of points contained in the spacetime trajectory, averaged over multiple spacetime trajectories.The motion characterized in this way may also include, but is not limited to, the measurement of the mean bond angle, which is defined by the angle formed by three continuous spatial coordinates averaged over multiple spatiotemporal trajectories. In certain embodiments, the motion of molecules can be measured through model-dependent analysis of multiple spatiotemporal trajectories. The motion characterized in this way is defined by the proportion of immobile molecules ("f") defined by fitting a two-state model. bound This may include, but is not limited to, ) ).

[0062] As used herein, the term “movement” encompasses changes in the direction in which the target is moving, as well as changes in both the speed and duration of the target’s movement, both increasing and decreasing. Thus, in certain embodiments, tracking motion may include determining whether the target is not moving, for example, whether the target is in a static constraint or is essentially in a static constraint. As described above, such movements can be characterized in various ways, including, but are not limited to, (a) the diffusion coefficients of multiple spatiotemporal trajectories obtained from the maximum likelihood estimator; (b) the geometric mean posterior diffusion coefficients of multiple spatiotemporal trajectories; (c) the median of the jump length distribution of multiple spatiotemporal trajectories; (b) the third quartile of the jump length distribution of multiple spatiotemporal trajectories; (c) the median of the turning radius of multiple spatiotemporal trajectories; (d) the mean posterior diffusion coefficient of multiple spatiotemporal trajectories; (f) the mean square displacement of multiple spatiotemporal trajectories; (g) the median of the joint angles of multiple spatiotemporal trajectories; (i) the spatiotemporal trajectory length of multiple spatiotemporal trajectories; (j) the anisotropic decay time; (k) the state occupation by inference; (l) the spatial range of detection; (m) the number and wavelength of conjugate fluorescent labels; (n) occupation in various diffusion states obtained from the state array; and / or (o) quantifying the polarization of the conjugate fluorescent labels.

[0063] As used herein, the term “spatiotemporal trajectory” refers to a set of spatial coordinates corresponding to the observation positions of temporally linked molecules. In certain embodiments, multiple spatiotemporal trajectories can be constructed algorithmically by linking multiple molecules whose positions have been determined at consecutive points in time. In certain embodiments, multiple spatiotemporal trajectories can be constructed conservatively by linking only spots within a fixed search radius when other links are not valid. In certain embodiments, multiple spatiotemporal trajectories can be constructed probabilistically.

[0064] In certain embodiments, molecular motion can be quantified by analyzing changes in the rotational motion of the molecule over a continuous period of time. For example, without limitation, motion characterized in this way can be determined as a measurement of fluorescence polarization. Fluorescence polarization measures the change in the orientation of the dipole moment of a fluorescent molecule (e.g., coupled to a target molecule or test molecule) over the period between absorption and emission events. For example, without limitation, when a fluorescent dye is excited with polarization, a slowly rotating molecule containing that dye will emit more light while retaining its original polarization than a faster rotating molecule. Therefore, by measuring fluorescence polarization, it is possible to determine the rotational motion of a target molecule or test molecule and use that information to calculate a measurement of rotational motion.

[0065] In certain embodiments, the rotational motion measurements described herein are calculated as a function of anisotropic decay time. The systems described herein enable the detection of anisotropic decay time, which can be calculated using strategies known in the art, such as those outlined in “Time-Dependent Anisotropy Decays” Lakowicz, JR (eds) Principles of Fluorescence Spectroscopy. Springer, Boston, MA. https: / / doi.org / 10.1007 / 978-0-387-46312-4_11 (which is incorporated as a whole by reference herein). In certain embodiments, the rotational motion measurements described herein are calculated as a function of the change in fluorescence polarization intensity. The systems described herein can detect the change in fluorescence polarization intensity, which can then be calculated using strategies known in the art. In certain embodiments, the rotational motion measurements described herein are calculated as a function of rotational diffusion relaxation time. The system described herein enables the detection of changes in the intensity of the relaxation time of rotational diffusion, which can then be calculated using strategies known in the art.

[0066] In certain embodiments, the movement of a target molecule can be quantified by analyzing changes in the conformation of the target molecule at a series of time points. In certain embodiments, such characterized movement may include, but is not limited to, the measurement of changes in the secondary, tertiary, and quaternary structures of the target protein, examples of which include folding and unfolding of the target protein and / or reconstruction of the internal conformation of the target protein.

[0067] In certain embodiments, molecular motion can be quantified by analyzing changes in the intermolecular bonding of a target molecule at a continuous time point. In certain embodiments, such characterized motion may include, but is not limited to, the measurement of homogeneous or heterogeneous bonding events (e.g., oligomerization processes).

[0068] When used herein, the detected motion includes, but is not limited to, any change in motion and may occur in response to any environmental or other factors (e.g., the presence of the test molecule). For example, but not limited to, motion or lack thereof may be caused by (A) the addition of molecules; (B) changes in temperature; (C) changes in oxygen concentration (e.g., introduction of a hypoxic state); (D) mechanical stress; (E) changes in pH; (F) changes in light exposure (e.g., increasing or decreasing the intensity); (G) changes in the composition of the solution, e.g., changes in viscosity and flow; and / or (H) changes in electrical or ionic stimulation.

[0069] As used herein, the term “multiple” refers to a number greater than one. In certain embodiments, the term “multiple target molecules” refers to a number of target molecules greater than one. For example, though not intended to be limiting, “multiple target molecules” may include at least about 10, at least about 50, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1,000, at least about 5,000, at least about 10,000, at least about 50,000, at least about 100,000, at least about 500,000, or at least about 1,000,000 target molecules. In certain embodiments, the term “multiple test molecules” refers to a number of test molecules greater than one. For example, though not intended to be limiting, multiple test molecules may contain at least approximately 10, at least approximately 50, at least approximately 100, at least approximately 200, at least approximately 300, at least approximately 400, at least approximately 500, at least approximately 600, at least approximately 700, at least approximately 800, at least approximately 900, at least approximately 1,000, at least approximately 5,000, at least approximately 10,000, at least approximately 50,000, at least approximately 100,000, at least approximately 500,000, or at least approximately 1,000,000 test molecules.

[0070] As used herein, “inherently disordered protein” (IDP) or “inherently disordered region” (IDR) refers to protein and peptide fragments of varying lengths that do not exist as a stably folded structure, as can be defined by classical structural biology tools such as X-ray crystallography and electron microscopy under physiological conditions. See, for example, Uversky and Dunker 2010 Biochimica et Biophysica Acta (1804: 1231-1264) (doi:10.1016 / j.bbapap.2010.01.017) and van der Lee et al., 2014 Chemical Reviews (114:6589-6631) (doi:10.1021 / cr400525m).

[0071] As used herein, the term “high-throughput” refers to the ability to rapidly process multiple samples in relation to the compositions, methods, and systems described herein. For example, but not limited thereto, the high-throughput compositions, methods, and systems described herein can be configured to utilize a variety of sample processing plates, including 6-well plates, 12-well plates, 24-well plates, 48-well plates, 96-well plates, 384-well plates, 1536-well plates, or other multi-well plates capable of holding any number of separated samples. Furthermore, the high-throughput compositions, methods, and systems of this disclosure can, in certain embodiments, be configured to utilize robotic sample and plate processing, as well as other computer-controlled sample and plate processing strategies. In certain embodiments, the high-throughput system includes, but is not limited thereto, a microfluidic flow chamber. In certain embodiments, the high-throughput method includes, but is not limited thereto, the use of a microfluidic flow chamber.

[0072] 2. Molecules This disclosure provides a high-throughput method for characterizing a single target molecule in a cell-free system. In certain embodiments, this disclosure provides a high-throughput method for characterizing a single target molecule in the presence of a test molecule in a cell-free system. In certain embodiments, the target molecule and / or test molecule analyzed by this disclosure may be any chemically defined entity.

[0073] In certain embodiments, the target molecule and / or test molecule may be an organic molecule with a magnitude of less than approximately 10 kDa, less than approximately 5 kDa, less than approximately 1 kDa, less than approximately 250 Da, or less than approximately 100 Da. In certain embodiments, the target molecule and / or test molecule may be an inhibitor or an agonist. For example, but not limited to, the target molecule and / or test molecule may be a kinase inhibitor (e.g., a tyrosine kinase inhibitor).

[0074] In certain embodiments, the target molecule and / or test molecule is a protein. In certain embodiments, the target molecule and / or test molecule is a protein containing a naturally occurring (e.g., wild-type) amino acid sequence or fragment thereof. In certain embodiments, the target molecule and / or test molecule is a fusion of two or more proteins or fragments thereof. In certain embodiments, the target molecule and / or test molecule is a protein containing one or more artificial (i.e., unnatural) amino acid sequences. In certain embodiments, the target molecule and / or test molecule is a protein containing natural and / or unnatural modifications, examples of which include lipid-protein conjugates, glycoproteins, and artificially modified proteins such as dye-conjugate proteins and drug-conjugate proteins, including antibody-drug conjugates. In certain embodiments, proteins that can be analyzed by the methods of the Disclosure may contain at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 1,000, at least about 5,000, at least about 10,000, at least about 15,000, at least about 20,000, or at least about 30,000 amino acids. In certain embodiments, proteins that can be analyzed using the high-throughput methods of the Disclosure may include any protein, which may include, but is not limited to, proteins containing one or more disulfide bonds, including multichain polypeptides containing one or more interchain and / or intrachain disulfide bonds, and further including multichain polypeptides that do not contain intrachain disulfide bonds.

[0075] In certain embodiments, the target protein and / or test protein of the Disclosure may be a signaling protein, such as a protein hormone, cytokine, kinase, phosphatase, receptor, or other enzyme or transcription factor. In certain embodiments, the target protein and / or test protein of the Disclosure is a receptor. In certain embodiments, the target protein and / or test protein of the Disclosure is an enzyme. In certain embodiments, the target protein and / or test protein of the Disclosure is a contractile protein. In certain embodiments, the target protein and / or test protein of the Disclosure is a structural protein. In certain embodiments, the target protein and / or test protein of the Disclosure is a storage protein. In certain embodiments, the target protein and / or test protein of the Disclosure is a transport protein. In certain embodiments, the target protein and / or test protein of the Disclosure is an ion channel. In certain embodiments, the target protein and / or test protein of the Disclosure is an antibody or antibody fragment.

[0076] In certain embodiments, the target protein and / or test protein may include one or more disordered domains. For example, but not limited to, the target protein and / or test protein may include at least one polypeptide segment lacking a stable three-dimensional structure. In certain embodiments, the target protein and / or test protein may include one or more structural domains and one or more disordered domains. In certain embodiments, the target protein and / or test protein may not include structural domains. Non-limiting examples of proteins containing disordered domains include glucocorticoid receptors, growth hormone receptors, cell tumor antigen p53, T cell surface glycoprotein CD4, SHC-converting protein 1, estrogen receptors, vitamin D3 receptors, cyclin H, serine / threonine protein kinase pim-1, aryl hydrocarbon receptors, androgen receptors, cryptochrome 2, T cell surface glycoprotein CD3 delta chain, sulfotransferase 2B1, DNA repair protein RAD52 homolog, huntingtin-interacting protein K, and Ras-related protein Ral-A. Additional non-exclusive examples of proteins containing disordered domains are disclosed in the databases and references listed in Uversky and Dunker 2010 Biochimica et Biophysica Acta (1804: 1231-1264) (doi:10.1016 / j.bbapap.2010.01.017) and van der Lee et al., 2014 Chemical Reviews (114:6589-6631) (doi:10.1021 / cr400525m), the full contents of which are disclosed herein by reference.

[0077] In certain embodiments, the target molecule and / or test molecule is a peptide. For example, but not limited to, peptides that can be analyzed by the methods of this disclosure may contain about 2, about 5, about 10, about 15, about 20, about 25, about 30, about 35, or about 40 amino acids. In certain embodiments, the target molecule and / or test molecule is a peptide containing non-natural amino acids or other modifications.

[0078] In certain embodiments, the target molecule and / or test molecule is a nucleic acid. In certain embodiments, the nucleic acid may have a defined sequence. In certain embodiments, the nucleic acid includes (A) ribonucleic acid (RNA) (e.g., including modified RNA); (B) deoxyribonucleic acid (DNA) (e.g., including modified DNA); and even (C) a combination of (A) and (B). In certain embodiments, the nucleic acid includes one or more categories of artificial nucleic acids, including, for example, morpholino, roch nucleic acid (LNA), peptide nucleic acid (PNA), and xeno nucleic acid (XNA). In certain embodiments, the nucleic acid may be single-stranded or double-stranded small interfering ribonucleic acid (e.g., double-stranded siRNA), antisense oligonucleotide, ribozyme, microRNA, or aptamer. In certain embodiments, the nucleic acid molecule may be linear or cyclic. In certain embodiments, the nucleic acid may contain natural or non-natural nucleotides. Non-limiting examples of non-natural nucleotides include modified nucleotide bases with derivatized sugar or phosphate backbone linkages, or chemically modified residues.

[0079] In certain embodiments, the target molecule and / or test molecule is a carbohydrate. In certain embodiments, the carbohydrate may be a monosaccharide (e.g., glucose, fructose, xylose, ribose, and galactose), a disaccharide (e.g., sucrose), or an oligosaccharide or polysaccharide (e.g., cellulose and starch).

[0080] In certain embodiments, the target molecule and / or test molecule is a lipid. For example, without limitation, the lipid may be a triglyceride, phospholipid, and / or sterol.

[0081] In certain embodiments, the target molecule and / or test molecule is a component of a mixture containing a bacterial extract, cell extract, tissue extract, plant extract, or animal extract. In certain embodiments, the bacterial extract, cell extract, tissue extract, plant extract, or animal extract is a lysate. In certain embodiments, the target molecule and / or test molecule is a component of a mixture containing serum, blood, and other biological samples. In certain embodiments, the target molecule and / or test molecule is a component of a mixture containing one or more buffers and / or glycerol.

[0082] In certain embodiments, the target molecule may be the wild-type form of the target molecule; for example, the target molecule may be a protein that is the wild-type form of a protein.

[0083] In certain embodiments, the target molecule may be a modified form of the target molecule. For example, for the purposes of this invention, the modified form of the target molecule may be a mutant form of the target molecule. For example, for the purposes of this invention, the target molecule may be a mutant form of a protein. In certain embodiments, the mutant form of a protein may include at least one amino acid substitution (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acid substitutions) compared to the wild type of the protein. In certain embodiments, the modified target molecule may be a post-translational modified form of the target protein. In certain embodiments, post-translational modifications include, but are not limited to, glycosylation, acetylation, phosphorylation, methylation, glycosylation, and lipidation. Additional non-limiting examples of post-translational modifications are disclosed in Ramazi and Zahiri, Database (Oxford):baab012 (2021), the entire contents of which are disclosed herein by reference. In certain embodiments, the modified target molecule may be a protein containing non-natural amino acids. In certain embodiments, the modified target molecule may be a protein containing artificially modified non-natural amino acids. In certain embodiments, the modified target molecule may include conjugates, examples of which include protein conjugates, fusion proteins, and proteins operably linked to non-protein compositions.

[0084] In certain embodiments, the target molecule is a homolog of the target molecule. In certain embodiments, the target molecule is an orthologue of the target molecule. In certain embodiments, the target molecule is a paralog of the target molecule.

[0085] In certain embodiments, the target molecule and / or test molecule may be present in a mixture of molecules, for example, in a mixture of a specified composition. For example, but not limited to, the target molecule may be present in a mixture (e.g., a sample) containing one or more different target molecules, such as one or more target proteins, one or more target peptides, one or more target organic molecules less than 1 kDa, one or more target carbohydrates, one or more target lipids, one or more target nucleic acids, one or more target proteins, one or more target peptides, one or more target organic molecules less than 1 kDa, one or more target carbohydrates, one or more target lipids, and / or one or more target nucleic acids. In certain embodiments, the sample or mixture may contain a first target molecule and a second target molecule. In certain embodiments, the first target molecule and the second target molecule are different species of target molecules. In certain embodiments, the first target molecule and the second target molecule are the same or different conformations of the target molecule. In certain embodiments, the distinction between the first target molecule and the second target molecule is achieved by differences in their labeling (e.g., fluorescent labeling). In certain embodiments, more than two target molecules are analyzed, for example, three or more target molecules. In certain embodiments, target molecules can be labeled. For example, for a limited purpose, target molecules can be fluorescently labeled so that they can be detected, for example, using the exemplary methods of this disclosure. In certain embodiments, target molecules can be labeled by adding a fluorescent protein or a tag that facilitates fluorescent labeling.Non-limiting examples of such fluorescent proteins include green fluorescent protein (GFP), Venus, monomer infrared fluorescent protein (mIFP), long-stokes shift monomer orange (LssmOrange), tagged red fluorescent protein 657 (TagRFP657), monomer orange 2 (mOrange2), monomer apple (mApple), sapphire, monomer tagged blue fluorescent protein (mTagBFP2), tdTomato, monomer cherry (mCherry), enhanced yellow fluorescent protein (EYFP), monomer cerulean 3 (mCerulean3), and enhanced green fluorescent protein (EGFP). Non-limiting examples of tags that can facilitate fluorescent labeling include Halo tags, SNAP tags, CLIP tags, TMP tags, and SunTag. In certain embodiments, target molecules can be labeled with fluorescent artificial materials (e.g., polystyrene beads, quantum dots, and / or nanodiamonds) to emit a fluorescent signal. In certain embodiments, the target molecule can be labeled with common tags used in recombinant protein expression (non-limiting examples include His tags, GST tags, FLAG tags, HA tags, and Avi tags). In certain embodiments, the target molecule can be biotinylated and labeled with avidin and its derivatives. In certain embodiments, the target molecule can be labeled with ligands, non-limiting examples of which include natural or non-natural ligands that can bind to proteins or tags. In certain embodiments, the target molecule can be labeled with antibodies. In certain embodiments, the target molecule can be labeled by chemical reactions, non-limiting examples of which include NHS-mediated and maleimide-mediated conjugates. In certain embodiments, the target molecule can be labeled by chemical reactions specific to the incorporated non-natural amino acids. In certain embodiments, the target molecule can be labeled by non-specific absorption. In certain embodiments, the target molecule can be labeled by conjugate with synthetic nanomaterials or polymers.

[0086] Alternatively or additionally, test molecules can be labeled. For example, for instance, test molecules can be fluorescently labeled so that they can be detected, for example, using the exemplary methods of this disclosure. In certain embodiments, test molecules can be labeled with fluorescent proteins. Non-limiting examples of such fluorescent proteins include green fluorescent protein (GFP), Venus, monomer infrared fluorescent protein (mIFP), long-stokes shift monomer orange (LssmOrange), tag red fluorescent protein 657 (TagRFP657), monomer orange 2 (mOrange2), monomer apple (mApple), sapphire, monomer tag blue fluorescent protein (mTagBFP2), tdTomato, monomer cherry (mCherry), enhanced yellow fluorescent protein (EYFP), monomer cerulean 3 (mCerulean3), and enhanced green fluorescent protein (EGFP). Non-limiting examples of tags that can facilitate fluorescent labeling include Halo tags, SNAP tags, CLIP tags, TMP tags, and SunTag. In certain embodiments, the test molecule can be labeled with a fluorescent artificial material (e.g., polystyrene beads, quantum dots, and / or nanodiamonds) to emit a fluorescent signal. In certain embodiments, the test molecule can be labeled with a ligand, non-limiting examples including natural or non-natural ligands. In certain embodiments, the test molecule can be labeled with an antibody. In certain embodiments, the test molecule can be labeled by a chemical reaction, non-limiting examples including NHS-mediated and maleimide-mediated conjugates. In certain embodiments, the test molecule can be labeled by non-specific absorption. In certain embodiments, the test molecule can be labeled with a synthetic nanomaterial or polymer conjugate.

[0087] In certain embodiments, the target molecule and / or test molecule can be purified. For example, but not limited to, the target molecule and / or test molecule can be purified from at least some of the components that were associated with it when it was first produced or generated. In certain embodiments, the purified target molecule and / or purified test molecule is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99% pure. For example, but not limited to, the target and / or test protein or peptide can be purified from the cell (and its components) in which it was expressed. In certain embodiments, the target molecule and / or test molecule can be partially purified. In certain embodiments, the target molecule and / or test molecule may be a partially purified extract from a biological sample. In certain embodiments, the target molecule and / or test molecule may be a crude extract from a biological sample. In certain embodiments, the target molecule and / or test molecule may be a crude biological sample, non-limiting examples of which include cell culture media, saliva, blood, serum, and other bodily fluids. In certain embodiments, the extract is a lysate (e.g., a cell or tissue lysate).

[0088] 3. SMT method for biophysical analysis This disclosure provides a method (e.g., a high-throughput method) for analyzing a single molecule in a cell-free sample. In certain embodiments, this disclosure provides a method for determining the motion of a target molecule. In certain embodiments, the motion of the target molecule is performed by tracking the target molecule over time to provide measurements of multiple spatiotemporal trajectories and / or rotational motions. Such motion may occur in or out of the presence of other sample components (e.g., test molecules or specific sample solutions).

[0089] In certain embodiments, the motion of a target molecule is determined by analysis of multiple spatiotemporal trajectories and / or rotational motion measurements detected by the method described herein. For example, without limitation, such analysis may address motion determined as follows: That is, (a) the diffusion coefficients of multiple spatiotemporal trajectories obtained from the maximum likelihood estimator; (b) the geometric mean posterior diffusion coefficients of multiple spatiotemporal trajectories; (c) the median of the jump length distribution of multiple spatiotemporal trajectories; (b) the third quartile of the jump length distribution of multiple spatiotemporal trajectories; (c) the median of the turning radius of multiple spatiotemporal trajectories; (d) the mean posterior diffusion coefficient of multiple spatiotemporal trajectories; (f) the mean square displacement of multiple spatiotemporal trajectories; (g) the median of the joint angles of multiple spatiotemporal trajectories; (i) the spatiotemporal trajectory lengths of multiple spatiotemporal trajectories; (j) the anisotropic decay time; (k) the inferred state occupation; (l) the spatial range of detection; (m) the number and wavelength of conjugated fluorescent labels; (n) the occupation in various diffusion states obtained from the state array; and / or (o) the polarization of the conjugated fluorescent labels.

[0090] The sensitivity of the methods disclosed herein provides the observation of changes in the motion of the target molecule compared to the motion of a reference target molecule. In certain embodiments, the motion of the reference target molecule may be the motion of the target molecule in the absence of the test molecule. In certain embodiments, the motion of the reference target molecule may be the motion of the target molecule in the presence of a reference solution. In certain embodiments, the motion of the reference target molecule may be the motion of a target molecule that is a different form of the target molecule analyzed by the methods of this disclosure. In certain embodiments, the motion of the reference target molecule may be the motion of a target molecule that is the same form as the target molecule analyzed by the methods of this disclosure.

[0091] In certain embodiments, the motion of a target molecule is determined as a diffusion coefficient associated with that target molecule. In certain embodiments, the motion of a target molecule (e.g., diffusion coefficient) determined by the method of this disclosure (e.g., a high-throughput method) can be used to determine the identity of the target molecule in a cell-free sample and / or to identify interactions between the target molecule and other molecules (e.g., test molecules or other solution components).

[0092] In certain embodiments, the motion of the target molecule and the motion of the reference target molecule are determined as diffusion coefficients. In certain embodiments, the change in the diffusion coefficient between the target molecule and the reference target molecule is at least about 0.001%. For example, but not limited to, the changes in the diffusion coefficient observed by the disclosed methods are at least about 0.001%, at least about 0.005%, at least about 0.01%, at least about 0.05%, at least about 0.1%, at least about 0.5%, at least about 1%, at least about 1.5%, at least about 2%, at least about 2.5%, at least about 3%, at least about 3.5%, at least about 4%, at least about 4.5%, at least about 5%, at least about 5.5%, at least about 6%, at least about 6.5%, at least about 7%, at least about 7.5%, at least about 8%, at least about 8.5%, at least about 9%, at least about 9.5%, or at least about 10%. In certain embodiments, the change in the diffusion coefficient compared to the reference diffusion coefficient is at least about 0.001%. In certain embodiments, the change in the diffusion coefficient compared to the reference diffusion coefficient is at least about 0.005%. In certain embodiments, the change in the diffusion coefficient compared to the reference diffusion coefficient is at least about 0.01%. In certain embodiments, the change in the diffusion coefficient compared to the reference diffusion coefficient is at least about 0.05%. In certain embodiments, the change in the diffusion coefficient compared to the reference diffusion coefficient is at least about 0.1%. In certain embodiments, the change in the diffusion coefficient compared to the reference diffusion coefficient is at least about 0.5%. In certain embodiments, the change in the diffusion coefficient compared to the reference diffusion coefficient is at least about 1%. In certain embodiments, the change in the diffusion coefficient compared to the reference diffusion coefficient is at least about 2%. In certain embodiments, the change in the diffusion coefficient compared to the reference diffusion coefficient is at least about 3%. In certain embodiments, the change in the diffusion coefficient compared to the reference diffusion coefficient is at least about 4%. In certain embodiments, the change in the diffusion coefficient compared to the reference diffusion coefficient is at least about 5%. In certain embodiments, the change in the diffusion coefficient compared to the reference diffusion coefficient is at least about 6%. In certain embodiments, the change in the diffusion coefficient compared to the reference diffusion coefficient is at least about 7%.In certain embodiments, the change in the diffusion coefficient compared to the reference diffusion coefficient is at least about 8%. In certain embodiments, the change in the diffusion coefficient compared to the reference diffusion coefficient is at least about 9%. In certain embodiments, the change in the diffusion coefficient compared to the reference diffusion coefficient is at least about 10%.

[0093] In certain embodiments, the expected diffusion coefficients of the target molecule and the test molecule are estimated. In certain embodiments, the diffusion coefficients of the target molecule and / or test molecule are measured using a tracking setting optimized for each frame rate (e.g., search radius). In certain embodiments, the diffusion coefficient of each molecule measured with the optimized tracking setting is used to fit the estimated Stokes radius of each molecule under experimental conditions. In certain embodiments, the estimated Stokes radius is used to estimate the expected diffusion coefficients of the target molecule and the test molecule. In certain embodiments, a comprehensive tracking model is used to estimate the expected diffusion coefficients of the target molecule and the test molecule. In certain embodiments, the comprehensive tracking model takes into account jump truncation, tracking error, and other effects. In certain embodiments, the comprehensive tracking model and the estimated Stokes radius are used to estimate the expected diffusion coefficients of the target molecule and the test molecule.

[0094] 3.1 Morphological analysis of target molecules This disclosure provides a method (e.g., a high-throughput method) for analyzing the movement of a target molecule to determine its morphology. As used herein, “morphology” is not limited to the three-dimensional conformation of the target molecule, but more accurately encompasses additional options. For example, a target molecule may have various morphologies depending on its properties. For instance, a protein target molecule may have a wild-type morphology, but may also have one or more modified morphologies, including, but not limited to, mutant morphologies, post-translational modified morphologies, alternative spliced ​​morphologies, or conjugated morphologies. For example, though not intended to be limiting, the methods of the present disclosure enable the determination of whether a target molecule is a post-translational modified form of the target molecule (e.g., a post-translational modified form of the target protein), whether a target molecule is an alternative splicing form of the target molecule (e.g., an alternative splicing form of the target protein), whether a target molecule is in a specific conformation (e.g., an open conformation of the target protein, or a closed conformation of the target protein), the wild-type form of the target molecule (e.g., the wild-type form of the target protein), a mutant form of the target molecule (e.g., a mutant form of the target protein), a homolog of the target molecule (e.g., a homolog of the target protein), an ortholog of the target molecule (e.g., an ortholog of the target protein), a paralog of the target molecule (e.g., a paralog of the target protein), or an artificially modified form of the target molecule (e.g., an artificially modified form of the target protein).

[0095] In certain embodiments, a method for analyzing the movement of a target molecule to determine its morphology (e.g., a high-throughput method) may include comparing the movement of the target molecule to the movement of a reference target molecule. In certain embodiments, the reference target molecule may be a different morphology of the target molecule being analyzed. In certain embodiments, the reference target molecule may be the same morphology as the target molecule being analyzed.

[0096] In certain embodiments, a method for determining the morphology of a target molecule may include (a) tracking multiple target molecules in a cell-free sample over time to provide measurements of multiple spatiotemporal trajectories and / or rotational motions; (b) analyzing the measurements of the multiple spatiotemporal trajectories and / or rotational motions to determine the motion of the target molecule; and (c) comparing the motion of the target molecule obtained in (b) with the motion of a reference target molecule, where the motion of the reference target molecule is the motion of one morphology of the target molecule, and (i) a change in the motion of the target molecule compared to the motion of the reference target molecule indicates that the target molecule has a different morphology from the reference target molecule, or (ii) no change in the motion of the target molecule compared to the motion of the reference target molecule indicates that the target molecule has the same morphology as the reference target molecule. In certain embodiments, the duration and / or reversibility of the change in the motion of the target molecule compared to the duration and / or reversibility of the change in the motion of the reference target molecule indicates that the target molecule has a different morphology from the reference target molecule. In certain embodiments, the duration of the change in the motion of the target molecule compared to the duration of the change in the motion of the reference target molecule indicates that the target molecule has a different morphology from the reference target molecule. In certain embodiments, the reversibility of the change in the movement of the target molecule compared to the reversibility of the change in the movement of the reference target molecule indicates that the target molecule has a different morphology from the reference target molecule.

[0097] In certain embodiments, the motion of a reference target molecule compared to the motion of a target molecule may correspond to the motion of the reference target molecule measured when the reference target molecule exists in a different form relative to the target molecule analyzed by the method of this disclosure. For example, without limitation, the motion of a reference target molecule may be measured under conditions where the reference target molecule exists in a different conformation relative to the conformation of the target molecule whose motion is being compared. In certain embodiments, the motion of a reference target molecule corresponds to the motion of the reference target molecule when it is in the same form as the target molecule analyzed by the method of this disclosure.

[0098] 3.2 Analysis of the interaction between the test solution and the target molecule This disclosure further provides a method (e.g., a high-throughput method) for analyzing the movement of target molecules in a test solution. In certain embodiments, the test solution is a solution of known composition. In certain embodiments, a cell-free sample comprises the test solution. In certain embodiments, the test solution is a biological sample taken from a subject. In certain embodiments, biological samples of varying purity levels comprise the test solution.

[0099] In certain embodiments, the test solution contains an agent that alters the conformation and / or oxidation or charge state of the target molecule. In certain embodiments, the test solution contains an agent that alters the conformational state of the target molecule (e.g., target protein).

[0100] In certain embodiments, the test solution contains one or more of the following: salts (e.g., sodium, magnesium, calcium), buffers (e.g., acetates, citrates, bistris, carbonates, CAPS, TAPS, bicine, tris, tricine, TAPSO, HEPES, TES, MOPS, PIPES, cacodylates, SSC, MES, succinic acid, or phosphates), amino acids, acids, bases, surfactants, detergents (e.g., SDS, Triton X-100, or Tween-20), chelating agents (e.g., ethylenediaminetetraacetic acid, phosphonates, or citric acid), preservatives, antibiotics, alcohols (e.g., methanol, ethanol, propanol, or isopropanol), reducing compounds, chaotropic agents, viscous agents, oxidizing compounds, dyes, or biomolecules (e.g., ATP, GTP, NADPH, nucleic acids, proteins, enzymes (e.g., RNase or protease K)).

[0101] In certain embodiments, the test solution contains an agent that reduces the structural stability of the target molecule. In certain embodiments, the test solution contains a chaotropic agent. Non-limiting examples of chaotropic agents include n-butanol, ethanol, acetone, acetonitrile, guanidinium chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and urea. In certain embodiments, the chaotropic agent is guanidinium chloride. In certain embodiments, the chaotropic agent is urea. In certain embodiments, the chaotropic agent is acetone. In certain embodiments, the chaotropic agent is acetonitrile.

[0102] In certain embodiments, the test solution contains an agent that gives viscosity to the solution (e.g., a viscous agent). In certain embodiments, the viscous agent is glycerol. In certain embodiments, the viscous agent (e.g., glycerol) is present in the solution at a concentration of about 1% volume / volume (%v / v) or more, about 2%v / v or more, about 5%v / v or more, about 10%v / v or more, about 15%v / v or more, about 20%v / v or more, about 25%v / v or more, about 30%v / v, about 35%v / v or more, about 40%v / v or more, about 45%v / v or more, about 50%v / v or more, about 55%v / v or more, about 60%v / v or more, about 65%v / v or more, about 70%v / v or more, about 75%v / v or more, about 80%v / v or more, about 85%v / v or more, about 90%v / v or more, or about 95%v / v or more.

[0103] In certain embodiments, the test solution includes a gradient. In certain embodiments, the gradient is a temperature gradient, a chemical gradient, or a combination thereof. In certain embodiments, the gradient is a temperature gradient, and for example, the solution may include a gradient from a higher temperature to a lower temperature. In certain embodiments, the temperature gradient is formed by the use of a laser. In certain embodiments, the gradient is a chemical gradient, and for example, the solution may include a gradient from a higher chemical concentration to a lower chemical concentration.

[0104] In certain embodiments, the test solution comprises at least two phases. In certain embodiments, the at least two phases comprise two liquid phases. In certain embodiments, the at least two phases comprise a liquid phase and a solid phase. For example, though not intended to be limiting, the test solution may comprise two liquid phases comprising an aqueous phase and an oil phase. Alternatively, the test solution may comprise two or more aqueous phases. In certain embodiments, the test solution comprises a liquid phase and a solid phase, where the liquid phase is an aqueous phase and the solid phase is a precipitate. In certain embodiments, the test solution comprises a liquid phase and a solid phase, where the liquid phase is an aqueous phase and the solid phase is an aggregate. In certain embodiments, the test solution comprises cells or other solid particles. In certain embodiments, the method of the present disclosure makes it possible to determine the movement of a target molecule at and / or across the interface between two phases of the solution.

[0105] In certain embodiments, the test solution flows actively through the inspection area of ​​the equipped device. For example, but not limited to, control of the test solution flow can be achieved by a flow-through microfluidic chip. In certain embodiments, the flow is controlled by a peristaltic pump or syringe pump. In certain embodiments, the microfluidic chip includes one or more channels, one or more inlets, one or more outlets, and one or more inspection areas. In certain embodiments, the microfluidic chip is composed of glass and / or polymer, but is not limited to these. In certain embodiments, the flow profile of the microfluidic is controlled by sheath flow, but is not limited to this.

[0106] In certain embodiments, the test solution is a homogeneous solution. For example, the test solution may have a uniform composition, though this is not intended to be limiting. In certain embodiments, the concentration of a component (e.g., a chemical or agent as illustrated herein) is consistent throughout the solution. In certain embodiments, the concentration of a viscous agent (e.g., glycerol) is consistent throughout the test solution.

[0107] In certain embodiments, the method of the present disclosure includes (a) tracking a plurality of target molecules in a test solution over time to provide measurements of a plurality of spatiotemporal trajectories and / or rotational motions; (b) analyzing the measurements of the plurality of spatiotemporal trajectories and / or rotational motions to determine the motion of the target molecules in the test solution; and (c) comparing the motion of the target molecules obtained in (b) with the motion of a reference target molecule, the motion of the reference target molecule being the motion of the target molecule in a reference solution, wherein (i) a change in the motion of the target molecule compared to the motion of the reference target molecule indicates that the target molecule is affected by the test solution, or (ii) no change in the motion of the target molecule compared to the motion of the reference target molecule indicates that the target molecule is not affected by the test solution.

[0108] In certain embodiments, the comparison in step (c) further includes comparing the duration and / or reversibility of the change in the motion of the target molecule with the duration and / or reversibility of the change in the motion of the reference target molecule. For example, for the purposes of this invention, comparing the duration of the change in the motion of the target molecule with the duration of the change in the motion of the reference target molecule may indicate that the target molecule is being affected by the test solution. In certain embodiments, comparing the reversibility of the change in the motion of the target molecule with the reversibility of the change in the motion of the reference target molecule may indicate that the target molecule is being affected by the test solution.

[0109] In certain embodiments, the method of the present disclosure includes (a) tracking a plurality of target molecules in a test solution over time to provide measurements of a plurality of spatiotemporal trajectories and / or rotational motions; (b) analyzing the measurements of the plurality of spatiotemporal trajectories and / or rotational motions to determine the motion of the target molecules in the test solution; and (c) comparing the motion of the target molecules obtained in (b) with the motion of a reference target molecule, where the motion of the reference target molecule is the motion of the target molecule in a reference solution, and the comparison in step (c) is used to determine the properties of the test solution. In certain embodiments, the comparison in step (c) further includes comparing the duration and / or reversibility of the change in the motion of the target molecules with the duration and / or reversibility of the change in the motion of the reference target molecule.

[0110] In certain embodiments, the determined properties of the test solution are pH, ionic concentration, organic molecule concentration, or viscoelastic properties. In certain embodiments, the viscoelastic property is viscosity. In certain embodiments, the properties of the test solution are used to diagnose a disease in the subject. In certain embodiments, the disease is cancer.

[0111] In certain embodiments, the reference solution may be a solution having the same composition as the test solution. In certain embodiments, the reference solution may be a solution having the same composition as the test solution except for one component (e.g., a viscous agent or a chaotropic agent). In certain embodiments, the reference solution may be a solution having a different composition than the test solution. In certain embodiments, the reference solution may be a second biological sample taken from the subject.

[0112] In certain embodiments, the interaction between the target protein and the test solution causes a conformational change in the target molecule. In certain embodiments, the interaction between the target protein and the test solution is reversible. In certain embodiments, the interaction between the target protein and the test solution is irreversible. In certain embodiments, the conformational change in the target molecule causes a change in the temperature stability of the target molecule.

[0113] 3.3 Analysis of the interaction between the target molecule and the test molecule Intermolecular interactions are essential to the function of biological molecules. These critical interactions include, but are not limited to, antibody-antigen, enzyme-coenzyme, enzyme-cofactor, enzyme-substrate, enzyme-allosteric / orthosteric regulator, ligand-receptor, ligand-coreceptor, protein-protein, protein-DNA, protein-RNA, protein-lipid, protein-cell surface, protein-viral particle, and protein-nanoparticle interactions. This disclosure provides methods, including high-throughput methods, for identifying interactions between a target molecule and a test molecule. In certain embodiments, the methods include detecting direct or indirect interactions between a target molecule and a test molecule.

[0114] In certain embodiments, a method for identifying interactions between a target molecule and a test molecule (e.g., a high-throughput method) includes: (a) contacting a cell-free sample containing multiple target molecules with multiple test molecules; (b) tracking the multiple target molecules over time to obtain measurements of multiple spatiotemporal trajectories and / or rotational motions; (c) analyzing the measurements of the multiple spatiotemporal trajectories and / or rotational motions to determine the movement of the target molecules in the presence of the test molecules; and (d) comparing the movement of the target molecules obtained in (c) with the movement of a reference target molecule, where the movement of the reference target molecule is the movement of the target molecule in the absence of the test molecule, and the change in the movement of the target molecule compared to the movement of the reference target molecule indicates an interaction between the target molecule and the test molecule. In certain embodiments, the duration and / or reversibility of the change in the movement of the target molecule compared to the duration and / or reversibility of the change in the movement of the reference target molecule indicates an interaction between the target molecule and the test molecule. In certain embodiments, the interaction between the target molecule and the test molecule is reversible. In certain embodiments, the interaction between the target molecule and the test molecule is irreversible. In certain embodiments, the interaction between the target protein and the test molecule results in oligomerization of the target protein.

[0115] In certain embodiments, the methods of the Disclosure (e.g., high-throughput methods) enable the identification of interactions between a target molecule and test molecules that induce conformational changes in the target molecule. For example, (not to limit) the methods of the Disclosure can be used to analyze a library of test molecules to identify test molecules that cause conformational changes in a target molecule. For example, (not to limit) the methods of the Disclosure can identify test molecules that allosterically bind to a target molecule. In certain embodiments, the methods of the Disclosure can identify test molecules that orthosterically bind to a target molecule. In certain embodiments, the method may include (a) contacting a cell-free sample containing multiple target molecules with multiple test molecules, each sample being contacted with a different test molecule; (b) tracking the multiple target molecules in each sample over time to provide measurements of multiple spatiotemporal trajectories and / or rotational motions; (c) analyzing the measurements of the multiple spatiotemporal trajectories and / or rotational motions to determine the movement of the target molecules in the presence of the test molecules; and (d) comparing the movement of the target molecules obtained in (c) with the movement of a reference target molecule, where the movement of the reference target molecule is the movement of the target molecule in the presence of a test molecule that does not induce a conformational change in the target molecule, and the change in the movement of the target molecule compared with the movement of the reference target molecule indicates an interaction between the target molecule and the test molecule that induces a conformational change in the target molecule. In certain embodiments, the duration and / or reversibility of the change in the movement of the target molecule compared with the duration and / or reversibility of the change in the movement of the reference target molecule indicates an interaction between the target molecule and the test molecule that induces a conformational change in the target molecule. In certain embodiments, conformational changes in the target molecule result in changes in the temperature stability of the target molecule.

[0116] This disclosure further includes a method (e.g., a high-throughput method) for determining the dose response of a target molecule to a test molecule. For example, but not limited thereto, this method could include: (a) contacting a plurality of cell-free samples containing a plurality of target molecules with a test molecule, wherein the plurality of cell-free samples are contacted with a range of test molecule doses; (b) tracking the plurality of target molecules over time in the presence of the range of test molecule doses to provide measurements of a plurality of spatiotemporal trajectories and / or rotational motions; (c) analyzing measurements of a plurality of spatiotemporal trajectories and / or rotational motions of the target molecules in the presence of the range of test molecule doses to determine the movement of the target molecules at each dose of the test molecule; and (d) comparing the movement of the target molecules obtained in (c) at various test molecule doses to determine the dose response of the target molecule to the test molecule.

[0117] This disclosure further includes a method (e.g., a high-throughput method) for determining the difference in dose response of two target molecules to a test molecule, the method comprising: (a) contacting a plurality of first cell-free samples containing a plurality of first target molecules with a test molecule, wherein the plurality of first cell-free samples are contacted with a range of test molecule doses; (b) tracking the plurality of target molecules over time in the presence of the range of test molecule doses to provide measurements of the plurality of spatiotemporal trajectories and / or rotational motions; and (c) tracking the plurality of target molecules over time in the presence of the range of test molecule doses. The process includes: (d) analyzing measurements of the spatiotemporal trajectory and / or rotational motion of the first target molecule to determine the movement of the first target molecule at each dose of the test molecule; (c) comparing the movement of the target molecule obtained in step (c) at various doses of the test molecule to determine the dose response of the target molecule to the test molecule; (e) repeating steps (a) to (d) with the second target molecule to determine the dose response of the second target molecule to the test molecule; and (f) comparing the dose response of the first target molecule with the dose response of the second target molecule to determine the difference in the responses of the first and second target molecules to the test molecule. In certain embodiments, more than two target molecules (e.g., three target molecules) can be analyzed.

[0118] In certain embodiments, the Disclosure provides a method (e.g., a high-throughput method) for identifying interactions between a test molecule and a target protein containing disordered domains. In certain embodiments, the target protein does not contain structured domains. In certain embodiments, the method includes (a) contacting a cell-free sample containing several target proteins with several test molecules; (b) tracking the several target proteins over time to obtain measurements of several spatiotemporal trajectories and / or rotational motions; (c) analyzing the measurements of several spatiotemporal trajectories and / or rotational motions to determine the movement of the target proteins in the presence of the test molecules; and (d) comparing the movement of the target proteins obtained in (c) with the movement of a reference target protein, where the movement of the reference target protein is the movement of the target protein in the absence of the test molecules, wherein the change in the movement of the target protein containing disordered domains compared with the movement of the reference target protein indicates an interaction between the target protein containing disordered domains and the test molecule. In certain embodiments, the interaction between a target protein containing disordered domains and the test molecule is demonstrated by comparing the duration and / or reversibility of the change in the movement of the target protein containing disordered domains with the duration and / or reversibility of the change in the movement of the reference target protein. In certain embodiments, the interaction between a target protein containing disordered domains and the test molecule is reversible. In certain embodiments, the interaction between a target protein containing disordered domains and the test molecule is irreversible. In certain embodiments, the interaction between a target protein containing disordered domains and the test molecule is demonstrated by a decrease in the diffusion coefficient of the target protein containing disordered domains compared to the diffusion coefficient of the reference target protein. In certain embodiments, the interaction between a target protein containing disordered domains and the test molecule is demonstrated by an increase in the diffusion coefficient of the target protein containing disordered domains compared to the diffusion coefficient of the reference target protein.

[0119] This disclosure further provides a method (e.g., a high-throughput method) for identifying test molecules that can distinguish between at least two target molecules. In certain embodiments, the method includes (a) contacting a cell-free sample containing a plurality of first target molecules with a plurality of test molecules; (b) tracking the plurality of first target molecules over time to obtain measurements of a plurality of spatiotemporal trajectories and / or rotational motions; (c) analyzing the measurements of the plurality of spatiotemporal trajectories and / or rotational motions to determine the movement of the first target molecules in the presence of the test molecules; (d) contacting a cell-free sample containing a plurality of second target molecules with a plurality of test molecules; (e) tracking the plurality of second target molecules over time to obtain measurements of a plurality of spatiotemporal trajectories and / or rotational motions; (f) analyzing the measurements of the plurality of spatiotemporal trajectories and / or rotational motions to determine the movement of the second target molecules in the presence of the test molecules; and (g) comparing the movements of the target molecules obtained in (c) and (f), demonstrating that the test molecules can distinguish between the two target molecules by changes in the movements of the first and second target molecules. In certain embodiments, the distinction between a first target molecule and a second target molecule is achieved by differences in their fluorescent labeling. In certain embodiments, more than two target molecules (e.g., three target molecules) can be analyzed. For example, but not limited to, this method may further include contacting a cell-free sample containing multiple third target molecules with multiple test molecules, tracking the multiple third target molecules over time to provide measurements of a third multiple spatiotemporal trajectory and / or rotational motion, analyzing the measurements of a third multiple spatiotemporal trajectory and / or rotational motion to determine the movement of the third target molecules in the presence of the test molecules, and comparing the movement of the third target molecules with the movement obtained in (c) and (f), demonstrating that the test molecule can distinguish between the three target molecules by changes in the movement of the third target molecules relative to the first and second target molecules.

[0120] The Disclosure further provides a method (e.g., a high-throughput method) for identifying a test molecule capable of distinguishing between at least two target molecules. In certain embodiments, the method includes (a) contacting a cell-free sample containing a plurality of first target molecules and a plurality of second target molecules with a plurality of test molecules; (b) tracking the plurality of first target molecules over time to obtain a plurality of spatiotemporal trajectory and / or rotational motion measurements; (c) analyzing the plurality of spatiotemporal trajectory and / or rotational motion measurements to determine the movement of the first target molecules in the presence of the test molecules; (d) tracking the plurality of second target molecules over time to obtain a plurality of spatiotemporal trajectory and / or rotational motion measurements; (e) analyzing the plurality of spatiotemporal trajectory and / or rotational motion measurements to determine the movement of the second target molecules in the presence of the test molecules; and (f) comparing the movements of the first and second target molecules obtained in (c) and (f), wherein the change in the movements of the first and second target molecules allows the test molecule to distinguish between the two target molecules.

[0121] 3.4 Single-molecule fluorescence polarization This disclosure further provides a method for characterizing the molecular dynamics of a target molecule by utilizing the orientation-dependent fluorescence properties of a fluorescent dye sufficiently firmly attached to the target molecule. In certain embodiments, the disclosed method includes detecting the orientation of the fluorescent dye and changes in its rotational degrees of freedom during oligomer formation. In certain embodiments, the disclosed method includes detecting how conformational changes affect the orientation and dynamics of a fluorescent dye attached to a specific site within a molecule. In certain embodiments, the disclosed method includes characterizing dynamic structural transitions (such as protein folding / unfolding events or nucleic acid conformational changes). In certain embodiments, the disclosed method includes time-resolved anisotropy measurements and polarization-sensitive imaging techniques coupled with advanced optical control. In certain embodiments, the disclosed method includes (a) time-correlated single-photon counting (TCSPC), (b) step-scan pump-probe techniques, (c) ultra-long-lived fluorescent dyes, and / or (d) advanced optical techniques.

[0122] In certain embodiments, TCSPCs are used to detect the arrival time of individual photons with picosecond precision. In certain embodiments, the TCSPC includes a 2D TCSPC via a single-photon avalanche diode (SPAD) array and a photon counting module. In certain embodiments, the TCSPC is used to resolve dynamics on a nanosecond scale. In certain embodiments, the TCSPC is paired with a polarization-sensitive readout to study the fast rotational dynamics and short fluorescence lifetimes typical of many biological fluorescent dyes. In certain embodiments, the TCSPC is used to sort between states and / or orientations of various fluorescent dyes.

[0123] In certain embodiments, the step-scan pump-probe technique involves modulating the polarization of excitation and probe light, and the resulting time-dependent fluorescence emission provides insights into the orientation / rotation and dynamics of the fluorescent dye. In certain embodiments, the step-scan pump-probe technique involves measuring polarization-dependent and time-correlated fluorescence depletion dynamics using step-scan pump-probe spectroscopic microscopy. In certain embodiments, the step-scan pump-probe technique is used to optically filter subpopulations of different molecular sizes, orientations, or dynamics, and / or to study dynamics in systems where the orientation of a fluorescent dye changes rapidly after excitation (e.g., in protein dynamics or in membrane structural changes).

[0124] In certain embodiments, ultra-long-lived fluorescent dyes exhibit fluorescence lifetimes longer than typical fluorescence decay times. In certain embodiments, ultra-long-lived fluorescent dyes exhibit fluorescence lifetimes several microseconds longer than typical fluorescence decay times. In certain embodiments, ultra-long-lived fluorescent dyes exhibit fluorescence lifetimes nanoseconds longer than typical fluorescence decay times. In certain embodiments, ultra-long-lived fluorescent dyes are used to significantly extend depolarization time beyond normal fluorescence time. Non-limiting examples of ultra-long-lived fluorescent dyes include carbon dots, quantum dots, and nanodiamonds. In certain embodiments, ultra-long-lived fluorescent dyes are used to measure rotational dynamics and depolarization processes over long time scales.

[0125] In certain embodiments, advanced optical techniques are used to precisely manipulate and control the polarization state of light spatially. In certain embodiments, the advanced optical techniques include wobble anisotropy and polarization filtering. In certain embodiments, the advanced optical techniques include advanced optical methodologies that include phase and polarization control. In certain embodiments, advanced optical techniques are used to achieve high-precision polarization-dependent measurements.

[0126] 4. Biophysical Analysis System 4.1 Image Acquisition System In certain embodiments, aspects of the subject matter can be implemented using an SMT workflow (e.g., an htSMT workflow), which incorporates a system for image acquisition. For example, such image acquisition can incorporate the imaging of a sample to generate a series of images and / or videos. In certain embodiments, an exemplary image acquisition system includes a light source configured to emit light relayed by one or more optical elements in an optical relay, the optical relay being configured to shape the light emitted from the light source and form a shaped beam such that the shaped beam has uniform intensity over the entire longitudinal dimension of a linear shape; an optical element (e.g., a Garbo mirror) which can be positioned either before or after the optical relay and configured to transform the shaped beam such that the shaped beam has uniform intensity over the entire longitudinal dimension of a linear shape; and one or more optical elements (e.g., a dichroic mirror (2-100)) configured to guide the shaped beam to an objective lens, thereby causing a portion of the sample plane to be illuminated by the inclined beam and light (e.g., a fluorescent emitter) from the sample, focused by the objective lens, to be emitted into the image acquisition system via a series of optical elements (e.g., lenses and absorption filters).

[0127] In certain embodiments, a microscope system for use in carrying out the method of the present disclosure may include: (a) a stage for supporting a cell-free sample, the cell-free sample containing a target molecule (e.g., a fluorescent molecule); (b) a light source for emitting a light beam capable of inducing a light-based response from the target molecule (e.g., a fluorescent molecule) in the cell-free sample; (c) an objective lens for focusing the light beam onto the cell-free sample in the sample plane, the target molecule (e.g., a fluorescent molecule) in the sample being positioned within the field of view in the sample plane; (d) a detection device for monitoring a light-based response from the target molecule (e.g., a fluorescent molecule) over a period of time (e.g., in the presence of a test molecule); (e) a memory; and (f) a processor communicating with the memory and the detection device. In certain embodiments, the processor may determine the diffusion coefficient of the target molecule (e.g., a fluorescent molecule) and compare its diffusion coefficient with a reference diffusion coefficient. In certain embodiments, the processor may determine the diffusion coefficient of the target molecule (e.g., a fluorescent molecule) in the presence of a test molecule. In certain embodiments, the reference diffusion coefficient is the diffusion coefficient of the target molecule (e.g., a fluorescent molecule) in the absence of the test molecule.

[0128] 4.2 Light source In certain embodiments, the system of the Disclosure includes a light source configured to emit light. In certain implementations of the image acquisition system disclosed herein, the light source may be configured to emit light of a single wavelength. In certain implementations of the image acquisition system disclosed herein, the light source may be configured to emit light of two, three, four, five, or more distinct wavelengths. In certain implementations, the wavelength(s) of light emitted from the light source are predetermined. For example, but not limited to, the wavelength(s) may be predetermined so that the emitted light induces fluorescence emission when it irradiates a sample, such as a sample containing a fluorescent molecule, such as a fluorescent target molecule and / or a fluorescent test molecule. In certain embodiments, the wavelength(s) employed in connection with the method described herein are in the range of 400 nm to 900 nm. In certain embodiments, the light source emits light having wavelengths of 400 nm to 408 nm, 550 nm to 565 nm, or 638 nm to 650 nm. In certain non-limiting implementations, the light source is configured to include three lasers having nominal center wavelengths of 405 nm, 560 nm, and 640 nm, which may vary within the absorption band of the fluorescent dye used. In certain embodiments, the 560 nm wavelength is used to excite the dye (e.g., JF549) attached to the Halo tag or saltase tag. In some cases, the dye attached to the HaloTag (e.g., JF 646 A wavelength of 642 nm or 646 nm is used to excite the ).

[0129] In certain non-limiting implementations, the light source is used to catalyze photochemical reactions. For example, though not intended as a limitation, the wavelength(s) and irradiation intensity may be such that chemical bonds are broken. As an additional example, and not intended as a limitation, the wavelength(s) and irradiation intensity may induce the adoption of a non-radiative dark state (i.e., "photobleached molecules"). As an additional example, and not intended as a limitation, the wavelength(s) and irradiation intensity may induce radiative or non-radiative energy transfer between fluorescent dyes in a sample.

[0130] In certain implementations of the image acquisition systems described herein, the light source can be configured to supply a predetermined amount of power to the back focal plane of the objective lens. For example, but not limited thereto, the light source may supply more than 10 mW of light for a particular wavelength (e.g., 405 nm) and / or more than 150 mW of light for another wavelength (e.g., 640 nm). Additionally or alternatively, if the light source includes three lasers emitting at wavelengths of 405 nm, 560 nm, and 640 nm, the light source can be configured to supply a predetermined amount of power to the back focal plane of the objective lens. For example, but not limited thereto, the 405 nm machine can be configured to supply more than 10 mW, the 560 nm machine can be configured to supply more than 150 mW, and the 640 nm machine can be configured to supply more than 50 mW.

[0131] In certain implementations of the image acquisition system described herein, the light source is configured to emit pulsed light. For example, for the purposes of this specification, the light source may be configured to emit strobo pulsed light. In certain implementations of the image acquisition system described herein, the light source is configured to emit pulsed light in sync with the start of image acquisition. In certain non-limiting implementations, the light source pulses at specific time intervals depending on the number of frames per second to be captured. For example, for the purposes of this specification, in a 200 frames per second (FPS) mode, the laser is on for 4ms and off for 1ms. In certain implementations of the SMT workflow, the light source is configured to transition to 90% to 10% power in less than approximately 0.4ms. In certain implementations of the SMT workflow, the light source is configured to transition to 90% to 10% power in less than approximately 0.2ms. In certain embodiments, the light source is configured to achieve 200, 400, 600, or 800 FPS.

[0132] In certain implementations of the image acquisition systems disclosed herein, the emission of light from a light source and the guidance of light to an optical relay can be facilitated using a single-mode fiber. Alternatively, in certain implementations of the image acquisition systems disclosed herein, a multimode fiber may be employed. For example, for the purposes of this disclosure, a multimode fiber may be configured in a predetermined shape for sample illumination.

[0133] In certain implementations of the image acquisition systems described herein, for example, with respect to systems configured for high-throughput sample analysis, the light source can be configured to have low drift in power output. In certain implementations, such low-drift configurations enhance the consistency of sample processing and facilitate high-throughput analysis. For example, but not limited to, such low-drift power output configurations maintain power output within a variation of approximately 0% to 15%, approximately 0% to 10%, approximately 10%, approximately 9%, approximately 8%, approximately 7%, approximately 6%, approximately 5%, approximately 4%, approximately 3%, approximately 2%, or approximately 1%.

[0134] In certain embodiments, such a low-drift power output configuration maintains power output within fluctuations of approximately 0% to approximately 15%, approximately 0% to approximately 10%, approximately 10%, approximately 9%, approximately 8%, approximately 7%, approximately 6%, approximately 5%, approximately 4%, approximately 3%, approximately 2%, or approximately 1% under varying ambient (room temperature) conditions (e.g., 17°C ± 5°C). In certain embodiments, this is achieved by using temperature sensors and / or closed-loop heaters to maintain a stable temperature of the internal light source (e.g., laser engine), thereby reducing output power drift. For example, but not limited to, the light source can be thermally isolated from ambient temperature fluctuations using an insulated enclosure design. Furthermore, or alternatively, closed-loop heaters can be strategically placed in specific locations within the system, e.g., on fiber couplers to reduce output drift. Furthermore, or alternatively, water jackets and / or cooling devices can be used to reduce heat buildup from the laser head. Furthermore, these thermal controls, used individually or in combination, reduce the warm-up time to reach a steady operating state and maintain a more stable internal operating temperature when the laser power supply is turned off and on.

[0135] 4.3 Optical elements and sample irradiation In certain embodiments, the system of the present disclosure includes a light source configured to emit light relayed by one or more optical elements in an optical relay, wherein the optical relay is configured to shape the light emitted from the light source and form a shaped beam. Specific optical elements of any particular optical relay implementation can be selected and configured to produce a suitable shaped beam and further provide a suitable conversion of that beam.

[0136] In certain non-limiting implementations of the optical relay in the image acquisition system disclosed herein, the optical relay includes one or more lenses and / or other optical elements. For example, but not limited to, the selection and orientation of the lenses and other optical elements in the optical relay are configured to appropriately shape the light beam directed to the sample. In certain non-limiting implementations, the optical relay includes an optical element (e.g., a collimator) for collimating the light emitted from the light source. Additionally or alternatively, the optical relay includes additional optical elements, examples of which include a Powell lens or other element adapted to produce a beam fan, one or more cylindrical lenses, one or more slits for adjusting the range of the light sheet, one or more achromatic lenses and / or one or more mirrors (one or more of which may be Garbo mirrors capable of converting light). Specific attributes of the optical elements are predetermined to produce a appropriately shaped light beam. For example, but not limited to, the SMT system of this disclosure can achieve not only a uniform horizontal FOV but also a uniform vertical FOV. This uniformity of horizontal and vertical FOV is in contrast to other strategies that provide non-uniform horizontal and / or non-uniform vertical FOV.

[0137] To achieve a uniform horizontal FOV and even a uniform vertical FOV, the optical relay of the SMT system described herein includes an optical element or assembly capable of producing an elongated beam along the X-plane and a narrow beam along the Y-plane, wherein the optical beam has uniform intensity over the entire longitudinal dimension of the linear shape. In certain non-limiting implementations, the optical relay of the SMT system described herein includes a Powell lens for shaping the optical beam so that it has uniform intensity over the entire longitudinal dimension of the linear shape. The optical relay of the SMT system described herein may include additional or alternative optical elements or assemblies for shaping the optical beam so that it has uniform intensity over the entire longitudinal dimension of the linear shape. For example, but not limited thereto, the optical relay of the SMT system described herein may include a diffracting element or assembly configured to shape the optical beam so that it has uniform intensity over the entire longitudinal dimension of the linear shape.

[0138] In certain non-limiting implementations of the optical relay of an image acquisition system disclosed herein, the optical relay includes one or more optical elements or assemblies configured to convert a light beam relative to the sample plane of the sample to be analyzed, for example, in a direction perpendicular to the longitudinal dimension of the light beam. For example, but not limited thereto, such optical elements or assemblies configured to convert a light beam relative to the sample plane of the sample to be analyzed may include a Garbo mirror or a piezoelectric element configured to convert the light beam. Additionally or alternatively, such optical elements or assemblies configured to convert a light beam relative to the sample plane of the sample to be analyzed may include a computer-controlled motor. In certain embodiments, the optical elements or assemblies configured to convert the light beam may be positioned before or after an optical element or assembly (e.g., a Powell lens) configured to shape the light beam so that it has uniform intensity over the entire longitudinal dimension of its linear shape.

[0139] In certain embodiments, the system includes an optical relay configured to shape light emitted from a light source to form a shaped beam, which is then guided by an optical element (e.g., a dichroic mirror) configured to guide the shaped beam to an objective lens, thereby illuminating the sample plane with an inclined beam.

[0140] In certain non-limiting implementations of the image acquisition system of this disclosure, the objective lens guides the inclined beam onto the plane of the sample to be analyzed. In certain non-limiting implementations of the image acquisition system of this disclosure, the objective lens is a water immersion objective lens. The use of a water immersion objective lens facilitates high-throughput sample analysis by eliminating the oil present in the use of an oil immersion objective lens, thereby enabling higher image quality and less distortion. The presence of oil poses problems in relation to automated systems, where oil may spread to components, including optical elements that can be contaminated by exposure to oil, and the refractive index of a water immersion objective lens is better suited to imaging aqueous samples. In certain non-limiting implementations, the objective lens is a 60X 1.27 NA water immersion objective lens (Nikon). In certain implementations of the workflow described herein, the water immersion objective lens is heated by a heating element. For example, such a heating element maintains the water immersion objective lens at a temperature sufficient to avoid inducing changes in the temperature of the sample contained in the sample plate.

[0141] 4.4. Image Acquisition In certain non-limiting implementations of the image acquisition system of this disclosure, the objective lens is also used to focus fluorescence emitted from the sample in response to illumination provided by the tilt beam. In certain non-limiting implementations, the fluorescent emitter focused on the target passes through an absorption filter (e.g., a bandpass absorption filter fitted to the spectrum of the fluorescent dye under observation and mounted on a fast filter wheel (Finger Lakes Instruments)) and is collected by a detector. In certain non-limiting implementations, the fluorescent emitter focused on the target is guided to an optical relay before being collected by the detector. For example, such an optical relay may include one or more lenses and one or more additional optical elements (e.g., elements configured to reject additional scattered light before collection by the detector). In certain non-limiting implementations, the fluorescent emitter focused on the target is guided through another dichroic mirror to split the emitter across multiple regions of the detector. In certain non-limiting implementations, the fluorescent emitter focused on the target is guided through another dichroic mirror to split the emitter across multiple detectors.

[0142] In certain non-limiting implementations of the image acquisition system of this disclosure, the detection device is configured to synchronize detection with the transformation of an inclined beam across the sample plane. For example, the detection device may be a CMOS camera (e.g., a back-illuminated CMOS camera (Hamamatsu Fusion BT)).

[0143] In certain implementations of the image acquisition system of this disclosure, the CMOS camera can be operated to collect a series of SMT frames for each field of view. For example, not limited to, but per field of view, 1 to 20,000 SMT frames, 1 to 15,000 SMT frames, 1 to 10,000 SMT frames, 1 to 5,000 SMT frames, 1 to 1,000 SMT frames, 2 to 500 SMT frames, 5 to 250 SMT frames, 10 to 200 SMT frames, 100 to 200 SMT frames, or 200 SMT frames may be collected. In certain implementations, the CMOS camera may be configured to operate at a frame rate of approximately 0.5 to approximately 2000 Hz. In certain implementations, the CMOS camera may be configured to operate at a frame rate of 0.5 to 1000 Hz. Or, in certain implementations, it may be configured to operate at 200 Hz. In certain embodiments, the CMOS camera can be configured to operate at frame rates from 100Hz to 1250Hz. For example, though not intended to be limiting, certain SMT implementations can be implemented at 200Hz. In certain embodiments, certain SMT implementations can be implemented at 400Hz. In certain embodiments, certain SMT implementations can be implemented at 800Hz. In certain embodiments, certain SMT implementations can be implemented at 1000Hz. In certain embodiments, certain SMT implementations can be implemented at 1200Hz. In certain embodiments, certain SMT implementations can be implemented at 1250Hz. In certain embodiments, certain SMT implementations can be implemented at 1400Hz. In certain embodiments, certain SMT implementations can be implemented at 1600Hz. In certain embodiments, certain SMT implementations can be implemented at 1800Hz. In certain embodiments, certain SMT implementations can be implemented at 2000Hz. In certain embodiments, a particular SMT implementation can be performed at frame rates of approximately 100Hz or higher, approximately 200Hz or higher, approximately 400Hz or higher, approximately 600Hz or higher, approximately 800Hz or higher, approximately 1000Hz or higher, approximately 1200Hz or higher, approximately 1400Hz or higher, approximately 1600Hz or higher, or approximately 1800Hz or higher.In certain embodiments, a specific SMT implementation can be performed at a maximum frame rate of approximately 1200 Hz. In certain embodiments, a specific SMT implementation can be performed at a maximum frame rate of approximately 1400 Hz. In certain embodiments, a specific SMT implementation can be performed at a maximum frame rate of approximately 1600 Hz. In certain embodiments, a specific SMT implementation can be performed at a maximum frame rate of approximately 1800 Hz. In certain embodiments, a specific SMT implementation can be performed at a maximum frame rate of approximately 2000 Hz. In certain embodiments, the CMOS camera is configured to achieve 200, 400, or 800 FPS.

[0144] In certain non-limiting implementations of the image acquisition system of this disclosure, the detection device is configured to transmit a signal in each frame to activate other elements of the imaging system. For example, for a limited time, the detection device may activate illumination from a light source to collect fluorescence emitters associated with a strobo laser pulse. For example, for a limited time, such fluorescence emission collection may be associated with frames of 10 to 100 ms and strobo laser pulses of 2 ms. In certain embodiments, fluorescence emission collection may be associated with strobo laser pulses of about 0.1 to about 1 ms. In certain embodiments, fluorescence emission collection is associated with strobe laser pulses of approximately 0.2–0.1 ms, 0.3–0.1 ms, 0.4–0.1 ms, 0.1–0.9 ms, 0.1–0.8 msec, 0.1–0.7 ms, 0.1–0.6 ms, 0.1–0.5 ms, 0.1–0.4 ms, 0.2–0.6 ms, 0.2–0.5 ms, 0.2–0.4 ms, or 0.3–0.5 ms. In certain embodiments, fluorescence emission collection is associated with strobe laser pulses of approximately 0.1–0.6 ms. In certain embodiments, fluorescence emission collection is associated with strobe laser pulses of approximately 0.1–0.5 ms. In certain embodiments, fluorescence emission collection is associated with strobe laser pulses of approximately 0.2–0.4 ms. In certain embodiments, fluorescence emission collection is associated with a strobolaser pulse of approximately 0.2 ms. In certain embodiments, fluorescence emission collection is associated with a strobolaser pulse of approximately 0.4 ms.

[0145] In certain implementations, the imaging acquisition system can be configured to acquire a predetermined field of view (FOV), for example, a detected FOV. In certain embodiments, the FOV, for example, a detected FOV, may have a size of first dimensions (approximately 150 μm to approximately 250 μm) × second dimensions (approximately 100 μm to approximately 210 μm). In certain embodiments, the FOV, for example, a detected FOV, may have a size of first dimensions (approximately 200 μm to approximately 250 μm) × second dimensions (approximately 150 μm to approximately 210 μm), or the FOV, for example, a detected FOV, may have a size of first dimensions (approximately 225 μm to approximately 250 μm) × second dimensions (approximately 175 μm to approximately 210 μm). For example, the FOV, for instance, the detected FOV, may have a size of a first dimension (approximately 250 μm) × a second dimension (approximately 190 μm), for example, as disclosed in Example 1.

[0146] In certain embodiments, a certain percentage of the FOV, for example, the detected FOV, provides usable data. In certain embodiments, at least 75% of the FOV, at least 80% of the FOV, at least 85% of the FOV, at least 90% of the FOV, at least 95% of the FOV, at least 96% of the FOV, at least 97% of the FOV, at least 98% of the FOV, at least 99% of the FOV, or 100% of the FOV, provides usable data. In certain embodiments, at least 75% of the FOV, for example, the detected FOV, provides usable data. In certain embodiments, at least 80% of the FOV, for example, the detected FOV, provides usable data. In certain embodiments, at least 85% of the FOV, for example, the detected FOV, provides usable data. In certain embodiments, at least 90% of the FOV, for example, the detected FOV, provides usable data. In certain embodiments, at least 95% of the FOV, for example, the detected FOV, provides usable data. In certain embodiments, at least 96% of the FOV, for example, the detected FOV, provides usable data. In certain embodiments, at least 97% of the FOV, for example, the detected FOV, provides usable data. In certain embodiments, at least 98% of the FOV, for example, the detected FOV, provides usable data. In certain embodiments, at least 99% of the FOV, for example, the detected FOV, provides usable data. In certain embodiments, 100% of the FOV, for example, the detected FOV, provides usable data. In certain embodiments, a percentage of about 75% or more of the FOV, for example, about 80% or more of the FOV, about 85% or more of the FOV, about 90% or more of the FOV, about 95% or more of the FOV, about 96% or more of the FOV, about 97% or more of the FOV, about 98% or more of the FOV, or about 99% or more of the FOV, provides usable data. In certain embodiments, a specific percentage of the FOV, for example, the detected FOV, achieves sufficient laser irradiation to track protein movement.For example, but not limited to, at least 75% of the FOV, at least 80% of the FOV, at least 85% of the FOV, at least 90% of the FOV, at least 95% of the FOV, at least 96% of the FOV, at least 97% of the FOV, at least 98% of the FOV, at least 99% of the FOV, or 100% of the FOV achieves sufficient laser irradiation to track protein movement. In a particular embodiment, at least 75% of the FOV, for example, the detected FOV, achieves sufficient laser irradiation to track protein movement. In a particular embodiment, at least 80% of the FOV, for example, the detected FOV, achieves sufficient laser irradiation to track protein movement. In a particular embodiment, at least 85% of the FOV, for example, the detected FOV, achieves sufficient laser irradiation to track protein movement. In a particular embodiment, at least 90% of the FOV, for example, the detected FOV, achieves sufficient laser irradiation to track protein movement. In a particular embodiment, at least 95% of the FOV, for example, the detected FOV, achieves sufficient laser irradiation to track protein movement. In certain embodiments, at least 96% of the FOV, for example, the detected FOV, achieves sufficient laser irradiation to track the protein's movement. In certain embodiments, at least 97% of the FOV, for example, the detected FOV, achieves sufficient laser irradiation to track the protein's movement. In certain embodiments, at least 98% of the FOV, for example, the detected FOV, achieves sufficient laser irradiation to track the protein's movement. In certain embodiments, at least 99% of the FOV, for example, the detected FOV, achieves sufficient laser irradiation to track the protein's movement. In certain embodiments, 100% of the FOV, for example, the detected FOV, achieves sufficient laser irradiation to track the protein's movement.In certain embodiments, a proportion of the FOV of 75% or more achieves sufficient laser irradiation to track protein movement, while proportions of approximately 80% or more of the FOV, 85% or more of the FOV, 90% or more of the FOV, 95% or more of the FOV, 96% or more of the FOV, 97% or more of the FOV, 98% or more of the FOV, or 99% or more of the FOV provide sufficient laser irradiation to track protein movement.

[0147] In certain implementations, the imaging acquisition system can be configured to acquire a predetermined image dimension for each frame, which is referred to herein as the region of interest (ROI). In certain implementations, the ROI varies depending on the frame rate employed. For example, at 200 FPS, the ROI is defined as 2304 × 768 pixels, and the sample plane is 248.832 × 82.944 μm. 2 At 400 FPS, the ROI is defined at 2304 × 432 pixels, and the sample plane is 248.832 × 46.6 μm. 2 At 800 FPS, the ROI is defined at 2304 × 192 pixels, and the sample plane is 248.832 × 20.928 μm. 2 In certain embodiments, the image acquisition system can be configured to achieve 200, 400, or 800 FPS.

[0148] In certain implementations, the imaging acquisition system can be configured to perform a predetermined sweep rate at a predetermined frame rate. For example, though not limiting, the sweep rate may be 82.94 μm / 4 ms, which corresponds to 20.7 μm / ms, or 2.07 cm / s. In certain embodiments, the imaging acquisition system can be configured to perform a sweep rate achieving 200, 400, 600, or 800 FPS.

[0149] In certain implementations, the detector can be used to collect fluorescent emitters at multiple wavelengths. For example, without limitation, fluorescent emitters from additional fluorescent dyes can be collected in the same field of view at the same or different frame rates to provide downstream registration of SMT tracks to other labeled components. Additional channels in the detector can be used as desired to increase the number of fluorescent emitters captured simultaneously in the same field of view, providing downstream registration of SMT tracks to other components in the sample (such as test molecules).

[0150] 4.5 SMT Software This disclosure provides an exemplary system of a high-throughput single-molecule imaging platform for measuring the movement of molecules in cell-free samples. Experiments can be performed to collect large amounts of data from multiple target molecules. Experiments may involve applying various identifiers (such as labels) to target molecules, which can then be detected by fluorescence or other means (e.g., using a laser or other light source). Samples forming part of such experiments can be organized in plates having multiple wells. Each well may have one or more associated fields of view (FOV). The FOV can be a location within a single well or a location corresponding to a single well. A series of images can be generated for each FOV to obtain one or more videos. These videos may include SMT videos and even non-SMT videos. Using SMT videos, the trajectory of individual labeled molecules, such as proteins, can be tracked to generate multiple spatiotemporal trajectories. Each spatiotemporal trajectory may consist of multiple spots containing the spatiotemporal coordinates of the labeled molecule at a particular time. Separately from, and possibly in parallel with, tracking, the videos can be used to identify molecules and generate masks by using machine learning and / or computer vision-based image segmentation. A mask is a spatial region within the field of view (FOV) produced by segmentation. Each mask can belong to a mask category.

[0151] In certain embodiments, tracking data and segmentation data can be combined to generate multiple evaluation metrics associated with various aspects of a sample (e.g., the location of phase changes, or other sample attributes). In other words, spatiotemporal trajectories (e.g., spatiotemporal trajectory data) can be combined with machine learning-processed image segmentation data and further analyzed using statistical / machine learning methods. The processing of the combined data can be used to generate evaluation metrics (such as hit scores associated with compounds and / or targets within a sample, which may be stored in a database structure).

[0152] This disclosure provides an exemplary data flow for a high-throughput single-molecule imaging platform for measuring protein movement in cell-free samples. Experimental specifications defining the experiment can be provided as data inputs via one or more clients. Experimental specifications can define various parameters of the experiment (e.g., dyes, compounds, treatments). In certain embodiments, the imaging system (e.g., the imaging system) can capture a series of images that generate one or more SMT videos and / or non-SMT videos or segmentation videos (e.g., video) characterizing molecular movement. SMT videos can characterize the movement of individual fluorescent molecules and / or may contain images of individual fluorescent molecules. Segmentation videos may include a series of images characterizing the movement of labeled molecules and / or their components.

[0153] SMT videos can be analyzed to perform operations related to molecular tracking. This may include detection, sub-pixel position estimation, and linking to identify the spatiotemporal trajectory of molecules across various images within the SMT video. More specifically, during detection, one or more spots within the SMT video can be detected or recovered. Each spot may have spatiotemporal coordinates. These spatiotemporal coordinates can be estimated using sub-pixel position estimation techniques. By performing linking on the spots, the spatiotemporal trajectory can ultimately be identified.

[0154] As used herein, a link is a potential association between two spots. Each link is directed, starting at one spot and ending at another. A “correct link” is one that connects two spots produced by the same emitter in different frames. Otherwise, the link is “incorrect”. One objective of the link algorithm is to estimate which links are correct. In this specification, a link is referred to in the form a:i→j, which is interpreted as meaning a link α that starts at spot i and ends at spot j. Links satisfy at least three of the following constraints: (a) a link moves forward in time, (b) a link cannot connect two spots that are further apart than a certain limit (referred to herein as the “search radius”), and (c) a link cannot connect two spots that are further apart in time than a certain limit (referred to herein as the “gap limit”). The spot-link graph is a graph of spots and links for a single SMT video. Spots are the vertices of this graph, and links are the edges of this graph. Since links advance over time, the spot-link graph is a directed acyclic graph. A matching is a subset of links in the spot-link graph, where no two links in this subset start or end at the same spot. Herein, spatiotemporal trajectory 715 is used to refer to a sequence of consecutive (end-to-end) links within the same matching. Multiple spatiotemporal trajectories can be used to determine dynamic evaluation metrics. Such parameters may include attributes of a spot that characterize the movement of that spot. Such parameters may include one or more of the velocity, diffusion coefficient, or anomaly parameters of each spot. The dynamic parameters of spot i are, herein, θ i This is referred to as Θ. In this specification, the set of dynamic parameters for all spots in a spot-link graph is called Θ.

[0155] Experimental information, such as dynamic evaluation metrics, image evaluation metrics, and any data from which any of the evaluation metrics are derived (such as segmentation information), can be provided to a data repository for storage. Such a data repository can store all experimental results, such as dynamic evaluation metrics, image evaluation metrics, and / or any data from which any of the evaluation metrics are derived. The data repository may include a local persistence server and / or a dedicated server accessed locally or by the cloud. The data repository may also store metadata associated with it and / or metadata associated with the experimental specifications. Experimental information (e.g., past experimental results and metadata) can be provided to the data repository via a repository application program interface (API). The repository API may also interface with a web-based graphical user interface frontend that provides such information for display to the client.

[0156] Examples of dynamic evaluation metrics can also include state arrays. State arrays are frameworks for learning interpretable dynamic models from the spatiotemporal trajectories of SMTs and may be used to gain additional insights into the movement of target proteins. In some variations, state arrays can be generated / augmented using segmentation information. State arrays can be used to quantify the relative occupancy of a molecule in different conformations (e.g., open or closed conformations). Fractional occupancy can be calculated by weighting the average of each state array bin by the occupancy in each bin and dividing by the total weighted average diffusion coefficient.

[0157] In certain embodiments, a computer implementation environment may include an imaging system that interacts with a computing architecture to implement various algorithms described herein. The imaging system may interface with one or more clients (e.g., clients via a web application having a graphical user interface). One or more clients may interface with one or more servers accessible via a network. One or more clients may host frame grabbers that capture images (e.g., video) from a camera. These images may be temporarily stored on one or more clients and periodically transferred via the network to one or more servers for remote storage. One or more servers may also contain, or access, one or more data stores for storing data collected and / or extracted from samples by the imaging system. In some variations, the network may include, or interface with, one or more network storage arrays for storing data such as captured images (e.g., video).

[0158] In some variations, the sample computing device architecture may be a client(s) and / or server(s), and some components described in relation to the diagram may be optional for the client(s) and / or server(s). A bus can function as an information highway interconnecting other illustrated components of the hardware. A processing system called a CPU (Central Processing Unit) (e.g., one or more computer processors / data processors on a given or multiple computers) can perform the calculations and logical operations necessary for program execution. Optionally or additionally, a processing system called a GPU (Graphics Processing Unit) (e.g., one or more computer processors / data processors on a given or multiple computers) can perform the calculations and logical operations necessary for program execution. Non-temporary processor-readable storage media, such as read-only memory (ROM) and random-access memory (RAM), can communicate with and / or the processing system and may contain one or more programming instructions for the operations specified herein. Optionally, program instructions can be stored on non-temporary computer-readable storage media, such as magnetic disks, optical disks, recordable memory devices, flash memory, solid-state drives, or other physical storage media.

[0159] In one example, the disk controller may interface to one or more optional removable storage or local storage to the system bus. Removable storage may be an external or internal disk drive, or a solid-state drive, or an external hard drive. Local storage may be an internal hard drive and / or memory. These various examples of removable storage, local storage, and disk controllers, as shown above, are optional devices. The system bus may also include at least one communication interface that enables communication with external devices, either physically connected to the computing system or available externally via a wired or wireless network, such as cloud storage and remote services. In some cases, at least one communication interface 1024 includes a network interface, or otherwise comprises a network interface.

[0160] In some variations, for example for client(s), the subject matter described herein can be implemented on a computing device to provide user interaction, which includes a display device (e.g., an LCD (liquid crystal display) or LED (light-emitting diode) monitor) for displaying information acquired from the bus to the user via a display interface, and an input device such as a keyboard and / or pointing device (e.g., a mouse or trackball) and / or touchscreen that allows the user to provide input to the computer. Other types of input devices can also be used to provide user interaction. For example, the feedback provided to the user may be any form of sensory feedback (e.g., visual feedback, auditory feedback via a microphone, or tactile feedback). Input from the user can also be received in any form, including acoustic, speech, or tactile input. The input device and microphone can be coupled to the bus via an input device interface to transmit information. As an example, the input device may be an imaging system configured to capture a series of images as described herein. A frame grabber can capture or grasp individual frames from analog or digital data encapsulating a series of images acquired from the bus. A frame grabber may include memory capable of storing individual frames or multiple frames. A frame grabber may also provide individual frames or multiple frames to the bus for further storage, for example, in local storage and / or removable storage.

[0161] One or more aspects or features of the subject matter described herein can be realized in digital electronic circuits, integrated circuits, specially designed application-specific integrated circuits (ASICs), field-programmable gate arrays (FPGAs), computer hardware, firmware, software, and / or combinations thereof. These various aspects or features may include implementations in one or more computer programs that are executable and / or interpretable on a programmable system including at least one programmable processor, the at least one programmable processor may be dedicated or general-purpose and coupled to receive data and instructions from and transmit data and instructions to a storage system, at least one input device, and at least one output device. The programmable system or computing system may include clients and servers. Clients and servers are generally far apart from each other and typically communicate through a communication network. The client-server relationship arises from computer programs running on each computer and from the client-server relationship they have with each other.

[0162] These computer programs, also called programs, software, software applications, applications, components, or code, include machine language instructions for a programmable processor and can be implemented in high-level procedural languages, object-oriented programming languages, functional programming languages, logic programming languages, and / or assembly / machine language. As used in this application, “machine-readable medium” means any computer program product, apparatus and / or device (e.g., magnetic disks, optical disks, memory, programmable logic circuits (PLDs), etc.) used to provide machine instructions and / or data to a programmable processor that includes a machine-readable medium that receives machine instructions as machine-readable signals. The term “machine-readable signals” means any signals used to provide machine instructions and / or data to a programmable processor. The machine-readable medium can store such machine instructions non-temporarily, for example, non-temporarily stored solid-state memory or magnetic hard drives, or any equivalent storage medium. The machine-readable medium can also store such machine instructions in a temporary manner, for example, a processor cache or other random-access memory associated with one or more physical processor cores.

[0163] 5. Examples The subject matter disclosed herein is not intended to be limiting, but will be better understood by referring to the following examples provided as illustrations of the subject matter disclosed herein. This section describes the experimental materials and methods used to generate the specific non-limiting illustrative data disclosed in Examples 1-5 herein.

[0164] Protein expression and purification Halo-tagged expression vectors (pH6HTN His6Halo-tagged® T7 Vectors) were purchased from Promega, and an expression vector for Halo protein 1 fusion was constructed by inserting a sequence encoding protein 1 at the C-terminus of the Halo tag immediately after the TEV cleavage site. Both plasmids were transfected into E. coli BL21 (DE3) and grown in LB medium at 37°C until the OD600 reached 0.6-0.8, followed by induction in 0.1 mM IPTG overnight at 16°C. Cells were collected by centrifugation, lysed with BugBuster® protein extraction reagent (EMD Millipore), and purified by the standard Ni-NTA method. The eluted proteins were further purified using a size exclusion column pre-equalized with a buffer containing (25 mM HEPES, pH 7.9, 100 mM KCl, 12.5 mM MgCl2, 0.1 mM EDTA, 10% glycerol, and 1 mM DTT). The pooled peak fractions were quantified by NanoDrop, aliquoted, snap-frozen in liquid nitrogen, and stored long-term at -80°C.

[0165] Protein 2 was subcloned into a pGEX4T-1 expression vector immediately following the thrombin cleavage site after the TEV cleavage site. The plasmid was transfected into E. coli BL21(DE3) and grown in LB medium at 37°C until OD600 reached 0.6–0.8, followed by induction overnight at 16°C with 0.1 mM IPTG. Cells were collected by centrifugation, lysed with BugBuster® protein extraction reagent (EMD Millipore), and purified by standard glutathione agarose beads. The GST tag was removed by thrombin cleavage, followed by a size exclusion column pre-equalized with a buffer containing (25 mM HEPES, pH 7.5, 150 mM NaCl, and 1 mM TCEP). Pooled peak fractions were quantified by NanoDrop, aliquoted with glycerol to 10% v / v, snap-frozen in liquid nitrogen, and stored long-term at -80°C.

[0166] DNA encoding protein 3 and protein 3 variants was cloned into the pET11a bacterial expression vector, along with an N-terminal 6x His tag, a subsequent TEV cleavage site, and three additional glycine residues (leaving a "GGGG" sequence at the N-terminus after TEV cleavage). The sequenced plasmids were transformed into chemically competent E. coli (BL21DE3). After single colony inoculation, the bacteria were grown in LB at 37°C until the OD600 reached 0.6–0.8, at which point 0.5 mM IPTG was added, and protein expression was induced overnight at 16°C. The bacterial cultures were resuspended in a buffer containing a newly supplemented protease inhibitor (25 mM HEPES, pH 8.0, 150 mM NaCl, 1 mM TCEP, 5% glycerol), then collected, mechanically sonicated, and incubated with Ni-NTA affinity resin for batch purification. The proteins were washed with a buffer containing 45 mM imidazole and eluted with a lysis buffer containing 300 mM imidazole. The affinity tag was cleaved overnight using TEV protease, followed by reverse affinity purification to obtain cleaved protein 3 and protein 3 variants. The final proteins were further purified by size exclusion chromatography for buffer exchange (25 mM HEPES, pH 7.5, 150 mM NaCl). The peak fractions were pooled, replenished with glycerol to 5%, snap-frozen in liquid nitrogen, and stored at -80°C.

[0167] The protein sequence of protein 4 was subcloned into a pET17b vector with an N-terminal 6x His tag. Transformed (BL21-DE3) E. coli were grown in a 37°C Turbo Broth until the OD600 reached 0.6. Protein expression was further induced at room temperature for 4 hours with the addition of 0.1 mM IPTG. The collected pellet was dissolved in buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM TCEP + 25 mM imidazole, 1 mM PMSF (supplemented with benzoase nuclease and protease inhibitors)) and loaded onto a Ni-NTA column pre-equilibriumated with lysis buffer. The proteins were eluted using a lysis buffer supplemented with 500 mM imidazole, and subsequently injected into a HiTrap Q-FF column pre-equilibriumated with buffer (50 mM HEPES, pH 8.0, 150 mM NaCl, 1 mM TCEP) and an S75 Superdex size exclusion column in buffer (50 mM HEPES, pH 8.0, 150 mM NaCl, 1 mM TCEP) for further purification. The peak fractions were pooled, supplemented with glycerol to 5%, snap-frozen in liquid nitrogen, and stored at -80°C.

[0168] The protein sequence encoding protein 5 was subcloned into a pFASTBAC1 insect expression vector having an N-terminal GST tag and TEV cleavage site, as well as C-terminal AviTag and StrepTag II affinity tags. This donor plasmid was then transformed into DH10Bac E. coli for bacmid integration and seeded onto Bluo-gal selective agar plates (Kan, Gen, Tet) for blue / white colony selection, where appropriate gene insertion products were verified by PCR. To generate baculovirus, bacmid was scaled up in LB units, purified using the Bacmid Maxiprep kit (Machery-Nagel), pre-conjugated with ExpiSf Expifectamine reagent (ThermoFisher Scientific), and introduced into transiently transfected ExpiSf9 cells (ThermoFisher Scientific). The collected baculovirus was then used to infect ExpiSf9 cells at a 1:50 dilution to induce protein expression. Cell pellets were collected and lysed at room temperature for at least 30 minutes using Insect Popculture detergent (Millipore Sigma) diluted with lysis buffer (50 mM HEPE, pH 8.0, 500 mM NaCl, 1 mM EDTA, 1 mM TCEP, 1 mM PMSF (supplemented with benzoase nuclease and protease inhibitors)). After clarification of the lysate, the soluble fraction was loaded onto StrepTactin affinity columns (IBA Biosciences), MonoQ anion exchange columns (Cytiva), and S200 Superdex size exclusion columns (Cytiva) to remove aggregates and contaminants, and then replaced with buffer containing (50 mM HEPES, pH 8.0, 250 mM NaCl, 1 mM TCEP). The pooled peak fractions were supplemented with glycerol to 10%, snap-frozen in liquid nitrogen, and stored at -80°C.

[0169] Protein labeling: 10–60 μM of Halo-tagged protein was mixed with 100–200 μM of Halo-JF549 dye (Tocris or tissue-synthesized) in a buffer containing (50 mM HEPES pH 7.4, 150 mM NaCl, 0.01% NP40 substitute, 0.5 mM EDTA, and 1 mM DTT) and incubated at room temperature (23°C) for 15–30 minutes. Free dye was removed by a size exclusion column pre-equalized with buffer (25 mM HEPES pH 7.9, 100 mM KCl, 12.5 mM MgCl2, 0.1 mM EDTA, 10% glycerol, and 1 mM DTT). Pooled peak fractions were quantified by NanoDrop, aliquoted, snap-frozen in liquid nitrogen, and stored long-term at -80°C.

[0170] In saltase-based enzyme ligation, the N-terminus is cleaved by either thrombin protease or TEV protease, exposing and labeling a free glycerin residue. The cleaved product is then labeled in labeling buffer (50 mM HEPE pH 7.5, 150 mM NaCl, 10 mM CalCl2) and 5 μM recombinant purified His-tagged saltase (SrtA). staph The reaction was incubated with pentamutant (DOI:10.1002 / cpps.38) and a 10 molar excess (peptide donor to protein substrate) of LPETGG-JF549 peptide (JF549 conjugated to the N-terminus of peptide LPETGG, Elim Biopharm) dissolved in water. After incubation at room temperature for at least 30–60 minutes, the reaction was cleaned up by magnetic separation using DYNABEADS® His tags, followed by injection into a size exclusion column to further separate the labeled product, peptide dye, and exchange buffer (50 mM HEPES, pH 8.0, 150 mM NaCl, 1 mM TCEP). Labeling efficiency (40%–70%) was estimated using the ratio of absorption at 549 nm and 280 nm. The final product was replenished with glycerol to 10% v / v and stored at -80°C.

[0171] Single-molecule tracking and data analysis: Labeled proteins were diluted to 4–200 pM in a buffer containing glycerol supplemented to specific concentrations and other additives such as specified chaotropic agents (25 mM HEPES, pH 7.9, 100 mM KCl, 12.5 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 0.01% NP40, 0.3 mg / mL BSA). Small molecule compounds (i.e., organic molecules less than 1 kDa) dissolved in DMSO were added to the reaction based on the DMSO tolerance of each protein being assayed. 30 μL of each reaction was alicoated into a 384-well glass-bottom CellVis plate and imaged under a single-molecule imaging microscope. The protein 1 peptide used to disrupt the protein 2-protein 1 interaction was custom synthesized (Elim Biopharm) and supplemented as specified. The molecule MX used for protein 3 was custom synthesized (CPC Scientific Inc.) and supplemented as specified. Pre-incubation before imaging may amplify changes caused by the presence of chaotropic agents and small molecule binders. These incubation steps were inserted into the specified experiments: 37°C for 1 hour followed by 4°C for 3 days (Figure 4D), 37°C for 2 hours (Figure 4E, center panel), 4°C for 3 days followed by 37°C for 1 hour (Figure 4E, bottom panel), and room temperature (25°C) for 2 hours (Figure 4F).

[0172] Single-molecule tracking (SMT) images were acquired by oblique scanning (OLS) microscopy at 200 Hz (Figures 2A–3B and 4B–6D) or 800 Hz (Figures 3C–3E). An exemplary OLS microscope is described in PCT / US2023 / 085589. Data were processed using a custom pipeline operating on the image sequences produced by the microscope. Briefly, individual emitters were detected by applying a generalized log-likelihood ratio test to each 11 × 11 subwindow in the image as described above (see the section below on the definition and quantification of the signal-to-noise ratio). Emitters were detected by identifying pixels with a log-likelihood ratio greater than 16. The detected emitters were localized to sub-pixel precision in a two-step procedure. First, the sub-pixel position was estimated by calculating the point of maximum radial symmetry. Next, using this estimate, we applied an iterative Levenberg-Marquardt fitting routine to a 2D integrated Gaussian within an 11x11 pixel subwindow centered on the detection location.

[0173] Using a modified version of Sbalzerini's hill-climbing algorithm, which estimates the uncertainty of data association using Gibbs sampling, the estimated emitters were temporally linked to produce spatiotemporal trajectories. In SMT, links longer than 2.5 μm (Figures 2A-3B and 4B-6D) or 1.5 μm (Figures 3C-3E), and links exceeding two gap frames were prohibited to limit association errors.

[0174] Definition and quantification of the signal-to-noise ratio The signal-to-noise ratio (SNR) is defined based on the likelihood ratio for hypothesis testing, which compares the case where the target is absent (the local image is modeled as the sum of a constant offset and independent Gaussian-distributed noise) with the case where the target is present (the local image is modeled as the sum of a centrally located Gaussian peak (whose width is known but whose amplitude is unknown), independent Gaussian-distributed noise, and a constant offset). The SNR is expressed as follows:

[0175]

Number

Number

[0176] Differential scanning fluorimetry (DSF): Unlabeled protein 4 was diluted to 3 μM with buffer (25 mM HEPES, pH 7.4, 150 mM NaCl), supplemented with only DMSO up to 0.5% (vector control), or supplemented with the test compound up to 50 μM . The melting temperature (Tm) was measured with a Prometheus Panta NanoDSF instrument, and the data were processed with the relevant software.

[0177] Fluorescence polarization (FP) assay: 20 nM of protein 4 was mixed in buffer containing 5 nM of FAM-labeled protein 6 peptide (which binds to the same site of protein 4 as molecule M3 and the C-terminus is capped with an amine, Elim Biopharm), and (10 mM HEPES pH 7.5, 150 mM NaCl, 50 mM EDTA, 0.005% Tween 20, 0.1 mg / ml BSA, and 1 mM DTT). 0.2% DMSO was added to the compounds. The signal was measured in a 384-well plate using an EnVision 2013 plate reader (Perkin Elmer). The half-maximal inhibitory concentration (IC50) was determined using Prism software (GraphPad).

[0178] Measurements of spatiotemporal trajectories: When reporting the number of spatiotemporal trajectories, singlets (spatiotemporal trajectories with one detection) were excluded because they do not contribute to most dynamic estimations.

[0179] The mean diffusion coefficient is calculated using the mean square displacement method (D est = MSD 2D This was calculated using σ ( / 4Δt). loc 2 It is expected that the diffusion coefficient will be overestimated by / Δt. Here, σ loc 2 Δt is the variance of the 1D position estimation error, and Δt is the frame interval.

[0180] To resolve the spatiotemporal trajectories in multiple dynamic states, the coefficients of the Brown mixing model on a grid of diffusion coefficient values ​​and position estimation error values ​​were inferred using a state array, which is a variational Bayesian routine based on Dirichlet process mixing. The components of the mixture ranged from 0.01 to 100 μm. 2 The Cartesian product of 100 diffusion coefficients with logarithmic intervals of / s and 31 position estimation error values ​​ranging from 0.02 to 0.08 μm (1D standard deviation) was selected. Occupancy is reported as the mean posterior probability of each diffusion coefficient marginalized over all values ​​of the position estimation error. For ease of handling of the inference, the inference was limited to 10,000 spatiotemporal trajectories randomly sampled from each well.

[0181] Bias estimation of a single-population sample was performed using analytical calculations that capture the probabilities of erroneous links and jump length distribution cuts within a finite search radius. The spatial distribution is uniform, and it is possible to estimate the probability of erroneous links based on the observed emitter concentration and nearest-neighbor link model. This probabilistic analysis also allows for the characterization of the jump length distribution of erroneous links. The single-population bias estimation incorporates the exclusion of these erroneous link artifacts and the observation of jump lengths beyond the search radius. As shown in Figure 6A, these bias equations are used to improve the accuracy of the diffusion coefficient estimation.

[0182] Empirical estimation of link accuracy A bootstrap procedure was used to estimate the accuracy of the linking algorithm. The detections from the first and second halves of the video were superimposed, and the tracking algorithm was run on the resulting set of detections, while blinding the origin of each detection. From this, the percentage of links generated at the point where individual detections from different halves of the video were joined was calculated. Since this percentage does not account for erroneous links between detections in the same half of the video, nor for the effects of photobleaching, this percentage forms a lower bound of the Error Rate of Links (ERLB).

[0183] 5.1. Example 1: SMT can distinguish proteins that have subtle differences depending on their movement. This example demonstrates the use of the method of the present disclosure to distinguish between target proteins by analyzing the movement of target proteins. This example further demonstrates that the method of the present disclosure can be used to measure the diffusion properties of purified proteins in solution.

[0184] As explained by the Stokes-Einstein equation, particle motion depends on temperature, the viscosity of the culture medium, and the particle's effective hydration radius. For example, size, shape, surface chemical / physical characteristics, and interactions with the surrounding culture medium can all contribute to and influence the effective hydration radius, and thus influence the movement of target molecules under specific conditions (such as temperature and culture medium). Therefore, tracking particle motion can provide valuable information about the particle's structural and functional properties.

[0185] To verify that the SMT system can reliably measure the movement of proteins in solution, we adjusted the viscosity by increasing the glycerol concentration while using JF 549 The diffusion of HisHalo labeled with was measured. His-Halo was diluted to 50 pM in imaging buffer consisting of 25 mM HEPES pH 7.4, 150 mM NaCl, 1 mM DTT, 0.03% (w / v) BSA, and 0.01% NP-40, with glycerol levels ranging from 0 to 40%. The dilutions were transferred to 384-well plates and incubated at 37°C for 15 minutes before imaging using OLS. Replications from 22 to 44 wells were captured for each concentration, and four FOVs per well were acquired at 200 frames per second. Glycerol titration experiments were performed on two plates using the same protein preparation. Viscosity calculations were performed. As shown in Figures 6A and 6B, the diffusion coefficients were estimated from the tracking results and corrected using analytical formulas to track bias, resulting in near agreement with the theoretical values ​​predicted by the Stokes-Einstein equations.

[0186] Further experiments were conducted to confirm that the SMT system could be used to distinguish between proteins. Halo tags and Halo tags fused to peptides derived from protein 1 (referred to herein as "Halo protein 1") were expressed and purified (Figure 1A). The diffusion of both Halo tags and Halo protein 1 was measured at 37°C in the presence of different glycerol concentrations.

[0187] To estimate the predicted diffusion coefficient, dynamic light scattering (DLS) was used to determine the hydration radii of Halo-tag and Halo-protein 1 in aqueous solution (50 mM HEPES, pH 7.5, 150 mM NaCl), yielding values ​​of 2.39 nm and 3.09 nm for Halo-tag and Halo-protein 1, respectively. These values ​​are in close agreement with estimates for Halo-tag using a publicly available resource (52.14.70.9 / Run_hullrad.html), which gives a hydration radius of 2.14 nm for the core domain of the Halo-tag protein. While not limited to any particular theory, this slight difference is likely due to the presence of an unstructured sequence at the end of the purified protein used in this experiment. Using another resource (www.met.reading.ac.uk / ~sws04cdw / viscosity_calc.html), the dynamic viscosity of glycerol was determined to be 0.00093 N*s / m for 10% v / v and 30% v / v glycerol, respectively. 2 and 0.00185 N*s / m 2 This was estimated. The Boltz constant is 1.38E. -23 In this case, at a temperature of 37°C, the diffusion coefficients of the Halo tag in 10% and 30% glycerol are 102.1 μm, respectively. 2 / s and 51.3μm 2 It is expected to be / s.

[0188] As shown in Figure 1B, when the Halo tag was measured under SMT conditions at 200 frames / second (FPS), the diffusion coefficients of the Halo tag in 10% and 30% glycerol were 52.4 and 34.2 μm, respectively. 2 It was determined to be / s. This is slower than expected overall (Figure 1B, "Theoretical value (μm) 2 (in the column "Estimated radius (μm / s)"). However, increasing the frame rate yields measurements that more closely reflect theoretical predictions. Similar results were observed with Halo Protein 1 (Figure 1B, "SMT measurement (μm)"). 2( / s), constant tracking settings (column). The remaining discrepancy between SMT measurements and theoretical values ​​may be due to two causes. First, optimizing the tracking settings (such as search radius) for each frame rate yielded better diffusion coefficient measurements (Figure 1B, last column). Second, the Stokes radius, or hydration radius, of a protein is highly dependent on the medium. The measured diffusion coefficient can be used with the optimized tracking settings to fit the estimated Stokes radius under experimental conditions. The Stokes radius of the Halo tag was estimated to be 3.6 nm, and the Stokes radius of the Halo tag-protein 1 was estimated to be 4.2 nm. Using these radii, the "Theoretical Value (μm)" in Figure 1B can be used. 2 The theoretical value is obtained in the column "Fitted Stokes Radius (μm / s)". Finally, the correction applied in Figure 6B is based on a comprehensive tracking model that takes into account jump truncation, tracking error, and other effects, and the estimated Stokes radius (Figure 1B, "Corrected prediction based on fitted radius (μm)". 2 Considering the / s) value, it can be used to estimate the diffusion coefficient measured in the assay. The values ​​in the "Corrected Prediction" column and the "SMT Measurement, Optimized Tracking Settings" column agree very well.

[0189] Figure 1C shows a plot of the mean diffusion coefficients - / +98% confidence interval of Halo Tag and Halo Protein 1 against the number of fields of view (FOV) used in the calculation, as a guideline for data collection. Interestingly, despite the close similarity of these two proteins, the SMT technique was able to distinguish them under each condition tested. This indicates that small changes in the amino acid sequence of a protein affect its movement (e.g., changes in hydration radius and interactions with surrounding molecules), and that SMT is significant enough to detect these kinds of otherwise subtle changes. Furthermore, despite the inherent difficulty of generating spatiotemporal trajectories of rapidly diffusing proteins in solution, measurements using the method disclosed herein were validated by the strong overall agreement between experimental measurements and the corresponding Stokes-Einstein theory.

[0190] 5.2. Example 2: SMT can identify protein-protein interactions. This example demonstrates the use of the method of the present disclosure to identify interactions between proteins and their ligands.

[0191] A direct application of the SMT method disclosed herein is the detection of protein-protein interactions. For example, when a labeled protein binds to a significantly larger partner, the mobility of the labeled protein should be greatly reduced. The high affinity binding of the N-terminus of protein 1 to protein 2 is well documented. To determine whether SMT can be used to identify protein-protein interactions, we analyzed the interaction of Halo protein 1 (41.5 kD) with recombinant protein 2 (85.6 kD).

[0192] GST protein 2 (dimer protein 2) was produced by bacterial expression as described above. The GST tag was removed by thrombin cleavage, and monomer protein 2 was obtained by purification using SEC. His-Halo protein 1 (1-53) was produced by bacterial expression as described above, and then JF 549 The samples were labeled and purified by SEC. The final concentration was determined by Nanodrop and the gel band intensity imaged on the Cy3 channel of ImageQuant800.

[0193] Halo Protein 1 and Protein 2 were diluted to final concentrations of 40 pM and 30 nM, respectively, in imaging buffer (25 mM HEPES-K pH=7.6, 0.1 M EDTA pH=8.0, 12.5 mM magnesium chloride, 100 mM potassium chloride, 0.2 mM PMSF, 1 mM DTT, 0.3 mg / ml BSA, 0.01% NP40, and 30% glycerol, pH=7.9). The compounds were printed onto 384-cell Vis glass-bottom plates via Echo 655 and then incubated with Protein 2 at room temperature for 10 minutes before adding Halo Protein 1. The protein solutions were distributed using Integra VIAFLO. The plates were then incubated at 37°C for 2 hours before imaging.

[0194] In the presence of 30% glycerol, titration of protein 2 gradually slowed the diffusion of Halo protein 1 (approximately 29.2 μm when measured at 40 pM). 2 / s~approx. 19.4μm 2 (Figures 2A and 6C). As shown in Figures 2A and 6C, it was observed that as the concentration of monovalent protein 2 increased, the diffusion of Halo protein 1 decreased in a concentration-dependent manner. The apparent kD of 0.9 nM is roughly consistent with previous reports, considering the differences in buffer composition and measurement temperature. Furthermore, when the divalent version of protein 2 was titrated, the diffusion of Halo protein 1 reached a minimum value of 15.08 μm. 2 The value shifted to / s, but when monovalent protein 2 was added, the value was 19.39 μm. 2 The response was / s. Furthermore, when the free, unlabeled protein 1 peptide was titrated in a mixture of 40 pM Halo protein 1 and 30 nM protein 2, the diffusion of Halo protein 1 reversed in a dose-dependent manner, suggesting competitive disruption (Figures 2B and 6D).

[0195] To demonstrate that the methods disclosed herein can be applied to drug discovery, a small molecule inhibitor and a protein 1 competitive peptide are combined with protein 2 in a JF 549The ability of labeled protein 1 to alter protein motility was tested. Molecular B1, a known inhibitor of protein 2, showed a dose-dependent response with a maximal effect similar to that of the competing peptide. 549 It was possible to increase the diffusion of labeled protein 1 (Figure 6D).

[0196] As these data demonstrate, SMT measurements of protein diffusion can detect protein-protein interactions, protein-ligand interactions, and even inhibition of such interactions by competing ligands in solution, with potential sensitivity in the picomolar range.

[0197] The relevance of the disclosed method was demonstrated by capturing the interaction between two proteins, protein 1 and protein 2. Furthermore, the functionality of the disclosed method was demonstrated by monitoring the disruption of this protein interaction with competing peptides and small molecule inhibitors, suggesting its potential application in drug discovery. The method disclosed herein requires picomolar purified protein, enabling assay development for proteins that are difficult to purify and requiring the sensitivity to measure sub-nanomolar affinity. It is hypothesized that ligand interactions can alter protein diffusion in ways beyond those caused by disruption of protein-protein interactions, including ligand-induced confirmation changes, changes in protein stability, and more subtle changes to the protein hydration shell, possibly resulting in small but measurable changes in diffusion.

[0198] 5.3. Example 3: Detection of protein conformational changes Many enzymes are allosterically regulated. This means that an effector binds to a site other than the enzyme's active site, inducing a structural / conformational change that controls the enzyme's function. Such changes are likely to affect diffusion by altering the protein's overall shape and surface features (chemical properties and topology), and consequently, the protein's effective hydration radius and its interactions with surrounding solution molecules. Protein 3 can be used as a model to test this feasibility. Protein 3 is a phosphatase known to undergo conformational changes throughout its activity cycle. Under resting conditions, it is mostly in a more closed, inactive state. Upon binding to an activated peptide, it returns to a more open, active conformation. Cancer cells often utilize this mechanism to gain a proliferative advantage by mutating key amino residues that control this process. One example is a protein 3 mutant, which results in an overwhelmingly open, active conformation.

[0199] Since fluorescent tags can sometimes contribute to the overall mobility characteristics of the target protein, multiple methods can be used for protein labeling. Halo tagging is a convenient autocatalytic process, but it requires the addition of a relatively large module of about 40 kD. As an alternative, saltase tagging requires a very small attachment consisting of only about 6 amino acid residues as the final product, allowing a small peptide with a fluorescent dye attached to it to be conjugated by the enzyme saltase. These two methods provide flexibility in the size and shape of the final labeled protein for the convenience of SMT. The diffusion of saltase-tagged protein 3 and protein 3 mutants was compared using SMT.

[0200] Significant differences were observed in the ensemble mean diffusion, with protein 3 being approximately 32 μm. 2 While the time was / s, the protein 3 mutant was approximately 42um. 2The value was / s (Figure 3A). When the occupation of these proteins in various diffusion states was estimated using a state array, it was observed that both protein 3 and protein 3 variants exist in two states. One has a peak diffusion coefficient of approximately 30 μm 2 / s, and the other one is about 100 μm 2 It is / s (Figure 3C). Protein 3 is overwhelmingly 30 μm 2 While existing in the / s state, the mutant shows more equal occupancy in both states, but 100 μm 2 The / s state shows a slightly higher occupancy. It should be noted that in SMT, the faster state is consistently undersampled, so although the protein 3 variant is likely overwhelmingly present in the faster state, the state array makes the occupancy of the two states appear more equal than it actually is.

[0201] As a result, the ensemble average diffusion coefficient of protein 3 is approximately 32 μm. 2 The coefficient of diffusion becomes / s, but the ensemble average diffusion coefficient of the mutant is higher (42 μm). 2 ( / s). Also, from this, the peak of the diffusion coefficient is approximately 30 μm. 2 The state / s represents a closed state, and the diffusion coefficient peak is approximately 100 μm. 2 The / s state represents an open state, leading to the hypothesis that both protein 3 and protein 3 variants exist in equilibrium between these two states.

[0202] The effective hydration radii of protein 3 and protein 3 mutants have been previously reported to be 2.62 nm and 2.92 nm, respectively. Based solely on radius, this suggests that protein 3 mutants need to diffuse approximately 10% slower. Since the opposite trend was observed, there are other factors besides pure hydration radius that contribute to the diffusion behavior of these proteins, and these factors can be further utilized to understand their structure and function.

[0203] To confirm that the diffusion changes caused by mutations are a direct reflection of conformational changes, we titrated protein 3 mutants with compounds (molecule M1 and molecule M2) that have been reported to revert them to a wild-type conformation. Indeed, dose-dependent mode of reversion was observed at the population level (Figure 3B), and the ranking of the two compounds tested is consistent with reports in the literature. This experiment demonstrated that SMT can be effectively used to detect organic molecules of less than 1 kDa that bind to proteins, and furthermore, to investigate structural consequences.

[0204] To further confirm that conformational changes of protein 3 can be detected, and that the two populations observed by the state array represent the two conformations known to be adopted by protein 3, the effects of molecule M1 and another molecule, molecule MX, were observed by the state array (Figure 3C). Molecule MX is a binding pocket interactor, not an allosteric interactor like molecule M1, and should bias protein 3 and protein 3 mutants towards a conformation more similar to the dominant conformation of the protein 3 mutant (i.e., the open conformation). Molecule MX actually shifts the occupation of protein 3 from an overwhelmingly slower state to an overwhelmingly faster state in a dose-dependent manner. This is consistent with the slower state representing the main (closed) conformation of protein 3 and the faster state representing the main (open) state of the protein 3 mutant. Similarly, in a dose-dependent manner, molecule M1 shifts the occupation of protein 3 mutants towards a slower state. This is consistent with the known effect of biasing protein 3 mutants towards a conformation more similar to that of protein 3. The reverse is not true. Molecular MX has minimal effect on the relative occupancy of protein 3 mutants, and in fact, molecular M1 biases protein 3 into an even slower state.

[0205] State arrays can provide an excellent method for quantifying the relative occupancy of each conformation in dose titration experiments. Calculating the "closed proportion" (for protein 3) or "open proportion" (for protein 3 variants) as a function of the concentration of molecular MX or M1 yields a larger signal window than looking at the ensemble-averaged diffusion coefficient (compare Figure 3D and Figure 3B). Here, the partial occupancy in each state is 10–50 μm. 2 / s (closed state) or 50-200 μm 2 The average of the state array bins in / s (open state) is taken and weighted by the occupancy of each bin, and the range is 10-200 μm. 2 It is determined by dividing by the total weighted average diffusion coefficient over / s. The error is the standard error across the entire replication.

[0206] Finally, this technique has been demonstrated to be usable to identify molecules that distinguish between these two conformational states. State array analysis shows that in the presence of a certain concentration of molecule M1 (which should bias protein 3 towards the slower state), a higher concentration of molecule MX is required to shift protein 3 occupation to the faster state than when molecule MX is added alone (Figure 3E). This indicates that molecule M1 selectively stabilizes the slower state. It should be noted that this is not a competitive effect, as molecules M1 and MX bind to various sites (allosteric pair binding pockets). Consistent with this, even at the highest concentration of molecule MX in the presence of molecule M1, the percentage of protein 3 in the closed state reaches a stable state at 0.7, which is much higher than the closed percentage of 0.2 at the highest dose in the absence of molecule M1. Since it is not possible to completely reverse the effect of molecule M1, molecule MX does not defeat molecule M1.

[0207] 5.4. Example 4: Detection of binding to proteins with a rigid structure This example demonstrates the use of the method of this disclosure to analyze the interaction between proteins and organic molecules with a caliber of less than 1 kDa.

[0208] Many protein-ligand interactions do not result in significant changes to the three-dimensional (3D) structure. Generally, conventional organic molecular drugs are in the range of 250 Da to 1 kDa, which, when totaled, amounts to only a few percent of the mass of many proteins. Given the limited overall mass added by such ligands binding to proteins, the impact of such binding on the protein's hydration shell may be minimal, and therefore, detecting this category of interactions using SMT can be challenging. As an example, recombinantly expressed protein 4 with a well-characterized organic molecular binder molecule M3 was selected for analysis. Unlike variants of protein 3, binding of protein 4 to the organic molecular ligand caused little to no change in the protein's 3D structure (Figure 4A). Saltase tags were attached to the protein and labeled with the JF549 fluorescent dye, and their diffusion was tracked in the presence of either DMSO (vector control) or molecule M3. The presence of molecule M3 did not cause any significant changes in the protein's diffusion (Figure 4B).

[0209] To explore buffer conditions that may allow for the detection of sub-1 kDa organic molecules bound to protein 4 by diffusion measurements, chaotropic agents known to destabilize protein structures (such as acetone, acetonitrile, DMSO, guanidinium, and urea) were analyzed (Figures 4C-4D). While not theoretically bound, it is hypothesized that chaotropic agents may partially unfold proteins, resulting in one or more intermediate or unfolded states, and that the presence of organic molecule binders may stabilize some structures more than others. Given that these structures are expected to have different effective hydration radii, this is expected to lead to differentiated diffusion. As shown in the figures (Figures 4C-4D), multiple chaotropic agents tested under multiple conditions increased the difference between the DMSO control (without molecule M3) and samples treated with molecule M3, with acetone, acetonitrile, urea, and guanidinium being more pronounced than DMSO.

[0210] To analyze the effects of various experimental conditions, the concentrations of chaotropic agents were titrated across a range of concentrations. Similar to the observations shown in Figures 4C-4D, a general trend of decreased diffusion was observed when urea, guanidinium, and acetone were titrated in Figure 4E. While not limited to any particular theory, one explanation is that protein unfolding leads to an increase in the effective hydration radius, thereby reducing diffusion. Furthermore, at certain concentrations, urea and guanidinium provided conditions that distinguished the diffusion of protein 4 treated with DMSO and molecule M3 within a larger dynamic window. More specifically, this was at 6-7 M for urea and approximately 1 M for guanidinium (Figure 4E). The overall trend was that molecule M3 resulted in increased diffusion, which is consistent with the hypothesis that it stabilizes the protein structure and reduces unfolding. Unlike the smooth transfer with urea, titration with guanidinium resulted in a significant decrease in the diffusion of DMSO-treated protein at approximately 1 M. The complexity of this titration curve indicates that additional factors are influencing the diffusion of the tested protein, and adjusting the buffer composition may provide new insights into the diffusion state of the tested protein.

[0211] To test whether this method is suitable for structure-activity relationship (SAR) analysis, dose titrations of a series of molecular M3 analogs against protein 4 were performed in the presence of 6M urea (Figure 4F). Upon addition of these compounds, a gradual increase in the diffusion of protein 4 was observed. Their potency was compared by determining the apparent EC50. These compounds were also characterized using other conventional assays, including fluorescence polarization (FP, measuring the substitution of fluorescently labeled probes that bind to the same pocket) and differential scanning fluorescence (DSF, measuring protein melting / unfolding) assays commonly used in SAR analysis. The potency ranking measured by SMT largely coincided with FP, and generally, molecules with higher potency measured by SMT showed a greater increase in melting temperature (Tm) measured by DSF. The results indicate that SMT is indeed suitable for use in SAR analysis.

[0212] 5.5. Example 5: Tracking the diffusion of an essentially disordered area This example demonstrates the use of the method of this disclosure to analyze the interaction between proteins containing inherently disordered regions (IDRs) and other molecules (e.g., organic molecules less than 1 kDa).

[0213] The human proteome contains a continuum of proteins / domains ranging from structured to unstructured. IDRs are abundant within this continuum and play a crucial role in biology. However, because IDRs lack stable structures, they are not well-suited to conventional biophysical approaches (e.g., crystallography and electron microscopy). In contrast to other proteins / domains that exist in relatively stable tertiary structures, IDRs generally exist as ensembles of interchangeable conformations. Since these conformational changes can lead to changes in the effective hydration radius of a protein, proteins containing IDRs are well-suited to motion-focused analysis. Tracking such motion can provide insights into the conformational profile of IDRs, including how it relates to the protein's biological function.

[0214] Protein 5 was analyzed as a model for IDRs, which are reported to have little secondary structure. The movement of protein 5 labeled with JF549 via an N-terminal saltase tag was tracked to determine its diffusion coefficient. Molecule M4 is a covalent modifier reported to bind to protein 5. Titration of the compound revealed a gradual increase in the diffusion of protein 5 (Figure 5). While not limited to any particular theory, these results indicate a change in the interaction between protein 5 and the buffer component (e.g., the direct interaction between the protein and molecule M4). Therefore, SMT provides an entirely new tool for studying IDRs and their interactions with ligands.

[0215] 5.6. Example 6: Single-molecule fluorescence polarization This example demonstrates a method using single-molecule fluorescence polarization to leverage the orientation-dependent fluorescence properties of a fluorescent dye firmly attached to a target biomolecule, thereby enabling the characterization of the latter's molecular dynamics. This approach is complemented by translational single-molecule tracking (SMT), providing insights into molecular size, conformational changes, and binding dynamics across diverse microenvironments. While translational SMT is influenced by factors such as local conditions (viscosity, matrix-like or cell boundaries, and microfluidic flow, etc.) in addition to molecular size, single-molecule fluorescence polarization, in its initial approximations, operates independently of its surroundings. Single-molecule fluorescence polarization can capture temporal dynamics through spatiotemporal correlation functions, providing valuable information about molecular size and changes due to binding and conformational events.

[0216] Methodology: Several methodologies can be used to perform fluorescence polarization of single molecules, namely (1) time-correlated single-photon counting (TCSPC), (2) step-scan pump-probe technique, (3) ultra-long-lived fluorescent dyes, and (4) advanced optical techniques.

[0217] Time-correlated single-photon counting (TCSPC) enables precise measurement of fluorescence lifetimes by detecting the arrival time of individual photons with picosecond precision. This can be crucial for elucidating nanosecond-scale dynamics. TCSPC achieves extremely high temporal resolution using 2D TCSPC via a single-photon avalanche diode (SPAD) array and photon counting module. When paired with polarization-sensitive readout, this method is ideal for studying the fast rotational dynamics and short fluorescence lifetimes typical of many biological fluorescent dyes. This enhances the ability to select various fluorescent dye states or orientations.

[0218] The step-scan pump-probe technique modulates the polarization of excitation and probe light, and the resulting time-dependent fluorescence emission provides insights into the orientation / rotation and dynamics of the fluorescent dye. The step-scan pump-probe technique uses step-scan pump-probe spectroscopic microscopy to measure polarization-dependent and time-correlated fluorescence depletion dynamics. This is useful for optically filtering subpopulations of different molecular sizes, orientations, or dynamics, and is also useful for studying the dynamics of systems where the orientation of a fluorescent dye changes rapidly after excitation (such as protein dynamics or membrane structural changes).

[0219] Ultra-long-lived fluorescent dyes exhibit fluorescence lifetimes (e.g., nanoseconds to microseconds or longer) that are far longer than the typical fluorescence decay times of organic fluorescent dyes. Using ultra-long-lived fluorescent dyes (e.g., carbon dots, quantum dots, nanodiamonds), depolarization times can be extended significantly beyond normal fluorescence lifetimes. Ultra-long-lived fluorescent dyes enable the measurement of rotational dynamics and depolarization processes over long timescales, allowing for the study of slow molecular motion or environments with restricted mobility.

[0220] Advanced optical techniques, such as stress engineering optics, enable precise spatial manipulation and control of the polarization state of light, which is crucial for techniques such as wobble anisotropy and polarization filtering. These advanced optical techniques include sophisticated optical methodologies, including phase and polarization control. These techniques enable high-precision polarization-dependent measurements, allowing for the extraction of detailed information regarding the orientation and dynamics of fluorescent dyes.

[0221] Consideration At the single-molecule and single-photon levels, single-molecule fluorescence polarization utilizes fluorescence properties governed by the alignment of the transition dipole moments of the fluorescent dye. For common fluorescent dyes such as rhodamine-based JF549, the probability of absorption and emission correlates with the orientation of the fluorescent dye relative to the polarization of the incident light; this is known as fluorescence anisotropy. This phenomenon is influenced by the rotational dynamics of the molecule within the lifetime of its excited state, providing temporal contrasts that reveal molecular size, intermolecular and intramolecular dynamics, and binding events.

[0222] Current camera-based translational SMTs face challenges due to integration times (>20 μs) that are significantly longer than typical fluorescence lifetimes (<100 ns) and rotational dynamics (<100 ns). To address this, the time-averaged fluorescence intensity dependence (i.e., wobble anisotropy) at specific polarizations can be measured using the method described herein.

[0223] In relation to oligomerization, the fluorescence polarization of a single molecule provides contrast by detecting changes in the orientation and rotational degrees of freedom of the fluorescent dye during oligomer formation. This contrast arises from the change in the anisotropic properties of fluorescence emission when a monomer transitions to the oligomer state.

[0224] Furthermore, single-molecule fluorescence polarization reveals how conformational changes affect the orientation and dynamics of fluorescent dyes attached to specific sites within a molecule. This capability enables the characterization of dynamic structural transitions (such as protein folding / unfolding events or nucleic acid conformational changes) with excellent sensitivity and spatial resolution.

[0225] The primary methodology involves time-resolved anisotropy measurements, which track changes in fluorescence polarization over time to elucidate the kinetics of oligomer formation, dissociation, and intramolecular conformational dynamics. Coupling polarization-sensitive imaging techniques with advanced optical control facilitates the spatial mapping of these events within complex biological environments.

[0226] By utilizing single-molecule fluorescence polarization, it becomes possible to characterize both oligomerization processes and intramolecular conformational dynamics at a level of detail difficult to achieve with ensemble averaging methods. This approach not only provides insights into the stoichiometry and structural dynamics of biomolecules but also offers a means to correlate these dynamics with functional changes in biological systems. Ultimately, single-molecule fluorescence polarization can elucidate the complexity of molecular assembly pathways and dynamic structural changes, advancing our understanding of biological functions and disease mechanisms.

[0227] 6. Exemplary Embodiments A. The present disclosure provides a method for identifying an interaction between a target molecule and a test molecule, the method comprising: (a) contacting a cell-free sample containing a plurality of the target molecules with a plurality of the test molecules; (b) tracking the plurality of the target molecules over time to obtain measurements of a plurality of spatiotemporal trajectories and / or rotational motions; (c) analyzing the measurements of the plurality of spatiotemporal trajectories and / or rotational motions to determine the movement of the target molecules in the presence of the test molecules; and (d) comparing the movement of the target molecules obtained in (c) with the movement of a reference target molecule, wherein the movement of the reference target molecule is the movement of the target molecules in the absence of the test molecules, and the change in the movement of the target molecules compared with the movement of the reference target molecule indicates an interaction between the target molecule and the test molecule.

[0228] A1. The method described above is a high-throughput method, the method according to A.

[0229] A2. The method according to A or A1, wherein the aforementioned change in motion is calculated as a change in (a) the diffusion coefficient obtained from the maximum likelihood estimator, (b) the geometric mean posterior diffusion coefficient, (c) the median of the jump length distribution, (d) the third quartile of the jump length distribution, (e) the median of the turning radius, (f) the mean posterior diffusion coefficient, (g) the mean square displacement, (h) the median of the bond angle, (i) the spatiotemporal trajectory length, (j) the anisotropic decay time, (k) the spatial range of detection, (l) the number and wavelength of conjugate fluorescent labels, (m) the occupation in various diffusion states obtained from the state array, (n) the polarization of the conjugate fluorescent labels, or (o) the state occupation by inference.

[0230] A3. The method according to any one of A to A2, wherein the change in the aforementioned motion is calculated as a change in the diffusion coefficient.

[0231] A4. The method according to any one of A to A3, wherein an increase or decrease in the movement of the target molecule compared to the movement of the reference target molecule indicates an interaction between the target molecule and the test molecule.

[0232] A5. The method according to any one of A to A4, wherein the target molecule is an organic molecule with a caliber of less than 1 kDa.

[0233] A6. The method according to any one of A to A5, wherein the target molecule is selected from the group consisting of peptides, proteins, nucleic acids, carbohydrates, and lipids.

[0234] A7. The method according to A6, wherein the protein is selected from the group consisting of antibodies and receptors and enzymes.

[0235] A8. The method according to A6 or A7, wherein the protein contains a disordered domain.

[0236] A9. The method according to A8, wherein the protein does not contain a structural domain.

[0237] A10. The method according to A6, wherein the peptide is a ligand.

[0238] A11. The method according to any one of A to A4, wherein the target molecule is a nanomaterial.

[0239] A12. The method according to any one of A to A4, wherein the target molecule is a synthetic polymer.

[0240] A13. The method according to any one of A to A12, wherein the test molecule is an organic molecule with a kDa of less than 1 kDa.

[0241] A14. The method according to any one of A to A13, wherein the test molecule is selected from the group consisting of peptides, proteins, nucleic acids, carbohydrates, and lipids.

[0242] A15. The method according to A14, wherein the protein is selected from the group consisting of antibodies and receptors and enzymes.

[0243] A16. The method according to A14 or A15, wherein the protein contains a disordered domain.

[0244] The method according to A16, wherein the protein does not contain a structured domain.

[0245] The method according to A14, wherein the peptide is a ligand.

[0246] The method according to any one of A to A12, wherein the test molecule is a nanomaterial.

[0247] The method according to any one of A to A12, wherein the test molecule is a synthetic polymer.

[0248] The method according to any one of A to A20, wherein the target molecule is labeled.

[0249] [[ID=NO=20]]The method according to any one of A to A~21, wherein the target molecule is fluorescently labeled.

[0250] The method according to any one of A to A22, wherein the test molecule is labeled.

[0251] The method according to any one of A to A23, wherein the test molecule is fluorescently labeled.

[0252] The method according to any one of A to A24, wherein the change in the movement of the target molecule compared to the movement of the reference target molecule indicates a direct or indirect interaction between the target molecule and the test molecule.

[0253] The method according to any one of A to A25, wherein the interaction between the target molecule and the test molecule causes a conformational change in the target molecule.

[0254] The method according to any one of A to A25, wherein the interaction between the target molecule and the test molecule increases the mass of the target molecule.

[0255] It should be noted that in the original text, there seems to be a typo in "[[ID=NO=20]]", which is likely a misspelling. I translated it as it is in the current context. If this is an important error, it may need to be corrected in the original source for a more accurate translation.A28. The cell-free sample comprises a solution, as described in any one of A to A27.

[0256] A29. The method according to A28, wherein the solution comprises a chaotropic agent, a carrier, a viscous agent, or a combination thereof.

[0257] A30. The method according to A29, wherein the solution contains a viscous agent.

[0258] A31. The method according to A30, wherein the viscous agent comprises glycerol.

[0259] A32. The method according to A31, wherein the solution contains at least about 30% glycerol.

[0260] A33. The method according to any one of A28 to A32, wherein the solution comprises a temperature gradient, a chemical gradient, at least two phases, or a combination thereof.

[0261] A34. The cell-free sample having a volume of approximately 0.1 μl to approximately 100 μl, according to any one of A to A33.

[0262] A35. The method according to any one of A to A34, wherein the change in the motion is measured as a change in the diffusion coefficient of the target molecule compared to the diffusion coefficient of the reference target molecule, and the change is at least about 0.001%, at least about 0.005%, at least 0.01%, at least about 0.05%, at least about 0.1%, at least about 0.5%, at least about 1%, at least about 1.5%, at least about 2%, or at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10%.

[0263] A36. The method according to any one of A to A35, wherein the duration of the change in the movement of the target molecule, compared to the duration of the change in the movement of the reference target molecule, indicates an interaction between the target molecule and the test molecule.

[0264] A37. The method according to any one of A to A36, wherein the reversibility of the change in the movement of the target molecule as compared with the reversibility of the change in the movement of the reference target molecule indicates the interaction between the target molecule and the test molecule.

[0265] A38. The method according to any one of A to A37, wherein oligomerization of the target molecule occurs due to the interaction between the target molecule and the test molecule.

[0266] A39. The method according to any one of A to A38, wherein the interaction between the target molecule and the test molecule is reversible.

[0267] A40. The method according to any one of A to A39, wherein the interaction between the target molecule and the test molecule is irreversible.

[0268] [[ID=十七]] A41. The method according to any one of A to A40, wherein the target molecule and / or the test molecule is a component of a mixture composed of a bacterial extract, a cell extract, a tissue extract, a plant extract or an animal extract.

[0269] A42. The method according to A41, wherein the bacterial extract, the cell extract, the tissue extract, the plant extract or the animal extract is a lysate.

[0270] [[ID=2Six]]A43. The method according to any one of A to A42, wherein the target molecule and / or the test molecule is a component of a mixture composed of serum, blood, and other biological samples.

[0271] A44. The method according to any one of A to A43, wherein the target molecule and / or the test molecule is a component of a mixture composed of a buffer solution and glycerol.

[0272] A45. The method according to any one of A to A44, wherein the target molecule and / or the test molecule is labeled with a fluorescent protein.

[0273] A46. The method according to any one of A to A45, wherein the target molecule and / or the test molecule are labeled by a conjugate of synthetic nanomaterial or polymer.

[0274] A47. The method according to any one of A to A46, wherein the target molecule and / or the test molecule is labeled with a fluorescent protein.

[0275] A48. The method according to any one of A to A47, wherein the multiple target molecules are conjugated with multiple fluorescent labels.

[0276] A49. The method according to any one of A to A48, wherein the target molecule and / or the test molecule are labeled by conjugation to a synthetic nanomaterial or polymer.

[0277] A50. A method according to any one of A to A49, wherein the interaction between the target molecule and the test molecule causes a change in the temperature stability of the target molecule.

[0278] B. The present disclosure provides a method for identifying an interaction between a test molecule and a target protein containing a disordered domain, the method comprising: (a) contacting a cell-free sample containing a plurality of the target proteins with a plurality of the test molecules; (b) tracking the plurality of the target proteins over time to obtain measurements of a plurality of spatiotemporal trajectories and / or rotational motions; (c) analyzing the measurements of the plurality of spatiotemporal trajectories and / or rotational motions to determine the movement of the target proteins in the presence of the test molecules; and (d) comparing the movement of the target proteins obtained in (c) with the movement of a reference target protein, wherein the movement of the reference target protein is the movement of the target protein in the absence of the test molecule, the method provides a method in which the interaction between the target protein containing the disordered domain and the test molecule is indicated by a change in the movement of the target protein containing the disordered domain compared with the movement of the reference target protein.

[0279] B1. The method described above is a high-throughput method, as described in B.

[0280] B2. The method according to B or B1, wherein the aforementioned change in motion is calculated as a change in (a) the diffusion coefficient obtained from the maximum likelihood estimator, (b) the geometric mean posterior diffusion coefficient, (c) the median of the jump length distribution, (d) the third quartile of the jump length distribution, (e) the median of the turning radius, (f) the mean posterior diffusion coefficient, (g) the mean square displacement, (h) the median of the bond angle, (i) the spatiotemporal trajectory length, (j) the anisotropic decay time, (k) the spatial range of detection, (l) the number and wavelength of conjugate fluorescent labels, (m) the occupation in various diffusion states obtained from the state array, (n) the polarization of the conjugate fluorescent labels, or (o) the state occupation by inference.

[0281] B3. The method according to any one of B to B2, wherein the change in the aforementioned motion is calculated as a change in the diffusion coefficient.

[0282] B4. The method according to any one of B to B3, wherein an increase or decrease in the movement of the target protein containing the disordered domain compared to the movement of the reference target protein indicates an interaction between the target protein containing the disordered domain and the test molecule.

[0283] B5. The method according to any one of B to B4, wherein the target protein containing the disordered domain is selected from the group consisting of antibodies, receptors, and enzymes.

[0284] B6. The method according to any one of B to B5, wherein the target protein containing the disordered domain does not contain a structured domain.

[0285] B7. The method according to any one of B to B6, wherein the test molecule is an organic molecule with a kDa of less than 1 kDa.

[0286] B8. The method according to any one of B to B7, wherein the test molecule is selected from the group consisting of peptides, proteins, nucleic acids, carbohydrates, and lipids.

[0287] B9. The method according to B8, wherein the protein is selected from the group consisting of antibodies and receptors and enzymes.

[0288] B10. The method according to B8 or B9, wherein the protein contains a disordered domain.

[0289] B11. The method according to B10, wherein the protein does not contain a structural domain.

[0290] B12. The method according to B8, wherein the peptide is a ligand.

[0291] B13. The method according to any one of B to B12, wherein the target protein containing the disordered domain is labeled.

[0292] B14. The method according to any one of B to B13, wherein the target protein containing the disordered domain is fluorescently labeled.

[0293] B15. The method according to any one of B to B14, wherein the test molecule is labeled.

[0294] B16. The method according to any one of B to B15, wherein the test molecule is fluorescently labeled.

[0295] B17. The method according to any one of B to B16, wherein a change in the movement of the target protein containing the disordered domain compared to the movement of the reference target protein indicates a direct or indirect interaction between the target protein containing the disordered domain and the test molecule.

[0296] B18. The method according to any one of B to B17, wherein the interaction between the target protein containing the disordered domain and the test molecule causes a conformational change in the target protein.

[0297] B19. The method according to any one of B to B17, wherein the mass of the target protein increases due to the interaction between the target protein containing the disordered domain and the test molecule.

[0298] B20. The cell-free sample comprises a solution, as described in any one of B to B19.

[0299] B21. The method according to B20, wherein the solution contains a viscous agent.

[0300] B22. The method according to B20 or B21, wherein the solution comprises a chaotropic agent, a carrier, a viscous agent, or a combination thereof.

[0301] B23. The method according to B22, wherein the viscous agent comprises glycerol.

[0302] B24. The method according to B23, wherein the solution contains at least about 30% glycerol.

[0303] B25. The method according to any one of B20 to B24, wherein the solution comprises a temperature gradient, a chemical gradient, at least two phases, or a combination thereof.

[0304] B26. The cell-free sample having a volume of approximately 0.1 μl to approximately 100 μl, according to any one of B to B25.

[0305] B27. The method according to any one of B to B26, wherein the change in the motion is measured as a change in the diffusion coefficient of the target protein compared to the diffusion coefficient of the reference target protein, and the change is at least about 0.001%, at least about 0.005%, at least 0.01%, at least about 0.05%, at least about 0.1%, at least about 0.5%, at least about 1%, at least about 1.5%, at least about 2%, or at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10%.

[0306] B28. The method according to any one of B to B27, wherein the duration of the change in the movement of the target protein, including the disordered domain, compared to the duration of the change in the movement of the reference target protein, indicates an interaction between the target protein and the test molecule.

[0307] B29. The method according to any one of B to B28, wherein the reversibility of the change in the movement of the target protein containing the disordered domain, compared to the reversibility of the change in the movement of the reference target protein, indicates an interaction between the target protein and the test molecule.

[0308] B30. The method according to any one of B to B29, wherein oligomerization of the target protein occurs due to the interaction between the target protein containing the disordered domain and the test molecule.

[0309] B31. The method according to any one of B to B30, wherein the interaction between the target protein containing the disordered domain and the test molecule is reversible.

[0310] B32. The method according to any one of B to B31, wherein the interaction between the target protein containing the disordered domain and the test molecule is irreversible.

[0311] B33. The method according to any one of B to B32, wherein the target protein containing the disordered domain and / or the test molecule is a component of a mixture consisting of a bacterial extract, a cell extract, a tissue extract, a plant extract, or an animal extract.

[0312] B34. The method according to B33, wherein the bacterial extract, cell extract, tissue extract, plant extract, or animal extract is a lysate.

[0313] B35. The method according to any one of B to B34, wherein the target protein containing the disordered domain and / or the test molecule is a component of a mixture consisting of serum, blood, and other biological samples.

[0314] B36. The method according to any one of B to B35, wherein the target protein containing the disordered domain and / or the test molecule is a component of a mixture comprising a buffer and glycerol.

[0315] B37. The method according to any one of B to B36, wherein the target protein and / or the test molecule is labeled with a fluorescent protein.

[0316] B38. The method according to any one of B to B37, wherein the target protein and / or the test molecule is labeled by a conjugate of synthetic nanomaterial or polymer.

[0317] B39. The method according to any one of B to B38, wherein the conformational change of the target protein results in a change in the temperature stability of the target protein.

[0318] C. The present disclosure provides a method for analyzing a target molecule in a test solution, the method comprising: (a) tracking a plurality of the target molecules in the test solution over time and providing measurements of a plurality of spatiotemporal trajectories and / or rotational motions; (b) analyzing the measurements of the plurality of spatiotemporal trajectories and / or rotational motions to determine the motion of the target molecules in the test solution; and (c) comparing the motion of the target molecules obtained in (b) with the motion of a reference target molecule, wherein the motion of the reference target molecule is the motion of the target molecule in a reference solution, and (i) a change in the motion of the target molecule compared to the motion of the reference target molecule indicates that the target molecule is affected by the test solution, or (ii) no change in the motion of the target molecule compared to the motion of the reference target molecule indicates that the target molecule is not affected by the test solution.

[0319] C1. The method described above is a high-throughput method, as described in C.

[0320] C2. The method according to C or C1, wherein the aforementioned change in motion is calculated as a change in (a) the diffusion coefficient obtained from the maximum likelihood estimator, (b) the geometric mean posterior diffusion coefficient, (c) the median of the jump length distribution, (d) the third quartile of the jump length distribution, (e) the median of the turning radius, (f) the mean posterior diffusion coefficient, (g) the mean square displacement, (g) the median of the bond angle, (i) the spatiotemporal trajectory length, (j) the anisotropic decay time, (k) the spatial range of detection, (l) the number and wavelength of conjugate fluorescent labels, (m) the occupation in various diffusion states obtained from the state array, (n) the polarization of the conjugate fluorescent labels, or (o) the state occupation by inference.

[0321] C3. The method according to any one of C to C2, wherein the change in the aforementioned motion is calculated as a change in the diffusion coefficient.

[0322] C4. The method according to any one of C to C3, wherein the target molecule is an organic molecule with a caliber of less than 1 kDa.

[0323] C5. The method according to any one of C to C4, wherein the target molecule is selected from the group consisting of peptides, proteins, nucleic acids, carbohydrates, and lipids.

[0324] C6. The method according to C5, wherein the protein is selected from the group consisting of antibodies and receptors and enzymes.

[0325] C7. The method according to C5 or C6, wherein the protein contains a disordered domain.

[0326] C8. The method according to C7, wherein the protein does not contain a structural domain.

[0327] C9. The method according to C5, wherein the peptide is a ligand.

[0328] C10. The method according to one of C to C3, wherein the target molecule is a nanomaterial.

[0329] C11. The method according to any one of C to C3, wherein the target molecule is a synthetic polymer.

[0330] C12. The method according to one of C to C11, wherein the target molecule is labeled.

[0331] C13. The method according to any one of C to C12, wherein the target molecule is fluorescently labeled.

[0332] C14. The method according to any one of C to C13, wherein the test solution contains a chaotropic agent.

[0333] C15. The method according to C14, wherein the chaotropic agent is urea.

[0334] C16. The method according to any one of C to C15, wherein the test solution contains a viscous agent.

[0335] C17. The method according to C16, wherein the viscous agent is glycerol.

[0336] C18. The method described in any one of C to C17, including the gradient of the test solution.

[0337] C19. The method according to C17, wherein the gradient is a temperature gradient, a chemical gradient, or a combination thereof.

[0338] C20. The method according to any one of C to C19, wherein the test solution comprises at least two phases, or a combination thereof.

[0339] C21. The method according to any one of C to C20, wherein the interaction between the target protein and the test solution causes a conformational change in the target molecule.

[0340] C22. The method according to any one of C to C21, wherein the duration of the change in the movement of the target molecule, compared to the duration of the change in the movement of the reference target molecule, indicates that the target molecule is being affected by the test solution.

[0341] C23. A method according to any one of C to C22, which indicates that the target molecule is affected by the test solution by comparing the reversibility of the change in the movement of the target molecule with the reversibility of the change in the movement of the reference target molecule.

[0342] C24. The method according to any one of C to C23, wherein the interaction between the target molecule and the test solution is reversible.

[0343] C25. The method according to any one of C to C24, wherein the interaction between the target molecule and the test solution is irreversible.

[0344] C26. The method according to any one of C to C25, wherein the target molecule and / or the reference target molecule is a component of a mixture consisting of a bacterial extract, a cell extract, a tissue extract, a plant extract, or an animal extract.

[0345] C27. The method according to C26, wherein the bacterial extract, cell extract, tissue extract, plant extract, or animal extract is a lysate.

[0346] C28. The method according to any one of C to C27, wherein the target molecule and / or the reference target molecule is a component of a mixture consisting of serum, blood, and other biological samples.

[0347] C29. The method according to any one of C to C28, wherein the target molecule and / or the reference target molecule is a component of a mixture comprising one or more buffers and glycerol.

[0348] C30. The method according to any one of C to C29, wherein the target molecule and / or the reference target molecule are labeled with a fluorescent protein.

[0349] C31. The method according to any one of C to C30, wherein the target molecule and / or the reference target molecule are labeled by a conjugate of synthetic nanomaterials or polymers.

[0350] D. The present disclosure provides a method for determining the morphology of a target molecule, the method comprising: (a) tracking a plurality of the target molecules in a cell-free sample over time and providing measurements of a plurality of spatiotemporal trajectories and / or rotational motions; (b) analyzing the measurements of the plurality of spatiotemporal trajectories and / or rotational motions to determine the motion of the target molecules; and (c) comparing the motion of the target molecules obtained in (b) with the motion of a reference target molecule, wherein the motion of the reference target molecule is the motion of one morphology of the target molecule, and (i) a change in the motion of the target molecule compared to the motion of the reference target molecule indicates that the target molecule has a different morphology from the reference target molecule, or (ii) no change in the motion of the target molecule compared to the motion of the reference target molecule indicates that the target molecule has the same morphology as the reference target molecule.

[0351] D1. The method described above is a high-throughput method, as described in D.

[0352] D2. The method according to D or D1, wherein the aforementioned change in motion is calculated as a change in (a) the diffusion coefficient obtained from the maximum likelihood estimator, (b) the geometric mean posterior diffusion coefficient, (c) the median of the jump length distribution, (c) the third quartile of the jump length distribution, (d) the median of the turning radius, (e) the mean posterior diffusion coefficient, (f) the mean square displacement, (h) the median of the bond angle, (i) the spatiotemporal trajectory length, (j) the anisotropic decay time, (k) the spatial range of detection, (l) the number and wavelength of conjugate fluorescent labels, (m) the occupation in various diffusion states obtained from the state array, (n) the polarization of the conjugate fluorescent labels, or (o) the state occupation by inference.

[0353] D3. The method according to any one of D to D2, wherein the change in the aforementioned motion is calculated as a change in the diffusion coefficient.

[0354] D4. The method according to any one of D to D3, wherein the target molecule is an organic molecule with a kDa of less than 1 kDa.

[0355] D5. The method according to any one of D to D4, wherein the target molecule is selected from the group consisting of peptides, proteins, nucleic acids, carbohydrates, and lipids.

[0356] D6. The method according to D5, wherein the protein is selected from the group consisting of antibodies and receptors and enzymes.

[0357] D7. The method according to D5 or D6, wherein the protein contains a disordered domain.

[0358] D8. The method according to D7, wherein the protein does not contain a structural domain.

[0359] D9. The method according to D5, wherein the peptide is a ligand.

[0360] D10. The method according to any one of D to D3, wherein the target molecule is a nanomaterial.

[0361] D11. The method according to any one of D to D3, wherein the target molecule is a synthetic polymer.

[0362] D12. The method according to any one of D to D11, wherein the target molecule is labeled.

[0363] D13. The method according to any one of D to D12, wherein the target molecule is fluorescently labeled.

[0364] D14. The cell-free sample is prepared according to any one of D to D13, including the solution.

[0365] D15. The method according to D14, wherein the solution comprises a chaotropic agent, a carrier, a viscous agent, or a combination thereof.

[0366] D16. The method according to D15, wherein the solution contains a chaotropic agent.

[0367] D17. The method according to D16, wherein the chaotropic agent is urea.

[0368] D18. The method according to any one of D14 to D17, wherein the solution contains a viscous agent.

[0369] D19. The method according to D18, wherein the viscous agent comprises glycerol.

[0370] D20. The method according to D19, wherein the solution contains at least about 30% glycerol.

[0371] D21. The method according to any one of D14 to D20, wherein the solution comprises a temperature gradient, a chemical gradient, at least two phases, or a combination thereof.

[0372] D22. The cell-free sample having a volume of approximately 0.1 μl to approximately 100 μl, according to any one of D to D21.

[0373] D23. The method according to any one of D to D22, wherein the change in the motion is measured as a change in the diffusion coefficient of the target molecule compared to the diffusion coefficient of the reference target molecule, and the change is at least about 0.001%, at least about 0.005%, at least 0.01%, at least about 0.05%, at least about 0.1%, at least about 0.5%, at least about 1%, at least about 1.5%, at least about 2%, or at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10%.

[0374] D24. The method according to any one of D to D23, wherein the form of the target molecule is a post-translational modified form of the target molecule.

[0375] D25. The method according to any one of D to D23, wherein the form of the target molecule is the wild-type form of the target molecule.

[0376] D26. The method according to any one of D to D23, wherein the form of the target molecule is a variant form of the target molecule.

[0377] D27. The method according to any one of D to D26, wherein the duration of the change in the movement of the target molecule, compared to the duration of the change in the movement of the reference target molecule, indicates that the target molecule is in a different form from the reference target molecule.

[0378] D28. A method according to any one of D to D27, wherein the reversibility of the change in the movement of the target molecule compared to the reversibility of the change in the movement of the reference target molecule indicates that the target molecule has a different form from the reference target molecule.

[0379] D29. The method according to any one of D to D28, wherein the target molecule and / or the reference target molecule is a component of a mixture consisting of a bacterial extract, a cell extract, a tissue extract, a plant extract, or an animal extract.

[0380] D30. The method according to D29, wherein the bacterial extract, cell extract, tissue extract, plant extract, or animal extract is a lysate.

[0381] D31. The method according to any one of D to D30, wherein the target molecule and / or the reference target molecule are components of a mixture consisting of serum, blood, and other biological samples.

[0382] D32. The method according to any one of D to D31, wherein the target molecule and / or the reference target molecule is a component of a mixture comprising a buffer and glycerol.

[0383] D33. The method according to any one of D to D32, wherein the target molecule and / or the reference target molecule are labeled with a fluorescent protein.

[0384] D34. The method according to any one of D to D33, wherein the target molecule and / or the reference target molecule are labeled by a conjugate of synthetic nanomaterials or polymers.

[0385] E. The present disclosure relates to a method for identifying a test molecule capable of distinguishing between at least two target molecules, the method comprising: (a) contacting a cell-free sample containing a plurality of first target molecules with a plurality of the test molecules; (b) tracking the plurality of the first target molecules over time to obtain measurements of a plurality of spatiotemporal trajectories and / or rotational motions; (c) analyzing the measurements of the plurality of spatiotemporal trajectories and / or rotational motions to determine the movement of the first target molecules in the presence of the test molecules; and (d) contacting a cell-free sample containing a plurality of second target molecules with a plurality of the test molecules. The method provides, comprising: (e) tracking a plurality of the second target molecules over time to obtain measurements of a plurality of spatiotemporal trajectories and / or rotational motions; (f) analyzing the measurements of the plurality of spatiotemporal trajectories and / or rotational motions to determine the movement of the second target molecules in the presence of the test molecule; and (g) comparing the movements of the first target molecule and the second target molecule obtained in (c) and (f), wherein the change in the movements of the first target molecule and the second target molecule indicates that the test molecule can distinguish between the two target molecules.

[0386] E1. The method described above is a high-throughput method, as described in E1.

[0387] E2. The method according to E or E1, wherein the aforementioned change in motion is calculated as a change in (a) the diffusion coefficient obtained from the maximum likelihood estimator, (b) the geometric mean posterior diffusion coefficient, (c) the median of the jump length distribution, (d) the third quartile of the jump length distribution, (e) the median of the turning radius, (f) the mean posterior diffusion coefficient, (g) the mean square displacement, (h) the median of the bond angle, (i) the spatiotemporal trajectory length, (j) the anisotropic decay time, (k) the spatial range of detection, (l) the number and wavelength of conjugate fluorescent labels, (m) the occupation in various diffusion states obtained from the state array, (n) the polarization of the conjugate fluorescent labels, or (o) the state occupation by inference.

[0388] E3. The method according to any one of E to E2, wherein the change in the aforementioned motion is calculated as a change in the diffusion coefficient.

[0389] E4. The method according to any one of E to E3, wherein the target molecule is an organic molecule with a caliber of less than 1 kDa.

[0390] E5. The method according to any one of E to E4, wherein the target molecule is selected from the group consisting of peptides, proteins, nucleic acids, carbohydrates, and lipids.

[0391] E6. The method according to E5, wherein the protein is selected from the group consisting of antibodies and receptors and enzymes.

[0392] E7. The method according to E5 or E6, wherein the protein contains a disordered domain.

[0393] E8. The method according to E7, wherein the protein does not contain a structural domain.

[0394] E9. The method according to E5, wherein the peptide is a ligand.

[0395] E10. The method according to any one of E to E3, wherein the target molecule is a nanomaterial.

[0396] E11. The method according to any one of E to E3, wherein the target molecule is a synthetic polymer.

[0397] E12. The method according to any one of E to E11, wherein the test molecule is an organic molecule with a kDa of less than 1 kDa.

[0398] E13. The method according to any one of E to E12, wherein the test molecule is selected from the group consisting of peptides, proteins, nucleic acids, carbohydrates, and lipids.

[0399] E14. The method according to E13, wherein the protein is selected from the group consisting of antibodies and receptors and enzymes.

[0400] E15. The method according to E13 or E14, wherein the protein contains a disordered domain.

[0401] E16. The method according to E15, wherein the protein does not contain a structural domain.

[0402] E17. The method according to E13, wherein the peptide is a ligand.

[0403] E18. The method according to any one of E to E17, wherein the target molecule is labeled.

[0404] E19. The method according to any one of E to E18, wherein the target molecule is fluorescently labeled.

[0405] E20. The test molecule is labeled, according to one of the methods described in E to E19.

[0406] E21. The method according to any one of E to E20, wherein the test molecule is fluorescently labeled.

[0407] E22. The method according to any one of E to E21, wherein the first target molecule and the second target molecule are related target molecules.

[0408] E23. The method according to E22, wherein at least one of the target molecules is the wild-type form of the target molecule.

[0409] E24. The method according to E22, wherein at least one of the target molecules is a variant form of the target molecule.

[0410] E25. The method according to E22, wherein the first target molecule and the second target molecule are the same or different conformations of the target molecule.

[0411] E26. The method according to E22, wherein at least one of the target molecules is a post-translational modified form of the target molecule.

[0412] E27. The method according to E22, wherein the first target molecule and the second target molecule are homologs, orthologs, or paralogs.

[0413] E28. The method according to any one of E to E21, wherein the first target molecule and the second target molecule are unrelated target molecules.

[0414] E29. The method according to any one of E to E28, further comprising: contacting a cell-free sample containing a plurality of third target molecules with a plurality of the test molecules; tracking the plurality of the third target molecules over time to provide measurements of a third plurality of spatiotemporal trajectories and / or rotational motions; analyzing the measurements of the third plurality of spatiotemporal trajectories and / or rotational motions to determine the motion of the third target molecules in the presence of the test molecules; and comparing the motion of the third target molecules with the motion obtained in (c) and (f), wherein the change in the motion of the third target molecules relative to the first and second target molecules indicates that the test molecules can distinguish between the three target molecules.

[0415] E30. The method according to any one of E to E29, wherein the duration of the change in the movement of the first target molecule and the second target molecule indicates that the test molecule can distinguish between the two target molecules.

[0416] E31. The method according to any one of E to E30, wherein the reversibility of the change in the movement of the first target molecule and the second target molecule indicates that the test molecule can distinguish between the two target molecules.

[0417] E32. The method according to any one of E to E31, wherein the first target molecule, the second target molecule and / or the test molecule are components of a mixture consisting of a bacterial extract, a cell extract, a tissue extract, a plant extract or an animal extract.

[0418] E33. The method according to E32, wherein the bacterial extract, cell extract, tissue extract, plant extract, or animal extract is a lysate.

[0419] E34. The method according to any one of E to E33, wherein the first target molecule, the second target molecule and / or the test molecule are components of a mixture consisting of serum, blood, and other biological samples.

[0420] E35. The method according to any one of E to E34, wherein the first target molecule, the second target molecule and / or the test molecule are components of a mixture comprising a buffer and glycerol.

[0421] E36. The method according to any one of E to E35, wherein the first target molecule, the second target molecule, and / or the test molecule are labeled with a fluorescent protein.

[0422] E37. The method according to any one of E to E36, wherein the first target molecule, the second target molecule and / or the test molecule are labeled by a conjugate of synthetic nanomaterial or polymer.

[0423] E38. The method according to any one of E to E37, wherein the first target molecule and the second target molecule are distinguished by differences in their fluorescent labeling.

[0424] F. The present disclosure provides a method for identifying one or more test molecules that induce conformational changes in a target molecule, comprising: (a) contacting a cell-free sample containing a plurality of the target molecules with a plurality of test molecules, each sample being contacted with a different test molecule; (b) tracking the plurality of target molecules in each sample over time to provide measurements of a plurality of spatiotemporal trajectories and / or rotational motions; (c) analyzing the measurements of the plurality of spatiotemporal trajectories and / or rotational motions to determine the movement of the target molecules in the presence of each of the different test molecules; and (d) comparing the movement of the target molecules obtained in (c) with the movement of a reference target molecule, the movement of the reference target molecule being the movement of the target molecule in the absence of the test molecules, wherein the change in the movement of the target molecules compared with the movement of the reference target molecule indicates an interaction between the target molecule and the one or more test molecules that induce conformational changes in the target molecule.

[0425] F1. The method described above is a high-throughput method, according to method F.

[0426] F2. The method according to F or F1, wherein the aforementioned change in motion is calculated as a change in (a) the diffusion coefficient obtained from the maximum likelihood estimator, (b) the geometric mean posterior diffusion coefficient, (c) the median of the jump length distribution, (b) the third quartile of the jump length distribution, (c) the median of the turning radius, (d) the mean posterior diffusion coefficient, (f) the mean square displacement, (g) the median of the bond angle, (i) the spatiotemporal trajectory length, (j) the anisotropic decay time, (k) the spatial range of detection, (l) the number and wavelength of conjugate fluorescent labels, (m) the occupation in various diffusion states obtained from the state array, (n) the polarization of the conjugate fluorescent labels, or (o) the state occupation by inference.

[0427] F3. The method according to any one of F to F2, wherein the change in the aforementioned motion is calculated as a change in the diffusion coefficient.

[0428] F4. The method according to any one of F to F3, wherein the target molecule is an organic molecule with a caliber of less than 1 kDa.

[0429] F5. The method according to any one of F to F3, wherein the target molecule is selected from the group consisting of peptides, proteins, nucleic acids, carbohydrates, and lipids.

[0430] F6. The method according to F5, wherein the protein is selected from the group consisting of antibodies and receptors and enzymes.

[0431] F7. The method according to F5 or F6, wherein the protein contains a disordered domain.

[0432] F8. The method according to F7, wherein the protein does not contain a structural domain.

[0433] F9. The method according to F5, wherein the peptide is a ligand.

[0434] F10. The method according to any one of F to F3, wherein the target molecule is a nanomaterial.

[0435] F11. The method according to any one of F to F3, wherein the target molecule is a synthetic polymer.

[0436] F12. The method according to any one of F to F11, wherein the test molecule is an organic molecule with a caliber of less than approximately 1 kDa.

[0437] F13. The method according to any one of F to F12, wherein the test molecule is selected from the group consisting of peptides, proteins, nucleic acids, carbohydrates, and lipids.

[0438] F14. The method according to F13, wherein the protein is selected from the group consisting of antibodies and receptors and enzymes.

[0439] F15. The method according to F13 or F14, wherein the protein contains a disordered domain.

[0440] F16. The method according to F15, wherein the protein does not contain a structural domain.

[0441] F17. The method according to F13, wherein the peptide is a ligand.

[0442] F18. The method according to any one of F to F17, wherein the target molecule is labeled.

[0443] F19. The method according to any one of F to F18, wherein the target molecule is fluorescently labeled.

[0444] F20. The method according to one of F to F19, wherein the test molecule is labeled.

[0445] F21. The method according to any one of F to F20, wherein the test molecule is fluorescently labeled.

[0446] F22. The cell-free sample comprising a solution, according to any one of F to F21.

[0447] F23. The method according to F22, wherein the solution comprises a chaotropic agent, a viscous agent, or a combination thereof.

[0448] F24. The method according to F23, wherein the viscous agent comprises glycerol.

[0449] F25. The method according to F24, wherein the solution contains at least about 30% glycerol.

[0450] F26. The method according to any one of F22 to F25, wherein the solution comprises a temperature gradient, a chemical gradient, at least two phases, or a combination thereof.

[0451] F27. The method according to any one of F to F26, wherein the sample has a volume of approximately 0.1 μl to approximately 100 μl.

[0452] F28. The method according to any one of F-F27, wherein the change in the motion is measured as a change in the diffusion coefficient of the target molecule in the presence of the test molecule and in the presence of a reference test molecule that does not induce a confirmation change, and the change is at least about 0.001%, at least about 0.005%, at least 0.01%, at least about 0.05%, at least about 0.1%, at least about 0.5%, at least about 1%, at least about 1.5%, at least about 2%, or at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10%.

[0453] F29. The method according to any one of F to F28, wherein the duration of the change in the movement of the target molecule, compared to the duration of the change in the movement of the reference target molecule, indicates an interaction between the target molecule and the one or more test molecules that induce a conformational change in the target molecule.

[0454] F30. The method according to any one of F to F29, wherein the reversibility of the change in the movement of the target molecule compared to the reversibility of the change in the movement of the reference target molecule indicates an interaction between the target molecule and the one or more test molecules that induce a conformational change in the target molecule.

[0455] F31. The method according to any one of F to F30, wherein the interaction between the target molecule and the one or more test molecules is reversible.

[0456] F32. The method according to any one of F to F31, wherein the interaction between the target molecule and the one or more test molecules is irreversible.

[0457] F33. The method according to any one of F to F32, wherein the target molecule, the reference target molecule and / or the one or more test molecules are components of a mixture consisting of a bacterial extract, a cell extract, a tissue extract, a plant extract or an animal extract.

[0458] F34. The method according to F33, wherein the bacterial extract, cell extract, tissue extract, plant extract, or animal extract is a lysate.

[0459] F35. The method according to any one of F to F34, wherein the target molecule, the reference target molecule and / or the one or more test molecules are components of a mixture consisting of serum, blood, and other biological samples.

[0460] F36. The method according to any one of F to F35, wherein the target molecule, the reference target molecule and / or the one or more test molecules are components of a mixture comprising a buffer and glycerol.

[0461] F37. The method according to any one of F to F36, wherein the target molecule, the reference target molecule and / or the one or more test molecules are labeled with a fluorescent protein.

[0462] F38. The method according to any one of F to F37, wherein the target molecule, the reference target molecule and / or the one or more test molecules are labeled by a conjugate of synthetic nanomaterial or polymer.

[0463] F39. The method according to any one of F to F38, wherein the conformational change in the target molecule is related to a change in the temperature stability of the target molecule.

[0464] G. The present disclosure provides a method for determining a dose response to an interaction between a target molecule and a test molecule, the method comprising: (a) contacting a plurality of cell-free samples containing a plurality of the target molecules with the test molecule, wherein the plurality of cell-free samples are contacted with a range of test molecule doses; (b) tracking the plurality of target molecules over time in the presence of the range of test molecule doses to provide a plurality of spatiotemporal trajectory and / or rotational motion measurements; (c) analyzing the plurality of spatiotemporal trajectory and / or rotational motion measurements of the target molecules in the presence of the range of test molecule doses to determine the movement of the target molecules at each dose of the test molecule; and (d) comparing the movement of the target molecules obtained in step (c) at various test molecule doses to determine the dose response of the target molecule to the test molecule.

[0465] G1. This disclosure relates to a method for determining the difference in dose response between two target molecules to a test molecule, comprising: (a) contacting a plurality of first cell-free samples containing a plurality of the first target molecules with the test molecule, wherein the plurality of first cell-free samples are contacted with a range of test molecule doses; (b) tracking the plurality of target molecules over time in the presence of the range of test molecule doses to provide measurements of the first plurality of spatiotemporal trajectories and / or rotational motions; and (c) analyzing the measurements of the plurality of spatiotemporal trajectories and / or rotational motions of the target molecules in the presence of the range of test molecule doses. The present invention provides a method comprising: (d) determining the movement of the first target molecule at various concentrations of the test molecule; (c) comparing the movement of the target molecule obtained in step (c) at various doses of the test molecule to determine the dose response of the target molecule to the test molecule; (e) repeating steps (a) to (d) using the second target molecule to determine the dose response of the second target molecule to the test molecule; and (f) comparing the dose response of the first target molecule with the dose response of the second target molecule to determine the difference in the responses of the first target molecule and the second target molecule to the test molecule.

[0466] G2. The method described above is a high-throughput method, as described in G or G1.

[0467] G3. The method according to G or G1, wherein the motion is calculated as (a) the diffusion coefficient of the plurality of spatiotemporal trajectories obtained from the maximum likelihood estimator, (b) the geometric mean posterior diffusion coefficient of the plurality of spatiotemporal trajectories, (c) the median of the jump length distribution of the plurality of spatiotemporal trajectories, (d) the third quartile of the jump length distribution of the plurality of spatiotemporal trajectories, (e) the median of the turning radius of the plurality of spatiotemporal trajectories, (f) the mean posterior diffusion coefficient of the plurality of spatiotemporal trajectories, (g) the mean square displacement of the plurality of spatiotemporal trajectories, (h) the median of the joint angle of the plurality of spatiotemporal trajectories, (i) the spatiotemporal trajectory length of the plurality of spatiotemporal trajectories, (j) the anisotropic decay time, (k) the spatial range of detection, (l) the number and wavelength of conjugate fluorescent labels, (m) occupation in various diffusion states obtained from the state array, (n) the polarization of the conjugate fluorescent labels, or (o) the state occupation by inference.

[0468] G4. The method according to any one of G to G3, wherein the aforementioned motion is calculated as the diffusion coefficient.

[0469] G5. The method according to any one of G to G4, wherein the target molecule is an organic molecule with a caliber of less than 1 kDa.

[0470] G6. The method according to any one of G to G5, wherein the target molecule is selected from the group consisting of peptides, proteins, nucleic acids, carbohydrates, and lipids.

[0471] G7. The method according to G6, wherein the protein is selected from the group consisting of antibodies and receptors and enzymes.

[0472] G8. The protein comprising a disordered domain, as described in G6 or G7.

[0473] G9. The method described in G8, wherein the protein does not contain a structural domain.

[0474] G10. The method according to G6, wherein the peptide is a ligand.

[0475] G11. The method according to any one of G to G4, wherein the target molecule is a nanomaterial.

[0476] G12. The method according to any one of G to G4, wherein the target molecule is a synthetic polymer.

[0477] G13. The method according to any one of G to G12, wherein the test molecule is an organic molecule with a caliber of less than 1 kDa.

[0478] G14. The method according to any one of G to G13, wherein the test molecule is selected from the group consisting of peptides, proteins, nucleic acids, carbohydrates, and lipids.

[0479] G15. The method according to G14, wherein the protein is selected from the group consisting of antibodies and receptors and enzymes.

[0480] G16. The protein comprising a disordered domain, as described in G14 or G15.

[0481] G17. The method described in G16, wherein the protein does not contain a structural domain.

[0482] G18. The method according to G14, wherein the peptide is a ligand.

[0483] G19. The method according to any one of G to G18, wherein the target molecule is labeled.

[0484] G20. The method according to one of G to G19, wherein the target molecule is fluorescently labeled.

[0485] G21. The test molecule is labeled, according to one of the methods in G to G20.

[0486] G22. The method according to any one of G to G21, wherein the test molecule is fluorescently labeled.

[0487] G23. The method according to any one of G1 to G22, wherein the first target molecule and the second target molecule are related target molecules.

[0488] G24. The method according to G23, wherein at least one of the target molecules is the wild-type form of the target molecule.

[0489] G25. The method according to G23, wherein at least one of the target molecules is a variant form of the target molecule.

[0490] G26. The method according to G23, wherein the first target molecule and the second target molecule are in the same or different conformational states as the target molecule.

[0491] G27. The method according to G23, wherein at least one of the target molecules is a post-translational modified form of the target molecule.

[0492] G28. The method according to G23, wherein the first target molecule and the second target molecule are homologs, orthologs, or paralogs.

[0493] G29. The method according to G1, wherein the first target molecule and the second target molecule are unrelated target molecules.

[0494] G30. The method according to G1, wherein the first cell-free sample and the second cell-free sample contain a solution comprising a chaotropic agent, a carrier, a viscous agent, or a combination thereof.

[0495] G31. The method according to G30, wherein the solution contains a viscous agent.

[0496] G32. The method according to G31, wherein the viscous agent comprises glycerol.

[0497] G33. The method according to G32, wherein the solution contains at least about 30% glycerol.

[0498] G34. The method according to any one of G30 to G33, wherein the solution comprises a temperature gradient, a chemical gradient, at least two phases, or a combination thereof.

[0499] G35. The method according to any one of G to G34, wherein comparing the movement of the target molecule obtained at various test molecular doses in step (c) further comprises comparing the duration of the change in the movement of the target molecule.

[0500] G36. The method according to any one of G to G35, further comprising comparing the movement of the target molecule obtained at various test molecular doses in step (c), wherein the comparison of the reversibility of the change in the movement of the target molecule is further included.

[0501] G37. The method according to any one of G to G36, wherein the interaction between the target molecule and the test molecule is reversible.

[0502] G38. The method according to any one of G to G37, wherein the interaction between the target molecule and the test molecule is irreversible.

[0503] G39. The method according to any one of G to G38, wherein the target molecule and / or the test molecule is a component of a mixture consisting of a bacterial extract, a cell extract, a tissue extract, a plant extract, or an animal extract.

[0504] G40. The method according to G39, wherein the bacterial extract, cell extract, tissue extract, plant extract, or animal extract is a lysate.

[0505] G41. The method according to any one of G to G40, wherein the target molecule and / or the test molecule is a component of a mixture consisting of serum, blood, and other biological samples.

[0506] G42. The method according to any one of G to G41, wherein the target molecule and / or the test molecule is a component of a mixture comprising a buffer and glycerol.

[0507] G43. The method according to any one of G to G42, wherein the target molecule and / or the test molecule is labeled with a fluorescent protein.

[0508] G44. The method according to any one of G to G43, wherein the target molecule and / or the test molecule are labeled by a conjugate of synthetic nanomaterial or polymer.

[0509] G45. The method according to any one of G to G44, wherein the first target molecule and the second target molecule are distinguished by differences in their fluorescent labeling.

[0510] G46. A method according to any one of G-G45, in which three or more target molecules are analyzed.

[0511] H. The present disclosure provides a method for identifying a test molecule capable of distinguishing between at least two target molecules, comprising: (a) contacting a cell-free sample containing a plurality of first target molecules and a plurality of second target molecules with a plurality of the test molecules; (b) tracking the plurality of the first target molecules over time to obtain a plurality of spatiotemporal trajectories and / or rotational motion measurements; (c) analyzing the plurality of spatiotemporal trajectories and / or rotational motion measurements to determine the movement of the first target molecules in the presence of the test molecule; (d) tracking the plurality of the second target molecules over time to obtain a plurality of spatiotemporal trajectories and / or rotational motion measurements; (e) analyzing the plurality of spatiotemporal trajectories and / or rotational motion measurements to determine the movement of the second target molecules in the presence of the test molecule; and (f) comparing the movements of the first target molecules and the second target molecules obtained in (c) and (f), wherein the change in the movements of the first target molecules and the second target molecules indicates that the test molecule is capable of distinguishing between the two target molecules.

[0512] H1. The method described above is a high-throughput method, as described in H1.

[0513] H2. The method according to H or H1, wherein the aforementioned change in motion is calculated as a change in (a) the diffusion coefficient obtained from the maximum likelihood estimator, (b) the geometric mean posterior diffusion coefficient, (c) the median of the jump length distribution, (d) the third quartile of the jump length distribution, (e) the median of the turning radius, (f) the mean posterior diffusion coefficient, (g) the mean square displacement, (h) the median of the bond angle, (i) the spatiotemporal trajectory length, (j) the anisotropic decay time, (k) the spatial range of detection, (l) the number and wavelength of conjugate fluorescent labels, (m) the occupation in various diffusion states obtained from the state array, (n) the polarization of the conjugate fluorescent labels, or (o) the state occupation by inference.

[0514] H3. The method according to any one of H to H2, wherein the change in the aforementioned motion is calculated as a change in the diffusion coefficient.

[0515] H4. The method according to one of H to H3, wherein the target molecule is an organic molecule with a caliber of less than 1 kDa.

[0516] H5. The method according to any one of H to H4, wherein the target molecule is selected from the group consisting of peptides, proteins, nucleic acids, carbohydrates, and lipids.

[0517] H6. The method according to H5, wherein the protein is selected from the group consisting of antibodies and receptors and enzymes.

[0518] H7. The method according to H5 or H6, wherein the protein contains a disordered domain.

[0519] H8. The method described in H7, wherein the protein does not contain a structural domain.

[0520] H9. The method described in H5, wherein the peptide is a ligand.

[0521] H10. The method described above for any one of H to H3, wherein the target molecule is a nanomaterial.

[0522] H11. The method according to one of H to H3, wherein the target molecule is a synthetic polymer.

[0523] H12. The method according to any one of H to H11, wherein the test molecule is an organic molecule with a kDa of less than 1 kDa.

[0524] H13. The method according to any one of H to H12, wherein the test molecule is selected from the group consisting of peptides, proteins, nucleic acids, carbohydrates, and lipids.

[0525] H14. The method according to H13, wherein the protein is selected from the group consisting of antibodies and receptors and enzymes.

[0526] H15. The protein comprising a disordered domain, as described in H13 or H14.

[0527] H16. The method described in H15, wherein the protein does not contain a structural domain.

[0528] H17. The method according to H13, wherein the peptide is a ligand.

[0529] H18. The method described in any one of H to H17, wherein the target molecule is labeled.

[0530] H19. The method according to one of H to H18, wherein the target molecule is fluorescently labeled.

[0531] H20. The test molecule is labeled, according to one of the methods described in H to H19.

[0532] H21. The test molecule is fluorescently labeled, according to the method described in one of H to H20.

[0533] H22. The method according to one of H to H21, wherein the first target molecule and the second target molecule are related target molecules.

[0534] H23. The method according to H22, wherein at least one of the target molecules is the wild-type form of the target molecule.

[0535] H24. The method according to H22, wherein at least one of the target molecules is a variant form of the target molecule.

[0536] H25. The method according to H22, wherein the first target molecule and the second target molecule are in the same or different conformational states as the target molecule.

[0537] H26. The method according to H22, wherein at least one of the target molecules is a post-translational modified form of the target molecule.

[0538] H27. The method according to H22, wherein the first target molecule and the second target molecule are homologs, orthologues, or paralogs.

[0539] H28. The method according to any one of H to H21, wherein the first target molecule and the second target molecule are unrelated target molecules.

[0540] H29. The method according to any one of H to H28, further comprising: contacting a cell-free sample containing a plurality of third target molecules with a plurality of test molecules; tracking the plurality of third target molecules over time to provide measurements of a third plurality of spatiotemporal trajectories and / or rotational motions; analyzing the measurements of the third plurality of spatiotemporal trajectories and / or rotational motions to determine the motion of the third target molecules in the presence of the test molecules; and comparing the motion of the third target molecules with the motion obtained in (c) and (f), wherein the change in the motion of the third target molecules relative to the first and second target molecules indicates that the test molecules can distinguish between the three target molecules.

[0541] H30. The method according to any one of H to H29, wherein the duration of the change in the movement of the first molecule, compared to the duration of the change in the movement of the second molecule, indicates that the test molecule can distinguish between the two target molecules.

[0542] H31. The method according to any one of H to H30, wherein the reversibility of the change in the movement of the first molecule compared to the reversibility of the change in the movement of the second molecule indicates that the test molecule can distinguish between the two target molecules.

[0543] H32. The method according to any one of H to H31, wherein the interaction between the target molecule and the test molecule is reversible.

[0544] H33. The method according to any one of H to H32, wherein the interaction between the target molecule and the test molecule is irreversible.

[0545] H34. The method according to any one of H to H33, wherein the first target molecule, the second target molecule and / or the test molecule are components of a mixture consisting of a bacterial extract, a cell extract, a tissue extract, a plant extract or an animal extract.

[0546] H35. The method according to H34, wherein the bacterial extract, cell extract, tissue extract, plant extract, or animal extract is a lysate.

[0547] H36. The method according to any one of H to H35, wherein the first target molecule, the second target molecule and / o...

Claims

1. A method for identifying interactions between a target molecule and a test molecule, (a) Contacting a cell-free sample containing multiple target molecules with multiple test molecules, (b) Tracking multiple target molecules over time to obtain multiple spatiotemporal trajectories and / or rotational motion measurements, (c) Analyzing the measured values ​​of the multiple spatiotemporal trajectories and / or rotational motions to determine the movement of the target molecule in the presence of the test molecule, (d)(c) Comparing the movement of the target molecule obtained in (d)(c) with the movement of a reference target molecule, wherein the movement of the reference target molecule is the movement of the target molecule in the absence of the test molecule, and the comparison is as follows: The method wherein the interaction between the target molecule and the test molecule is demonstrated by a change in the movement of the target molecule compared to the movement of the reference target molecule.

2. The method according to claim 1, wherein the method is a high-throughput method.

3. The changes in the aforementioned movement are (a) Diffusion coefficient obtained from the maximum likelihood estimator, (b) Geometric mean apost-diffusion coefficient, (c) Median of the jump length distribution, (d) The third quartile of the jump length distribution, (e) Median of turning radius, (f) Average post-dispersion coefficient, (g) Mean square displacement, (h) Median of bond angles, (i) Space-time trajectory length, (j) Anisotropic decay time, (k) Spatial range of detection, (l) Number and wavelength of conjugated fluorescent labels, (m) Occupancy in various diffusion states obtained from the state array, (n) Polarization of conjugated fluorescent label, or (o) State occupation by inference, The method according to claim 1 or 2, which is calculated as a change in

4. The method according to any one of claims 1 to 3, wherein the change in the aforementioned motion is calculated as a change in the diffusion coefficient.

5. The method according to any one of claims 1 to 4, wherein an increase or decrease in the movement of the target molecule compared to the movement of the reference target molecule indicates an interaction between the target molecule and the test molecule.

6. The method according to any one of claims 1 to 5, wherein the interaction between the target molecule and the test molecule is reversible or irreversible.

7. The method according to any one of claims 1 to 6, wherein the duration and / or reversibility of the change in the movement of the target molecule, compared to the duration and / or reversibility of the change in the movement of the reference target molecule, indicates an interaction between the target molecule and the test molecule.

8. The method according to any one of claims 1 to 7, wherein the target molecule and / or the test molecule is a component of a mixture comprising a bacterial extract, a cell extract, a tissue extract, a plant extract, or an animal extract.

9. The method according to claim 8, wherein the bacterial extract, cell extract, tissue extract, plant extract, or animal extract is a lysate.

10. The method according to any one of claims 1 to 9, wherein the target molecule and / or the test molecule is a component of a mixture comprising serum, blood, and / or other biological samples.

11. The method according to any one of claims 1 to 10, wherein the target molecule and / or the test molecule is a component of a mixture comprising a buffer and glycerol.

12. The method according to any one of claims 1 to 11, wherein the target molecule and / or the test molecule is an organic molecule with a strength of less than 1 kDa.

13. The method according to any one of claims 1 to 12, wherein the target molecule and / or the test molecule is selected from the group consisting of peptides, proteins, nucleic acids, carbohydrates, and lipids.

14. The method according to claim 13, wherein the protein is selected from the group consisting of antibodies and receptors and enzymes.

15. The method according to claim 13 or 14, wherein the protein includes a disordered domain and / or does not include a structured domain.

16. The method according to claim 13, wherein the peptide is a ligand.

17. The method according to any one of claims 1 to 11, wherein the target molecule and / or the test molecule is a nanomaterial or a synthetic polymer.

18. The method according to any one of claims 1 to 17, wherein the target molecule and / or the test molecule is labeled.

19. The method according to any one of claims 1 to 18, wherein the target molecule and / or the test molecule is fluorescently labeled.

20. The method according to any one of claims 1 to 19, wherein the target molecule and / or the test molecule is labeled with a fluorescent protein.

21. The method according to any one of claims 1 to 20, wherein the plurality of target molecules and / or the plurality of test molecules are conjugated with a plurality of fluorescent labels.

22. The method according to any one of claims 1 to 21, wherein the target molecule and / or the test molecule is labeled by conjugation to a synthetic nanomaterial or polymer.

23. The method according to any one of claims 1 to 22, wherein the change in the movement of the target molecule compared to the movement of the reference target molecule indicates a direct or indirect interaction between the target molecule and the test molecule.

24. The method according to any one of claims 1 to 23, wherein the interaction between the target molecule and the test molecule results in a conformational change in the target molecule, an increase in the mass of the target molecule, a change in the temperature stability of the target molecule, and / or oligomerization of the target molecule.

25. The method according to any one of claims 1 to 24, wherein the cell-free sample comprises a solution.

26. The method according to claim 25, wherein the solution comprises a chaotropic agent, a carrier, a viscous agent, or a combination thereof.

27. The method according to claim 26, wherein the viscous agent comprises glycerol.

28. The method according to claim 27, wherein the solution comprises at least about 30% glycerol.

29. The method according to any one of claims 25 to 28, wherein the solution comprises a temperature gradient, a chemical gradient, at least two phases, or a combination thereof.

30. The cell-free sample has a volume of about 0.1 μl to about 100 μl, according to any one of claims 1 to 29.

31. The method according to any one of claims 1 to 30, wherein the change in the motion is measured as a change in the diffusion coefficient of the target molecule compared to the diffusion coefficient of the reference target molecule, and the change is at least about 0.001%, at least about 0.005%, at least 0.01%, at least about 0.05%, at least about 0.1%, at least about 0.5%, at least about 1%, at least about 1.5%, at least about 2%, or at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10%.

32. A method for identifying interactions between a test molecule and a target protein containing a disordered domain, (a) Contacting a cell-free sample containing multiple target proteins with multiple test molecules, (b) Tracking multiple target proteins over time to obtain multiple spatiotemporal trajectories and / or rotational motion measurements, (c) Analyzing the measured values ​​of the multiple spatiotemporal trajectories and / or rotational motions to determine the movement of the target protein in the presence of the test molecule, (d) Comparing the movement of the target protein obtained in (c) with the movement of a reference target protein, wherein the movement of the reference target protein is the movement of the target protein in the absence of the test molecule, and the comparison is as follows: The method wherein the interaction between the target protein containing the disordered domain and the test molecule is demonstrated by a change in the movement of the target protein containing the disordered domain compared to the movement of the reference target protein.

33. The method according to claim 32, wherein the method is a high-throughput method.

34. The changes in the aforementioned movement are (a) Diffusion coefficient obtained from the maximum likelihood estimator, (b) Geometric mean apost-diffusion coefficient, (c) Median of the jump length distribution, (d) The third quartile of the jump length distribution, (e) Median of turning radius, (f) Average post-dispersion coefficient, (g) Mean square displacement, (h) Median of bond angles, (i) Space-time trajectory length, (j) Anisotropic decay time, (k) Spatial range of detection, (l) Number and wavelength of conjugated fluorescent labels, (m) Occupancy in various diffusion states obtained from the state array, (n) Polarization of conjugated fluorescent label, or (o) State occupation by inference, The method according to claim 32 or 33, which is calculated as a change in

35. The method according to any one of claims 32 to 34, wherein the change in the aforementioned motion is calculated as a change in the diffusion coefficient.

36. The method according to any one of claims 32 to 35, wherein an increase or decrease in the movement of the target protein containing the disordered domain compared to the movement of the reference target protein indicates an interaction between the target protein containing the disordered domain and the test molecule.

37. The method according to any one of claims 32 to 36, wherein the interaction between the target protein containing the disordered domain and the test molecule is reversible or irreversible.

38. The method according to any one of claims 32 to 37, wherein the duration and / or reversibility of the change in the movement of the target protein containing the disordered domain, compared to the duration and / or reversibility of the change in the movement of the reference target protein, indicates an interaction between the target molecule and the test molecule.

39. The method according to any one of claims 32 to 38, wherein the target protein and / or test molecule containing the disordered domain is a component of a mixture comprising a bacterial extract, a cell extract, a tissue extract, a plant extract, or an animal extract.

40. The method according to claim 39, wherein the bacterial extract, cell extract, tissue extract, plant extract, or animal extract is a lysate.

41. The method according to any one of claims 32 to 40, wherein the target protein containing the disordered domain and / or the test molecule is a component of a mixture comprising serum, blood, and other biological samples.

42. The method according to any one of claims 32 to 41, wherein the target protein and / or test molecule containing the disordered domain is a component of a mixture containing a buffer and glycerol.

43. The method according to any one of claims 32 to 42, wherein the target protein containing the disordered domain is selected from the group consisting of antibodies, receptors, and enzymes.

44. The method according to any one of claims 32 to 43, wherein the target protein containing the disordered domain does not contain a structured domain.

45. The method according to any one of claims 32 to 44, wherein the test molecule is an organic molecule with a kDa of less than 1 kDa.

46. The method according to any one of claims 32 to 45, wherein the test molecule is selected from the group consisting of peptides, proteins, nucleic acids, carbohydrates, and lipids.

47. The method according to claim 46, wherein the protein is selected from the group consisting of antibodies and receptors and enzymes.

48. The method according to claim 46 or 47, wherein the protein includes a disordered domain and / or does not include a structured domain.

49. The method according to claim 46, wherein the peptide is a ligand.

50. The method according to any one of claims 32 to 49, wherein the target protein and / or the test molecule containing the disordered domain is labeled.

51. The method according to any one of claims 32 to 50, wherein the target protein and / or the test molecule containing the disordered domain is fluorescently labeled.

52. The method according to any one of claims 32 to 51, wherein the target protein and / or the test molecule is labeled with a fluorescent protein.

53. The method according to any one of claims 32 to 52, wherein the target protein and / or the test molecule is labeled by a conjugate of synthetic nanomaterial or polymer.

54. The method according to any one of claims 32 to 53, wherein the change in the movement of the target protein containing the disordered domain compared to the movement of the reference target protein indicates a direct or indirect interaction between the target protein containing the disordered domain and the test molecule.

55. The method according to any one of claims 32 to 54, wherein the interaction between the target protein containing the disordered domain and the test molecule results in a conformational change in the target protein, a change in the temperature stability of the target protein, an increase in the mass of the target protein, and / or oligomerization of the target protein.

56. The method according to any one of claims 32 to 55, wherein the cell-free sample comprises a solution.

57. The method according to claim 56, wherein the solution comprises a chaotropic agent, a carrier, a viscous agent, or a combination thereof.

58. The method according to claim 57, wherein the viscous agent comprises glycerol.

59. The method according to claim 58, wherein the solution contains at least about 30% glycerol.

60. The method according to any one of claims 56 to 59, wherein the solution comprises a temperature gradient, a chemical gradient, at least two phases, or a combination thereof.

61. The cell-free sample has a volume of about 0.1 μl to about 100 μl, according to any one of claims 32 to 60.

62. The method according to any one of claims 32 to 61, wherein the change in the motion is measured as a change in the diffusion coefficient of the target protein compared to the diffusion coefficient of the reference target protein, and the change is at least about 0.001%, at least about 0.005%, at least 0.01%, at least about 0.05%, at least about 0.1%, at least about 0.5%, at least about 1%, at least about 1.5%, at least about 2%, or at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10%.

63. A method for analyzing target molecules in a test solution, (a) Tracking multiple target molecules in the test solution over time and providing multiple spatiotemporal trajectories and / or rotational motion measurements, (b) Analyzing the measured values ​​of the plurality of spatiotemporal trajectories and / or rotational motions to determine the movement of the target molecule in the test solution, (c)(b) Comparing the movement of the target molecule obtained in (c)(b) with the movement of a reference target molecule, wherein the movement of the reference target molecule is the movement of the target molecule in a reference solution, and the comparison is as follows: (i) a change in the movement of the target molecule compared to the movement of the reference target molecule indicates that the target molecule is affected by the test solution, or (ii) no change in the movement of the target molecule compared to the movement of the reference target molecule indicates that the target molecule is not affected by the test solution.

64. The method according to claim 63, wherein the method is a high-throughput method.

65. The changes in the aforementioned movement are (a) Diffusion coefficient obtained from the maximum likelihood estimator, (b) Geometric mean apost-diffusion coefficient, (c) Median of the jump length distribution, (d) The third quartile of the jump length distribution, (e) Median of turning radius, (f) Average post-dispersion coefficient, (g) Mean square displacement, (g) Median of bond angles, (i) Space-time trajectory length, (j) Anisotropic decay time, (k) Spatial range of detection, (l) Number and wavelength of conjugated fluorescent labels, (m) Occupancy in various diffusion states obtained from the state array, (n) Polarization of conjugated fluorescent label, or (o) The method according to claim 63 or 64, which is calculated as a change in state occupation by inference.

66. The method according to any one of claims 63 to 65, wherein the change in the aforementioned motion is calculated as a change in the diffusion coefficient.

67. The method according to any one of claims 63 to 66, wherein the duration and / or reversibility of the change in the movement of the target molecule, compared to the duration and / or reversibility of the change in the movement of the reference target molecule, indicates that the target molecule is being affected by the test solution.

68. The method according to any one of claims 63 to 67, wherein the target molecule and / or the reference target molecule is a component of a mixture comprising a bacterial extract, a cell extract, a tissue extract, a plant extract, or an animal extract.

69. The method according to claim 68, wherein the bacterial extract, cell extract, tissue extract, plant extract, or animal extract is a lysate.

70. The method according to any one of claims 63 to 69, wherein the target molecule and / or the reference target molecule is a component of a mixture comprising serum, blood, and other biological samples.

71. The method according to any one of claims 63 to 70, wherein the target molecule and / or the reference target molecule is a component of a mixture comprising a buffer and glycerol.

72. The method according to any one of claims 63 to 71, wherein the target molecule and / or the reference target molecule is an organic molecule with a strength of less than 1 kDa.

73. The method according to any one of claims 63 to 72, wherein the target molecule and / or the reference target molecule are selected from the group consisting of peptides, proteins, nucleic acids, carbohydrates, and lipids.

74. The method according to claim 73, wherein the protein is selected from the group consisting of antibodies and receptors and enzymes.

75. The method according to claim 73 or 74, wherein the protein includes a disordered domain and / or does not include a structured domain.

76. The method according to claim 73, wherein the peptide is a ligand.

77. The method according to any one of claims 63 to 72, wherein the target molecule and / or the reference target molecule is a nanomaterial or a synthetic polymer.

78. The method according to any one of claims 63 to 77, wherein the target molecule and / or the reference target molecule are labeled.

79. The method according to any one of claims 63 to 78, wherein the target molecule and / or the reference target molecule are fluorescently labeled.

80. The method according to any one of claims 63 to 79, wherein the target molecule and / or the reference target molecule are labeled with a fluorescent protein.

81. The method according to any one of claims 63 to 80, wherein the target molecule and / or the reference target molecule are labeled by conjugation to a synthetic nanomaterial or polymer.

82. The method according to any one of claims 63 to 81, wherein the test solution comprises a chaotropic agent and / or a viscous agent.

83. The method according to claim 82, wherein the chaotropic agent is urea.

84. The method according to claim 82 or 83, wherein the viscous agent is glycerol.

85. The method according to any one of claims 63 to 84, wherein the test solution includes a gradient.

86. The method according to claim 85, wherein the gradient is a temperature gradient, a chemical gradient, or a combination thereof.

87. The method according to any one of claims 63 to 86, wherein the test solution comprises at least two phases, or a combination thereof.

88. The method according to any one of claims 63 to 87, wherein the interaction between the target protein molecule and the test solution causes a conformational change in the target molecule.

89. The method according to any one of claims 63 to 88, wherein the interaction between the target molecule and the test solution is reversible or irreversible.

90. A method for determining the morphology of a target molecule, (a) Track multiple target molecules in a cell-free sample over time and provide measurements of multiple spatiotemporal trajectories and / or rotational motions, (b) Analyzing the measured values ​​of the multiple spatiotemporal trajectories and / or rotational motions to determine the motion of the target molecule, (c) Comparing the motion of the target molecule obtained in (b) with the motion of a reference target molecule, wherein the motion of the reference target molecule is the motion of one form of the target molecule, the comparison includes: (i) A change in the movement of the target molecule compared to the movement of the reference target molecule indicates that the target molecule has a different morphology from the reference target molecule, or (ii) No change in the movement of the target molecule compared to the movement of the reference target molecule indicates that the target molecule has the same morphology as the reference target molecule.

91. The method according to claim 90, wherein the method is a high-throughput method.

92. The changes in the aforementioned movement are (a) Diffusion coefficient obtained from the maximum likelihood estimator, (b) Geometric mean apost-diffusion coefficient, (c) Median of the jump length distribution, (d) The third quartile of the jump length distribution, (e) Median of turning radius, (f) Average post-dispersion coefficient, (g) Mean square displacement, (h) Median of bond angles, (i) Space-time trajectory length, (j) Anisotropic decay time, (k) Spatial range of detection, (l) Number and wavelength of conjugated fluorescent labels, (m) Occupancy in various diffusion states obtained from the state array, (n) Polarization of conjugated fluorescent label, or (o) The method according to claim 90 or 91, calculated as a change in state occupation by inference.

93. The method according to any one of claims 90 to 92, wherein the change in the aforementioned motion is calculated as a change in the diffusion coefficient.

94. The method according to any one of claims 90 to 93, wherein the duration and / or reversibility of the change in the movement of the target molecule, compared to the duration and / or reversibility of the change in the movement of the reference target molecule, indicates that the target molecule is in a different form from the reference target molecule.

95. The method according to any one of claims 90 to 94, wherein the target molecule and / or the reference target molecule is a component of a mixture comprising a bacterial extract, a cell extract, a tissue extract, a plant extract, or an animal extract.

96. The method according to claim 95, wherein the bacterial extract, cell extract, tissue extract, plant extract, or animal extract is a lysate.

97. The method according to any one of claims 90 to 96, wherein the target molecule and / or the reference target molecule is a component of a mixture comprising serum, blood, and other biological samples.

98. The method according to any one of claims 90 to 97, wherein the target molecule and / or the reference target molecule is a component of a mixture comprising a buffer and glycerol.

99. The method according to any one of claims 90 to 98, wherein the target molecule and / or the reference target molecule is an organic molecule with a strength of less than 1 kDa.

100. The method according to any one of claims 90 to 99, wherein the target molecule and / or the reference target molecule is selected from the group consisting of peptides, proteins, nucleic acids, carbohydrates, and lipids.

101. The method according to claim 100, wherein the protein is selected from the group consisting of antibodies and receptors and enzymes.

102. The method according to claim 100 or 101, wherein the protein comprises a disordered domain and / or does not comprise a structured domain.

103. The method according to claim 100, wherein the peptide is a ligand.

104. The method according to any one of claims 90 to 98, wherein the target molecule and / or the reference target molecule is a nanomaterial or a synthetic polymer.

105. The method according to any one of claims 90 to 104, wherein the target molecule and / or the reference target molecule are labeled.

106. The method according to any one of claims 90 to 105, wherein the target molecule and / or the reference target molecule are fluorescently labeled.

107. The method according to any one of claims 90 to 106, wherein the target molecule and / or the reference target molecule are labeled with a fluorescent protein.

108. The method according to any one of claims 90 to 107, wherein the target molecule and / or the reference target molecule are labeled by a conjugate of synthetic nanomaterials or polymers.

109. The method according to any one of claims 90 to 108, wherein the cell-free sample comprises a solution.

110. The method according to claim 109, wherein the solution comprises a chaotropic agent, a carrier, a viscous agent, or a combination thereof.

111. The method according to claim 110, wherein the chaotropic agent is urea.

112. The method according to claim 110 or 111, wherein the viscous agent comprises glycerol.

113. The method according to claim 112, wherein the solution contains at least about 30% glycerol.

114. The method according to any one of claims 109 to 113, wherein the solution comprises a temperature gradient, a chemical gradient, at least two phases, or a combination thereof.

115. The cell-free sample has a volume of about 0.1 μl to about 100 μl, according to any one of claims 90 to 114.

116. The method according to any one of claims 90 to 115, wherein the change in the motion is measured as a change in the diffusion coefficient of the target molecule compared to the diffusion coefficient of the reference target molecule, and the change is at least about 0.001%, at least about 0.005%, at least 0.01%, at least about 0.05%, at least about 0.1%, at least about 0.5%, at least about 1%, at least about 1.5%, at least about 2%, or at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10%.

117. The method according to any one of claims 90 to 116, wherein the form of the target molecule is the post-translational modified form of the target molecule, the wild-type form of the target molecule, or the mutant form of the target molecule.

118. A method for identifying a test molecule that can distinguish between at least two target molecules, (a) Contacting a cell-free sample containing multiple first target molecules with multiple test molecules, (b) Tracking multiple first target molecules over time to obtain multiple spatiotemporal trajectories and / or rotational motion measurements, (c) Analyzing the measured values ​​of the plurality of spatiotemporal trajectories and / or rotational motions to determine the movement of the first target molecule in the presence of the test molecule, (d) Contacting a cell-free sample containing multiple secondary target molecules with multiple test molecules, (e) Tracking multiple second target molecules over time to obtain multiple spatiotemporal trajectories and / or rotational motion measurements, (f) Analyzing the measured values ​​of the plurality of spatiotemporal trajectories and / or rotational motions to determine the movement of the second target molecule in the presence of the test molecule, (g)(c) and (f) compare the movements of the first target molecule and the second target molecule obtained in (g), The method, wherein the change in the movement of the first target molecule and the second target molecule demonstrates that the test molecule can distinguish between the two target molecules.

119. The method according to claim 118, wherein the method is a high-throughput method.

120. The changes in the aforementioned movement are (a) Diffusion coefficient obtained from the maximum likelihood estimator, (b) Geometric mean apost-diffusion coefficient, (c) Median of the jump length distribution, (d) The third quartile of the jump length distribution, (e) Median of turning radius, (f) Average post-dispersion coefficient, (g) Mean square displacement, (h) Median of bond angles, (i) Space-time trajectory length, (j) Anisotropic decay time, (k) Spatial range of detection, (l) Number and wavelength of conjugated fluorescent labels, (m) Occupancy in various diffusion states obtained from the state array, (n) Polarization of conjugated fluorescent label, or (o) The method according to claim 118 or 119, which is calculated as a change in state occupation by inference.

121. The method according to any one of claims 118 to 120, wherein the change in the aforementioned motion is calculated as a change in the diffusion coefficient.

122. The method according to any one of claims 118 to 121, wherein the duration and / or reversibility of the change in the movement of the first target molecule and the second target molecule indicates that the test molecule can distinguish between the two target molecules.

123. The method according to any one of claims 118 to 122, wherein the first target molecule, the second target molecule and / or the test molecule are components of a mixture comprising a bacterial extract, a cell extract, a tissue extract, a plant extract or an animal extract.

124. The method according to claim 123, wherein the bacterial extract, cell extract, tissue extract, plant extract, or animal extract is a lysate.

125. The method according to any one of claims 118 to 124, wherein the first target molecule, the second target molecule and / or the test molecule are components of a mixture comprising serum, blood, and other biological samples.

126. The method according to any one of claims 118 to 125, wherein the first target molecule, the second target molecule and / or the test molecule are components of a mixture comprising a buffer and glycerol.

127. The method according to any one of claims 118 to 126, wherein the first target molecule, the second target molecule and / or the test molecule are organic molecules with a strength of less than 1 kDa.

128. The method according to any one of claims 118 to 127, wherein the first target molecule, the second target molecule and / or the test molecule are selected from the group consisting of peptides, proteins, nucleic acids, carbohydrates and lipids.

129. The method according to claim 128, wherein the protein is selected from the group consisting of antibodies and receptors and enzymes.

130. The method according to claim 128 or 129, wherein the protein comprises a disordered domain and / or does not comprise a structured domain.

131. The method according to claim 128, wherein the peptide is a ligand.

132. The method according to any one of claims 118 to 126, wherein the first target molecule, the second target molecule and / or the test molecule are nanomaterials or synthetic polymers.

133. The method according to any one of claims 118 to 132, wherein the first target molecule, the second target molecule and / or the test molecule are labeled.

134. The method according to any one of claims 118 to 133, wherein the first target molecule, the second target molecule and / or the test molecule are fluorescently labeled.

135. The method according to any one of claims 118 to 134, wherein the first target molecule, the second target molecule and / or the test molecule are labeled with a fluorescent protein.

136. The method according to any one of claims 118 to 135, wherein the first target molecule, the second target molecule and / or the test molecule are labeled by a conjugate of synthetic nanomaterial or polymer.

137. The method according to any one of claims 118 to 136, wherein the first target molecule and the second target molecule are distinguished by differences in their fluorescent labeling.

138. The method according to any one of claims 118 to 137, wherein the first target molecule and the second target molecule are related target molecules.

139. The method according to claim 138, wherein at least one of the target molecules is the wild-type form of the target molecule, at least one of the target molecules is a mutant form of the target molecule, and / or at least one of the target molecules is a post-translational modified form of the target molecule.

140. The method according to any one of claims 118 to 138, wherein the first target molecule and the second target molecule are the same or different conformations of the target molecule, the first target molecule and the second target molecule are homologs, orthologues or paralogs, and / or the first target molecule and the second target molecule are unrelated target molecules.

141. The method according to any one of claims 118 to 140, further comprising: contacting a cell-free sample containing a plurality of third target molecules with a plurality of test molecules; tracking the plurality of third target molecules over time to provide measurements of a third plurality of spatiotemporal trajectories and / or rotational motions; analyzing the measurements of the third plurality of spatiotemporal trajectories and / or rotational motions to determine the motion of the third target molecules in the presence of the test molecules; and comparing the motion of the third target molecules with the motion obtained in (c) and (f), wherein the change in the motion of the third target molecules relative to the first target molecule and the second target molecule indicates that the test molecule can distinguish between the three target molecules.

142. A method for identifying one or more test molecules that induce a conformational change in a target molecule, (a) Contacting a cell-free sample containing multiple target molecules with multiple test molecules, wherein each sample is contacted with a different test molecule, (b) Tracking multiple target molecules in each sample over time to provide measurements of multiple spatiotemporal trajectories and / or rotational motions, (c) Analyzing the measured values ​​of the multiple spatiotemporal trajectories and / or rotational motions to determine the movement of the target molecule in the presence of each of the different test molecules, (d) Comparing the movement of the target molecule obtained in (c) with the movement of a reference target molecule, wherein the movement of the reference target molecule is the movement of the target molecule in the presence of a test molecule that does not induce a conformational change in the target molecule, and the comparison includes: The method wherein the interaction between the target molecule and one or more test molecules that induce a conformational change in the target molecule is demonstrated by a change in the movement of the target molecule compared to the movement of the reference target molecule.

143. The method according to claim 142, wherein the method is a high-throughput method.

144. The changes in the aforementioned movement are (a) Diffusion coefficient obtained from the maximum likelihood estimator, (b) Geometric mean apost-diffusion coefficient, (c) Median of the jump length distribution, (b) The third quartile of the jump length distribution, (c) Median value of turning radius, (d) Average post-dispersion coefficient, (f) Mean square displacement, (g) Median of bond angles, (i) The spatiotemporal trajectory length, (j) Anisotropic decay time, (k) Spatial range of detection, (l) Number and wavelength of conjugated fluorescent labels, (m) Occupancy in various diffusion states obtained from the state array, (n) Polarization of conjugated fluorescent label, or (o) The method according to claim 142 or 143, calculated as a change in state occupation by inference.

145. The method according to any one of claims 142 to 144, wherein the change in the aforementioned motion is calculated as a change in the diffusion coefficient.

146. The method according to any one of claims 142 to 145, wherein the conformational change in the target molecule is related to a change in the temperature stability of the target molecule.

147. The method according to any one of claims 142 to 146, wherein the interaction between the target molecule and the one or more test molecules is reversible or irreversible.

148. The method according to any one of claims 142 to 147, wherein the duration and / or reversibility of the change in the movement of the target molecule, compared to the duration and / or reversibility of the change in the movement of the reference target molecule, indicates an interaction between the target molecule and the one or more test molecules that induce a conformational change in the target molecule.

149. The method according to any one of claims 142 to 148, wherein the target molecule, the reference target molecule and / or the one or more test molecules are components of a mixture comprising a bacterial extract, a cell extract, a tissue extract, a plant extract or an animal extract.

150. The method according to claim 149, wherein the bacterial extract, cell extract, tissue extract, plant extract, or animal extract is a lysate.

151. The method according to any one of claims 142 to 150, wherein the target molecule, the reference target molecule and / or the one or more test molecules are components of a mixture comprising serum, blood, and other biological samples.

152. The method according to any one of claims 142 to 151, wherein the target molecule, the reference target molecule and / or the one or more test molecules are components of a mixture comprising a buffer and glycerol.

153. The method according to any one of claims 142 to 152, wherein the target molecule, the reference target molecule, and / or the one or more test molecules are organic molecules with a kDa of less than 1 kDa.

154. The method according to any one of claims 142 to 153, wherein the target molecule, the reference target molecule and / or the one or more test molecules are selected from the group consisting of peptides, proteins, nucleic acids, carbohydrates and lipids.

155. The method according to claim 154, wherein the protein is selected from the group consisting of antibodies and receptors and enzymes.

156. The method according to claim 154 or 155, wherein the protein comprises a disordered domain and / or does not comprise a structured domain.

157. The method according to claim 154, wherein the peptide is a ligand.

158. The method according to any one of claims 142 to 152, wherein the target molecule, the reference target molecule, and / or the one or more test molecules are nanomaterials or synthetic polymers.

159. The method according to any one of claims 142 to 158, wherein the target molecule, the reference target molecule and / or the one or more test molecules are labeled.

160. The method according to any one of claims 142 to 159, wherein the target molecule, the reference target molecule and / or the one or more test molecules are fluorescently labeled.

161. The method according to any one of claims 142 to 160, wherein the target molecule, the reference target molecule and / or the one or more test molecules are labeled with a fluorescent protein.

162. The method according to any one of claims 142 to 161, wherein the target molecule, the reference target molecule and / or the one or more test molecules are labeled by a conjugate of synthetic nanomaterials or polymers.

163. The method according to any one of claims 142 to 162, wherein the cell-free sample comprises a solution.

164. The method according to claim 163, wherein the solution comprises a chaotropic agent, a viscous agent, or a combination thereof.

165. The method according to claim 164, wherein the viscous agent comprises glycerol.

166. The method according to claim 165, wherein the solution contains at least about 30% glycerol.

167. The method according to any one of claims 163 to 166, wherein the solution comprises a temperature gradient, a chemical gradient, at least two phases, or a combination thereof.

168. The method according to any one of claims 142 to 167, wherein the sample has a volume of about 0.1 μl to about 100 μl.

169. The method according to any one of claims 142 to 168, wherein the change in the motion is measured as a change in the diffusion coefficient of the target molecule in the presence of the test molecule and in the presence of a reference test molecule that does not induce a conformational change, and the change is at least about 0.001%, at least about 0.005%, at least 0.01%, at least about 0.05%, at least about 0.1%, at least about 0.5%, at least about 1%, at least about 1.5%, at least about 2%, or at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10%.

170. A method for determining the dose-response relationship between a target molecule and a test molecule, (a) Contacting multiple cell-free samples containing multiple target molecules with the test molecule, wherein the multiple cell-free samples are contacted with a certain range of test molecule doses, (b) Tracking multiple target molecules over time in the presence of doses of the test molecule within the range described above to provide measurements of multiple spatiotemporal trajectories and / or rotational motions, (c) Analyzing the measured values ​​of the multiple spatiotemporal trajectories and / or rotational motions of the target molecule in the presence of doses of the test molecule within the range, to determine the motion of the target molecule at each dose of the test molecule, (d) The method comprising comparing the movement of the target molecule obtained in step (c) at various test molecule doses to determine the dose response of the target molecule to the test molecule.

171. A method for determining the difference in dose response between two target molecules to a test molecule, (a) Contacting a plurality of first cell-free samples containing a plurality of first target molecules with the test molecule, wherein the plurality of first cell-free samples are contacted with a certain range of test molecule doses, (b) Tracking a plurality of target molecules over time in the presence of doses of the test molecule within the range described above to provide measurements of a first plurality of spatiotemporal trajectories and / or rotational motions, (c) Analyzing the measured values ​​of the multiple spatiotemporal trajectories and / or rotational motions of the target molecule in the presence of doses of the test molecule within the range, to determine the motion of the first target molecule at each concentration of the test molecule, (d) In step (c), the movement of the target molecule obtained at various test molecular doses is compared to determine the dose response of the target molecule to the test molecule, (e) Repeat steps (a) to (d) using the second target molecule to determine the dose response of the second target molecule to the test molecule, (f) The method comprising comparing the dose response of the first target molecule with the dose response of the second target molecule to determine the difference in the responses of the first target molecule and the second target molecule to the test molecule.

172. The method according to claim 170 or 171, wherein the method is a high-throughput method.

173. The aforementioned movement, (a) The diffusion coefficients of the multiple spatiotemporal trajectories obtained from the maximum likelihood estimator, (b) Geometric mean apost-diffusion coefficient of the plurality of spatiotemporal trajectories, (c) The median of the jump length distribution of the plurality of spatiotemporal trajectories, (d) The third quartile of the jump length distribution of the plurality of spatiotemporal trajectories, (e) The median of the turning radii of the plurality of spacetime trajectories, (f) The average post-diffusion coefficient of the plurality of spatiotemporal trajectories, (g) Mean square displacement of the plurality of spacetime trajectories, (h) The median of the combined angle of the plurality of spatiotemporal trajectories, (i) The spatiotemporal trajectory lengths of the plurality of spatiotemporal trajectories, (j) Anisotropic decay time, (k) Spatial range of detection, (l) Number and wavelength of conjugated fluorescent labels, (m) Occupancy in various diffusion states obtained from the state array, (n) Polarization of conjugated fluorescent label, or (o) The method according to any one of claims 170 to 172, which is calculated as state occupancy by inference.

174. The method according to any one of claims 170 to 173, wherein the aforementioned motion is calculated as a diffusion coefficient.

175. The method according to any one of claims 170 to 174, wherein the interaction between the target molecule and the test molecule is reversible or irreversible.

176. The method according to any one of claims 170 to 175, further comprising comparing the movement of the target molecule obtained at various test molecular doses in step (c) to compare the duration and / or reversibility of the change in the movement of the target molecule.

177. The method according to any one of claims 170 to 176, wherein the first target molecule, the second target molecule and / or the test molecule are components of a mixture comprising a bacterial extract, a cell extract, a tissue extract, a plant extract or an animal extract.

178. The method according to claim 177, wherein the bacterial extract, cell extract, tissue extract, plant extract, or animal extract is a lysate.

179. The method according to any one of claims 170 to 178, wherein the first target molecule, the second target molecule and / or the test molecule are components of a mixture comprising serum, blood, and other biological samples.

180. The method according to any one of claims 170 to 179, wherein the first target molecule, the second target molecule and / or the test molecule are components of a mixture comprising a buffer and glycerol.

181. The method according to any one of claims 170 to 180, wherein the first target molecule, the second target molecule and / or the test molecule are organic molecules with a strength of less than 1 kDa.

182. The method according to any one of claims 170 to 181, wherein the first target molecule, the second target molecule and / or the test molecule are selected from the group consisting of peptides, proteins, nucleic acids, carbohydrates and lipids.

183. The method according to claim 182, wherein the protein is selected from the group consisting of antibodies and receptors and enzymes.

184. The method according to claim 182 or 183, wherein the protein comprises a disordered domain and / or does not comprise a structured domain.

185. The method according to claim 182, wherein the peptide is a ligand.

186. The method according to any one of claims 170 to 180, wherein the first target molecule, the second target molecule and / or the test molecule are nanomaterials or synthetic polymers.

187. The method according to any one of claims 170 to 186, wherein the first target molecule, the second target molecule and / or the test molecule are labeled.

188. The method according to any one of claims 170 to 187, wherein the first target molecule, the second target molecule and / or the test molecule are fluorescently labeled.

189. The method according to any one of claims 170 to 188, wherein the first target molecule, the second target molecule and / or the test molecule are labeled with a fluorescent protein.

190. The method according to any one of claims 170 to 189, wherein the first target molecule, the second target molecule and / or the test molecule are labeled by a conjugate of synthetic nanomaterials or polymers.

191. The method according to any one of claims 170 to 190, wherein the first target molecule and the second target molecule are distinguished by differences in their fluorescent labeling.

192. The method according to claim 171, wherein the first target molecule and the second target molecule are related target molecules.

193. The method according to claim 192, wherein at least one of the target molecules is the wild-type form of the target molecule, at least one of the target molecules is a mutant form of the target molecule, and / or at least one of the target molecules is a post-translational modified form of the target molecule.

194. The method according to any one of claims 171 to 192, wherein the first target molecule and the second target molecule are the same or different conformations of the target molecule, the first target molecule and the second target molecule are homologs, orthologues or paralogs, and / or the first target molecule and the second target molecule are unrelated target molecules.

195. The method according to claim 171, wherein the first cell-free sample and the second cell-free sample comprise a solution containing a chaotropic agent, a carrier, a viscous agent, or a combination thereof.

196. The method according to claim 195, wherein the viscous agent comprises glycerol.

197. The method according to claim 196, wherein the solution contains at least about 30% glycerol.

198. The method according to any one of claims 195 to 197, wherein the solution comprises a temperature gradient, a chemical gradient, at least two phases, or a combination thereof.

199. The method according to any one of claims 170 to 198, wherein three or more target molecules are analyzed.

200. A method for identifying a test molecule that can distinguish between at least two target molecules, (a) Contacting a cell-free sample containing multiple first target molecules and multiple second target molecules with multiple test molecules, (b) Tracking multiple first target molecules over time to obtain multiple spatiotemporal trajectories and / or rotational motion measurements, (c) Analyzing the measured values ​​of the plurality of spatiotemporal trajectories and / or rotational motions to determine the movement of the first target molecule in the presence of the test molecule, (d) Tracking multiple second target molecules over time to obtain multiple spatiotemporal trajectories and / or rotational motion measurements, (e) Analyzing the measured values ​​of the plurality of spatiotemporal trajectories and / or rotational motions to determine the movement of the second target molecule in the presence of the test molecule, (f)(c) and (f) include comparing the movements of the first target molecule and the second target molecule obtained in (f), The method, wherein the change in the movement of the first target molecule and the second target molecule demonstrates that the test molecule can distinguish between the two target molecules.

201. The method according to claim 200, wherein the method is a high-throughput method.

202. The changes in the aforementioned movement are (a) Diffusion coefficient obtained from the maximum likelihood estimator, (b) Geometric mean apost-diffusion coefficient, (c) Median of the jump length distribution, (d) The third quartile of the jump length distribution, (e) Median of turning radius, (f) Average post-dispersion coefficient, (g) Mean square displacement, (h) Median of bond angles, (i) Space-time trajectory length, (j) Anisotropic decay time, (k) Spatial range of detection, (l) Number and wavelength of conjugated fluorescent labels, (m) Occupancy in various diffusion states obtained from the state array, (n) Polarization of conjugated fluorescent label, or (o) The method according to claim 200 or 201, calculated as a change in state occupation by inference.

203. The method according to any one of claims 200 to 202, wherein the change in the aforementioned motion is calculated as a change in the diffusion coefficient.

204. The method according to any one of claims 200 to 203, wherein the duration and / or reversibility of the change in the movement of the first molecule, compared to the duration and / or reversibility of the change in the movement of the second molecule, indicates that the test molecule can distinguish between the two target molecules.

205. The method according to any one of claims 200 to 204, wherein the first target molecule, the second target molecule and / or the test molecule are components of a mixture comprising a bacterial extract, a cell extract, a tissue extract, a plant extract, or an animal extract.

206. The method according to claim 205, wherein the bacterial extract, cell extract, tissue extract, plant extract, or animal extract is a lysate.

207. The method according to any one of claims 200 to 206, wherein the first target molecule, the second target molecule and / or the test molecule are components of a mixture comprising serum, blood, and other biological samples.

208. The method according to any one of claims 200 to 207, wherein the first target molecule, the second target molecule and / or the test molecule are components of a mixture comprising a buffer and glycerol.

209. The method according to any one of claims 200 to 208, wherein the first target molecule, the second target molecule and / or the test molecule are organic molecules with a strength of less than 1 kDa.

210. The method according to any one of claims 200 to 209, wherein the first target molecule, the second target molecule and / or the test molecule are selected from the group consisting of peptides, proteins, nucleic acids, carbohydrates and lipids.

211. The method according to claim 210, wherein the protein is selected from the group consisting of antibodies and receptors and enzymes.

212. The method according to claim 210 or 211, wherein the protein includes a disordered domain and / or does not include a structured domain.

213. The method according to claim 210, wherein the peptide is a ligand.

214. The method according to any one of claims 200 to 208, wherein the first target molecule, the second target molecule and / or the test molecule are nanomaterials or synthetic polymers.

215. The method according to any one of claims 200 to 214, wherein the first target molecule, the second target molecule and / or the test molecule are labeled.

216. The method according to any one of claims 200 to 215, wherein the first target molecule, the second target molecule and / or the test molecule are fluorescently labeled.

217. The method according to any one of claims 200 to 216, wherein the first target molecule, the second target molecule and / or the test molecule are labeled with a fluorescent protein.

218. The method according to any one of claims 200 to 217, wherein the first target molecule, the second target molecule and / or the test molecule are labeled by a conjugate of synthetic nanomaterial or polymer.

219. The method according to any one of claims 200 to 218, wherein the first target molecule and the second target molecule are related target molecules.

220. The method according to claim 219, wherein at least one of the target molecules is the wild-type form of the target molecule, at least one of the target molecules is a mutant form of the target molecule, and / or at least one of the target molecules is a post-translational modified form of the target molecule.

221. The method according to any one of claims 200 to 219, wherein the first target molecule and the second target molecule are the same or different conformations of the target molecule, the first target molecule and the second target molecule are homologs, orthologues or paralogs, and / or the first target molecule and the second target molecule are unrelated target molecules.

222. The method according to any one of claims 200 to 221, further comprising: contacting a cell-free sample containing a plurality of third target molecules with a plurality of test molecules; tracking the plurality of third target molecules over time to provide measurements of a third plurality of spatiotemporal trajectories and / or rotational motions; analyzing the measurements of the third plurality of spatiotemporal trajectories and / or rotational motions to determine the motion of the third target molecules in the presence of the test molecules; and comparing the motion of the third target molecules with the motion obtained in (c) and (f), wherein the change in the motion of the third target molecules relative to the first target molecule and the second target molecule indicates that the test molecule can distinguish between the three target molecules.

223. A method for analyzing a test solution containing a target molecule, (a) Tracking multiple target molecules in the test solution over time and providing measurements of multiple spatiotemporal trajectories and / or rotational motions, (b) Analyzing the measured values ​​of the plurality of spatiotemporal trajectories and / or rotational motions to determine the movement of the target molecule in the test solution, (c)(b) Comparing the movement of the target molecule obtained in (c)(b) with the movement of a reference target molecule, wherein the movement of the reference target molecule is the movement of the target molecule in a reference solution, and the comparison is as follows: The comparison in step (c) is the method used to determine the properties of the test solution.

224. The method according to claim 223, wherein the method is a high-throughput method.

225. The movement of the aforementioned target molecule is (a) Diffusion coefficient obtained from the maximum likelihood estimator, (b) Geometric mean apost-diffusion coefficient, (c) Median of the jump length distribution, (d) The third quartile of the jump length distribution, (e) Median of turning radius, (f) Average post-dispersion coefficient, (g) Mean square displacement, (g) Median of bond angles, (i) Space-time trajectory length, (j) Anisotropic decay time, (k) Spatial range of detection, (l) Number and wavelength of conjugated fluorescent labels, (m) Occupancy in various diffusion states obtained from the state array, (n) Polarization of conjugated fluorescent label, or (o) The method according to claim 223 or 224, which is calculated as state occupancy by inference.

226. The method according to any one of claims 223 to 225, wherein the change in the aforementioned motion is calculated as a change in the diffusion coefficient.

227. The method according to any one of claims 223 to 226, wherein comparing the movement of the target molecule obtained in (b) with the movement of a reference target molecule further comprises comparing the duration and / or reversibility of the change in the movement of the target molecule with the duration of the change in the movement of the reference target molecule.

228. The method according to any one of claims 223 to 227, wherein the target molecule and / or the reference target molecule is a component of a mixture comprising a bacterial extract, a cell extract, a tissue extract, a plant extract, or an animal extract.

229. The method according to claim 228, wherein the bacterial extract, cell extract, tissue extract, plant extract, or animal extract is a lysate.

230. The method according to any one of claims 223 to 229, wherein the target molecule and / or the reference target molecule is a component of a mixture comprising serum, blood, and other biological samples.

231. The method according to any one of claims 223 to 230, wherein the target molecule and / or the reference target molecule is a component of a mixture comprising a buffer and glycerol.

232. The method according to any one of claims 223 to 231, wherein the target molecule and / or the reference target molecule is an organic molecule with a strength of less than 1 kDa.

233. The method according to any one of claims 223 to 232, wherein the target molecule and / or the reference target molecule are selected from the group consisting of peptides, proteins, nucleic acids, carbohydrates, and lipids.

234. The method according to claim 233, wherein the protein is selected from the group consisting of antibodies and receptors and enzymes.

235. The method according to claim 233 or 234, wherein the protein includes a disordered domain and / or does not include a structured domain.

236. The method according to claim 233, wherein the peptide is a ligand.

237. The method according to any one of claims 223 to 231, wherein the target molecule and / or the reference target molecule is a nanomaterial or a synthetic polymer.

238. The method according to any one of claims 223 to 237, wherein the target molecule and / or the reference target molecule are labeled.

239. The method according to any one of claims 223 to 238, wherein the target molecule and / or the reference target molecule are fluorescently labeled.

240. The method according to any one of claims 223 to 239, wherein the test solution comprises a chaotropic agent and / or a viscous agent.

241. The method according to claim 240, wherein the chaotropic agent is urea.

242. The method according to claim 240 or 241, wherein the viscous agent is glycerol.

243. The method according to any one of claims 223 to 242, wherein the test solution includes a gradient.

244. The method according to claim 243, wherein the gradient is a temperature gradient, a chemical gradient, or a combination thereof.

245. The method according to any one of claims 223 to 244, wherein the test solution comprises at least two phases, or a combination thereof.

246. The method according to any one of claims 223 to 245, wherein the interaction between the target protein molecule and the test solution causes a conformational change in the target molecule.

247. The method according to any one of claims 223 to 246, wherein the interaction between the target molecule and the test solution is reversible or irreversible.

248. The method according to any one of claims 223 to 247, wherein the characteristic of the test solution is pH, ion concentration, organic molecule concentration, or viscoelastic properties.

249. The method according to claim 248, wherein the viscoelastic properties are viscous.

250. The method according to any one of claims 223 to 249, wherein the test solution is a biological sample taken from the subject.

251. The method according to any one of claims 223 to 250, wherein the properties of the test solution are used to diagnose a disease in the subject.

252. The method according to claim 251, wherein the disease is cancer.

253. The method according to any one of claims 1 to 252, wherein the change in the motion is calculated as a change in the polarization of the conjugated fluorescent label, and determining the change in the polarization of the conjugated fluorescent label includes time-correlated single-photon counting.

254. The method according to any one of claims 1 to 253, wherein the change in the motion is calculated as a change in the polarization of the conjugated fluorescent label, and determining the change in the polarization of the conjugated fluorescent label includes step-scan pump-probe spectroscopic microscopy.

255. The method according to any one of claims 1 to 254, wherein the target molecule and / or the test molecule is labeled with an ultra-long-lived fluorescent dye.

256. The method according to any one of claims 1 to 255, wherein the change in the motion is calculated as a change in the polarization of the conjugate fluorescent label, and determining the change in the polarization of the conjugate fluorescent label involves advanced optical techniques.

257. The method according to any one of claims 1 to 256, wherein the change in the motion is calculated as a change in the polarization of the conjugate fluorescent label, and determining the change in the polarization of the conjugate fluorescent label includes wobble anisotropy and / or polarization filtering.

258. A system for carrying out the method described in any one of claims 1 to 257.

259. The system according to claim 258, comprising a microfluidic device.

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