Method and apparatus for measuring the phase transition properties of polymers

Abstract

A method for measuring phase transition properties of a polymer is provided, the method comprising generating a stream of microdroplets comprising at least one component, one of the at least one component comprising a polymer, varying conditions within the microdroplets, and measuring the relative concentrations of the components of the microdroplets and the phase of the polymer present in the microdroplets.

Description

[[Technical Field]]

[0001] The project leading to the present application has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 841466.

[0002] The present invention relates to a method and an apparatus for measuring phase transition properties of polymers. Specific embodiments of the present invention relate to liquid-liquid phase separation (LLPS). [[Background Art]]

[0003] Liquid-liquid phase separation (LLPS), which is the spontaneous demixing from a polymer solution (e.g., a polymer solution of polymers such as proteins, peptides, and nucleic acids) into a mixed state of a concentrated phase and a dilute phase, has become the subject of intense interest as new important insights into the role of this process in the regulation of biological functions have recently been achieved. For example, phase-separated protein (e.g., biomolecular protein) condensates act as microreactors by discretely organizing cellular space and localizing biomolecules. Condensates are essential for a variety of fundamental biochemical processes, including the regulation of transcription, the regulation of translation, and the regulation of cellular stress responses. These condensates are also heavily implicated in protein misfolding diseases, including the pathogenesis of motor neuron diseases and cancer, making them attractive targets for therapeutic intervention.

[0004] Phase separation, which has currently been demonstrated for hundreds of cellular proteins and other biological macromolecules (including nucleic acids and peptides), can be triggered by slight changes in environmental conditions, such as variations in ionic strength, pH, temperature, molecular crowding, and the presence or absence of small molecules. Owing to this sensitivity to physicochemical parameters, many phase separation systems are currently gaining strong interest as fundamentally novel drug targets for ameliorating human diseases. However, at present, the demand for quantification of the physical parameters that regulate phase separation behavior in condensed systems remains unmet.

[0005] The fundamental measure for describing the thermodynamics of condensed matter is the phase diagram, which is generated by systematically analyzing whether or not LLPS (Low-Liquid Phase Separation) is occurring as a function of polymer concentration and polymer solution conditions. The phase diagram clearly summarizes the phase behavior of an LLPS system by identifying the position of the phase boundary in chemical space and elucidating the position of chemical equilibrium between LLPS and homogeneous morphology. By changing the solution conditions within the LLPS system, the phase boundary can be directly altered, resulting in insights into the thermodynamic processes that promote biomolecular condensation. Therefore, generating a phase diagram is an important step in understanding the phase separation behavior of polymers.

[0006] However, given the diverse range of proteins that undergo LLPS and the environmental conditions that regulate their behavior, there is an urgent need for experimental methods that can rapidly and with high resolution characterize LLPS phase diagrams. Typically, these methods are produced by uneconomical and laborious processes that involve stepwise coupling of reagents to induce essential changes in solution conditions and observation under individual conditions using a microscope. Microfluidic technology is now established as an effective means of improving assay throughput, parallelization, and miniaturization in biochemical experiments, and new applications are being found in quantifying the phase behavior of biomolecules on chips. However, techniques for rapidly and high-throughput generation of LLPS phase diagrams have yet to be demonstrated.

[0007] Known methods for assaying the phase behavior of LLPS systems in the presence of drug candidates involve performing reactions under different conditions using multiwell plates, either manually or with the help of robotics. However, these methods are excessively slow. The best automated / robot-assisted multiwell plate assays are limited to fewer than 100,000 conditions per day. [Overview of the project]

[0008] The present invention aims to solve the above-mentioned problems at least partially. For example, embodiments of the present invention can achieve data acquisition much faster than current technologies in terms of phase separation, high-resolution data, a significant reduction in reagent consumption, and directed feedback from real-time data acquisition.

[0009] According to one aspect of the present invention, a method for measuring the phase transition properties of a polymer is provided, which comprises generating a flow of microdroplets containing at least one component, wherein one of the at least one component contains a polymer; changing the conditions within the microdroplets; and measuring the relative concentration of the components of the microdroplets and the phase of the polymer present in the microdroplets.

[0010] Optionally, the conditions within the microdroplets are altered by changing the relative concentrations of the polymer-containing components and at least one further component within the microdroplets.

[0011] Alternatively, or additionally, the conditions within the microdroplet can be altered by changing the microdroplet's temperature. Optionally, the microdroplet's temperature can be altered by controlling the temperature of the channel through which the microdroplet flows.

[0012] According to one aspect of the present invention, a method for measuring the phase transition properties of a polymer is provided, which comprises generating a stream of microdroplets containing at least two components, wherein one of the at least two components contains a polymer, and the microdroplets contain the same component at different relative concentrations; and measuring the relative concentrations of the components in the microdroplets and the phase of the polymer present in the microdroplets.

[0013] Optionally, the flow of microdroplets is continuous.

[0014] Optionally, the measurement is performed continuously with respect to the flow of microdroplets.

[0015] Microdroplets are selected at will, and measurements are performed on the selected microdroplets.

[0016] Optionally, a flow of microdroplets is generated by injecting a flow of a first fluid containing at least two components into a flow of a second fluid, the second fluid being immiscible with the first fluid.

[0017] Optionally, the flows of at least two constituent components within the microdroplet merge to form a first fluid flow.

[0018] Optionally, the relative concentrations of the components of a microdroplet are changed by varying the relative flow rates of each of at least two components of the microdroplet.

[0019] These flows are selectively routed through the channels of a microfluidic system.

[0020] Optionally, the relative concentrations of the components of the microdroplets are measured by a first optical means. Optionally, the first optical means illuminates the microdroplets with illumination light and detects the response. Optionally, the relative concentrations of the components of the microdroplets are determined based on the response of each component to the illumination light. Optionally, each component exhibits a different response to the illumination light. Optionally, each component contains a separate fluorescent dye molecule that emits light of a specific wavelength in response to the illumination light.

[0021] Optionally, the polymer phase present in the microdroplet is measured by a second optical means. Optionally, an image of the microdroplet is acquired by the second optical means, and the polymer phase present in the microdroplet is determined based on the characteristics of the image indicating a specific phase. Alternatively, a light scattering profile of the microdroplet is acquired by the second optical means, and the polymer phase present in the microdroplet is determined based on the characteristics of the light scattering profile indicating a specific phase.

[0022] Optionally, the relative concentrations of the components of the microdroplet are varied based on the measured relative concentrations of the components of the microdroplet and the measured phase of the polymer present in the microdroplet.

[0023] Optionally, the relative concentrations of the components of the microdroplet are systematically varied to produce microdroplets having conditions that are, or approximately approximate, conditions at which the polymer transitions from a first phase to a second phase.

[0024] Optionally, the method further comprises analyzing the measurements to identify the point at which the polymer transitions from the first phase to the second phase.

[0025] Optionally, the polymer comprises one or more of a protein or a nucleic acid. Optionally, the at least two components further comprise one or more of a pH buffer, a phase separator, saline, or a therapeutic agent / candidate agent. Optionally, the therapeutic agent / candidate agent is a small molecule drug or a biologic.

[0026] According to a second aspect of the present invention, there is provided a method of screening candidate therapeutic agents, the method comprising the steps of the method according to any preceding aspect, wherein at least one component other than the polymer in the microdroplet comprises a candidate agent. Optionally, candidate therapeutic agents that are unable to alter phase transition properties in a predefined desired manner are discarded.

[0027] According to a third aspect of the present invention, there is provided an apparatus for measuring phase transition properties of a polymer, the apparatus comprising: a microfluidic system configured to generate a flow of microdroplets comprising at least two components, wherein one of the at least two components comprises the polymer, and the microfluidic system is configured to vary the relative concentration of the components; a first optical system configured to measure the relative concentration of the components of the microdroplets generated by the microfluidic system; and a second optical system configured to measure the phase of the polymer present in the microdroplets generated by the microfluidic system.

[0028] Optionally, the first optical system and the second optical system are configured to continuously measure the flow of microdroplets. Optionally, the first optical system and the second optical system are arranged adjacent to a measurement region of the microfluidic system.

[0029] Optionally, the microfluidic system comprises: at least two inlets configured to inject a respective component flow of at least two components; a first flow channel configured to allow a first fluid flow to pass therethrough, wherein the first fluid comprises the at least two components coming from the at least two inlets; a second flow channel configured to allow a second fluid flow to pass therethrough, wherein the second fluid is immiscible with the first fluid; and the first flow channel comprises a nozzle that opens into the second flow channel and is configured to inject the first fluid flow into the second fluid flow to generate microdroplets made of the first fluid within the second fluid.

[0030] Optionally, the device further comprises at least two pumps corresponding to the at least two inlets, and the at least two pumps are configured to change the relative flow rates of the respective component flows so as to change the relative concentrations of the at least two components in the generated microdroplets.

[0031] Optionally, the device further comprises a controller configured to control the pump to change the relative concentration of the components of the microdroplets based on the measured relative concentration of the components of the microdroplets and the measured phase of a polymer present in the microdroplets. Optionally, the controller is configured to control the pump to systematically change the relative concentration of the components of the microdroplets, so as to generate microdroplets having conditions at which the polymer transitions from the first phase to the second phase, or conditions that approximately approximate such conditions.

[0032] Optionally, the first optical means includes a light source configured to illuminate a microdroplet with illumination light, and a detector configured to detect the response of the microdroplet to the illumination light. Optionally, the light source includes a plurality of light-emitting units, each configured to emit light of a different wavelength. Optionally, the detector includes a plurality of photodetectors, each configured to detect light of a different wavelength. Optionally, the apparatus further includes a processor configured to determine the relative concentrations of the components of the microdroplet based on the response of two or more components to the illumination light.

[0033] Optionally, the second optical means includes an image sensor configured to acquire an image of a microdroplet. Optionally, the apparatus further includes a processor configured to determine the phase of a polymer present in the microdroplet based on the characteristics of the acquired image indicating a particular phase. Alternatively, the second optical means includes a light source configured to illuminate a microdroplet and a detector configured to acquire a light scattering profile of the light from the light source scattered by the microdroplet. Optionally, the apparatus further includes a processor configured to determine the phase of a polymer present in the microdroplet based on the characteristics of the light scattering profile indicating a particular phase.

[0034] In any of the prior embodiments, the polymer-containing component optionally includes one or more of the polymer itself, cells, intracellular organelles, and cell lysates.

[0035] In any of the prior embodiments, the phase transition properties of two or more polymers are optionally measured simultaneously, wherein at least one component comprises a further polymer. [Brief explanation of the drawing]

[0036] [Figure 1] An exemplary system according to the present invention is schematically shown. [Figure 2] An exemplary measurement system of the apparatus according to the present invention is schematically shown. [Figure 3]An example of a phase diagram showing the liquid-liquid phase transition as a function of salt concentration and protein concentration is shown. [Figure 4] An exemplary system according to the present invention is schematically shown. [Figure 5] The images show epifluorescence microscope images of droplets containing FUS-GFP (left) and 1,6-hexanediol at various concentrations, with the concentration of the latter indicated by fluorescence from co-encapsulated ALEXA647 (right). [Figure 6] The phase diagram of FUS-GFP as a function of protein concentration and 1,6-hexanediol concentration is shown, with dots representing the solution conditions measured for individual microdroplets (N=322), and the presence or absence of phase separation indicated by white or black circles, respectively, while the dashed lines are identification guides indicating the approximate location of the LLPS phase boundary. [Figure 7] The plots show the flow rates of the buffer solution, 10% by volume of 1,6-hexanediol, and 23 µm of FUS solution. [Figure 8] Figure 7 shows plots of 1,6-hexanediol and FUS concentrations from the flow rate profiles. [Figure 9] An exemplary system according to the present invention is schematically shown. [Figure 10] The plot shows the fluctuating flow rate. [Figure 11] This shows the formation of microdroplets. [Figure 12] The images show fluorescence diagrams illustrating the different constituent components (left, center) and droplets depending on whether or not phase separation is present. [Figure 13] This shows the boundaries of condensation over time. [Figure 14] The phase diagram obtained using the system shown in Figure 9 is shown. [Modes for carrying out the invention]

[0037] Further features of the present invention will be described below, as non-limiting examples, with reference to the accompanying drawings. To measure the phase transition properties of polymers according to the present invention, a flow of microdroplets is required. This flow is preferably substantially continuous, but may be intermittent or in other forms. The microdroplets form a dispersed phase within a fluid that forms a continuous phase (the term "phase" here is used in the context of microfluidic systems and should not be confused with the term "phase" in the context of polymer phase transitions). Microdroplets generally refer to droplets with a diameter of less than 1 mm, and also include droplets with dimensions on the micrometer scale, and droplets with a diameter of less than 1 micrometer, i.e., nanodroplets.

[0038] Microdroplets must contain at least two components, one of which should be a polymer. Preferably, the polymer is a polymer, and more preferably, it contains one or more proteins or nucleic acids. In some embodiments, the polymer may be part of a cell, an intracellular organelle (e.g., nucleus or mitochondria), or a cell lysate, and the components may include a cell, an intracellular organelle, or a cell lysate. Further components may include one or more of a pH buffer, a phase separator, saline solution, a cell, a cell lysate, or a therapeutic agent / candidate drug. As will be further described below, some or all of the components (e.g., components whose relative concentration is measured, including a polymer) may additionally contain an optical marker (also referred to as a "barcode") (for the components described above). The pH buffer may not be optically characterized. The phase separator may contain a flocculant that induces phase separation (biogenic and non-biogenic polymers, e.g., PEG and dextran), a protein, a nucleic acid, various salts, or a low molecular weight. The therapeutic agent / candidate drug may include, but is not limited to, proteins, nucleic acids, lipids, peptides, or antibodies, and may also be a small molecule formulation or a biologic.

[0039] Furthermore, the microdroplets must contain the same components at different relative concentrations. To achieve this, the microfluidic system 1 shown in Figure 1 may be used.

[0040] The exemplary microfluidic system 1 shown in Figure 1 comprises two main parts: a generation unit 2 and an incubation unit 3. Microdroplets containing the same components at different relative concentrations, i.e., different reaction conditions, are generated in the generation unit 2. The microdroplets flow through the incubation unit 3 for a predetermined time, causing the components to react and, in some cases, polymers to condense.

[0041] In the exemplary microfluidic system 1 shown in Figure 1, the generation unit 2 includes three component inlets 21A, 21B, and 21C configured to inject a flow of each component of a microdroplet. In this embodiment, there are three components: a first component containing a protein (polymer), a second component containing a drug candidate, and a third component containing a phase separator. In this embodiment, each of these components is optically characterized with a different marker. In this embodiment, each of these components is mixed with a pH buffer injected through buffer inlets 22A, 22B, and 22C. However, this mixing is optional. In this embodiment, the pH buffer is not optically characterized. In alternative embodiments, different components and / or different numbers of components may be used.

[0042] Although not shown in the figure, the generation unit 2 may further include pumps corresponding to the component inlets 21A, 21B, and 21C. These pumps may be configured to change the relative flow rate of each component, and therefore the relative concentration of the components in the microdroplets. It should be understood that these pumps must be of a type suitable for use in a microfluidic system, such as a micropump. By using a pressure-controlled fluid flow, the conditions of the microdroplets can be changed on a millisecond timescale.

[0043] As shown in Figure 1, the components flow through subchannels 23A, 23B, and 23C, which communicate with component inlets 21A, 21B, and 21C. These subchannels 23A, 23B, and 23C converge into a first channel 24. Thus, the first channel 24 carries a flow of a first fluid containing all the components from inlets 21A, 21B, and 21C. It should be understood that all channels referred to herein, unless otherwise specified, are microchannels, i.e., channels having at least one dimension less than 1 mm. Naturally, alternative configurations may be used to mix the components.

[0044] In the exemplary microfluidic system 1 shown in Figure 1, the generating unit 2 further includes a continuous phase inlet 25 configured to inject a flow of a second fluid (the first fluid containing all its components). The second fluid forms a continuous phase in which microdroplets are dispersed. The second fluid may be immiscible with the first fluid. For example, the first fluid may be an aqueous solution, and the second fluid may be an oil such as fluorinated oil. The second fluid may optionally contain a surfactant.

[0045] As shown in Figure 1, the continuous phase inlet 24 communicates with a second flow path 26 configured for the flow of the second fluid. In Figure 1, the second flow path 26 is loop-shaped, and the second fluid flows from the continuous phase inlet 25 in two opposing directions, but the second flow path 26 may have a different configuration. For example, the second flow path 26 may be linear (i.e., the second fluid may be restricted to flow along a single path, but that path is not necessarily linear). Naturally, alternative configurations may be used instead.

[0046] As shown in Figure 1, the first flow path 24 includes a nozzle 27 that opens into the second flow path 26. The nozzle 27 is configured to inject the flow of the first fluid into the flow of the second fluid and to generate microdroplets of the first fluid (dispersed phase) within the second fluid (continuous phase). Of course, alternative configurations may be used to mix the first and second fluids to form microdroplets.

[0047] As shown in Figure 1, the second flow path 26 includes an opening 28. As shown, this opening may face the nozzle 27. Alternatively, other configurations may be used. The opening 28 communicates with the incubation section 3 of the microfluidic system 1 and is configured to allow microdroplets to flow from the generation section 2 into the incubation section 3.

[0048] As shown in Figure 1, the incubation section 3 includes an inlet 31 communicating with the opening 28 of the second channel, a third channel 32, and an outlet 33. The outlet 33 is configured to discharge the first and second fluids from the microfluidic system 1. The measurement area 34 of the third channel 32 is located upstream of the outlet 33. The measurement area 34 is where the microdroplets are analyzed. For reasons that will become apparent below, at least the measurement area 34 of the microfluidic system 1 may be transparent.

[0049] The third channel 32 is configured to have a predetermined length from the inlet 31 to the measurement area 34. This predetermined length determines the incubation time of the microdroplets before any measurement is performed, given a given flow rate of microdroplets passing through the third channel 32. The incubation time may be, for example, 10 MS to 10 minutes, and preferably 1 to 20 seconds. To minimize the dimensions of this microfluidic system, the third channel 32 may have a meandering shape as shown in Figure 1. Of course, alternative configurations may be used instead.

[0050] As shown in Figure 1, the third channel 31 may be linear (in that the microdroplets are restricted to flow along a single path, but that path is not necessarily physically linear). Naturally, alternative configurations may be used. For example, the third channel 31 may be divided into any number of parallel channels (in that the microdroplets flow simultaneously through each channel like a parallel electrical circuit, but these channels are not necessarily physically parallel to one another). Each parallel channel may contain its own measurement area, which allows multiple sets of measurements to be performed in parallel, resulting in further improved throughput. In this embodiment, the parallel channels may converge upstream of the outlet 33, or they may remain divided, each having its own corresponding outlet.

[0051] The system may further include means for controlling the temperature in the third channel 32. For example, the incubation section may include a heater such as a heat block, or an incubation chamber for the microfluidic system 1. In some exemplary systems, temperature may be the only variable; that is, the temperature may fluctuate, but the relative concentrations of the components of the microdroplets may not. Therefore, the behavior of polymers at different temperatures can be measured.

[0052] To measure the phase transition properties of the polymer according to the present invention, it is necessary to measure the relative concentrations of the constituent components of the microdroplets. For example, imaging may be performed using standard epifluorescence microscopy techniques, for example, by (I) capturing droplets on a chip, regardless of whether the apparatus is the same as or different from the apparatus used for droplet generation, or by (II) imaging droplets outside the microfluidic environment, such as in an array or reservoir, such as a multiwell plate or coverslide. On the other hand, it is preferable that the measurement be performed continuously with respect to the flow of microdroplets. To achieve this, the measurement system 4 shown in Figure 2 may be used.

[0053] In the exemplary measurement system 4 shown in Figure 2, the relative concentrations of the components of the microdroplets are measured by a first optical system 5. The use of optical means can increase the detection speed, thereby promoting higher throughput. The measurement system shown in Figure 2 is configured for use in a microfluidic system where some or all of the components of the microdroplets may contain separate optical markers, such as fluorescent dye molecules. The fluorescent dye molecules may include low-molecular-weight fluorescent dyes, high-molecular-weight fluorescent proteins such as GFP and RFP, or fluorescent particles such as quantum dots. Each fluorescent dye molecule may be configured to emit light of a specific wavelength when illuminated. As shown in Figure 2, the first optical system 5 includes a light source 51 and a detector 52.

[0054] The light source 51 is configured to illuminate microdroplets with illumination light. As shown in the figure, the light source 51 includes a plurality (in this case, three) light-emitting units 51A, 51B, and 51C. Each of the light-emitting units 51A, 51B, and 51C is configured to emit light of a different wavelength. Each of the light-emitting units 51A, 51B, and 51C may be configured to emit light having substantially a single wavelength, such as laser light. The light-emitting units 51A, 51B, and 51C may include, for example, LEDs.

[0055] The wavelengths of the light-emitting sections 51A, 51B, and 51C may correspond to the wavelengths absorbed by the respective fluorescent dye molecules coupled to the optically characterized components in the microdroplets. These wavelengths may correspond to, for example, red, green, and blue visible light. Alternatively, any wavelength may be used. The emitted light from the light-emitting sections 51A, 51B, and 51C may be guided using appropriate optical elements such as lenses, mirrors, or optical fibers.

[0056] The detector 52 is configured to detect the response of microdroplets to illumination light. As shown in the figure, the detector 52 may include a plurality of (in this case, three) photodetectors 52A, 52B, 52C corresponding to a plurality of light-emitting units 51A, 51B, 51C. The photodetectors 52A, 52B, 52C may include photodiodes. The photodetectors 52A, 52B, 52C may optionally include one or more filters for separating the received light into light of different wavelengths for detection. The wavelengths detected by the photodetectors 52A, 52B, 52C may correspond to the wavelengths emitted by fluorescent dye molecules bound to each component of the microdroplet. The received light may be guided to the photodetectors 52A, 52B, 52C using appropriate optical elements such as lenses, mirrors, or optical fibers.

[0057] The relative concentrations of the components in a microdroplet can be determined based on the detection response of each component to illumination light. Assuming proper calibration, the relative concentrations can be determined based on the relative signals output by the photodetectors 52A, 52B, and 52C, as shown in the example in Figure 1. For example, some processing of raw data by a processor may be necessary to determine the relative concentrations of the components in the microdroplet.

[0058] To measure the phase transition characteristics of the polymer according to the present invention, it is necessary to measure the phase of the polymer present in the microdroplet. To achieve this, the measurement system 4 shown in Figure 2 may be used.

[0059] In the exemplary measurement system 4 shown in Figure 2, the polymer phase present in the microdroplet is measured by a second optical system 6. The use of optical means can facilitate the achievement of high throughput. As shown in Figure 2, the second optical system 6 includes an image sensor 61 configured to acquire an image of the microdroplet. This acquisition may include acquiring a fluorescence image and / or a bright-field image. The image sensor 61 may include, for example, a high-speed camera. In the embodiment shown in Figure 2, the polymer phase present in the microdroplet is determined based on the characteristics of the acquired image that show a specific phase. This image processing may be performed by a processor (for example, the same processor described above with respect to measuring the relative concentrations of the components of the microdroplet). This image processing may include a pattern recognition algorithm and / or a machine learning algorithm.

[0060] In alternative embodiments not shown in the figures, the second optical system includes a light source configured to illuminate the microdroplets and a detector configured to acquire a light scattering profile of the light from the light source scattered by the microdroplets. For example, the second optical system may be an interference scattering microscope system. The polymer phase present in the microdroplets may be determined based on the characteristics of the light scattering profile that indicate a particular phase. The image processing may be performed by a processor (e.g., the same processor described above with respect to measuring the relative concentrations of the components of the microdroplets). The image processing may include pattern recognition algorithms and / or machine learning algorithms.

[0061] The data obtained by the above measurements, namely the relative concentrations of the constituent components and the present polymer phases for each microdroplet being analyzed, may be stored in a memory device, such as RAM. This data may be stored in a tabular format, where rows represent each analyzed droplet and columns represent the concentrations of the constituent components and the presence or absence of the polymer phase.

[0062] By analyzing the acquired data, the phase boundaries of polymers in chemical space can be identified. That is, the chemical conditions (relative concentrations of constituent components) under which a polymer transitions from a first phase to a second phase can be determined. Phase boundaries can be identified by any suitable mathematical method, many of which are well known in the art. For example, a regression curve can be mathematically determined based on the data, and the regression is weighted towards data points that are closely adjacent in different phases.

[0063] To acquire data points in the chemical space, the relative concentrations of the components of the microdroplets may be systematically changed. This change may be predetermined according to a specific path in the chemical space, or it may be dynamic based on real-time data, as further described below.

[0064] In one specific example of a predetermined change in the relative concentrations of the components of a microdroplet, the concentrations of the components may vary periodically between a minimum and a maximum concentration. Furthermore, the concentrations of at least two different components may vary periodically at different periods. This period can be measured by the time or number of microdroplets, i.e., the data points. This period may remain constant or may vary between cycles. The minimum and maximum values ​​may also vary between cycles. These variations can be achieved by pre-programming a flow control system that controls the fluid flow into the microfluidic system (e.g., a syringe driver or pressure control system). The control system may be programmed directly by the device or controlled by an external computer. The system may be controlled to periodically change the relative flow rates of the components while keeping the total flow rate constant. This ensures uniform droplet formation.

[0065] Therefore, as shown in Figures 7 and 8, the surface or volume in the chemical space can be investigated in detail. As shown in Figures 7 and 8, data points in the chemical space are generated with short periods relative to a first axis in the chemical space (e.g., the concentration of a first component) and with long periods relative to a second axis in the chemical space (e.g., the concentration of a second component), and as a result, the two-dimensional surface in the chemical space can be investigated in detail. The concentrations of some different components can be changed within the same period. Therefore, the relative concentrations of these different components can be kept constant. For example, as shown in Figure 7, the relative concentration of the buffer to the diol remains constant.

[0066] Using the identified phase boundaries, a phase diagram such as the one shown in Figure 3 may be generated. In Figure 3, the upper left portion represents the homogeneous liquid phase, and the lower right portion represents the condensed liquid phase. Figure 3 further illustrates how the phase boundaries may be displaced (in the direction of the arrows) due to the presence of the agent.

[0067] Data processing may be performed by a processor. This processor may be the same processor described above for measuring the relative concentrations of the components of the microdroplets and for measuring the phases present in the microdroplets.

[0068] All of the above processes may be performed in real time, i.e., as the microdroplets are generated and then measured. Therefore, the relative concentrations of the components of the microdroplets may change based on the measured relative concentrations of the components of the microdroplets and the measured phase of the polymer present in the microdroplets. Such a feedback loop is shown in Figure 1.

[0069] In a specific embodiment, the method may be initiated by changing the relative concentrations of the components of microdroplets in a predetermined manner along a predetermined path in a chemical space, as described above, for example in relation to Figures 7 and 8. Once data is acquired and analyzed, the path in the chemical space may be modified based on the analytical data to converge substantially to a specified phase boundary. That is, the relative concentrations of the components of the microdroplets may be systematically changed to generate microdroplets having conditions for the polymer to transition from a first phase to a second phase, or conditions substantially approximating this. In this way, the most useful data points, i.e., data points closest to the phase boundary, can be acquired more efficiently. This further promotes improved throughput. This can be achieved by adjusting the minimum and / or maximum concentrations, and / or the period of the periodically changing concentrations. For example, to investigate a particular region of the chemical space in more detail, the concentration range defined by the minimum and maximum concentrations may be reduced, and / or the cycle period may be extended.

[0070] The microfluidic system 1, the first optical system 5, and the second optical system 6 may be components of a single device. The device may further include a controller for controlling the microfluidic system 1. The device may further include a storage device for storing measurement data and processing means necessary for acquiring the measurement data. The device may further include processing means for processing the measurement data to identify phase boundaries and / or for controlling the microfluidic system based on the measurement data, as described above. For example, the device may include a microfluidic chip forming the microfluidic system 1, and / or appropriate electronic and optical components forming the first optical system 5 and the second optical system 6, and / or integrated circuits forming the processing means and control means. Thus, the device may be a lab-on-a-chip type. In some embodiments, the processing means and / or control means may be provided by a separate device such as a computer.

[0071] The following describes an exemplary system for characterizing the phase behavior of FUS proteins, which significantly improves assay throughput and reduces sample consumption compared to conventional experiments. The method described can generally be applied to characterizing the liquid-liquid phase separation behavior of proteins.

[0072] This method utilizes droplet microfluidics to rapidly generate numerous microdroplets, each of which can be considered a separate microenvironment for investigating protein phase separation. By changing the injection solution conditions, a wide range of phase separation environments can be rapidly generated to map LLPS behavior across a broad chemical space. The acquisition of a phase diagram (Figure 6) of protein FUS, a central protein in the pathogenesis of amyotrophic lateral sclerosis, is demonstrated. Using the microfluidic platform, it is determined that the phase boundary between phase-separated homogeneous FUS solutions is regulated by the low-molecular-weight 1,6-hexanediol, which is known to strongly interfere with LLPS behavior.

[0073] Figure 4 shows an example of a microfluidic droplet generation unit 102 for microencapsulating FUS-GFP (GFP-labeled FUS) under various solution conditions. The generation unit 102 functions by mixing a water-soluble protein mixture with a continuous phase of immiscible fluorinated oil containing a surfactant to prevent droplet coalescence at the T-binding unit 127. Prior to the binding unit 127, buffer, protein, and 1,6-hexanediol solutions are mixed in different ratios to define the concentration ranges of protein and 1,6-hexanediol that will be investigated in detail by the microfluidic platform. The flow rate control and mixing ratios for this example are shown in Figures 7 and 8.

[0074] The dye ALEXA647 (10 µm) was mixed with a 1,6-hexanediol solution to impart a fluorescent marker indicating the droplet concentration of 1,6-hexanediol. A pre-programmed syringe pump 107 was used to control the injection flow rate of the water-soluble droplet component, enabling automated sampling of the chemical space. Laminar flow prevented mixing of the assay components before encapsulation, before rapid mixing occurred after droplet formation. The droplets were then collected off-tip under a mineral oil layer to prevent evaporation. The samples were held for 3 minutes before fluorescence imaging analysis.

[0075] FUS present in each droplet G156E The concentrations of EGFP and 1,6-hexanediol were determined by integrating volume-normalized EGFP (green fluorescent protein) and ALEXA647 fluorescence, respectively. Phase separation was observed due to discrete spots in the GFP fluorescence, while the uniform fluorescence of the droplets indicated that phase separation did not occur (Figure 5). FUS G156E -FUS is a method that combines the measured concentrations of EGFP and 1,6-hexanediol droplets with the presence or absence of phase separation. G156E A phase diagram for the -EGFP / 1,6-hexanediol system was generated (Figure 6).

[0076] 1,6-Hexanediol is known to strongly inhibit protein condensate formation, and as expected, phase separation was only observable at low concentrations (less than 1% by volume) of 1,6-hexanediol, while FUS-GFP showed a homogeneous phase at higher diol concentrations. Furthermore, a positive gradient was observed at the phase boundary, indicating that LLPS tends to occur more frequently at higher FUS-GFP concentrations in that region of the phase space.

[0077] Using this method, the location of the LLPS phase boundary was estimated by generating more than 300 independent measurements of FUS-GFP behavior within 5 minutes (3 minutes for droplet generation and 2 minutes for imaging). This assay processing volume is significantly higher than that achievable by manual experiments, and the subsequent increase in the number of data points may improve the fit to the location of the protein phase boundary.

[0078] Figure 9 shows an example of a microfluidic droplet generation unit 202 for microencapsulating FUS-EGFP (EGFP-labeled FUS) under various solution conditions. Droplets are generated using a flow-focusing microfluidic device controlled by an automated syringe pump 207 and then imaged in well 230 by a fluorescence microscope. In the droplet generation coupling unit 227, the aqueous solution is mixed under laminar flow before droplet formation. Figure 10 shows how the flow rate of the aqueous solution is controlled by a syringe pump system programmed to allow detailed investigation of the concentrations of various proteins and regulatory factors. Figure 11 shows bright-field microscope images of droplet generation (left) and combined fluorescence images of droplet generation (right) showing the fluorescence of EGFP (green / light gray) and ALEXA647 (red-purple / dark gray) barcodes for FUSG156E and PEG, respectively.

[0079] Figure 12 (left / center) shows epifluorescence microscope images of captured microdroplets, where EGFP fluorescence and ALEXA647 fluorescence correspond to the concentrations of FUSG156E and PEG, respectively. Figure 12 (right) shows the classification of droplets as a separated phase (dashed outline) or a homogeneous phase (solid outline) based on the distribution of EGFP fluorescence. Figure 13 shows how the liquid condensates mix over time (1 hour) within the microdroplets, demonstrating their liquid properties.

[0080] Figure 14 shows a phase diagram of EGFP-FUSG156E concentration versus PEG 4000 concentration. The red / light gray (upper right) and blue / dark gray (lower left) data points in the scatter plot correspond to individual separated or homogeneous phase droplets, respectively. The heatmap corresponds to the probability of phase separation determined by an SVM classifier trained on the droplet scatter plot.

[0081] The number, size, and shape of condensates within each microfluidic droplet are measured using a microscope and compared with the total volume of the droplet to determine the volume fraction of the condensed phase within the droplet's microenvironment. These parameters can be used to characterize the condensation system, and can also be determined as a function of the variable components characterized within each microdroplet.

[0082] The unification of liquid-liquid phase separated condensates can be observed over time within a droplet as a function of the barcoded chemical variables constituting each droplet. The unification time allows for the understanding of the physical properties of the condensates, such as surface tension and viscosity.

[0083] Biomolecular condensates exist as complex mixtures composed of different molecular components. By labeling these components separately, it is possible to observe how different molecules co-localize within the same condensate. Such molecules may include proteins, nucleic acids, and small molecules. Furthermore, it is possible to observe the microstructure within the condensate, where the local concentrations of condensate components differ depending on the volume of the condensate. The relative amounts of co-localized molecules contained both inside and outside the condensate (i.e., the partition coefficient) are useful parameters that describe the affinity of molecules to the condensed phase. The partition coefficient can be determined drop by drop as a function of system parameters.

[0084] Further optical techniques can be applied to analyze biomolecular condensates within the system, including (but not exhaustive): • Fluorescence recovery after photobleaching (FRAP). This technology allows for the determination of the diffusion rate of labeled molecules within condensates, which indicates the liquid state and local viscosity of the condensate. • Fluorescence polarization spectroscopy. • Förster resonance energy transfer (FRET). Brillouin microscopy.

[0085] Summary: Biophysical characterization of condensates as a function of phase scanning parameters • Size, number, volume fraction, aspect ratio (shape) ·Combining speed • Colocalization of different molecules within the same condensate. 〇Distribution 〇 Multiphase characteristic evaluation FRAP, polarization spectroscopy, etc. • Material properties and viscoelastic properties

[0086] According to one embodiment, the above-described method and system may be used to screen for therapeutic drug candidates. In this case, at least one component other than the polymer in the microdroplet must contain the drug candidate. Therapeutic drug candidates that cannot alter the phase transition properties in a predefined desired manner are discarded. On the other hand, drug candidates that do indeed alter the phase transition properties in a predefined desired manner may be retained for further investigation. Specifically, the intended treatment may be for the treatment of protein misfolding disorders such as motor neuron disease, Alzheimer's disease and / or certain cancers.

[0087] Although the present invention has been described above with reference to examples, the present invention is not limited thereto. It should be understood that modifications or alterations can be made to the described examples without departing from the scope of the present invention as defined by the claims.

Claims

1. A method for measuring the phase transition properties of a polymer, wherein the method is The method for generating a flow of multiple microdroplets, wherein each of the multiple microdroplets contains multiple components, and one of the multiple components contains the polymer, The conditions in the plurality of microdroplets are changed by changing a plurality of relative concentrations, including the relative concentration of the component containing the polymer and the relative concentration of at least one further component, with respect to the plurality of components in the plurality of microdroplets, wherein each of the component containing the polymer and the at least one further component comprises an optical marker, and the conditions in the plurality of microdroplets are changed by changing a plurality of relative concentrations, wherein each of the component containing the polymer and the at least one further component comprises an optical marker. For each of the plurality of microdroplets in the flow of the plurality of microdroplets, the plurality of relative concentrations, including the relative concentration of the constituent components containing the polymer in the plurality of microdroplets and the relative concentration of at least one further constituent component, are measured by a first optical means based on the optical marker, and the phase of the polymer present in each of the plurality of microdroplets in the flow of the plurality of microdroplets is further measured. Methods that include...

2. The method according to claim 1, wherein the conditions within the plurality of microdroplets are changed by changing the temperature of the plurality of microdroplets.

3. The method according to claim 2, wherein the temperature of the plurality of microdroplets is changed by controlling the temperature of the channel through which the plurality of microdroplets flow.

4. The method according to any one of claims 1 to 3, wherein the flow of the plurality of microdroplets is a continuous flow.

5. The method according to any one of claims 1 to 4, wherein the measurement is performed continuously with respect to the flow of the plurality of microdroplets.

6. The method according to any one of claims 1 to 5, wherein the plurality of microdroplets are collected and measurements are performed on the plurality of collected microdroplets.

7. The method according to any one of claims 1 to 6, wherein the flow of the plurality of microdroplets is generated by injecting a flow of a first fluid containing the plurality of components into a flow of a second fluid, and the second fluid is immiscible with the first fluid.

8. The method according to any one of claims 1 to 7, wherein the plurality of relative concentrations are changed by changing the relative flow rates of each of the flows of at least two constituent components of the plurality of microdroplets.

9. The method according to any one of claims 1 to 8, wherein the plurality of relative concentrations are measured by a first optical means.

10. The method according to claim 9, wherein the first optical means illuminates the plurality of microdroplets with illumination light to detect a response.

11. The method according to claim 10, wherein the plurality of relative concentrations are determined based on the response of each of the plurality of constituent components to the illumination light.

12. The method according to claim 11, wherein each of the plurality of constituent components to be measured at the plurality of relative concentrations includes a separate fluorescent dye molecule that emits light of a specific wavelength in response to the illumination light.

13. The method according to any one of claims 1 to 12, wherein the phase of the polymer present in the plurality of microdroplets is measured by a second optical means.

14. The method according to claim 13, wherein images of the plurality of microdroplets are acquired by the second optical means, and the phase of the polymer present in the plurality of microdroplets is determined based on the characteristics of the image that show a specific phase.

15. The method according to claim 13, wherein the light scattering profiles of the plurality of microdroplets are obtained by the second optical means, and the phase of the polymer present in the plurality of microdroplets is determined based on the characteristics of the light scattering profile that indicate a specific phase.

16. The method according to any one of claims 1 to 15, wherein the plurality of relative concentrations change based on the plurality of measured relative concentrations of the plurality of microdroplets and the measured phase of the polymer present in the plurality of microdroplets.

17. The method according to claim 16, wherein the plurality of relative concentrations are systematically changed to generate a plurality of microdroplets having conditions for the polymer to transition from a first phase to a second phase, or conditions substantially approximating thereto.

18. The method according to any one of claims 1 to 17, wherein the polymer comprises one or more proteins or nucleic acids.

19. The method according to any one of claims 1 to 18, wherein the at least two components further comprise one or more of a pH buffer, a phase separator, saline solution, or a therapeutic agent / candidate drug.

20. A method for screening therapeutic drug candidates, wherein the method comprises the method described in any one of claims 1 to 19, wherein at least one component other than the polymer in the plurality of microdroplets contains a therapeutic drug candidate.

21. A system for measuring the phase transition properties of polymers, wherein the system is A microfluidic system that generates a flow of multiple microdroplets, each of which comprises multiple components, one of which comprises the polymer, and varies a plurality of relative concentrations in which the components in the multiple microdroplets are relative to the polymer-containing component and the relative concentration of at least one further component, each of which comprises an optical marker. A first optical system is configured to measure, for each of the plurality of microdroplets in the flow of the plurality of microdroplets generated by the microfluidic system, the plurality of relative concentrations, including the relative concentration of the constituent components including the polymer in the plurality of microdroplets and the relative concentration of at least one further constituent component, based on the optical marker, A second optical system configured to measure the polymer phase present in each of the plurality of microdroplets in the flow of the plurality of microdroplets generated by the microfluidic system, A control processor configured to control the microfluidic system to measure the phase of the polymer at or approximately thereto at the boundary where the polymer transitions from a first phase to a second phase, by changing the plurality of relative concentrations, including the relative concentrations of the constituent components containing the polymer in the plurality of microdroplets and the relative concentrations of the at least one further constituent component. A system equipped with these features.

22. The microfluidic system is At least two inlets configured to inject the flow of each component of at least two components, A first flow path configured to allow a first fluid to flow, the first flow path comprising the at least two components through which the first fluid arrives from the at least two inlets, A second flow path configured to allow a second fluid to flow, wherein the second fluid is immiscible with the first fluid, comprising: The system according to claim 21, wherein the first flow path includes a nozzle that is open into the second flow path and configured to inject the flow of the first fluid into the flow of the second fluid to generate a plurality of microdroplets of the first fluid in the second fluid.

23. The system according to claim 22, further comprising at least two pumps corresponding to the at least two inlets, wherein the at least two pumps are configured to change the relative flow rate of each of the plurality of components so as to change the plurality of relative concentrations of the at least two components of the plurality of microdroplets generated.

24. The system according to any one of claims 21 to 23, wherein the control processor is configured to change the plurality of relative concentrations of the constituent components of the plurality of microdroplets based on the plurality of measured relative concentrations of the plurality of constituent components of the plurality of microdroplets and the phase of the polymer present in the plurality of microdroplets.

25. The system according to claim 24, wherein the control processor is configured to systematically change the relative concentrations of the plurality of constituent components of the plurality of microdroplets, and generates a plurality of microdroplets having conditions for the polymer to transition from a first phase to a second phase or conditions substantially similar thereto.

26. The first optical system is, A light source configured to illuminate the plurality of microdroplets with illumination light, A detector configured to detect the response of the plurality of microdroplets to the illumination light, The system according to any one of claims 21 to 25, including the system described in any one of claims 21 to 25.

27. The system according to claim 26, wherein the light source includes a plurality of light-emitting units, each configured to emit light of a different wavelength.

28. The system according to claim 26 or 27, wherein the detector includes a plurality of photodetectors configured to detect light of different wavelengths.

29. The system according to any one of claims 21 to 28, wherein the second optical system includes an image sensor configured to acquire images of the plurality of microdroplets.

30. The second optical system described above, A light source configured to illuminate the plurality of microdroplets, A detector configured to acquire the light scattering profile of light from the light source scattered by the plurality of microdroplets, The system according to any one of claims 21 to 28, including the system described in any one of claims 21 to 28.

31. The method according to any one of claims 1 to 20, wherein the component comprising the polymer further comprises one or more of a cell, an intracellular organelle, or a cell lysate.

32. The method according to any one of claims 1 to 20, wherein the phase transition properties of two or more polymers are measured simultaneously, and at least one of the components comprises a further polymer.

33. The system according to any one of claims 21 to 30, wherein the component comprising the polymer further comprises one or more of cells, intracellular organelles, and cell lysates.

34. The system according to any one of claims 21 to 30, wherein the phase transition properties of two or more polymers are measured simultaneously, and at least one of the components comprises a further polymer.

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