Methods, apparatus, components and systems suitable for determining the characteristic properties of molecular interactions

Abstract

This invention relates to methods, components, and systems for determining characteristic properties of molecular interactions. The method includes providing a liquid sample comprising particles capable of being in equilibrium and non-equilibrium states. The particles comprise a marker in at least one of their equilibrium and non-equilibrium states. The method further includes subjecting the sample to a conditional transition including a temperature jump and / or a pressure jump, bringing the particles to a non-equilibrium state; reading out the marker as a function of time during at least a portion of the relaxation time of the particles; and determining the characteristic properties of the molecular interactions.

Description

Technical Field

[0001] This invention relates to a method for determining the characteristic properties of molecular interactions, as well as apparatus, components, and systems suitable for determining the characteristic properties of molecular interactions. Background Technology

[0002] Molecular interactions are important in various fields, including protein folding, drug design, materials science, sensors, nanotechnology, separation, and the origin of life. Rapid and reliable determination of molecular interactions is particularly crucial in medical science and medicinal chemistry.

[0003] Biochemical and biophysical concepts of molecular interactions between ligands and their receptors are crucial, for example, in drug discovery and / or drug design. Many drugs are small ligand molecules that interact with macromolecules. The affinity and specificity of ligand binding are properties used to determine the potential effects of chemical compounds or molecules.

[0004] Many methods and apparatuses have been provided for determining the properties of molecular interactions.

[0005] US2016011180 discloses a method for determining the biological response of a target to a soluble candidate substance, the method comprising providing a concentration distribution of the candidate substance in a laminar flow and introducing a concentration distribution of the target and a scan combination to detect an optical signal representing the biological response of the target to the soluble candidate substance.

[0006] US2002 / 0090644 discloses a method and apparatus for determining the presence or concentration of analyte particles in a medium, comprising: means for contacting a first medium containing analyte particles with a second medium containing binding particles capable of binding the analyte particles; wherein at least one of the analyte particles or binding particles is capable of diffusing into a medium containing another of the analyte particles or binding particles; and means for detecting the presence of the diffused particles. The apparatus may, for example, include a T-shaped flow device for placing the first and second media in adjacent laminar flow. Polinkovsky, M., Gambin, Y., Banerjee, P. et al., Ultrafast cooling reveals microsecond-scale biomolecular dynamics. Nat Commun 5, 5737 (2014). https: / / doi.org / 10.1038 / ncomms6737 discloses an apparatus for measuring conformational changes of DNA hairpins using a microfluidic cell, wherein a square wave of temperature is applied and the amplitude of the conformational changes of the DNA hairpins as a function of the frequency of the temperature wave is measured. The square wave temperature is induced using an IR laser to heat a microscopic small volume. Cooling of the heated region is accelerated by using a sapphire substrate with high thermal conductivity.

[0007] Another system for studying protein folding is described in the following article: The use of pressure-jump relaxation kinetics to study protein folding landscapes. Biochimica et biophysica acta 2006;1764(3):489-96.

[0008] US9,310,359 discloses a method for dispersion analysis using flow-induced dispersion analysis (FIDA) to quantify analytes such as, for example, antigens, toxins, nucleotides (DNA, RNA), etc. For pressure-driven flow of a single substance, FIDA corresponds to previously observed Taylor dispersion of pressure-driven flow in a tube or capillary.

[0009] New and reliable methods and equipment are still needed for determining the characteristic properties of molecular interactions.

[0010] Disclosure of the invention

[0011] One object of the present invention is to provide a relatively rapid and reliable method for determining the characteristic properties of molecular interactions, as well as an apparatus for performing such determinations.

[0012] In one embodiment, the aim is to provide a relatively simple method for determining the characteristic properties of molecular interactions, which is relatively fast and cost-effective.

[0013] In one embodiment, the aim is to provide an apparatus, component, and / or system suitable for the reliable determination of at least one characteristic property of molecular interactions, said apparatus, component, and / or system preferably operating relatively quickly, being durable, and / or relatively simple to operate.

[0014] These and other objectives have been achieved by the inventions or embodiments thereof as defined in the claims and as described herein below.

[0015] It has been discovered that the present invention or embodiments thereof have many other advantages, which will be apparent to those skilled in the art from the following description.

[0016] Molecular interactions are also called non-covalent interactions, or intermolecular and / or intramolecular interactions.

[0017] The phrase “molecular interaction” refers to any non-covalent interaction between molecules or within one or more molecules.

[0018] In one implementation, molecular interactions include liquid-liquid phase interactions that result in liquid-liquid phase separation (LLPS). LLPS is also known as aqueous two-phase systems, biomolecular condensates, or membrane lesscompartmentalization.

[0019] The term "particle" is used herein to refer to any part of a substance that contains at least one molecule, such as an organic or inorganic molecule. A particle may, for example, comprise an aggregate, a cluster, a complex, or any combination of one or more of these. The term "particle" also includes a plurality of identical or different molecules, such as molecules of a liquid mixture, which may undergo liquid-liquid phase separation following a change in conditions.

[0020] The term “binding coupler” is used in this document to refer to any molecule or group of molecules capable of non-covalent interaction with particles.

[0021] The term "marker" is used herein to refer to any intrinsic or extrinsic marker that can be detected by a reader device. In one embodiment, a marker comprises an element, a group of elements, a portion, and / or any combination of one or more of these, wherein the marker can be detected directly by the reader device and / or after being affected by external and / or internal sources.

[0022] The term "reader device" refers to any detector or detector system capable of detecting signals, such as optical signals and / or electrochemical signals, associated with binding couplers and / or particles. A reader device may include, for example, an image acquisition unit combined with an optical reader configured to read optical signals, such as those of a marker, and / or an electrical reader configured to read electrochemical signals.

[0023] The term "matter" is used to specify any substance that is uncountable, that is, not in the form of different articles. A substance may include a homogeneous or non-homogeneous mixture of components and / or elements.

[0024] The term "buffer solution" refers to an aqueous solution that resists changes in pH when a buffer is used. Buffer solutions advantageously contain aqueous solutions of weak acids and their salts or weak bases and their salts.

[0025] Unless otherwise stated, the pH of the buffer solution is determined at 20°C.

[0026] The terms “test” and “trial” are used interchangeably.

[0027] The terms "equilibrium" and "chemical equilibrium" are used interchangeably.

[0028] It should be emphasized that, when used in this document, the term “comprises” should be interpreted as an open term, meaning that it should be understood as specifying the presence of (multiple) features of a specific statement, such as (multiple) elements, (multiple) units, (multiple) integers, (multiple) steps, (multiple) components, and (multiple) combinations thereof, but does not exclude the presence or addition of features of one or more other statements.

[0029] The reference to "some embodiments" or "implementation" means that the specific features(s), structures(s), or characteristics(s) described in conjunction with such embodiments(s) are included in at least one embodiment of the disclosed subject matter. Therefore, the appearance of the phrase "in some embodiments" or "in one embodiment" throughout the specification does not necessarily refer to the same embodiments(s). Furthermore, those skilled in the art will understand that specific features, structures, or characteristics can be combined in any suitable manner within the scope of the invention as defined in the claims.

[0030] The term “basically” should be understood in this article to include common product variations and tolerances.

[0031] Throughout the specification or claims, unless the context otherwise requires, the singular includes the plural.

[0032] All features of the invention described herein and embodiments thereof, including the scope and preferred scope, may be combined in various ways within the scope of the invention unless there is a specific reason not to combine such features.

[0033] It has been found that methods and apparatus for determining the characteristic properties of molecular interactions can provide very accurate measurements, and furthermore, embodiments of the methods can be used to perform different and complex measurements, such as determining the characteristic properties or multiple characteristic properties of macroscopic particles with desired high accuracy.

[0034] The method of the present invention includes

[0035] • Provide a liquid sample comprising particles capable of being in equilibrium and non-equilibrium states, said particles containing markers indicating at least one of their equilibrium and non-equilibrium states.

[0036] • By subjecting the sample to a sudden change in conditions, the particles are placed in a non-equilibrium state.

[0037] • During at least a portion of the particle's relaxation time, read out markers as a function of time, and

[0038] • Determine the characteristic properties of molecular interactions,

[0039] The step of subjecting the sample to a conditional change may advantageously include subjecting the sample to a temperature change from at least one first temperature to a second temperature, and / or subjecting the sample to a pressure change from a first pressure to a second pressure.

[0040] The method of measuring very fast reaction rates using temperature jumps, also known as T-jumps, is one of a class of chemical relaxation methods pioneered by the German physical chemist Manfred Eigen in the 1950s. In these methods, a reaction system initially in equilibrium is rapidly perturbed and then observed as it relaxes back to equilibrium.

[0041] Advantageously, conditional transitions and readouts are performed within the capillary channels of the microfluidic unit, as further described below. For example, conditional transitions may occur in a first portion of the capillary channel (e.g., the inlet portion), and readouts may occur in a second portion of the capillary channel (the readout portion).

[0042] Generally, it is desirable that, during at least a portion of the particle's relaxation time, the readings of markers as a function of time include at least two, preferably at least five, such as at least eight, readings as a function of time, starting from the point in time when the particle undergoes a conditional transition, preferably without any intermediate conditional transitions.

[0043] In one implementation, the method includes

[0044] • Provide a liquid sample comprising particles capable of being in equilibrium and non-equilibrium states, said particles containing markers indicating at least one of their equilibrium and non-equilibrium states.

[0045] • By subjecting the sample to a conditional transition including a temperature jump from at least one first temperature to a second temperature, the particles are placed in a non-equilibrium state.

[0046] • During at least a portion of the particle's relaxation time, read out markers as a function of time, and

[0047] • Determine the characteristic properties of molecular interactions,

[0048] Temperature jumps occur via conduction and / or convection, preferably via conduction and / or convection within a microfluidic unit.

[0049] The inventors of this invention have discovered that very uniform heating can be obtained when temperature jumps occur via conduction and / or convection, which helps to improve the accuracy of the measured characteristic properties. For example, when a sample is heated by subjecting it to high-voltage discharge pulses and / or optical discharge pulses, the sample may have localized hot spots, which may reduce the accuracy of some measurements. It has been found that, in particular, laser heating induces unwanted hot spots, which may degrade measurements or even damage the sample.

[0050] Preferred methods for achieving temperature jumps via conduction and / or convection are described below.

[0051] In one implementation, the method includes

[0052] • Provide a liquid sample comprising particles capable of being in equilibrium and non-equilibrium states, said particles containing markers indicating at least one of their equilibrium and non-equilibrium states.

[0053] • By subjecting the sample to a sudden change in conditions, the particles are placed in a non-equilibrium state.

[0054] • During at least a portion of the particle's relaxation time, read out markers as a function of time, and

[0055] • Determine the characteristic properties of molecular interactions,

[0056] The conditional transition includes subjecting the sample to a temperature transition from at least one first temperature to a second temperature, and the method further includes maintaining the second temperature during at least a portion of the label readout, preferably in a microfluidic unit during at least a portion of the label readout.

[0057] Advantageously, maintaining the second temperature during at least a portion of the reading of the marker includes maintaining the temperature within a temperature range of about 2°C from the second temperature, such as within a temperature range of about 1°C from the second temperature, such as within a temperature range of about 0.5°C from the second temperature, such as within a temperature range of about 0.1°C from the second temperature.

[0058] The inventors of this invention have discovered that maintaining a second temperature for at least a portion of the time during which the marker is read can improve the accuracy of the measured characteristic properties, because otherwise the sample temperature may begin to change, for example, change back to the first temperature, which could provide altered equilibrium conditions and thus potentially reduce accuracy. A preferred method for maintaining the second temperature for at least a portion of the time during which the marker is read is described below.

[0059] In one implementation, the method includes

[0060] • Provide a liquid sample comprising particles capable of being in equilibrium and non-equilibrium states, said particles containing markers indicating at least one of their equilibrium and non-equilibrium states.

[0061] • By subjecting the sample to a conditional jump including a temperature jump from at least one first temperature to a second temperature and / or by subjecting the sample to a conditional jump including a pressure jump from a first pressure to a second pressure, the particles are placed in a non-equilibrium state.

[0062] • During at least a portion of the particle's relaxation time, read out markers as a function of time, and

[0063] • Determine the characteristic properties of molecular interactions,

[0064] The readouts include readouts as a function of time, which include making two or more readouts that are time-transferred and from different parts of the sample that have undergone conditional transitions, preferably in a microfluidic unit.

[0065] The inventors of this invention have discovered that when readouts, as a function of time, involve two or more reads from different portions of a sample, the risk of sample and / or sample marker degradation can be reduced. When readings are performed on the same portion of the sample, that portion or a portion thereof may degrade, leading to reduced accuracy.

[0066] This degradation effect is particularly relevant when the reader device includes optical readout. Such optical readout can lead to sample degradation, such as the degradation of sample markers by photobleaching. The risk of photobleaching can be reduced by performing two or more readouts from different portions of the sample. Advantageously, at least about half of the readouts are performed from different portions of the sample.

[0067] Advantageously, each read is performed on a “new” portion of the sample that has not been read before.

[0068] Preferred methods for performing two or more readings from different parts of a sample are described below.

[0069] In one implementation, the conditional jump includes a pressure jump. Using a pressure jump to bring the particles to a non-equilibrium state requires a relatively large pressure jump, depending on the particle and molecular interactions in question.

[0070] Advantageously, the difference between the first pressure and the second pressure is at least about 1 bar, such as at least about 3 bar, such as at least about 10 bar, such as at least about 25 bar.

[0071] In practice, a pressure jump of less than 1 bar will not be sufficient to bring the particles into a non-equilibrium state. A suitable pressure jump is preferably in the range of about 5 bar to about 200 bar, such as about 20 bar to about 150 bar.

[0072] In one implementation, the particles can be in equilibrium or non-equilibrium states because the sample contains a binding pair of the particles, or because the particles have a structure that depends on temperature and / or pressure. The particles and binding pairs can virtually include any interacting molecules, where this relates to determining the characteristic nature of the molecular interactions between the particles and the binding pairs.

[0073] The particles may, for example, contain a drug or a toxin or a candidate drug, and the conjugate may, for example, be a biological compound naturally occurring in an organism, such as a mammal. In another embodiment, the conjugate may contain a drug or a toxin or a candidate drug, and the particles may be a biological compound naturally occurring in an organism, such as a mammal.

[0074] In one embodiment, the particles have a structure that depends on temperature and / or pressure, wherein the particles have a structure in equilibrium under a second condition that differs from their structure before the conditional abrupt change.

[0075] Advantageously, the change in particle structure from before the conditional jump to the structure that the particle will have when it is in equilibrium under the second condition is at least partially reversible.

[0076] In one implementation, the particle has an equilibrium conformation under a second condition, which is different from its conformation before the conditional transition.

[0077] In this paper, conformational change is used to refer to the shape change of molecules induced by conditional transitions, such as macromolecules.

[0078] Macromolecules are typically flexible and dynamic. They can change their shape in response to changes in their environment or other factors; each possible shape is called a conformation, and the transitions between them are called conformational changes. In one embodiment, conformational changes induced by conditional hopping are structural changes, such as changes in folding when a particle contains a protein.

[0079] In embodiments of a method in which a sample contains a binding coupler of particles, it may be desirable for at least one of the particles or binding couplers to contain one or more markers. The markers may be any markers capable of being read by a reader device.

[0080] Examples of suitable markers are described further below.

[0081] Prior to the conditional transition, the particle or particle and its conjugate may be in or not in equilibrium. Advantageously, the conditional transition is sufficient to cause the particle or particle and its conjugate to change towards an equilibrium state that differs from the equilibrium state under the conditions prior to the conditional transition.

[0082] In a preferred embodiment, the liquid sample comprises particles and bound pairs that are in chemical equilibrium at the start of the conditional transition, or particles that are in chemical equilibrium. This allows for better control of the step of bringing the particles to a non-equilibrium state, and the determination of characteristic properties can be more accurate. Furthermore, characteristic properties can be determined more quickly compared to cases where the particles and bound pairs are not in chemical equilibrium at the start of the conditional transition.

[0083] Advantageously, the method includes holding the sample at a constant temperature for at least about 30 seconds before the temperature jump. This allows the particles / particles and their binding partners to be in equilibrium or near equilibrium. Preferably, the method includes holding the sample at a constant temperature for at least about 1 minute, such as at least about 5 minutes, such as at least about 10 minutes, before the temperature jump.

[0084] The time to reach equilibrium can range from seconds to hours, depending on the particles, the optional binding couple, and the transitions that lead to equilibrium, such as conformational changes.

[0085] Particles can be any kind of particle capable of undergoing at least partial chemical or structural transformations, such as conformational changes, either alone or together with a conjugated partner.

[0086] The liquid sample preferably comprises a liquid buffer system containing particles or particles together with bound couples. The buffer system is advantageously selected to have a pH value that does not impair or degrade the particles or, optionally, the bound couples. The pH value of the buffer system can be advantageously selected based on the molecular interactions to be examined. In one embodiment—particularly in the case where the particles contain biopolymers—the pH value is from about 4 to about 9, such as from about 5 to about 8.

[0087] In one embodiment, the particles comprise organic molecules, molecular clusters, molecular aggregates, nanoparticles, liposome vesicles, micelles, or any combination of one or more of these.

[0088] In one embodiment, the particles comprise biomolecules; proteins, such as antibodies (monoclonal or polyclonal), nanobodies, antigens, enzymes and / or hormones; nucleotides; nucleosides; nucleic acids, such as RNA, DNA, PNA or any fragment thereof and / or any combination containing at least one of these.

[0089] Nanobodies are antibody fragments composed of single monomeric variable antibody domains. Like complete antibodies, they can selectively bind to specific antigens.

[0090] In one implementation, molecular interactions include liquid-liquid phase interactions, such as liquid-liquid phase separation (LLPS). Liquid-liquid phase separation is a phenomenon present in various biological systems and is of great importance to biological function. For example, many non-membrane organelles in living cells and structures are formed through liquid-liquid phase separation.

[0091] The list of cell compartments believed to form via the LLPS process is growing rapidly and involves a wide range of cellular functions. In addition to punctate membraneless bodies, other subcellular structures are also formed via LLPS and share similar basic interactions and physical properties.

[0092] Understanding the biophysical principles underlying the formation of LLPS (Liquid-Liquid Phase Separation Systems) in biomolecules is crucial for the study of a wide range of biological processes and the physiology and pathophysiology of biological systems. Furthermore, for diagnostic and industrial purposes—such as in the food and pharmaceutical industries—there is a need for improved, rapid, and simpler identification and characterization of diverse biological and non-biological liquid-liquid phase separation systems.

[0093] As described and illustrated below, the methods of embodiments of the present invention provide an improved, faster, and simpler method for the identification and characterization of liquid-liquid phase separation systems.

[0094] In cases where molecular interactions involve liquid-liquid phase separation, the conditional transition advantageously includes a temperature transition from at least one first temperature to a second temperature, wherein the particles comprise at least two different molecules and optionally an additional solvent, the molecules being capable of forming a liquid-liquid phase separation under conditions prior to or after the temperature transition.

[0095] For example, at least two different molecules may include at least one protein, such as an antibody or enzyme; at least one polymer, such as polyethylene glycol (PEG) or a PEGylated molecule; at least one lipid, such as a phospholipid or cholesterol; and / or at least one sugar, such as a dextran. In one embodiment, one or more of the two or more different molecules are biomolecules. In one embodiment, at least one of the two or more different molecules is a salt in the dissociation phase.

[0096] The solvent can be an organic solvent, water, or an organic solvent-water mixture. Advantageously, the organic solvent in the solvent-water mixture is partially or completely miscible with water under conditions prior to a temperature jump.

[0097] Advantageously, the liquid sample is in a single-phase condition just before it is subjected to a temperature jump. This simplifies ensuring that the sample transferred and used is representative. If the sample is in two or more phases, it may be difficult to transfer a representative amount of each phase from the parent sample to be applied when the sample undergoes a temperature jump.

[0098] To ensure that the sample is in single-phase condition before undergoing a temperature jump, it is desirable that the temperature jump is from a higher temperature to a lower temperature. For example, the sample can be in single-phase condition at a higher temperature and can undergo liquid-liquid phase separation when subjected to a temperature jump to a lower temperature, such as a temperature jump within a temperature range where the sample does not freeze or boil, such as 90°C to 5°C, such as a temperature jump across 5°C to 40°C, such as 15°C to 30°C, such as a temperature jump of 20-25°C, such as a temperature jump of 50°C to 25°C.

[0099] Induced liquid-liquid phase separation may include at least locally forming a first liquid phase having an interface with a second liquid phase.

[0100] When conducting experiments involving liquid-liquid phase separation, starting from a first, higher temperature where the sample is under single-phase conditions and subjected to a temperature jump to a lower temperature, the first indication of liquid-liquid phase separation may appear as small droplets and / or bubbles of one phase in the remaining portion of the sample. These bubbles can gradually increase as a function of time from the temperature jump, for example, until complete phase separation.

[0101] In one embodiment, the sample under single-phase conditions is a sample removed from a parent sample that remains stable at a higher temperature. The parent sample may be subjected to stirring or shaking, for example, to maintain the sample under single-phase conditions.

[0102] Labels, such as those described elsewhere herein, can be incorporated into or inherent to one or more components of a sample. It has been found that during the formation of droplets and / or bubbles, detectable signals, such as fluorescence intensity, reflect this formation, for example, through spikes in the signal and / or changes in signal level, such as intensity. This allows for the determination of characteristic properties of liquid-liquid phase separations in various samples under diverse conditions. This provides a very rapid and attractive method for examining the formation and stability of liquid-liquid phase separations, such as those of biomolecules, LLPS.

[0103] The first liquid phase, the second liquid phase, and other liquid phases can be different from each other in any way. For example, the phases can differ in the concentration and / or presence of at least one molecule, such as one of at least two molecules, or in the concentration of dissolved salts. The phases can have the same or different solvents, different pH values, and / or different hydrophilicity / hydrophobicity. In one embodiment, the lipid concentration in one phase is higher than the lipid concentration in the other phase. In one embodiment, the protein concentration in one phase is higher than the protein concentration in the other phase.

[0104] In one embodiment, the content of the sample is known, and the purpose of the test is to determine at least one characteristic of the sample.

[0105] In one embodiment, the content of the sample is unknown, and the purpose of the test is to determine at least a portion of its content by measuring at least one characteristic of the sample and comparing it with a measured characteristic of a known sample.

[0106] The characteristic properties of liquid-liquid phase separation may include, for example, one or more of the following: the ability to separate liquid-liquid phases based on temperature, the concentration of one or more molecules, the presence of one or more other molecules, pH value, and the concentration of salts in dissociated forms.

[0107] In one embodiment, when the content of the sample is unknown, the method may include identifying sample portions capable of forming liquid-liquid phase separation under selected conditions following a temperature jump, the sample being, for example, a non-uniform sample.

[0108] The method may further include separating a target portion of the sample from the remaining portion of the sample, wherein the target portion of the sample is a portion exhibiting at least one indication of liquid-liquid phase separation formation. Therefore, even in cases of sample inhomogeneity, a portion with a high capacity for forming LLPS can be obtained.

[0109] When a sample undergoes a temperature jump in the channel of a microfluidic unit and is read out in the channel, the sample can advantageously be supplied into the channel at a selected pressure that ensures the sample is within the channel. The speed can be easily adjusted, for example, according to the liquid-liquid phase separation state determined by the readout.

[0110] The method may also include acquiring an image of at least a local portion of the channel. For example, the formation of spikes and / or bubbles can be imaged. It may be desirable to reduce the flow rate or stop the flow completely when acquiring the image.

[0111] The sample volume may be relatively small, so preparing a larger volume master sample may be simpler, which can then be used for several checks on particles in the sample. In one embodiment, the method includes preparing at least one master sample and removing a sample from the master sample.

[0112] The sample volume is advantageously relatively small. This makes conditional jumps simpler and faster, especially when the conditional jumps include temperature jumps. Furthermore, the temperature jump can be a jump to a uniform second temperature throughout the sample, which contributes to high accuracy in determining characteristic properties.

[0113] Advantageously, the volume of the sample is from about 0.1 nl to about 1 ml, such as from about 0.1 μl to about 0.5 ml, such as from about 1 μl to about 0.1 ml.

[0114] In one embodiment, the method includes performing a temperature jump from at least one first temperature to a second temperature and / or a pressure jump from a first pressure to a second pressure with a time-prolonged jump time, the jump time being less than the time required for the sample to reach equilibrium under the second condition, preferably less than twice the time required for the sample to reach equilibrium, preferably about 1 minute or less, such as about 30 seconds or less, such as about 10 seconds or less.

[0115] In principle, the time extension for conditional transitions should be as short as possible. The shorter the time extension for conditional transitions, the longer the time from the conditional transition to equilibrium under the second condition will be. As a result, the time available for reading can be longer, which can help to achieve the desired high accuracy relatively quickly.

[0116] It has been found that time extensions for conditional transitions of 0.1 to 10 seconds are very effective.

[0117] The conditional jump time can be determined from the time from the onset of a temperature jump and / or a pressure jump to when the entire sample reaches a second temperature and / or a second pressure.

[0118] To ensure a relatively long time for reading, it has been found that conditional jumps within a microfluidic unit are desirable. Therefore, in one embodiment, temperature and / or pressure jumps of the sample occur within the microfluidic unit, the method comprising introducing the sample into the microfluidic unit, wherein the microfluidic unit is preferably at least partially located in a temperature-controlled holding compartment.

[0119] The microfluidic unit may include, for example, an introduction portion in which a sample is introduced. The introduction portion may advantageously have at least one narrow dimension to ensure that conditional changes in the sample within the introduction portion can occur relatively rapidly.

[0120] The introduced portion may advantageously include a cross-sectional dimension of about 1 mm or less, such as about 0.5 mm or less, such as about 0.1 mm or less, such as about 75 μm or less.

[0121] In one embodiment, the introduction portion includes a flat chamber, a channel, two or more interconnected channels, or any combination of one or more of these.

[0122] A flat chamber is advantageously a chamber with a height dimension that is 50% or less of at least one of its width and length.

[0123] The introduction portion preferably has a volume at least as large as the sample volume. Additionally, it is desirable that the introduction portion not be significantly larger than the sample. Advantageously, its volume corresponds to the sample volume, or is up to approximately 20% larger than the sample volume.

[0124] The volume of the introductory portion of the microfluidic unit can be, for example, from about 0.1 nl to about 1 ml, such as from about 0.1 μl to about 0.5 ml, such as from about 1 μl to about 0.1 ml.

[0125] In one embodiment, the volume of the introduction portion defines the volume of the sample and / or the introduction portion is defined by the volume of the sample. That is, the volume of the microfluidic unit filled by the sample during a conditional transition is defined as the introduction portion of the microfluidic unit.

[0126] Advantageously, during at least a portion of the relaxation time, preferably during at least a portion of the readout time, the temperature-controlled holding compartment is maintained at a second temperature and / or a second pressure, thereby ensuring stable second conditions.

[0127] Temperature-controlled holding compartments can control their temperature, for example, by means of blowing air, preferably air having a second temperature. It should be understood that any gas other than air can be used in place of air or in combination with air.

[0128] In one embodiment, the temperature-controlled holding compartment controls the temperature by including filling the compartment completely or partially with a liquid and / or vapor, preferably having a second temperature.

[0129] In one embodiment, the temperature jump is performed by a method including blowing air or flowing liquid through a container containing the sample, for example, wherein the container forms part of or includes at least a portion of the microfluidic unit as explained above.

[0130] In one embodiment, the temperature jump can be performed by a method including applying a high voltage to the sample (e.g., using pulsed and / or Joule heating), preferably when the sample is in a container, such as a container forming part of a microfluidic unit or including at least a portion of a microfluidic unit, such as when the sample is in the inlet portion of a microfluidic unit, the temperature jump can be performed by a method including applying a high voltage to the sample (e.g., using pulsed and / or Joule heating).

[0131] High voltage can be applied as a discharge pulse at high voltage. As explained above, using a discharge pulse at high voltage may result in the formation of local hot spots in the sample. However, for some molecular interactions, the time from a temperature jump to equilibrium is relatively long, and by ensuring that the sample volume is relatively small, the thermal alternation of local hot spots can dissipate relatively quickly throughout the sample, thus ensuring that measurements can be performed with acceptable and even relatively high accuracy.

[0132] In one embodiment, the temperature jump is achieved by a method comprising applying a Joule heating element (e.g., applying a substantially continuous high voltage through the sample for at least 0.1 seconds until the desired temperature is reached), a resistive element, and / or a Peltier element to conduct heat to the sample. Heat conduction to the sample is advantageously performed when the sample is located in a container, such as a container forming part of or including at least a portion of a microfluidic unit, such as when the sample is located in the inlet portion of the microfluidic unit. Preferably, the Joule heating element, resistive element, and / or Peltier element are positioned in physical contact with the container.

[0133] Joule heating elements, resistive elements, and Peltier elements are known to those skilled in the art, and they will be able to select appropriate Joule heating elements, resistive elements, and / or Peltier elements based on the teachings presented herein.

[0134] In one embodiment, the pressure jump is performed by a method comprising placing a sample in a container comprising a membrane, such as a polyimide membrane (e.g., a Kapton membrane), wherein a piezoelectric stack is arranged to press down the membrane, and wherein the pressure jump is performed by activating the piezoelectric stack to increase pressure or deactivating the piezoelectric stack to decrease pressure. The container used as the microfluidic unit in which the conditional jump is performed as a pressure jump is advantageously made of a robust material, such as sapphire, for example synthetic sapphire (crystalline alumina). The sample can be injected to allow it to flow through the membrane into the microfluidic unit and can be optically read out, for example, via the sapphire.

[0135] In one embodiment, the temperature jump is performed by mixing the sample with another liquid at a selected temperature different from the first temperature. This method can be carried out in a T-shaped flow cell, such as a microscale channel cell described in US 5,972,710, which serves as a microfluidic device.

[0136] In one embodiment, the additional liquid is preferably free of particles and bound particles. Thus, the sample becomes a diluted sample.

[0137] In one embodiment, the method includes providing a sample in the form of two or more sub-samples having different first temperatures, wherein the temperature jump is performed by means including bringing the two or more sub-samples together, for example, in adjacent laminar flows or by mixing. The two or more sub-samples may have the same or different concentrations of particles and / or bound couples.

[0138] In one embodiment, the relative concentrations of particles and bound couplers are the same in each subsample; preferably, the concentrations of particles and bound couplers are substantially the same in each subsample; more preferably, the chemical composition of the subsamples is the same.

[0139] Temperature jumps from at least one first temperature to a second temperature advantageously include providing a temperature jump of at least about 2°C, such as at least about 5°C, such as at least about 10°C, such as at least about 15°C.

[0140] The minimum temperature jump that would bring a particle to a non-equilibrium state depends on the molecular interactions examined and the concentrations of the particle and, optionally, the binding coupler.

[0141] For many molecular interactions, a temperature jump from about 5°C to about 30°C may be suitable. For LLPS experiments, a temperature jump from high to low temperature, such as from 40-50°C to about 20-25°C, may be advantageous.

[0142] For molecular interaction analysis, a second temperature may be important for determining the characteristic properties to be measured. If, for example, the characteristic properties are related to the properties of particles within a specific temperature range—such as the properties of drugs in vivo—then the second temperature is advantageously chosen within that specific temperature range.

[0143] The second temperature can be higher or lower than at least one first temperature. In many cases, such as when using a heating element to perform a temperature jump, it may be simpler to perform a temperature jump from a lower temperature to a higher temperature.

[0144] The second temperature can advantageously be about 5°C to about 50°C, such as about 10°C to about 45°C, such as about 20°C to about 42°C, such as about 35°C to about 40°C, for example 25-37°C.

[0145] In fact, it may be desirable to be at a temperature 5°C away from the organism's natural temperature or at a second temperature within 5°C of the organism's natural temperature.

[0146] In one embodiment, the method includes introducing a sample into a microfluidic unit at a pressure difference of at least about 0.1 bar, such as at least about 0.2 bar, such as at least about 0.3 bar, such as at least about 0.4 bar, such as at least about 0.5 bar, such as at less than 1 bar, such as less than 0.9 bar.

[0147] In one embodiment, the method includes introducing a sample into a microfluidic unit at a pressure of about 0.5 to about 3 bar.

[0148] The sample is advantageously introduced into the microfluidic unit, for example, relatively rapidly into the introduction section of the microfluidic unit, where the sample undergoes a conditional jump, such as a temperature jump. The microfluidic unit can be preheated so that the temperature jump begins immediately when the sample is introduced into the microfluidic unit.

[0149] Microfluidic units can, in principle, have any shape, but the shape described herein is advantageous. In one embodiment, a microfluidic unit includes a flat chamber, a channel, two or more interconnected channels, or any combination of one or more of these.

[0150] In one embodiment, the microfluidic unit includes channels, and is preferably in the form of tubes or sheets, wherein the channels preferably have a cross-sectional dimension of about 1 mm or less, such as about 0.5 mm or less, such as about 0.1 mm or less, such as about 75 μm or less, and preferably, the channels have a maximum cross-sectional dimension of about 1 mm or less, such as about 0.5 mm or less, such as about 0.1 mm or less, such as about 75 μm or less. The microfluidic unit can, for example, be shaped as a tube having a constant diameter along its entire length. Such a tube is also called a capillary.

[0151] In one embodiment, the microfluidic unit includes, for example, an inlet portion and a readout portion as described above. The inlet portion and the readout portion may be directly connected to each other in length.

[0152] In one implementation, the introduction and readout portions at least partially overlap. Readout can be performed when the sample is at the same location where it has already undergone a conditional transition.

[0153] In a preferred embodiment, the introductory portion and the readout portion are different portions.

[0154] In an advantageous embodiment, the method includes causing at least a portion of the sample to flow from the introduction portion to the reading portion.

[0155] In one embodiment, readout includes reading the sample when it is stationary (non-flowing) within the microfluidic unit. As described above, readout is preferably performed from different portions of the sample. This can be done, for example, by moving the reader device and the microfluidic unit relative to each other.

[0156] In a preferred embodiment, readout includes reading the sample while it flows within the microfluidic unit. Preferably, readout as a function of time includes reading from different portions of the sample while it flows within the reading portion of the microfluidic unit. Thus, the reader device can read from different portions of the sample without requiring movement of the reader device and the microfluidic unit relative to each other. Typically, moving elements in a device can increase its complexity and cost. Therefore, a method including reading the sample while it flows within the microfluidic unit is provided to improve the cost-effectiveness of the method and the device used to perform it.

[0157] The flow rate of the sample in the readout section can be advantageously adjusted to the readout rate, allowing for the desired number of reads.

[0158] Advantageously, the method includes adjusting the flow velocity at the readout locations to up to about 50 cm / s, such as up to about 25 cm / s, such as up to about 10 cm / s, such as up to about 2 cm / s, such as up to about 1 cm / s, such as up to about 0.1 cm / s.

[0159] The read rate can be, for example, at least about 5 reads per minute, such as at least about 10 reads per minute, such as at least about 30 reads per minute, such as at least about 60 reads per minute, such as at least about 120 reads per minute.

[0160] A read rate of approximately 1 to 30 reads per second is likely suitable for most measurements.

[0161] Advantageously, the readout as a function of time includes continuous reading from different parts of the sample as each sample portion passes the readout position of the microfluidic unit.

[0162] The method can advantageously include introducing a sample into the microfluidic unit at a first relatively high pressure, such as, for example, a pressure difference of up to 1 bar as described above. A conditional jump can be performed after or during the introduction. If the conditional jump occurs after the sample has been fully introduced, the pressure difference can be reduced or terminated, causing the sample to stop flowing during the conditional jump. This embodiment is advantageous when the conditional jump includes a temperature jump.

[0163] If the conditional jump includes a temperature jump, it is advantageous to perform the temperature jump during the introduction of the sample into the introduction section. The microfluidic unit can advantageously be preheated. After the conditional jump, the method advantageously includes reducing the pressure to a second, lower pressure.

[0164] The second lower pressure can be as described above. For example, the second lower pressure is advantageously at least about 10% lower than the first higher pressure, such as at least about 25% lower than the first higher pressure, such as at least about 50% lower than the first higher pressure, such as at least about 75% lower than the first higher pressure, such as at least about 90% lower than the first higher pressure, such as at least about 95% lower than the first higher pressure, such as at least about 99% lower than the first higher pressure.

[0165] The marker can be any marker that can be read by a reader device, such as those described above. The marker can be an intrinsic marker, an extrinsic marker, or a combination thereof.

[0166] When particles contain biomolecules, it is often desirable to use intrinsic markers, such as intrinsic tryptophan fluorescence or absorbance.

[0167] Advantageously, the marker is sensitive to molecular interactions, such as to conformational changes in the particles. Preferably, the marker alters the signal according to the conformation of the particles and its conformational changes, such as changes in binding / dissociation and / or structural changes.

[0168] In one implementation, the marker is sensitive to protein interactions, for example, the signal changes during binding / dissociation.

[0169] In one embodiment, the marker is an optically readable marker, such as a light-absorbing marker and / or a fluorescent marker, preferably operating in the ultraviolet / visible wavelength range of about 190 nm to about 700 nm.

[0170] The marker may include, for example, a quencher.

[0171] Especially when the marker requires excitation, there is a risk of photobleaching if the same sample portion is read multiple times. Therefore, it is preferable to ensure that the method includes reading from different portions of the sample two or more times, as described elsewhere herein.

[0172] In one embodiment, the label is an electrochemically readable label, such as an electroactive label. A non-limiting example of an electrochemically readable label is an osmium tetroxide label.

[0173] The reading of the marker as a function of time during at least a portion of the relaxation time advantageously includes multiple consecutive readings of the marker. The readings preferably include multiple readings of the electrode potential, multiple readings of the intensity of one or more wavelengths, and / or multiple readings of changes in one or more wavelengths.

[0174] A change in one or more wavelengths can be, for example, a wavelength shift.

[0175] In one implementation, fluorescence resonance energy transfer (FRET) and / or bioluminescent resonance energy transfer (BRET) are used to monitor the distance between two markers, one of which is on or bound to a particle, and the other of which is on or bound to a binding partner.

[0176] Multiple reads advantageously include at least 5 reads, such as at least 10 reads, such as at least 50 reads, such as at least 50 reads or more.

[0177] Advantageously, the method involves making multiple consecutive reads of the marker until the consecutive read variation from one read to the next is less than about 25%, such as until the consecutive read variation is less than about 10%, such as until the consecutive read variation is less than about 5%, such as until the consecutive read variation is less than about 1%, preferably until relaxation is achieved. It may not be necessary to continue reading until full relaxation; however, in practice, continuing reading until full relaxation may be simpler and / or safer.

[0178] In one embodiment, the method further includes performing the method once or multiple times using different temperature jumps and / or using (multiple) different concentrations of particles and / or conjugate couples, and preferably determining additional characteristic properties of molecular interactions.

[0179] The method can be applied to determine any conformational changes, such as protein folding and / or any kinetic reactions between particles and their binding partners.

[0180] In one embodiment, the method includes determining at least one of the following: kinetic parameters, such as Kd; partition parameters, such as the formation / deformation of liposomes or micelles; degradation parameters; oligomerization parameters; folding parameters, such as unfolding or refolding; and multiple binding parameters, such as parameters representing multiple bindings by different time scales.

[0181] In one embodiment, the method includes determining the characteristic properties of molecular interactions between particles and two or more binding partners and / or between two or more particles and binding partners.

[0182] Characteristic properties of molecular interactions may include, for example, determining at least one kinetic parameter, such as the equilibrium constant (Kd value) of at least one particle and / or at least one particle and at least one binding partner, such as determining the affinity between at least one particle and at least one binding partner, and / or determining one of two kinetic rate constants kon / koff.

[0183] Examples of measurable characteristic properties include any kinetic parameters such as Kd, kon, and koff; partitioning, such as entry into and exit from liposomes or micelles, LLPS systems; degradation; oligomerization; unfolding; refolding; and multiple binding by different time scales and / or particle concentrations.

[0184] The methods described herein can be combined with other tests, such as diffusion tests of one or more particles or particles and their bound pairs. Diffusion tests can be applied, for example, to determine the concentration equilibrium of particles / bound pairs, which may be desirable for use in the methods described herein, for example, where abrupt changes in conditions may have a significant impact on the equilibrium / non-equilibrium state of particles and bound pairs.

[0185] Diffusion tests can be used, for example, to determine the hydrodynamic radius of particles.

[0186] In one embodiment, the diffusion test is performed at different (multiple) concentrations of at least one particle and / or binding coupler to determine the concentration at which at least one kinetic rate constant kon / koff is sensitive to change.

[0187] The present invention also includes apparatus suitable for determining the characteristic properties of molecular interactions.

[0188] The device includes

[0189] • A sample compartment for containing at least one liquid mother sample;

[0190] • A transfer device arranged for removing a sample from at least one parent sample stored in a sample compartment.

[0191] • Conditional transition device, and

[0192] • At least one reader device for reading at least one marker that is a function of time.

[0193] The conditional transition device is advantageously arranged to perform the conditional transition as described above.

[0194] In one embodiment, the device includes

[0195] • A sample compartment for containing at least one liquid mother sample;

[0196] • A transfer device arranged for removing a sample from at least one parent sample stored in a sample compartment.

[0197] • A conditional transition device arranged to perform a temperature jump of a sample from at least one first temperature to a second temperature, and

[0198] • At least one reader device for reading at least one marker that is a function of time.

[0199] The device is adapted to achieve temperature jumps via conduction and / or convection, and preferably has a sample contained in a microfluidic unit.

[0200] As explained above, the device is adapted to achieve temperature jumps via conduction and / or convection, ensuring that very uniform sample heating can be obtained.

[0201] In one embodiment, the device includes

[0202] • A sample compartment for containing at least one liquid mother sample;

[0203] • An extraction device arranged for removing a sample from at least one parent sample stored in a sample compartment.

[0204] • A conditional transition device arranged to perform a temperature jump of a sample from at least one first temperature to a second temperature, and

[0205] • At least one reader device for reading at least one marker that is a function of time.

[0206] The device further includes a holding compartment for holding the sample under a second condition during marker readout, preferably containing the sample within a microfluidic unit.

[0207] The device is advantageously adapted to maintain the temperature within a temperature range of about 2°C from the second temperature, such as within a temperature range of about 1°C from the second temperature, such as within a temperature range of about 0.5°C from the second temperature, such as within a temperature range of about 0.1°C from the second temperature.

[0208] As explained above, adapting the device to maintain a second temperature during at least a portion of the readout ensures that the accuracy of the measured characteristic properties can be improved.

[0209] In one embodiment, the device includes

[0210] • A sample compartment for containing at least one liquid mother sample;

[0211] • A transfer device arranged for removing a sample from at least one parent sample stored in a sample compartment.

[0212] • A conditional transition device arranged for performing a temperature jump of a sample from at least one first temperature to a second temperature, and / or arranged for performing a pressure jump from a first pressure to a second pressure, and

[0213] • At least one reader device for reading at least one marker that is a function of time.

[0214] The device is adapted to perform readouts as a function of time by taking two or more reads from different portions of the sample, and preferably has a sample contained in a microfluidic unit.

[0215] As explained above, adapting the device to readouts as a function of time by performing two or more reads from different portions of the sample ensures that the risk of sample and / or sample marker degradation can be reduced.

[0216] The device can be advantageously adapted to perform the methods as described in the claims and as stated above.

[0217] Advantageously, the sample compartment includes at least one temperature control device for selecting and controlling the temperature of at least one mother sample located in the mother sample chamber of the sample compartment. The sample compartment may be adapted to or include two or more mother sample chambers, wherein the device is adapted to individually or jointly select and control the temperature of each mother sample located in each mother sample chamber. Thus, the device can be applied, for example, to perform tests on several identical or different samples one after another without requiring refilling or changing the mother samples(s).

[0218] In one embodiment, the transfer device includes tools, such as manual handling tools, for removing a sample from a sample and delivering it to the inlet of a microfluidic unit.

[0219] The tool may include, for example, a pipette, which allows the user to pick up a sample (e.g., a drop) and manually move it to the inlet of the microfluidic unit.

[0220] This implementation scheme may be advantageous for users who only need to perform a small number of measurements, as it can reduce the cost of the equipment.

[0221] Advantageously, the transfer device forms part of or is in fluid communication with the microfluidic unit.

[0222] The transfer device may advantageously include a pump device adapted to move (flow) the sample from the sample compartment to the microfluidic unit. The pump device can be any device capable of delivering the sample from the sample compartment to the microfluidic unit. Preferably, the pump device includes an electrically driven pump device and / or a pressure-driven pump device, such as a suction pump arranged for drawing the sample into the microfluidic unit and / or a pressure pump arranged for pumping the sample into the microfluidic unit.

[0223] Examples of electrically driven pump units can be found, for example, in Devasenathipathy S, Santiago JG (2004), “Electrokinetic flow diagnostics”, Springer, New York, Berlin, Heidelberg.

[0224] The transfer device may include a tube for transferring samples from a sample compartment. The tube may be multi-branched and have several inlets, which may be arranged to transfer samples from various parent sample chambers. In one embodiment, one or more tube ends are adapted to move from a parent sample container to a parent sample container between sample transfers.

[0225] Electrically driven flow phenomena include electroosmosis, electrophoresis, and flow potential.

[0226] The transfer device can be adapted to transfer samples from a single master sample chamber.

[0227] In one embodiment, the transfer device is adapted to transfer samples from two or more mother sample chambers.

[0228] The transfer device can be advantageously configured to supply a sample to the inlet of a microfluidic unit under a supply pressure, wherein the supply pressure is adjustable, such as manually adjustable or controlled by a computer system. The computer system can be programmed to control the sample rate based on the time of conditional transitions and / or based on readout signals, preferably controlling the sample rate in real time.

[0229] Computer systems can be programmed to control the rate at which signals are read out in real time. The phrase "real time" is used herein to refer to a delay of less than one second. For example, when signal changes exceed a preset threshold, the computer can be programmed to slow down image acquisition and / or improve reading accuracy.

[0230] The device may include an image acquisition unit positioned to acquire an image of at least a portion of a sample located downstream of a position where the sample undergoes a conditional transition. The image acquisition unit may also be positioned to acquire an image of at least a local portion of a channel, such as a local portion located downstream of a readout position.

[0231] The conditional transition device can be integrated at least partially with a microfluidic unit. For example, the microfluidic unit may include two or more inlets adapted to contact subsamples removed from individual parent sample chambers, for example, by arranging subsamples into a layered (e.g., laminar) flow or by mixing subsamples as further described above.

[0232] Advantageously, the condition change device includes heating and / or cooling devices adapted to perform a temperature change from a first temperature to a second temperature.

[0233] In one embodiment, the condition change device includes a pressurization or depressurization device adapted to perform a pressure change from a first pressure to a second pressure.

[0234] The device is advantageously adapted to perform conditional transitions relatively quickly, for example, with a transition time as described above.

[0235] Advantageously, the conditional jump device is arranged to perform temperature and / or pressure jumps on the sample in the microfluidic unit. The conditional jump device is preferably located at least partially in a temperature-controlled holding compartment.

[0236] The condition change device and / or holding compartment preferably include a temperature control device. The temperature control device may include, for example, a blower for blowing air at a selected temperature and / or a liquid sprayer for spraying liquid at a selected temperature and / or a liquid filler for completely or partially filling the holding compartment with liquid at a selected temperature.

[0237] In one embodiment, the conditional transition device includes a Joule heating device arranged to apply a high voltage to the sample, preferably when the sample is in a container, such as a container forming part of a microfluidic unit or including at least a portion of a microfluidic unit, such as when the sample is in a microfluidic unit, for example in the inlet portion of the microfluidic unit.

[0238] In one embodiment, the conditional transition device includes a Joule heating element, a resistive element, and / or a Peltier element arranged to conduct heat to the sample, preferably when the sample is located in a container, such as a container forming part of a microfluidic unit or including at least a portion of a microfluidic unit. When the sample is located within a microfluidic unit, the conditional transition device includes a Joule heating element, a resistive element, and / or a Peltier element arranged to conduct heat to the sample. Preferably, the Joule heating element, the resistive element, and / or the Peltier element are positioned in physical contact with the container.

[0239] The reader device can be as described above.

[0240] In one implementation, the reader device can be any kind of reader that does not make undesirable changes to the interaction being analyzed.

[0241] At least one reader device includes an optical reader device and / or an electrochemical reader device.

[0242] Advantageously, at least one reader device is adapted to perform multiple reads as a function of time, preferably at a read rate of at least about 5 reads per minute, such as at least about 10 reads per minute, such as at least about 30 reads per minute, such as at least about 60 reads per minute, such as at least about 120 reads per minute.

[0243] Advantageously, at least one reader device is fixedly located in the device, and the reader device is advantageously adapted to read the markers of the sample portion as the sample portion passes through the reader device, preferably as the sample portion flows through the reader device in the microfluidic unit.

[0244] By making the reader device fixed in place, the cost of devices such as those described above can be reduced.

[0245] The device can be advantageously adapted to control the flow rate.

[0246] The reader device is preferably positioned for reading from the microfluidic unit in the holding compartment, and preferably, at least one reading head of the reader device is located in the holding compartment.

[0247] The invention also includes a component comprising the device as described in claims and herein, in combination with a microfluidic unit. The microfluidic unit is preferably located at least partially in a temperature-controlled holding compartment.

[0248] Microfluidic units can advantageously be as described herein and include, for example, flat chambers, channels, two or more interconnected channels, or any combination of one or more of these.

[0249] In one embodiment, the microfluidic unit is adapted to be enclosed and includes a membrane wall portion and means for moving the membrane, such as using a piezoelectric crystal stack to change the pressure within the microfluidic unit.

[0250] Microfluidic units advantageously include channels. The channels preferably have a length of at least about 1 cm, such as at least about 10 cm, such as at least about 25 cm, such as at least about 50 cm, such as at least about 75 cm, such as at least about 1 m or longer. In principle, the channels can be as long as desired, but for most measurements, a channel length of 1 cm to 2 m may be sufficient. The channels can be folded, coiled, or bent into any other desired configuration.

[0251] In one embodiment, the microfluidic unit includes an inlet portion and a readout portion. The inlet portion and the readout portion may at least partially overlap, or the inlet portion and the readout portion may be different portions.

[0252] Advantageously, the reader device is positioned to read from a fixed reading position of the microfluidic unit.

[0253] In one embodiment, the device includes a pump assembly, such as the pump assembly described above.

[0254] The pump device can, for example, be adapted to introduce a sample into the microfluidic unit at a first higher pressure differential and reduce the pressure differential to a second lower pressure differential. The pump device can preferably be adapted to maintain the second lower pressure differential during at least a portion of the readout. The pump device can advantageously include a pressure pump and / or a suction pump.

[0255] The present invention also includes a system suitable for determining the characteristic properties of molecular interactions. The system includes the apparatus as described in claims and / or as described herein, or the components as described in claims and / or as described herein, and a computer system. The computer system is configured for...

[0256] • Controlled transfer device

[0257] • Device for controlling temperature jumps and diffusion

[0258] • Control reader device and / or

[0259] • Determine the characteristic properties of molecular interactions.

[0260] The system is advantageously suited for determining the characteristic properties of molecular interactions, wherein the molecular interactions include changes in particle structure and / or changes in the binding between particles and their binding partners, preferably wherein the molecular interactions include conformational changes.

[0261] In one embodiment, the computer system is configured to determine at least one of the following: kinetic parameters, such as Kd; partition parameters, such as liposome formation / deformation, micelle formation / deformation and / or liquid-liquid phase separation or merging; degradation parameters; oligomerization parameters; folding parameters, such as unfolding or refolding; and multiple binding parameters, such as parameters representing multiple bindings by different time scales.

[0262] In one embodiment, the computer system is configured to determine the characteristic properties of molecular interactions between particles and two or more binding partners and / or between two or more particles and binding partners.

[0263] In one embodiment, the computer system is configured to determine at least one kinetic parameter, such as the equilibrium constant (Kd value) of at least one particle and / or at least one particle and at least one binding couple, such as determining the affinity between at least one particle and at least one binding couple, and / or determining one of two kinetic rate constants kon / koff.

[0264] In one embodiment, the computer system is configured to control the execution of the method according to any one of claims 1-60.

[0265] All features of the invention(s) and its embodiments, including the scope and preferred scope, can be combined in various ways within the scope of this invention, unless there is a specific reason for not combining such features.

[0266] Brief Description of Embodiments and Drawings

[0267] The present invention will be further described below with reference to examples, embodiments, and accompanying drawings. The drawings are schematic and may not be drawn to scale. The examples and embodiments provided are merely illustrative and should not be construed as limiting the scope of the invention.

[0268] Figure 1 An embodiment of the system of the present invention is described, the system comprising a computer system and components of devices and microfluidic units.

[0269] Figure 2 illustrate Figure 1 A variation of the implementation plan.

[0270] Figures 3a-3e Examples of microfluidic units suitable for use in the devices of the present invention are shown.

[0271] Figure 4a and 4b It is a graph showing the fluorescence intensity as a function of time, as described in Example 1.

[0272] Figures 5a-5g It is a graph showing the fluorescence intensity as a function of time, as described in Examples 2a-2g.

[0273] Figure 1 The system includes an apparatus 1 suitable for determining the characteristic properties of molecular interactions and a microfluidic unit 4. The apparatus includes a holding compartment 2 and a sample compartment 3 separated by a partition wall 14 having channels of the microfluidic unit 4.

[0274] The sample compartment 3 includes a plurality of mother sample chambers 7 arranged in a support unit 7a. The support unit 7a advantageously includes a temperature controller for controlling the temperature of the mother samples in each mother sample chamber 7 to a selectable temperature. The sample compartment 3 includes a transfer device comprising a pump device 5 connected to a plurality of transfer tubes 6. Each tube advantageously includes a needle adapted to penetrate a covering membrane on the respective mother sample chamber 7. The respective tubes 6 can be manually inserted into the desired mother sample chamber by penetrating the membrane of the mother sample chamber with the needles at their ends. In one embodiment, the device 1 includes a robotic arm adapted to insert the tube(s) 6 into the selected mother sample(s) 6(s).

[0275] In a variation of this implementation, the transfer device includes a single transfer tube.

[0276] The device 1 includes a hinged cover 1b 1a that leads into the sample compartment 3, providing an inlet to the sample compartment 3.

[0277] In this embodiment, the microfluidic unit 4 is, for example, a tube with a narrow diameter as described above. The tube 4 is connected to a pumping device such that the pump can pump the transferred master sample into the microfluidic unit 4 at a desired pressure differential.

[0278] The compartment 2 includes a computer unit 9 adapted to control the components of the device 1. The computer 9 is connected to the reader device 11.

[0279] The holding compartment 2 includes a conditional changeover device 8, which is adapted to cause a temperature jump by conduction and / or convection, as described above. The conditional changeover device 8 may include, for example, a blower or a Peltier element. A temperature controller device 8a is connected to the conditional changeover device 8 such that the temperature controller device 8a can control the operation of the conditional changeover device 8 and control the temperature in the holding compartment 2.

[0280] The lumbar chamber 10 is positioned to collect used samples and optional cleaning fluid passing through the microfluidic unit 4.

[0281] The microfluidic unit 4 has an inlet portion 4a, which is arranged adjacent to the condition transition device 8. The microfluidic unit 4 also has a readout portion 4b, which in this embodiment is a single location of the microfluidic unit.

[0282] In use, the sample is transferred from one or more selected mother sample containers 7 through the transfer device (multiple) tubes 6 and pump device 5.

[0283] The sample is supplied to the microfluidic unit 4 and enters the inlet section 4a under a relatively high pressure differential to ensure relatively rapid sample introduction. Once the sample has reached the inlet section 4a via the pump device 5, the pressure supplied by the pump device 5 is reduced or completely stopped. In the inlet section 4a, the condition jump device 8 heats the sample very rapidly to ensure the desired temperature jump.

[0284] Subsequently, the pump device 5 pumps the sample to the readout section 4b. The pressure is reduced so that the sample passes through the readout section 4b at a desired low speed to ensure the desired long reading time. As the sample passes through the readout section 4b, the reader device 11 performs multiple reads at, for example, the desired reading rate as described above.

[0285] Figure 2 The variant of the system shown includes a personal computer 12 with a screen 12a. The personal computer 12 is data-connected to a computer 9 integrated in device 1. The computer system includes the personal computer 12 and the computer 9.

[0286] Figure 3a This illustrates an implementation scheme for a suitable microfluidic unit in the form of a long, essentially straight tube with a narrow inner diameter.

[0287] Figure 3b This illustrates an implementation scheme for a suitable microfluidic unit in the form of a long, coiled tube with a narrow inner diameter.

[0288] Figure 3c An embodiment of a suitable microfluidic unit in the form of a microfluidic device 21 having a flat chamber 22 and an inlet 23 leading to the chamber 22 is shown.

[0289] Figure 3d This illustration shows an embodiment of a suitable microfluidic unit in the form of a microfluidic device 28 having a long, coiled channel 29a. The channel has an inlet 29c with a guide inlet portion 29d, at which the sample can be subjected to a temperature jump. The channel also has a readout portion 29b.

[0290] Figure 3e An embodiment of a suitable microfluidic unit in the form of a chamber provided by crystalline alumina 24, having a membrane cap 25 and a bottom, is shown. A sample can be introduced into the chamber via a tube 26. The figure also illustrates a portion of a conditional transition device suitable for performing pressure transitions. The conditional transition device includes a piezoelectric crystal stack 27 and a holding arm 27a adapted to hold the piezoelectric crystal stack 27 against the membrane 25.

[0291] Example 1 - HSA-fluorescein binding mating body test

[0292] A sample containing 83 micromoles of human serum albumin (HSA) and 10 nanomoles of HSA binding couplers, the HSA binding couplers being fluorescein (fl) in a buffer solution at pH 7.4.

[0293] Combination Figure 1 The experiment was conducted as described, wherein the temperature jump was a 10-degree jump from 5°C to 15°C. The resulting readings were plotted and displayed. Figure 4a middle.

[0294] Combination Figure 1 Another experiment was conducted as described, in which the temperature jump was a 20-degree jump from 5°C to 25°C. The resulting readings were plotted and displayed. Figure 4b middle.

[0295] exist Figure 4a In the process, the final temperature is 15℃, and the relaxation to equilibrium is controlled by the rate constant at 15℃. Figure 4b In this model, the final temperature is 25°C, and the relaxation to equilibrium is controlled by the rate constant at 25°C. The kinetic rate constant is higher at higher temperatures compared to lower temperatures. The relaxation kinetics can be described by the relaxation time, expressed in terms of tau.

[0296] S = a + b(1 - exp(-t / tau))

[0297] S is the signal obtained from the reader (in this case, a fluorescence reader), a is a constant describing the detection offset and / or background, b is the magnitude of the signal change between the initial and final states, and it is time.

[0298] Tau is quantified by the data and appropriately fitted to it. In more advanced data analysis, several Tau values ​​can be used to model relaxation, with several relaxation processes in progress.

[0299] Tau is associated with the rate constant, which is related to the molecular properties under study. For example, a 1-1 non-covalent interaction where A greatly exceeds I (A + I = AI) can be associated with tau according to the following equation:

[0300] tau=1 / (kon[A]+koff)

[0301] Kon and Koff are rate constants related to the formation and dissociation of the AI ​​complex.

[0302] Example 2a - LLPS Test

[0303] Prepare the master sample (a).

[0304] The following materials were used in this embodiment or in the following embodiments:

[0305] Fl-glucan: A fluorescently labeled glucan with a molecular weight of approximately 7,000 Daltons.

[0306] Dextran: Unlabeled dextran with a molecular weight of approximately 200,000 Daltons.

[0307] PEG: Poly(ethylene glycol), with a molecular weight of approximately 6,000 Daltons.

[0308] Water: Pure water (Type II).

[0309] Fl-HSA: Fluorescently labeled human serum albumin.

[0310] Aqueous master samples (a) were prepared from water, PEG and fl-glucan with a PEG concentration of 5% by mass and a fl-glucan concentration of 20 nM.

[0311] Combination Figure 1 The experiment was conducted as described.

[0312] The prepared master sample (a) is applied to the sample chamber 7 of sample compartment 3, and the temperature of the master sample is set to 50°C. The sample is removed from the master sample (a) and pumped into the inlet portion of the tube in the holding compartment, where it undergoes a 25-degree temperature jump from 50°C to 25°C. As the sample passes through, the fluorescence intensity is read at the readout portion.

[0313] The readings obtained in the readout section are as follows: Figure 5a As shown.

[0314] The symbol "s" indicates the start of the readout. During the first few seconds of the readout, the sample does not fully reach the readout section. When the sample reaches the readout section, the signal rises to its maximum level and remains substantially stable for the remainder of the readout time until the end of the data (DE). This leads to the conclusion that a single phase remained in place from the start to the end of the experiment; that is, no liquid-liquid phase separation occurred.

[0315] Example 2b - LLPS Test

[0316] A master sample (b) was prepared using the same materials as those listed in Example 2a.

[0317] Aqueous master sample (b) was prepared from water, dextran, PEG and fl-glucan with a PEG concentration of 5% by mass, a dextran concentration of 1% by mass and a fl-glucan concentration of 20 nM.

[0318] The experiment was conducted as described in Example 2a.

[0319] The readings obtained in the readout section are as follows:Figure 5b As shown.

[0320] The curve obtained in 5b is very similar to Figure 5a The curve, however, exhibits a slight instability immediately after reaching its maximum level, as indicated by label 32.

[0321] in addition, Figure 5b The maximum level reached was slightly lower than Figure 5a The level reached in China.

[0322] These characteristics indicate that the single phase of the sample has become unstable and show signs of liquid-liquid phase separation, such as the formation of droplets or bubbles of the separated phase.

[0323] Example 2c-LLPS test

[0324] A master sample (c) was prepared using the same materials as those listed in Example 2a.

[0325] Aqueous master sample (c) was prepared from water, dextran, PEG and fl-glucan with a PEG concentration of 5% by mass, a dextran concentration of 2% by mass and a fl-glucan concentration of 20 nM.

[0326] The experiment was conducted as described in Example 2a.

[0327] The readings obtained in the readout section are as follows: Figure 5c As shown.

[0328] As indicated by label 33a, in the curve obtained in 5c, a clear spike is visible immediately after the signal reaches its maximum level. Following the spike 33a, the signal strength drops to a lower level 33b, which is also lower than... Figure 5a and 5b The typical maximum intensity level shown in the figure.

[0329] These characteristics indicate that the sample has begun liquid-liquid phase separation. The instability of the signal intensity at a lower level of 33b also indicates the formation of droplets or bubbles of the separated phase.

[0330] Example 2 d-LLPS test

[0331] The master sample (d) was prepared using the same materials as those listed in Example 2a.

[0332] Aqueous master samples (d) were prepared from water, dextran, PEG and fl-glucan with a PEG concentration of 5% by mass, a dextran concentration of 3% by mass and a fl-glucan concentration of 20 nM.

[0333] The experiment was conducted as described in Example 2a.

[0334] The readings obtained in the readout section are as follows:Figure 5d As shown.

[0335] The curve obtained in 5d shows a very significant peak 34a, and the instability of the intensity level 34b increases after the peak 34a.

[0336] In addition, it can be observed that the intensity level after peak 34a is generally lower than the intensity level after peak in previous LLPS tests with lower amounts of dextran.

[0337] These characteristics indicate clear liquid-liquid phase separation of the sample, and that droplets or bubbles of the separated phase have formed.

[0338] Example 2 e-LLPS test

[0339] The master sample (e) was prepared using the same materials as those listed in Example 2a.

[0340] Aqueous master samples (e) were prepared from water, dextran, PEG and fl-glucan with a PEG concentration of 5% by mass, a dextran concentration of 4% by mass and a fl-glucan concentration of 20 nM.

[0341] The experiment was conducted as described in Example 2a.

[0342] The readings obtained in the readout section are as follows: Figure 5e As shown.

[0343] The curve obtained in 5e shows a very significant peak 35a. Furthermore, the intensity level 35b following peak 35a is significantly lower than, for example, as in Example 2d / Figure 5d Previous LLPS experiments using lower amounts of dextran. Figure 5e The intensity level 35b following the peak 35a is... Figure 2 Comparing the intensity levels of 34b after the peak of d34a, the intensity level in 5e is almost 30% lower.

[0344] These characteristics indicate that the formation of droplets or bubbles in the separated phase in Example 2e is greater than that in Example 2d.

[0345] Example 2 f-LLPS test

[0346] A master sample (f) was prepared using the same materials as those listed in Example 2a.

[0347] An aqueous master sample (f) was prepared from water, dextran, PEG and fl-glucan with a PEG concentration of 5% by mass, a dextran concentration of 5% by mass and a fl-glucan concentration of 20 nM.

[0348] The experiment was conducted as described in Example 2a.

[0349] The readings obtained in the readout section are as follows: Figure 5f As shown.

[0350] The curve obtained in 5f shows a very significant peak 36a. Furthermore, the intensity level 36b following peak 35a is even lower than in Example 2e / Figure 5e The intensity level following the mid-peak. This indicates that the liquid-liquid phase separation is even more complete, and that larger droplets or bubbles of the separated phase have formed.

[0351] Example 2 g-LLPS test

[0352] A master sample (g) was prepared from the same materials as listed in Example 2a.

[0353] Aqueous master samples (g) were prepared from water, dextran, PEG and fl-HSA with a PEG concentration of 5% by mass, a dextran concentration of 4% by mass and a fl-dextran concentration of 50 nM.

[0354] The experiment was conducted as described in Example 2a.

[0355] The readings obtained in the readout section are as follows: Figure 5g As shown.

[0356] The curve obtained at 5g shows a very high and significant peak at 37, clearly indicating that liquid-liquid phase separation occurred a few minutes after the temperature jump. After peak 37, the intensity level drops by about 45%, and the intensity signal becomes increasingly unstable over time, clearly indicating the formation of droplets or bubbles in the separated phase.

Claims

1. A method for determining a characteristic property of a molecular interaction, the method comprising providing a liquid sample, the liquid sample comprising a particle capable of being in an equilibrium state and in a non-equilibrium state in the liquid sample, the particle comprising a label in at least one of its equilibrium state and non-equilibrium state, bringing the particle into the non-equilibrium state by subjecting the sample to a condition jump comprising a temperature jump from at least one first temperature to a second temperature, reading out the label as a function of time during at least a portion of the relaxation time of the particle, and determining the characteristic property of the molecular interaction, wherein the temperature jump is performed by conduction and / or convection; wherein the particle is capable of being in an equilibrium state and in a non-equilibrium state because the sample comprises a binding partner of the particle, or because the particle has a temperature dependent structure; wherein the condition jump comprises subjecting the sample to a temperature jump from at least one first temperature to a second condition at a second temperature, and the method further comprises maintaining the second temperature during at least a portion of the reading out of the label; wherein the reading out comprises a reading out as a function of time comprising two or more readings from different portions of the sample in a microfluidic unit.

2. The method of claim 1, wherein the condition jump further comprises a pressure jump, wherein the difference between the first pressure and the second pressure is at least 1 bar.

3. The method of claim 1, wherein the particle has a temperature dependent structure, wherein the particle has an equilibrium structure under the second condition that is different from its structure prior to the condition jump.

4. The method of claim 3, wherein the change from the particle structure prior to the condition jump to the equilibrium structure under the second condition is a reversible change.

5. The method of claim 1, wherein the particle is a protein.

6. The method of claim 1, wherein the particle has an equilibrium conformation under the second condition that is different from its conformation prior to the condition jump.

7. The method of claim 1, wherein the sample comprises a binding partner of the particle, and at least one of the particle or binding partner comprises the label.

8. The method of claim 1, wherein the liquid sample comprises the particle and the binding partner in chemical equilibrium at the time the condition jump is initiated.

9. The method of claim 1, wherein the method comprises maintaining the sample at a constant temperature for at least 30 seconds prior to performing the temperature jump.

10. The method of claim 1, wherein the particle comprises an organic molecule, a molecular cluster, a molecular aggregate, a nanoparticle, a liposome vesicle, a micelle, or any combination comprising one or more of these.

11. The method of claim 1, wherein the particle comprises a biomolecule; a protein; a nucleoside; a nucleic acid or any fragment thereof and / or any combination comprising at least one of these. ​ ​ ​ ​ ​ ​ ​ ​ 12. The method of claim 1, wherein the method comprises preparing at least one master sample and removing the sample from the master sample.

13. The method of claim 1, wherein the method comprises performing the temperature jump from at least one first temperature to a second temperature in a jump time having a time extension that is less than the time required for the sample to reach equilibrium under the second conditions.

14. The method of claim 1, wherein the jump in temperature of the sample is performed in a microfluidic unit, the method comprising introducing the sample into the microfluidic unit, wherein the microfluidic unit is at least partially located in a temperature-controlled holding compartment.

15. The method of claim 14, wherein the microfluidic unit comprises an introduction portion into which the sample is introduced, the introduction portion comprising a cross-sectional dimension of 1 mm or less.

16. The method of claim 15, wherein the introduction portion comprises a flat chamber, a channel, two or more interconnected channels, or any combination comprising one or more of these.

17. The method of claim 15, wherein the introduction portion has a volume that is at least as large as the sample volume.

18. The method of any one of claims 14-17, wherein the temperature-controlled holding compartment is held at the second temperature during at least a portion of the relaxation time and during at least a portion of the readout.

19. The method of claim 18, wherein the temperature-controlled holding compartment is controlled in temperature by a method comprising blowing air.

20. The method of claim 18, wherein the temperature-controlled holding compartment is controlled in temperature by a method comprising fully or partially filling the compartment with a liquid and / or vapor.

21. The method of claim 1, wherein the temperature jump is performed by a method comprising blowing air or flowing a liquid through a container containing the sample, wherein, the container forms a portion of or comprises at least a portion of the microfluidic unit.

22. The method of claim 1, wherein the temperature jump is performed by a method comprising applying a joule heating element, a resistive element, and / or a Peltier element to conduct heat to the sample when the sample is located in the container.

23. The method of claim 22, wherein the temperature jump is performed when the joule heating element, the resistive element, and / or the Peltier element is positioned in physical contact with the container.

24. The method of claim 1, wherein the temperature jump from the at least one first temperature to the second temperature comprises providing a temperature jump of at least 2 °C.

25. The method of claim 1, wherein the second temperature is higher than the at least one first temperature.

26. The method of claim 1, wherein the second temperature is lower than the at least one first temperature.

27. The method of claim 1, wherein the second temperature is from 5 °C to 50 °C.

28. The method of claim 1, wherein the microfluidic unit comprises a flat chamber, a channel, two or more interconnected channels, or any combination comprising one or more of these.

29. The method of claim 1, wherein the microfluidic unit comprises a channel in the form of a tube or a chip, wherein the channel has a cross-sectional dimension of 1 mm or less.

30. The method of claim 1, wherein the microfluidic unit comprises an introduction portion and a readout portion.

31. The method of claim 30, wherein the introduction portion and the readout portion at least partially overlap.

32. The method of claim 30, wherein the introduction portion and the readout portion are distinct portions.

33. The method of claim 32, wherein the method comprises flowing at least a portion of the sample from the introduction portion to the readout portion.

34. The method of claim 1, wherein the reading comprises performing the reading of the sample while the sample is stationary in the microfluidic cell, wherein, The reading is performed from different portions of the sample by moving the reader device and the microfluidic unit relative to each other.

35. The method of claim 30, wherein the readout comprises performing a reading of the sample as the sample flows in the microfluidic unit.

36. The method of claim 35, wherein the readout as a function of time comprises performing the two or more readings from different portions of the sample as the sample flows in the readout portion of the microfluidic unit.

37. The method of claim 35 or claim 36, wherein the method comprises adjusting the flow velocity at the readout location to up to 50 cm / s.

38. The method of claim 1, wherein the readout as a function of time comprises performing successive readings from different portions of the sample as the respective sample portions pass through a readout location of the microfluidic unit.

39. The method of claim 1, wherein the label is an intrinsic label and / or an extrinsic label.

40. The method of claim 1, wherein the label is sensitive to molecular interactions, wherein, The label changes a signal as a function of the conformation of the particle and changes thereof.

41. The method of claim 1, wherein the label is an optically readable label that functions in the ultraviolet / visible wavelength range of 190 nm to 700 nm.

42. The method of claim 1, wherein the label is an electrochemically readable label.

43. The method of claim 1, wherein the readout of the label as a function of time during at least a portion of the relaxation time comprises performing a plurality of successive readings of the label, the readings comprising a plurality of readings of electrode potential, a plurality of readings of intensity at one or more wavelengths, and / or a plurality of readings of change at one or more wavelengths.

44. The method of claim 1, wherein the method comprises performing a plurality of successive readings of the label until successive readings vary by less than 25% from one reading to the next.

45. The method of claim 1, wherein the method comprises determining at least one of: a kinetic parameter; a partition parameter; a degradation parameter; an oligomerization parameter; a folding parameter; and a multiplexing parameter.

46. The method of claim 1, wherein the method comprises determining a characteristic property of a molecular interaction between a particle and two or more binding partners and / or between two or more particles and binding partners.

47. The method of claim 1, wherein said characteristic property of said molecular interaction comprises determining at least one kinetic parameter, including an equilibrium constant of said at least one particle and / or said at least one particle and said at least one binding partner, an affinity between said at least one particle and said at least one binding partner and / or determining one or both of two kinetic rate constants kon / koff.

48. The method of claim 1, wherein said method further comprises performing said method one or more additional times using different temperature jumps and / or using different concentrations of said particle and / or said binding partner.

49. The method of claim 1, wherein said method further comprises performing a diffusion assay and determining at least one diffusion parameter between a solution of said particle and a solution of said binding partner.

50. The method of claim 49, wherein said diffusion assay is performed on different concentrations of at least one particle and / or binding partner to determine a concentration at which at least one kinetic rate constant kon / koff is sensitive to changes.

51. The method of claim 1, wherein said molecular interaction comprises a liquid-liquid phase separation, said condition jump is a temperature jump comprising a temperature jump from at least one first temperature to a second temperature, and wherein said particle comprises at least two different molecules and optionally an additional solvent, said molecules being capable of forming a liquid-liquid phase separation under conditions prior to or after the temperature jump.

52. The method of claim 51, wherein said at least two different molecules comprise at least one polymer and / or at least one lipid.

53. The method of claim 51, wherein said solvent is an organic solvent and / or water.

54. The method of claim 51, wherein said liquid sample is in a single phase condition at a time immediately prior to subjecting the sample to the temperature jump.

55. The method of claim 51, wherein said temperature jump is a jump from a higher temperature to a lower temperature, wherein said sample is in a single phase condition at the higher temperature.

56. The method of claim 51, wherein said liquid-liquid phase separation comprises at least a partial formation of a first liquid phase having an interface with a second liquid phase.

57. The method of claim 56, wherein said first liquid phase and said second liquid phase differ from each other in concentration of at least one molecule.

58. The method of claim 51, wherein the content of said sample is known or unknown, and said characteristic property of said liquid-liquid phase separation comprises at least one of an ability to form a liquid-liquid phase separation.

59. The method of claim 51, wherein the content of said sample is unknown, and said method comprises identifying a portion of the sample that is capable of forming a liquid-liquid phase separation under selected conditions after the temperature jump.

60. The method of claim 51, wherein said method comprises separating a target portion of said sample from a remaining portion of said sample, wherein said target portion of said sample is a portion having at least one indication of a liquid-liquid phase separation formation.

61. The method of claim 51, wherein the sample is subjected to a temperature jump in a channel of the microfluidic cell and readout is performed in the channel, wherein the sample is fed to the channel under a pressure that ensures a selected velocity of the sample in the channel, wherein, said velocity is adjustable in accordance with a liquid-liquid phase separation state determined by said readout.

62. The method of claim 61, wherein the method further comprises acquiring an image of at least one partial cross-section of the channel.

63. An apparatus suitable for determining a characteristic property of a molecular interaction according to the method of claim 1, the apparatus comprising a sample compartment for holding at least one liquid master sample; a pipetting device arranged for pipetting a sample from at least one master sample stored in the sample compartment, a pump device adapted to transport the sample to a microfluidic cell, a condition jump device arranged for performing a temperature jump of the sample in the microfluidic cell from at least one first temperature to a second temperature, and at least one reader device for reading at least one marker as a function of time, wherein the apparatus is adapted to perform the temperature jump by conduction and / or convection with the sample contained in the microfluidic cell; wherein the apparatus is adapted to perform the read-out as a function of time by performing two or more readings in the microfluidic cell from different parts of the sample; wherein the apparatus further comprises a holding compartment for holding the sample at the second condition during the read-out of the marker with the sample contained in the microfluidic cell.

64. The apparatus of claim 63, wherein the sample compartment comprises at least one temperature control device for selecting and controlling the temperature of at least one master sample located in a master sample chamber of the sample compartment.

65. The apparatus of claim 63, wherein the pipetting device forms part of or is in fluid communication with the microfluidic cell.

66. The apparatus of claim 63, wherein the pipetting device comprises means for pipetting a sample from a sample and transporting it to an inlet of the microfluidic cell.

67. The apparatus of claim 63, wherein the pipetting device comprises a pump device.

68. The apparatus of claim 63, wherein the pipetting device is adapted to pipette a sample from one single master sample chamber.

69. The apparatus of claim 63, wherein the pipetting device is adapted to pipette a sample from two or more master sample chambers.

70. The apparatus of claim 63, wherein the condition jump device comprises a heating and / or cooling device adapted to perform a temperature jump from the first temperature to the second temperature.

71. The apparatus of claim 63, wherein the condition jump device is arranged for performing the jump of the temperature of the sample in the microfluidic cell, wherein the condition jump device is at least partially located in a temperature controlled holding compartment.

72. The apparatus of claim 63, wherein the condition jump device and / or the holding compartment comprises a temperature controller device comprising an air blower for blowing air at a selected temperature and / or a liquid sprayer for spraying a liquid at a selected temperature and / or a liquid filler for completely or partially filling the holding compartment with a liquid at a selected temperature. ​ ​ ​ ​ ​ ​ ​ ​ 73. The apparatus of claim 63, wherein, The condition jump device comprises a Joule heating element, a resistive element and / or a Peltier element arranged to conduct heat to the sample when the sample is located in a container forming part of or comprising at least part of the microfluidic unit.

74. The device of claim 63, wherein the at least one reader device comprises an optical reader device and / or an electrochemical reading device.

75. The device of claim 63, wherein the at least one reader device is adapted to perform multiple readings as a function of time.

76. The device of claim 63, wherein the at least one reader device is fixedly located in the device, wherein the reader device is adapted to perform a reading of a marker of a sample portion as the sample portion passes the reader device.

77. The device of claim 63, wherein the pipetting device is configured to feed the sample to an inlet of the microfluidic unit under a feed pressure, wherein the feed pressure is adjustable.

78. The apparatus of claim 63, wherein the microfluidic unit comprises a channel, and the apparatus comprises an image acquisition unit positioned to acquire an image of at least a portion of the sample downstream of the location at which the sample is subjected to the conditional jump, wherein, The image acquisition unit is positioned to acquire at least one local portion of the channel downstream of the readout position.

79. An assembly comprising the device of claim 63 in combination with a microfluidic unit, wherein the microfluidic unit is at least partially located in the temperature- controlled holding compartment.

80. The assembly of claim 79, wherein the microfluidic unit comprises a flat chamber, a channel, two or more interconnected channels, or any combination comprising one or more of these.

81. The assembly of claim 79, wherein the microfluidic unit comprises a channel in the form of a tube or a sheet, wherein the channel has a cross-sectional dimension of 1 mm or less.

82. The assembly of claim 79, wherein the microfluidic unit comprises an introduction portion adapted to introduce the sample therein, the introduction portion comprising a cross-sectional dimension of 1 mm or less.

83. The assembly of claim 79, wherein the microfluidic unit comprises an introduction portion and a readout portion.

84. The assembly of claim 83, wherein the introduction portion and the readout portion at least partially overlap.

85. The assembly of claim 83, wherein the introduction portion and the readout portion are different portions.

86. The assembly of claim 79, wherein a reader device is positioned to read out from a fixed readout position of the microfluidic unit.

87. The assembly of claim 79, wherein the device comprises a pump device.

88. A system for determining a characteristic property of a molecular interaction, comprising the device of claim 63 or the assembly of claim 79 and a computer system, wherein the computer system is configured to control the pipetting device, control the temperature jump and diffusion device, control the reader device, and / or determine the characteristic property of the molecular interaction.

89. The system of claim 88, wherein the molecular interaction comprises a change in the structure of the particle and / or a change in binding between the particle and a binding partner of the particle, wherein, The molecular interaction comprises a change in conformation.

90. The system of claim 88, wherein the computer system is configured to determine at least one of: a kinetic parameter; a partitioning parameter; a degradation parameter; an oligomerization parameter; and a folding parameter.

91. The system of claim 88, wherein the computer system is configured to determine a kinetic parameter.

92. The system of claim 88, wherein the computer system is configured to determine a partitioning parameter.

93. The system of claim 88, wherein the computer system is configured to determine a degradation parameter.

94. The system of claim 88, wherein the computer system is configured to determine an oligomerization parameter.

95. The system of claim 88, wherein the computer system is configured to determine a folding parameter.

96. The system of claim 88, wherein the computer system is configured to determine a kinetic parameter and a partitioning parameter.

97. The system of claim 88, wherein the computer system is configured to determine a kinetic parameter and a degradation parameter.

98. The system of claim 88, wherein the computer system is configured to determine a kinetic parameter and an oligomerization parameter.

99. The system of claim 88, wherein the computer system is configured to determine a kinetic parameter and a folding parameter.

100. The system of claim 88, wherein the computer system is configured to determine a partitioning parameter and a degradation parameter.

101. The system of claim 88, wherein the computer system is configured to determine a partitioning parameter and an oligomerization parameter.

102. The system of claim 88, wherein the computer system is configured to determine a partitioning parameter and a folding parameter.

103. The system of claim 88, wherein the computer system is configured to determine a degradation parameter and an oligomerization parameter.

104. The system of claim 88, wherein the computer system is configured to determine a degradation parameter and a folding parameter.

105. The system of claim 88, wherein the computer system is configured to determine an oligomerization parameter and a folding parameter.

106. The system of claim 88, wherein the computer system is configured to determine a kinetic parameter, a partitioning parameter, a degradation parameter, an oligomerization parameter, and a folding parameter.

91. The system of claim 88, wherein the computer system is configured to determine a characteristic property of a molecular interaction between a particle and two or more binding partners and / or between two or more particles and binding partners.

92. The system of claim 88, wherein the computer system is configured to determine at least one kinetic parameter selected from the group consisting of an equilibrium constant of the at least one particle and / or of the at least one particle and the at least one binding partner, an affinity between the at least one particle and the at least one binding partner and / or one of two kinetic rate constants kon / koff.

93. The system of claim 88, wherein the computer system is configured to control the performance of the method according to claim 1.

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