Method of determining a binding parameter between a first particle and a second particle

Abstract

A method for determining binding parameters between a first particle (L) and a second particle (A) capable of non-covalently interacting with the first particle. The method includes acquiring N sets of raw data, each set of raw data acquired by performing a Taylor dispersion determination, and processing each of at least one set of raw data. Each Taylor dispersion determination for acquiring the corresponding set of raw data includes i) supplying a liquid portion comprising a sample portion containing the second particle (A) and at least one supplementary portion into a microfluidic channel, such that the sample portion and at least one supplementary portion form an interfacial contact in the microfluidic channel, wherein at least one of the liquid portions comprises a certain concentration of the first particle (L); ii) supplying the liquid portion to a flow in the microfluidic channel; and iii) acquiring the set of raw data, which includes reading a marked signal intensity s(t) as a function of time. Each set of raw data includes at least one imbalance readout, and processing each of the N sets of raw data includes determining the apparent diffusivity D and the hydrodynamic radius R. h At least one of them, and thus determine and will be the fitting equation for each of the N sets of original data.

Description

Technical Field

[0001] This invention relates to a method for determining binding parameters between a first particle and a second particle, wherein the first particle and the second particle are capable of non-covalent bonding to form a complex. The method of this invention is particularly suitable for determining at least one kinetic binding parameter between the first particle and the second particle. Background Technology

[0002] The complete or partial characterization of particle association, especially when at least one of the first and second particles is a biomolecule, is important for many technologies, such as in various chemical processes, for example in pharmaceuticals, food and diagnostics, for understanding, developing and administering drugs, for understanding and controlling chemical processes of food, for understanding biological systems and / or for performing diagnostic tests and optionally for identifying biochemical disorders.

[0003] The article “How to measure and evaluate binding affinities” (by Jarmoskaite et al., eLife 2020;9:e57264, DOI: https: / / doi.org / 10.7554 / eLife.57264) describes the equilibrium constant K between the protein Puf4 and its homologous RNA sequence. D The determination of epigenetic K was achieved by mixing Puf4 with trace amounts of tagged RNA at different temperatures and incubation times, followed by non-denaturing gel electrophoresis to determine the fraction of bound RNA. The article concludes that more than 4.5 hours of incubation is required to reach equilibrium, and that only 30 minutes of incubation provides 7-fold higher epigenetic K compared to 24 hours of incubation. D Value. The article discusses many available K values. D The value may be incorrect because the equilibration time may be too short. Furthermore, the article indicates that temperature can significantly affect K. D value.

[0004] The article "Study of Binding between Protein A and Immunoglobulin G Using a Surface Tension Probe" (authors Yang et al., Journal of Biophysics, Vol. 84, January 2003, 509-522) discloses a method for determining molecular interactions through surface tension measurements. It describes how surface tension measurements can quantify the molecular binding ratio (or molecular binding capacity) between interacting species.

[0005] The article “Time, the Forgotten Dimension of Ligand Binding Teaching” (by Javier Orzo, Biochemistry and Molecular Biology Education, Vol. 34, No. 6, pp. 413-416, 2006) discusses the kinetic parameter constant k. on and k off The importance of determining a single constant k through surface plasmon resonance. on and k off .

[0006] New and reliable methods are still needed to determine the binding parameters between the first particle (A) and the second particle, which is capable of non-covalent interaction. Summary of the Invention

[0007] The purpose of this invention is to provide a relatively fast and reliable method for determining the binding parameters between a first particle and a second particle capable of interacting with the first particle.

[0008] In one embodiment, the aim is to provide a relatively fast and reliable method for determining the binding parameters between a first molecule and a second molecule capable of non-covalent interaction.

[0009] In one embodiment, the aim is to provide a relatively rapid and reliable method for determining binding parameters between a first molecule and a second molecule capable of non-covalent interaction, wherein the binding parameters include at least one kinetic binding parameter, preferably including at least one of the following: k off (Dissociation rate), k on (Association rate), k obs (The rate constant at which equilibrium is reached), K D (Equilibrium dissociation constant), R AL (hydrodynamic radius of AL), R A (The hydrodynamic radius of A), D A (Diffusion rate of unbound A), D AL (AL diffusion rate) and / or any binding parameter obtained from one or more of the mentioned binding parameters.

[0010] These and other objectives have been achieved by the invention as defined in the claims or by embodiments thereof, as described below.

[0011] It has been found that the present invention or its embodiments have many additional advantages, which will become clear to those skilled in the art from the following description.

[0012] Abbreviations and symbols used in this article:

[0013] K D or K d Equilibrium dissociation constant

[0014] k off Dissociation rate

[0015] k on Association rate

[0016] k obs The rate constant at which equilibrium is reached.

[0017] R A The hydrodynamic radius of A

[0018] R AL : Hydrodynamic radius of AL

[0019] R h Apparent hydrodynamic radius of A+AL

[0020] t ½ : Reaction half-life (association)

[0021] k(t): Time-dependent dispersion coefficient

[0022] t: time

[0023] D(t): Time-dependent diffusivity constant

[0024] D: Apparent diffusion rate of the second particle A (free A and bound A+L complex)

[0025] : Diffusion rate of unbound A

[0026] : Diffusion rate of AL

[0027] R c : Capillary radius, or if it is non-circular, then = 2 * cross-sectional area / perimeter

[0028] t R Peak occurrence time

[0029] : Tortuous diffusion rate

[0030] L: First Particle

[0031] A: The second particle

[0032] S(t): Signal strength as a function of time

[0033] a, p, and q: Fitting constants

[0034] σ 2Signal peak variance

[0035] : Peak width as a function of time

[0036] η: viscosity

[0037] Linear flow rate

[0038] T: Kelvin temperature

[0039] A f (t): Time dilution factor

[0040] Average dilution factor

[0041] Pr: Operating pressure

[0042] Pi: Injection pressure

[0043] C A Baseline concentration of A (concentration of A in the sample portion)

[0044] C L Baseline concentration of L (concentration of L in the sample portion)

[0045] S L : Base concentration of L (concentration of L in the supplementary section)

[0046] : The total concentration of A in the sample / dispersion region without dilution ~ C A

[0047] L T : Concentration of all L in the sample / dispersion region without dilution ~ C L

[0048] The average dilution concentration of all A molecules (the average concentration of A in the dispersed region).

[0049] The average dilution concentration of all L (the average concentration of L in the dispersion region).

[0050]

[0051] S: The concentration of [LA] or [AL] in the sample (dispersion region) at time 0.

[0052] [L]: Concentration of unbound L

[0053] [A]: Concentration of unbound A

[0054] [LA] or [AL]: Concentration of the AL / LA complex

[0055] C(x,t): Particle concentration (solute concentration) at position x at time t.

[0056] x: Axial position in front of the dispersion region

[0057] TDA: Taylor dispersion assay

[0058] The phrase “molecular interaction” means any non-covalent interaction between a first particle and a second particle, where the particles, which are independent of each other, can be molecules, molecular clusters, or molecular complexes.

[0059] The term "particle" is used herein to mean any part of a substance that includes at least one molecule, such as an organic or inorganic molecule. A particle may include, for example, an aggregate, a cluster, a complex, or any combination of one or more of these.

[0060] The terms "first particle L", "first particle", and the abbreviation "L" are used interchangeably.

[0061] The terms "second particle A", "second particle", and the abbreviation "A" are used interchangeably.

[0062] The phrase “concentration of the first particle L” in this document means the total concentration of the first particle under both bound and unbound conditions, unless otherwise stated or clearly indicated from the context.

[0063] The phrase “concentration of the second particle L” in this document means the total concentration of the first particle under both bound and unbound conditions, unless otherwise stated or clearly indicated from the context.

[0064] All concentrations are molar concentrations unless otherwise stated or clearly indicated from the context.

[0065] The term “binding partner” in this article means any molecule or group of molecules that can interact non-covalently with a particle.

[0066] Microfluidic channels are also known as "capillaries".

[0067] The terms “mark” or “tag” are used interchangeably and herein are used to mean any intrinsic or extrinsic mark / tag that can be detected by a reader device. In one embodiment, a mark / tag includes an element, a group of elements, a portion, and / or any combination of one or more of these, wherein the mark can be detected directly by a reader device and / or after being affected by external and / or internal sources. Similarly, the terms “marked” and “labeled” are used interchangeably.

[0068] The term "reader device" means any detector or detector system capable of detecting signals (such as optical and / or electrochemical signals) associated with binding partners and / or particles.

[0069] The term "buffer" means an aqueous solution that resists changes in pH in an environment where the buffer is used. Buffers advantageously include aqueous solutions of either a weak acid and its salt or a weak base and its salt.

[0070] It should be emphasized that the term “includes / contains” as used herein is interpreted as an open-ended term, that is, it should be understood as specifying the presence of a particular statement feature (such as an element, unit, integer, step, component, or combination thereof), but does not exclude the presence or addition of one or more other statement features.

[0071] The reference to "some embodiments" or "one embodiment" means that a particular feature, structure, or characteristic described in conjunction with these embodiments is 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 embodiment. Furthermore, those skilled in the art will understand that, within the scope of the invention as defined by the claims, particular features, structures, or characteristics can be combined in any suitable manner.

[0072] Throughout the specification or claims, the singular includes the plural, unless otherwise stated or required by the context.

[0073] All features of the invention and its embodiments described herein, 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 these features.

[0074] The inventors of this invention have recognized that by applying a combination of Taylor dispersion determination (TDA) data processing techniques using one or more of the acquired raw data, one or more binding parameters associated with the non-covalent interaction between the first particle L and the second particle A can be found in an alternative and highly efficient manner. Taylor dispersion determination is well known in the art for determining the diffusion coefficient of a class of particles and thus the hydrodynamic radius of that class. Taylor dispersion determination is based on the dispersion of a sample plug comprising a class of particles (e.g., between buffer plugs) in a narrow channel under laminar Poiseuille flow (pressure-induced flow). This dispersion is due to the combined effect of the parabolic velocity profile of the dispersion and the molecular diffusion of the class of particles, which causes the molecules to redistribute within the cross-section of the channel. The Taylor dispersion signal profile can be recorded as a function of time, S(t). TDA is described, for example, in US9310359 and WO22237946, under which conditions the dispersion of a solute in a solvent flow can be used to measure molecular diffusion. By SIR Geoffrey Taylor (1954) https: / / royalsocietypublishing.org / , February 27, 2023, or in “Microfluidics and the quantification of biomolecular interactions” (authors Otzen et al., Frontiers in Structural Biology, 2021, 70:8-15, https: / / doi.org / 10.1016 / j.sbi.2021.02.006).

[0075] The method of the present invention aims to determine at least one binding parameter between a first particle (L) and a second particle (A) capable of non-covalently interacting with the first particle, wherein the second particle includes a selected marker. This marker may conveniently be or include an intrinsic marker and / or an extrinsic marker when desired. The method includes acquiring N sets of raw data, each set of raw data being acquired by performing a Taylor dispersion determination, and processing each of at least one set of raw data. Each Taylor dispersion determination used to acquire the corresponding set of raw data includes:

[0076] i. A liquid portion comprising a sample portion containing a second particle (A) and at least one supplementary portion is supplied into a microfluidic channel such that the sample portion and at least one supplementary portion form an interfacial contact in the microfluidic channel, wherein at least one of the liquid portions comprises a certain concentration of the first particle (L).

[0077] ii. Provide a liquid component to the flow in the microfluidic channel;

[0078] iii. Obtain the raw data set, which includes reading the signal strength s(t) as a function of time;

[0079] The terms “supply” and “injection” are used interchangeably.

[0080] The formation of an interfacial contact between the sample portion and at least one supplementary portion in the microfluidic channel can provide modification of the sample portion to the modified sample portion, and can introduce a concentration jump of at least one of the first particle L and the second particle A into the modified sample portion. Therefore, the concentrations of the second particle A and the at least one supplementary portion can be different from each other, and advantageously, the supplementary portion should be free of the second particle A, or conveniently, the concentration of the second particle should be very low, such as at most 50%, at most 10%, or at most 1% of the concentration of the second particle A in the sample portion.

[0081] If the first particle L and the second particle A, as well as the interaction between them, become unbalanced, a concentration jump can induce an association reaction or a dissociation reaction.

[0082] Each set of raw data includes multiple intensity reads of the marker for the second particle A, wherein each set of raw data includes at least one imbalance read, and preferably multiple imbalance reads.

[0083] The processing of each of the N sets of raw data includes determining the apparent diffusivity D and the hydrodynamic radius R. h At least one of them,

[0084] The apparent diffusivity D is determined by applying Equation 12a.

[0085] (12)

[0086] and / or

[0087] By applying Equation 23, the hydrodynamic radius R... h Sure

[0088] (twenty three)

[0089] And will be determined for each of the N sets of original data. Fit to Equation 21

[0090] (twenty one)

[0091] One or more of a, p, and q are fitting parameters, and at least one binding parameter is determined by one or more of the fitting parameters, wherein the binding parameter preferably includes at least one of a kinetic parameter or an affinity parameter.

[0092] The inventors discovered that the fitting parameters can be described as equations, where the equation to be applied depends on whether the concentration jump induces an association or dissociation reaction. Furthermore, the equation to be applied can depend on whether the corresponding concentrations of the first particle L and the second particle A in the dispersion region can be considered constant.

[0093] In one embodiment, at least one of the N original datasets is obtained from a Taylor dispersion assay, wherein a concentration jump induces an association reaction, and the method includes determining at least one binding parameter by applying one or more of equations E1, E2, and / or E3 from one or more fitting parameters a, p, and q.

[0094] (E1)

[0095] (E2)

[0096] (E3)

[0097] Where the concentration of L in the dispersion region can be considered constant, L X It is L T And when the concentration of L in the dispersion region can not be considered constant, L X Yes, it's L. d And where the concentration of A in the dispersion region can be considered constant, A X It is 0, and when the concentration of A in the dispersion region can not be considered constant, A X It is A d Equations E1, E2, and E3 are also referred to as a set of equations E1, E2, and E3.

[0098] In one embodiment, at least one of the N original datasets is obtained from a Taylor dispersion assay, wherein a concentration jump induces a dissociation reaction, and the method includes determining at least one binding parameter by applying one or more of the following equations from one or more fitting parameters a, p, and q.

[0099] (50)

[0100] (51)

[0101] (52)

[0102] In this document, the first particle is also referred to as L, and the second particle is also referred to as A. The first particle L and the second particle A may be molecules, clusters, aggregates, or complexes, independently of each other. Advantageously, one or both of the first particle L and the second particle A are molecules. Equations 50, 51, and 52 are also referred to as a set of equations 50, 51, and 52.

[0103] Taylor dispersion determination can also be described as dispersion determination for which Taylor conditions apply.

[0104] An imbalance read is a read that occurs when the interaction between the first and second particles is unbalanced.

[0105] Referring to this set of raw data means at least one of the N sets of raw data. As further described below and / or as shown in the examples, determine... The processing of each of the N sets of original data can be the same or different.

[0106] Any method, such as those known in the art, can be used to determine the apparent diffusivity D from the raw data set, as described in Poulsen et al., “Flow induced dispersion analysis rapidly quantifies proteins in human plasma samples”, DOI: 10.1039 / C5AN00697J. Analyst, 2015, 140, 4365-4369; in Jensen et al., “Flow Induced Dispersion Analysis Quantifies Noncovalent Interactions in Nanoliter Samples”, JAMSCI Journal, 132(12):4070-1, March 2010, DOI: 10.1021 / ja100484d and / or as described in US9310359.

[0107] The apparent diffusivity D can preferably be determined by a method including fitting the set of original data to the following equation.

[0108] (12b)

[0109] here It is the detector / background offset, and It is a constant related to the fluorescence intensity and response factor of the detector. During the Taylor dispersion assay, the raw data S(t) is recorded as a function of time t, and the parameters are determined by fitting the raw data to Equation 12b. D and .

[0110] The hydrodynamic radius R can be determined from the raw data using any method (such as that described in the articles specified above and / or WO21180289). h In one embodiment, the hydrodynamic radius R is determined based on the apparent diffusivity D by applying Equation 22. h

[0111] (twenty two)

[0112] The processing of the corresponding N sets of raw data may include preprocessing steps such as filtering the raw data, including removing outliers, signal noise, etc.

[0113] The liquid portion is, or advantageously includes, a buffer as a base liquid, wherein one or more of the mentioned particles can be dispersed, and in one embodiment, at least one supplementary portion may be a pure buffer. The buffer is advantageously selected according to the first particle L and the second particle A to provide the desired pH level for the interaction between the particles.

[0114] After extensive effort and thorough analysis, the inventors of this invention have achieved a method for determining one or more bonding properties in a relatively simple, rapid, and reliable manner. In particular, it aims to determine kinetic bonding parameters, which specifically include k. off (Dissociation rate), k on (Association rate) and k obs (The rate constant at which equilibrium is reached), these parameters are cumbersome and difficult to determine precisely enough using existing methods. However, other combined parameters, such as the kinetic parameter K, can be used. D (Equilibrium dissociation constant), R AL (hydrodynamic radius of AL), R A (The hydrodynamic radius of A), D A (Diffusion rate of unbound A), D AL The diffusion rate of AL can be determined with relatively high accuracy by applying the method of the present invention, which is also very beneficial.

[0115] Advantageously, at least one binding parameter includes one or more of the following: k off (Dissociation rate), k on (Association rate), k obs(The rate constant at which equilibrium is reached), R AL (hydrodynamic radius of AL), R A (The hydrodynamic radius of A), D A (Diffusion rate of unbound A), D AL (AL diffusion rate) and / or any derived parameter of one or more of the mentioned binding parameters.

[0116] The underlying principles and effects of this invention can be explained within the following theoretical framework.

[0117] We assume that binding kinetics can be evaluated by achieving conditions where no equilibrium is established. Here, we assume that a simple concentration change of one of the equilibrium substances can be applied.

[0118] Taylor dispersion measurements have been found to be applicable to providing information on rapid changes in particle concentration in microfluidic capillary flow systems.

[0119] This can be provided as described above, including a sample portion containing a second particle (A) and at least one supplementary portion and an optional influence portion of liquid portion being supplied to and provided to a laminar flow in a microfluidic channel, wherein at least one liquid portion comprises a certain concentration of the first particle (L).

[0120] Although there are many variations in the content of A and L in the sample portion and in one or more (usually two—leading supplement and following supplement) and optional effect portion, there are mainly four basic types of Taylor dispersion determinations for inducing imbalance states.

[0121] Type 1, known as "capmix", consists of a sample portion containing a certain concentration of unbound second particles A and no first particles L, and a supplementary portion containing a certain concentration of first particles L and essentially no second particles A.

[0122] Type 2, known as "premix", includes a sample portion consisting of a certain concentration of second particle A and a certain concentration of first particle at equilibrium, and a supplementary portion consisting of a certain concentration of first particle L but without second particle A.

[0123] Type 3, known as "dismix", consists of a sample portion containing a certain concentration of a second particle A and a certain concentration of a first particle at equilibrium, and a supplementary portion consisting primarily of a pure buffer free of both A and L.

[0124] Type 4, known as “cap-dis-mix”, further includes an influence portion supplied to the microfluidic channel to form an interface with the sample portion, wherein the sample portion includes a certain concentration of second particle A and substantially contains no first particle L, the influence portion includes a certain concentration of first particle L and substantially contains no second particle A, and the supplement portion substantially contains neither the first particle L nor the second particle A.

[0125] In the premix assay, the first particle L and the second particle A are mixed to ensure equilibrium is established before performing the Taylor dispersion assay. Further, L is introduced into the supplementary fraction. When the concentration of L in the supplementary fraction differs from the concentration of L in the sample fraction, the concentration of L in the modified sample fraction rapidly becomes equal to the concentration of L in the supplementary fraction, thus inducing a concentration jump in the modified sample fraction under conditions of induced imbalance, especially when A is initially in excess in the sample fraction. When A is in excess in the sample fraction and the concentration of L in the supplementary fraction is greater than that in the sample fraction, the concentration jump induces an association reaction. When A is in excess in the sample fraction and the concentration of L in the supplementary fraction is less than that in the sample fraction, the concentration jump induces a dissociation reaction.

[0126] In capmix assays, at time 0, i.e., before the liquid portion is supplied to the microfluidic channel, there is no binding between A and L. Mixing and binding occur between A and L as the sample passes through the capillary (microfluidic channel)—that is, the concentration jump in capmix assays induces an association reaction. Ideally, assay conditions can be selected such that the injected sample is only a very small portion of the capillary, i.e., the volume of the sample portion is much smaller than the total volume of the replenished portion. Under such conditions, L is rapidly mixed into the modified sample portion, and it can be assumed that L is uniformly distributed in the capillary at times greater than 0.

[0127] Suppose that the apparent diffusion coefficient describing dispersion is the time-dependent weighted average of the diffusion rates of bound A and unbound A. It has been found that, under this condition, a k-dependent coefficient can be obtained. on and k off The solution is a dispersed, closed-form solution.

[0128] When the dynamics are combined slowly enough to perform an imbalance read, it has been found that the data can be analyzed in a way that extracts dynamic information.

[0129] LA is a complex in solution composed of L and A.

[0130] LA = L + A, K in equilibrium state d = [L][A] / [LA](1)

[0131] k off

[0132] LA -> LA + A(2)

[0133] k on

[0134] L + A -> LA(3)

[0135] K d = k off / k on (4)

[0136] Different constraints may depend on the assemblage dynamics. It is useful to define the relaxation dynamic constant as follows:

[0137] k obs = k off + k on *[A](5)

[0138] In terms of kinetics, a convenient estimate of the system can be studied when the reaction half-life is defined as:

[0139] t ½ = ln2 / k obs (6)

[0140] Taylor dispersion assays, also known as flow-induced dispersion analysis (FIDA), can be conveniently performed in microfluidic channels in the form of fine capillaries, with peak occurrence times as low as 10 seconds or even less. Based on changes in size (or labeling, such as fluorescence) at binding time, a rough estimate of the binding kinetics obtainable would correspond to tg times of 1–5 seconds and longer. ½ conditions.

[0141] On the other hand, using very low flow rates / long capillaries could potentially make the peak time reach approximately 1 hour (3600 seconds) and possibly even longer.

[0142] In the case where L exceeds A, referred to as the first case, a first theoretical framework for capmix-type determinations is elucidated. Assume t ½ Between 1 second and approximately 3600 seconds (60 minutes).

[0143] The concentration used for fitting to L (L) T The appearance of the function R) h In the following equation for the binding curve, the concentration of L in the supplementary portion exceeds the concentration of A in the sample portion.

[0144] Due to rapid mixing (small sample volume), it can be assumed that the concentration of L is constant in the dispersion region (including the region of the microfluidic channels of the modified sample). Under these conditions, the kinetics can be considered independent of the precise concentration of A. Furthermore, it is assumed that the Taylor conditions apply.

[0145] The Taylor condition implies that the quality transmission problem is significantly simplified. This means that the signal shape can be described by the following differential equation:

[0146] (7)

[0147] Where k(t) is the dispersion coefficient, which is obtained as follows:

[0148] (8)

[0149] in

[0150] (9)

[0151] It is the capillary radius, and t R This refers to the time when the peak occurs.

[0152] In the limiting case of constant diffusion rate, k(t) (and D(t)) are simply constants.

[0153] However, for the current case, the diffusivity is modeled as a weighted average of bound and unbound A. Therefore, the apparent diffusivity is time-dependent (controlled by the kinetics of complex formation) but decoupled from spatial diffusion.

[0154] Conveniently convert the tortuous diffusivity Defined as:

[0155] (10)

[0156] It can be considered as the average dispersion during the Fida experiment.

[0157] By changing the variables, equation 7 can be rewritten as follows:

[0158] (11)

[0159] Therefore, the original data appears to be fit to a solution similar to that used under Taylor conditions, with boundary conditions corresponding to pulse injection.

[0160] According to the general Gaussian solution With peak variance (at t) R The relevant information (for the location) is as follows:

[0161] (12)

[0162] Where D is the apparent diffusivity obtained from fitting the original data (S(t), fluorescence intensity relative to time). Under the condition that the Taylor condition applies, the original data can be fitted to the following equation:

[0163] (12b)

[0164] here It is the detector / background offset, and It is a constant related to the fluorescence intensity and response factor of the detector. The raw data S(t) is recorded as a function of time t, and the parameters are determined from the raw data to Equation 12b. D and .Then, It can be obtained from Equation 12.

[0165] The apparent diffusivity varies depending on the binding fraction of A. Initially, A binds 0% with L, but during the experiment, the binding fraction increases as described in the binding kinetics.

[0166] Under these conditions, the time-dependent diffusivity D(t) is related to the binding fraction according to the following:

[0167] D(t) = x(t)·D I + (1-x(t))·D AL (13)

[0168] The rate equations for AL can be solved (corresponding to equations 2 and 3), and for the boundary condition that [AL] is 0 at time 0, the solution is:

[0169] (14)

[0170] Where A T It is the concentration of all A in the dispersed region, and L T It represents the concentration of all ligands.

[0171] Then there is:

[0172] (15)

[0173] Combining equations 15 and 13, we get:

[0174] (16)

[0175] Note that the first two (time-independent) terms correspond to case 1, and the normal 1-1 equilibrium associative model was used to obtain K in the Fida experiment. d .

[0176] Equation 16 can be simplified using the following parameters, which can then be applied as fitting parameters as described below:

[0177] (17)

[0178] (18)

[0179] (19)

[0180] Therefore, equations 17, 18 and 19 correspond to equations E1, E2 and E3, where the concentrations of L and A in the dispersion region can be considered constant.

[0181] Equations 17, 18, and 19 are also referred to as a set of equations 17, 18, and 19.

[0182] Next, in t R Evaluation at time (Equation 10) is given by the following formula:

[0183] (20)

[0184] This integral can be solved analytically and has a solution:

[0185] (twenty one)

[0186] Through this set of equations With D A D AL K d and dynamic parameters (k) off and k on This is related to the measurement according to Equation 12. Change L in titration T Then, it is fitted to Equation 21, in principle obtaining all the above parameters. In practice, two or more experiments are conducted, including given D. A D AL and K D Better accuracy can be obtained through premix experiments and capmix experiments that can fit the remaining unknown kinetic parameters. Alternatively, or additionally, experiments can be performed at different t... R Several Capmix experiments are executed at the specified time.

[0187] In many cases, the hydrodynamic radius (R) is used. h It is more convenient than using the diffusivity. The diffusivity can be converted to the hydrodynamic radius using the Stokes-Einstein equation (Equation 22).

[0188] (twenty two)

[0189] Combining equations 22 and 12, we can determine R based on equation 23. h Associated with :

[0190] (twenty three)

[0191] therefore With R A R AL K d and dynamics (k off and k on The R-values ​​are correlated with, and as mentioned above, obtained from the original data using an appropriate fitting procedure. h It can be used to obtain the hydrodynamic radius (R) of A. A The hydrodynamic radius of AL (R) AL ), K d and dynamics (k off and k on ).

[0192] Examples of implementation may include different L T The apparent R value (i.e., the concentration of L in the supplementary portion, assuming the concentration is the same in the modified sample / dispersion region) is determined from the original dataset of the original data. h The fitting parameters are determined by one or more of a, p, and q, and the corresponding parameters are fitted. To obtain the dynamic parameters related to the bonding.

[0193] In the second case below, a second theoretical framework for capmix type determinations is presented. Here, it is also assumed that t... ½ Between 1 second and approximately 3600 seconds (60 minutes).

[0194] The concentration used for fitting to L (L) T The appearance of the function R) h In the following equation for the binding curve, the concentration of L in the supplementary portion does not exceed the concentration of A in the sample portion.

[0195] Although the second case is similar to the first, some differences can be considered. When the concentration of A is high, it cannot generally be assumed that the concentration of free L [L] can be used with L. T The reason for this approximation is that a significant portion of L binds to A.

[0196] L T = [L]+[AL](24)

[0197] The amount of L combined with A can be determined by the interaction K.d And the concentration of A is determined. To handle the second case, the concentration of A is estimated. At time 0, the total concentration of A is C. A Furthermore, it can be estimated based on readable markings on the sample portion before injection into the capillary.

[0198] During capmix measurements, A (unbound A and AL) is diluted due to the dispersion process (Taylor dispersion) in the capillary. Therefore, the concentration of A in the dispersion region is not constant. To describe this second case, it is convenient to describe the dilution process that occurs during the experiment. It has been found that the raw data itself can be a good measure of how the sample was diluted during the experiment, since the Gaussian signal is essentially a time-concentration profile.

[0199] The initial dispersion (region A) spatial width (δ) i ) can be obtained through the linear flow rate (u) during sample injection. i Multiply by the injection time (t) i Get:

[0200] (25)

[0201] During the experiment, as described by Taylor theory, the sample region exhibited a Gaussian distribution. The temporal dispersion was determined by the peak width Z. The description, where Z is a scaling factor that can be assumed to be 2, and therefore can be used to calculate at t R Spatial width of the time-dispersion region (δ) s ):

[0202] (26)

[0203] in It is the linear flow rate during the actual experiment, and:

[0204] (27)

[0205] In Equation 27, it is assumed that D is the average diffusion coefficient during the experiment, which can be obtained from the raw data as described above.

[0206] Then time dilution factor A f (t) is given by the following formula:

[0207] (28)

[0208] In the experiment, P i It is the injection pressure, P r It refers to operating pressure. Here we utilize the fact that, according to the Hagen-Poiseuille equation, u and P are directly proportional.

[0209] Equations 28 and 27 can be combined and rewritten as:

[0210] (29)

[0211] Where b is given by the following:

[0212] (30)

[0213] Note that b consists of system parameters and experimental parameters, which are specific to the experiment (and are known) or can be obtained directly from the raw data.

[0214] As shown in Equation 29, the dilution factor is time-dependent. It should also be noted that t R With P r Proportional. Therefore, A f The dilution factor (t) is proportional to sqrt(t), which means that the dilution factor increases (dilution decreases) at high t values. This is because diffusion will offset flow-induced dispersion over time.

[0215] In the following text, it is assumed that during the experiment, the average dilution factor (A) was used. f The dilution effect of A is well described, see below.

[0216] (31)

[0217] The integral in equation 31 can be solved analytically. The result is:

[0218] (32)

[0219] It can be noted that it is possible to obtain data from the original data (D and ) and calculated directly from known experimental parameters .

[0220] Using the results of Equation 32, the total indicator concentration during the experiment can therefore be simply given as follows:

[0221] (33)

[0222] Where C A It is the total concentration of A in the sample portion.

[0223] The rate equations for the second case can then be solved in a manner similar to that of simple homogeneous dynamics.

[0224] The solution is:

[0225] (34)

[0226] From this point onward, the second case is considered similar to the first case, although with slightly different definitions of a, p, and q compared to the first case:

[0227] (35)

[0228] (36)

[0229] (37)

[0230] Therefore, equations 36, 37, and 35 correspond to equations E1, E2, and E3, where the concentration of L in the dispersion region can be considered constant, while the concentration of A is considered non-constant.

[0231] Equations 36, 37, and 35 are also referred to as a set of equations 36, 37, and 35.

[0232] In the third and fourth cases, the third and fourth theoretical frameworks for dismix-type determinations are discussed, respectively. Here, it is also assumed that t... ½ Between 1 second and approximately 3600 seconds (60 minutes).

[0233] In the third case, the concentration of L in the sample exceeds the concentration of A in the sample. In the fourth case, the concentration of L in the sample does not exceed the concentration of A in the sample.

[0234] During Taylor dispersion determination, the dilution of both unbound A and / or unbound L in the dispersion region is considered as a kinetically independent average dilution, and vice versa. However, kinetics are indirectly taken into account because dilution can be derived from the binding fraction. This means that, when Taylor conditions apply, the concentrations of unbound A and unbound L can be assumed to be adequately described by average concentrations. This conveniently simplifies the solution to the mass transport equation describing the particle distribution.

[0235] Therefore, it is possible to obtain a general solution even though different boundary conditions are used for the solutions to capmix applied in the first and second cases.

[0236] Generally speaking:

[0237] (38)

[0238] (39)

[0239] in and These are the average dilution concentrations of all A and L, respectively.

[0240] In the third case, we can assume LT = [L] (i.e., L is in excess).

[0241] When t=0, the concentration is determined by K. d Decide:

[0242] (40)

[0243] Where A T and L T These are the total concentrations of the first and second particles in the sample portion when the sample is injected.

[0244] (41)

[0245] (42)

[0246] According to equation 38, at t=0 we obtain:

[0247] (43)

[0248] Derivation:

[0249] (44)

[0250] According to equations 38 and 44, we obtain:

[0251] (45)

[0252] Then there is:

[0253]

[0254] (46)

[0255] (47)

[0256] The expression for S is obtained by solving the corresponding second-order equation at t=0:

[0257]

[0258]

[0259] The physically meaningful solution is:

[0260]

[0261] Then there is:

[0262]

[0263] (46)

[0264] (47)

[0265]

[0266] (48)

[0267] (49)

[0268] Derivation

[0269] (50)

[0270] (51)

[0271] (52)

[0272] In a corresponding manner, equations can be determined from the fitting parameters a, q, and p used to determine the desired binding parameters from the Taylor dispersed signals measured in the third and fourth cases.

[0273] The average concentration of A (A) d (As described above)

[0274] When L exceeds A, L can be obtained in a similar procedure. d .

[0275] (53)

[0276] Where L d1 This refers to the limiting case, and the diffusion rate of L is used in the calculation of the constant b, from which A is obtained in equations 30 and 31. f (L).

[0277] For the limiting case where L does not exceed A: L T ≈ [AL], the dilution factor of A can be used to determine L. d .

[0278] (55)

[0279] In general, the dilution factor of L can be used as a weighted average of the two extreme cases.

[0280] (56)

[0281] in It is the average binding fraction of L, and It is the average score excluding L.

[0282] (57)

[0283] (58)

[0284] The above equation can be easily solved using numerical algorithms. However, using average concentrations is also accurate, which significantly simplifies the method.

[0285] Using the average concentration, we obtain:

[0286] (59)

[0287] (60)

[0288] in It is the average AL concentration during the experiment.

[0289] This can be found from the experimental measurement of the binding fraction of A during the experiment. :

[0290] (61)

[0291] Substituting equations 59 and 60, we get:

[0292] (62)

[0293] Where D is the measured apparent diffusion rate.

[0294] Combining equations 56-62, we get:

[0295]

[0296] (63)

[0297] Rearranging them yields the following second-order equation:

[0298] (64)

[0299] Solving equation 64, we get:

[0300] (65)

[0301] Only equation 66 is a physically meaningful solution:

[0302] (66)

[0303] It is recognized that the hydrodynamic radii of L and AL can also be used to calculate the dilution factor, since, according to the Stokes-Einstein equation (Equation 22), the hydrodynamic radius is related to the diffusion rate.

[0304] The above-defined type 4 determination of "cap-dis-mix" can be described as a mixture of capmix and dismix types, in which a concentration jump induces an association reaction, and in which the concentrations of both A and L in the dispersion region are not considered constant. The total volume of the sample portion and the affected portion is advantageously relatively small, preferably less than 5% of the total volume of the liquid portion, such as less than 2%, and ideally 1% or less.

[0305] Under these conditions, also referred to here as the fifth case, the above applies to A. d and L d The equation holds, and the boundary conditions at time 0 are similar to those in cases 1 and 2. Therefore, the fitting parameters a, p, and q can be expressed by the following equations 57, 58, and 59.

[0306] (67)

[0307] (68)

[0308] (69)

[0309] In a corresponding manner, an equation can be determined from the fitting parameters a, q, and p used to determine the desired binding parameters from the Taylor dispersed signal measured in the fifth case.

[0310] Equations 67, 68, and 69 are also referred to as a set of equations 67, 68, and 69.

[0311] In one embodiment, at least one binding parameter includes one or more of the following: k off (Dissociation rate), k on (Association rate); k obs (The rate constant at which equilibrium is reached), R AL (hydrodynamic radius of AL), R A (The hydrodynamic radius of A), D A (Diffusion rate of unbound A), D AL (AL diffusion rate) and / or any derived parameter of one or more of the mentioned binding parameters. Preferred binding parameters determined by embodiments of the invention may be as described above.

[0312] Advantageously, the formation of an interfacial contact between the sample portion and at least one supplementary portion in the microfluidic channel provides modification of the sample portion to the modified sample portion dispersed between the sample portion and at least one supplementary portion, thereby inducing a concentration jump of at least one of a first particle L and a second particle A in the modified sample portion, wherein the concentration jump causes the first particle and the second particle to become unbalanced in the modified sample portion, and wherein the concentration jump induces an association reaction or a dissociation reaction.

[0313] The region along the microfluidic channel that includes the second particle initially in the sample portion is called the dispersion region.

[0314] The association reaction induces the first particle L and the second particle A in the sample portion to bind together to form bound first and second particles (AL). Given that the bound first and second particles (AL) are already present in the modified sample portion, the association reaction induces an increase in the concentration of the bound first and second particles (AL).

[0315] The dissociation reaction induces the bound first and second particles (AL) to dissociate into a first particle L and a second particle A, thereby reducing the concentration of the bound first and second particles (AL).

[0316] The modified sample portion is defined by a second particle A carrying a selected marker (intrinsic or extrinsic) that is read during Taylor dispersion assays. The modified sample portion within the microfluidic channel is also referred to as the dispersion region.

[0317] In one embodiment, at least one of the N original datasets is obtained from a Taylor dispersion assay, wherein second particles (A) of the sample portion are dispersed to form a dispersion region in a microfluidic channel, and wherein the molar concentration of the second particles in the dispersion region is equal to or less than the molar concentration of the first particles, and the method includes determining at least one binding parameter by applying one or more of equations 17, 18 and / or 19 from one or more fitting parameters a, p and q.

[0318] (17)

[0319] (18)

[0320] (19),

[0321] Preferably, at least half or all or N original datasets are each obtained from a Taylor dispersion determination, wherein the second particles (A) of the sample portion are dispersed to form a dispersion region in a microfluidic channel, and wherein the molar concentration of the second particles in the dispersion region is equal to or less than the molar concentration of the first particles.

[0322] In one embodiment, at least one of the N original datasets is obtained from a Taylor dispersion determination, wherein a second particle (A) of the sample portion is dispersed to form a dispersion region in the microfluidic sample, and wherein the molar concentration of the second particle in the dispersion region is equal to or greater than the molar concentration of the first particle, and wherein the method includes determining at least one binding parameter by applying one or more of equations 35, 36 and / or 37 from one or more fitting parameters a, p and q.

[0323] (35)

[0324] (36)

[0325] (37),

[0326] Preferably, at least half or all or N original datasets are each obtained from a Taylor dispersion determination, wherein the second particles (A) of the sample portion are dispersed to form a dispersion region in the microfluidic sample, and wherein the molar concentration of the second particles in the dispersion region is equal to or greater than the molar concentration of the first particles.

[0327] In one embodiment, at least one binding parameter is determined from one or more of the fitting parameters by applying one or more of Equations 17-19, and at least one binding parameter is determined from one or more of the fitting parameters by applying one or more of Equations 35-37, after which the average value of at least one binding parameter can be determined, thereby determining at least one binding parameter with higher precision. This may be particularly desirable when N sets of raw data include at least one set of raw data obtained from a Taylor dispersion determination (where the molar concentration of the second particle in the dispersion region is equal to or lower than the molar concentration of the first particle) and at least one set of raw data obtained from a Taylor dispersion determination (where the molar concentration of the second particle in the dispersion region is equal to or higher than the molar concentration of the first particle). The N raw datasets may include one or more datasets obtained from corresponding one or more capmix-type Taylor dispersion determinations, one or more datasets obtained from corresponding one or more dismix-type Taylor dispersion determinations, one or more datasets obtained from corresponding one or more cap-dis-mix-type Taylor dispersion determinations, and / or one or more datasets obtained from corresponding one or more premix-type Taylor dispersion determinations.

[0328] The interfacial contact between the sample portion and at least one supplementary portion in the microfluidic channel provides modification of the sample portion to the modified sample portion dispersed between the sample portion and at least one supplementary portion. This can induce a concentration jump in at least one of a first particle L and a second particle A within the sample portion. The concentration jump causes an imbalance between the first and second particles in the modified sample portion, wherein the concentration jump induces an association reaction, or wherein the concentration jump induces a dissociation reaction.

[0329] To ensure the desired large concentration jump, it is desirable that the volume of the sample portion is 5% or less of the total volume of the sample portion and at least one supplementary portion. Preferably, when supplied, the volume of the sample portion is 1% or less of the total volume of the sample portion and at least one supplementary portion, such as 0.5% or less, such as 0.1% or less, such as 0.05% or less.

[0330] In reality, the sample portion may only be a few nanoliters (nL) because microfluidic channels are typically quite narrow to ensure laminar flow and establish the desired Taylor conditions.

[0331] Microfluidic channels may 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 even less. Microfluidic units can be conveniently shaped into tubular structures with a constant diameter along their entire length. Such tubular structures are also referred to as capillary tubes or simply capillaries. Examples of suitable instruments for performing Taylor dispersion assays are disclosed in WO21180289 and / or sold by FIDA Biosystems, such as the instrument sold under the trade name FIDA 1.

[0332] Microfluidic channels can conveniently have lengths of 10 cm or longer, such as 20 cm to several meters, depending on the run time required for the measurement. Therefore, for applications with relatively slow reaction kinetics (e.g., t...),... ½ For measurements that take a long time, microfluidic channels can be advantageously relatively long, such as up to 5m, up to 2m, or up to 1m.

[0333] In one embodiment, the total molar amount of the second particle in the total volume of the sample portion and at least one supplementary portion is less than the total molar amount of the first particle in the total volume of the sample portion and at least one supplementary portion. Preferably, the total molar amount of the second particle in the total volume of the sample portion and at least one supplementary portion is less than 5%, such as less than 1%, such as 0.5% or less. Because the second particle A is the particle being read, this embodiment can provide a read that is easier to process.

[0334] In one embodiment, there is only one replenishment portion, and this single replenishment portion may be supplied to the microfluidic channel before or after the sample portion. However, it is generally preferred that the at least one replenishment portion comprises at least two replenishment portions.

[0335] In one embodiment, the at least one replenishment portion includes a leading replenishment portion and a following replenishment portion, wherein supplying the liquid portion to the microfluidic channel includes providing the leading replenishment portion and the following replenishment portion on either side of the sample portion.

[0336] The leading supplementary part includes a supplementary part adapted to lead the flow in the microfluidic channel, i.e., in front of the dispersion region, while the following supplementary part includes a supplementary part adapted to follow the flow in the microfluidic channel, i.e., after the dispersion region.

[0337] The leading and following replenishment portions may have the same volume and / or different contents. Advantageously, the leading and following replenishment portions have the same contents when supplied to the microfluidic channel.

[0338] In one embodiment, at least one of the N original datasets is obtained from a Taylor dispersion assay, wherein when a sample portion is supplied to a microfluidic channel, the sample portion includes a base concentration of second particles, and wherein when at least one supplementary portion is supplied to the microfluidic channel, the at least one supplementary portion contains no second particles or has a concentration lower than the base concentration of the second particles, and wherein the formation of an interfacial contact between the sample portion and the at least one supplementary portion in the microfluidic channel modifies the sample portion to provide a modified sample portion comprising the dispersion between the sample portion and the at least one supplementary portion, thereby inducing a concentration jump of at least a first particle L in the modified sample portion, wherein the concentration jump causes an imbalance between the first particle and the second particle in the modified sample portion, and wherein the concentration jump induces an association reaction.

[0339] The Taylor dispersion assay in this embodiment can be a capmix type assay, wherein at least one supplementary portion does not contain the second particle A, or a capmix variant assay, wherein a capmix variant assay means that at least one supplementary portion does not contain the second particle A, but has a concentration lower than the baseline concentration of the second particle when the at least one supplementary portion is supplied to the microfluidic channel.

[0340] A concentration jump can provide modification to a sample portion to form a dispersed region, wherein the dispersed region includes a first particle of a certain concentration dispersed from at least one supplementary portion, thereby inducing a concentration jump of at least the first particle in the sample portion. When acquiring a dataset from a capmix-type assay or a capmix variant assay, the data obtained from a set of data... This could be very helpful in obtaining the fitting parameters, based on which k can be determined with the desired very high accuracy. on .

[0341] Because the sample portion is small in volume relative to at least one supplementary portion, the molar concentration of the first particle (L) in the modified sample portion will reach the molar concentration of the first particle (L) very quickly, which can be considered to be the same as the molar concentration of the first particle (L) in at least one supplementary portion.

[0342] When the supplementary portion includes a second particle, but its concentration is lower than that of the second particle in the sample portion, it is desirable that the concentration of the second particle in at least one supplementary portion is less than 50% of the baseline concentration of the second particle in the sample portion, such as less than 10%, less than 5%, or less than 1%. In the processing of the raw data, such as in the preprocessing step of filtering the raw data, the signal of the second particle initially present in at least one supplementary portion can be extracted as background noise.

[0343] In one embodiment, the Taylor dispersion assay is a capmix-type assay or a capmix variant assay, at least one supplementary portion is free of second particles, or the second particles in the supplementary portion are free of selected markers, and / or the concentration of the second particles is less than 10% of the baseline concentration of the second particles when the sample portion is supplied to the microfluidic channel, such as less than 1% of the baseline concentration of the second particles when the sample portion is supplied to the microfluidic channel.

[0344] In a preferred embodiment, at least one of the N original datasets is obtained from a Taylor dispersion assay, a capmix-type assay, wherein when a sample portion is supplied to the microfluidic channel, the sample portion includes a base concentration of second particles A and contains no first particles L, and wherein when at least one supplementary portion is supplied to the microfluidic channel, the at least one supplementary portion contains no second particles and has a certain concentration of first particles L. The formation of an interfacial contact between the sample portion and at least one supplementary portion in the microfluidic channel modifies the sample portion, providing a modified sample portion comprising the dispersion between the sample portion and at least one supplementary portion, thereby inducing a concentration jump of at least the first particles L in the modified sample portion. This concentration jump causes an imbalance between the first and second particles in the modified sample portion, wherein the concentration jump triggers an association reaction.

[0345] In one embodiment, at least one of the N original datasets is obtained from a Taylor dispersion assay, wherein when a sample portion is supplied to the microfluidic channel, the sample portion comprises a base concentration of second particles (A) and a base concentration of first particles (L), and wherein when at least one supplementary portion is supplied to the microfluidic channel, at least one supplementary portion either does not contain the first particles (in which case, the Taylor dispersion assay is a dismix type assay) or has a different concentration of L, such as a concentration higher than the base concentration of the first particles (in which case, the Taylor dispersion assay is a premix type assay), or when at least one supplementary portion is supplied to the microfluidic channel, it contains a different concentration of L. The concentration A, such as a concentration lower than the base concentration of the second particle (in which case, the Taylor dispersion assay is a premix variant assay), and the formation of an interfacial contact between the sample portion and at least one supplementary portion in the microfluidic channel, provides modification of the sample portion to the modified sample portion comprising the dispersion between the sample portion and at least one supplementary portion, thereby inducing a concentration jump of at least one of the first particle L and the second particle A in the modified sample portion, wherein the concentration jump causes the first particle and the second particle to become unbalanced in the modified sample portion, wherein the concentration jump induces an association reaction or a dissociation reaction depending on the concentration of the particles in the supplementary portion relative to the sample portion.

[0346] Advantageously, at least one supplementary portion, wherein when the sample portion is supplied to the microfluidic channel, the at least one supplementary portion has a first particle of a different concentration, such as a higher concentration of first particles, which differs from the baseline concentration of the first particles L of the sample portion by at least 50%, such as at least 90%.

[0347] In a preferred embodiment, at least one of the N original datasets is obtained from a Taylor dispersion assay, which is a dismix type assay, wherein when a sample portion is supplied to a microfluidic channel, the sample portion includes a base concentration of second particles (A) and a base concentration of first particles (L), and wherein when at least one supplementary portion is supplied to the microfluidic channel, the at least one supplementary portion contains neither the first particles nor the second particles, and wherein the formation of an interfacial contact between the sample portion and at least one supplementary portion in the microfluidic channel modifies the sample portion to provide a modified sample portion comprising the dispersion between the sample portion and at least one supplementary portion, thereby inducing a concentration jump of at least one of the first particles L and the second particles A in the modified sample portion, wherein the concentration jump causes the first particles and the second particles to become unbalanced in the modified sample portion, and wherein the concentration jump induces a dissociation reaction.

[0348] When obtaining the dataset from a dismix-type measurement, the data obtained from this set of data... This is highly beneficial for obtaining fitting parameters, based on which k can be determined with the desired very high accuracy. off .

[0349] In a dismix-type assay, it is preferable that the first and second particles of the sample portion are in equilibrium when the sample portion is supplied to the microfluidic channel.

[0350] In one embodiment, at least one supplementary portion comprises a first particle L and / or a second particle A, and it is desirable that the leading supplementary portion has a leading portion concentration of the first particle, and the following supplementary portion has a following portion concentration of the first particle, wherein the concentration of the leading portion of the first particle and the concentration of the following portion of the first particle are equal. In variations thereof, the leading portion concentration may differ from the following portion concentration, wherein preferably, the leading portion concentration of the first particle and the following portion concentration of the first particle are equal to or differ from the average concentration of the following portion concentration of the first particle and the leading portion concentration of the first particle by less than 10%, such as differing from the average concentration by less than 1%.

[0351] In one embodiment, the leading supplement portion contains no second particles or has a second particle concentration of the leading portion, and the following supplement portion contains no second particles or has a second particle concentration of the following portion, wherein the leading supplement portion concentration of the second particle and the following portion concentration of the second particle are equal or different, preferably, the leading portion concentration of the second particle and the following portion concentration of the second particle are equal or differ from the average concentration of the following portion concentration of the second particle and the leading portion concentration of the second particle by less than 10%, such as differing from the average concentration by less than 1%.

[0352] The leading supplementary portion has a leading portion volume, and the following supplementary portion has a following portion volume, wherein the leading portion volume and the following portion volume may be the same or different. Typically, it is desirable, preferably, that each of the leading portion volume and the following portion volume is larger than the sample volume, such as being 5 times larger than the sample volume, such as being 10 times larger than the sample volume, such as being 20 times larger than the sample volume.

[0353] In one embodiment, at least one of the N original datasets is obtained from a type 3 (dismix) Taylor dispersion assay. When a sample portion is supplied to a microfluidic channel, the sample portion includes a base concentration of second particles (A) and a base concentration of first particles (L). When at least one supplementary portion is supplied to the microfluidic channel, the at least one supplementary portion contains neither the first particles nor the second particles. The formation of an interfacial contact between the sample portion and the at least one supplementary portion in the microfluidic channel modifies the sample portion by providing a modified sample portion comprising the dispersion between the sample portion and the at least one supplementary portion, thereby inducing a concentration jump of at least one of the first particles L and the second particles A in the modified sample portion. The concentration jump causes an imbalance between the first particles and the second particles in the modified sample portion, and the concentration jump induces a dissociation reaction.

[0354] In one embodiment, where at least one of the N original datasets is obtained from a type 3 (dismix) Taylor dispersion determination, the method may advantageously include determining at least one binding parameter by applying one or more of the fitting parameters a, p, and q.

[0355] (50)

[0356] (51)

[0357] (52)

[0358] Preferably, at least half or all or N original datasets, such as all of N original datasets, each obtained from a type 2 (premix) or type 3 (dismix) Taylor dispersion determination.

[0359] In one embodiment, at least one of the N original datasets is obtained from a cap-dis-mix Taylor dispersion assay, wherein the liquid portion further includes an influence portion supplied to a microfluidic channel to form an interfacial contact with the sample portion, wherein when the sample portion is supplied to the microfluidic channel, the sample portion includes a base concentration of second particles A and substantially contains no first particles L, and when the influence portion is supplied to the microfluidic channel, the influence portion includes a base concentration of first particles L and substantially contains no second particles A, and wherein when at least one supplementary portion is supplied to the microfluidic channel, the supplementary portion substantially contains neither first particles L nor second particles A, and wherein the formation of interfacial contacts between the sample portion and the influence portion and / or between the sample portion and at least one supplementary portion in the microfluidic channel modifies the sample portion by providing a modified sample portion comprising the sample portion, the influence portion and at least one supplementary portion, thereby inducing a concentration jump of at least first particles L in the modified sample portion, wherein the concentration jump causes an imbalance between the first particles and the second particles in the modified sample portion, and wherein the concentration jump induces an association reaction.

[0360] In one embodiment, at least one of the N original datasets is obtained from a type 4 (cap-dis-mix) Taylor dispersion determination, and the method advantageously includes determining at least one binding parameter by applying one or more of the fitting parameters a, p, and q.

[0361] (67)

[0362] (68)

[0363] (69)

[0364] Preferably, at least half or all or N original datasets, such as all of N original datasets, are obtained from a type 4 (cap-dis-mix) Taylor dispersion determination.

[0365] In one embodiment, at least one set of equations 17, 18, 19, 36, 37, and 35, 50, 51, and 52, and 67, 68, and 69 is applied to determine at least one binding parameter by one or more of the fitting parameters a, p, and q.

[0366] In one embodiment, two, three, or all four sets of equations 36, 37, and 35, 50, 51, and 52, and 67, 68, and 69 are applied to determine at least one binding parameter by one or more of the fitting parameters a, p, and q.

[0367] The number of reads for each set of data is advantageously sufficient to generate a Gaussian representation, such as a Taylor plot. In one embodiment, acquiring each set of raw data includes performing multiple reads of signal intensity at the readout section of the microfluidic channel, wherein the multiple reads preferably include at least 5 intensity reads, including at the peak occurrence time (t). R At least one intensity reading at the time of peak occurrence (t) R At least one intensity reading prior to the peak occurrence time (t) R At least one intensity read after that, preferably, acquiring each set of raw data includes recording intensity at a recording rate of at least 1 Hz (such as 5 to 12 Hz or higher), such as acquiring a complete Gaussian readout profile (i.e., a Taylor plot).

[0368] The method conveniently includes acquiring N sets of raw data, wherein the corresponding N sets of raw data are obtained from corresponding N Taylor dispersion measurements, where N is an integer of at least 1. N can advantageously be from 1 to 50, such as from 2 to 20, such as from 3 to 10.

[0369] In one embodiment, at least the parameter K is combined D (Equilibrium dissociation constant), D A (Diffusion rate of unbound A) and / or D AL The diffusivity of AL is a known binding parameter, and one or more of these known parameters are applied to determine at least one other binding parameter by one or more of the fitting parameters p, q, and a. In this embodiment, in particular, at least one of the N datasets is based on a dismix-type determination, and the required number of datasets may be relatively low. For example, N may be 5 or less, such as 1, 2, or 3. Preferably, at least one of the N sets of raw data is obtained from a Taylor dispersion determination, wherein the sample portion comprises a first particle L at a baseline concentration and a second particle A at a baseline concentration in equilibrium.

[0370] In a preferred embodiment, N is 2 or greater, and the N sets of raw data include: at least one set of raw data obtained from Taylor dispersion assays, wherein concentration jumps induce association reactions, and at least one set of raw data obtained from Taylor dispersion assays, wherein concentration jumps induce dissociation reactions. It has been found that this combination can provide the determination of one or more binding parameters with very high accuracy.

[0371] In one embodiment, N is 2 or greater, and the N sets of raw data include: at least one set of raw data obtained from a Taylor dispersion assay, wherein the second particles (A) of the sample portion are dispersed to form a dispersion region in a microfluidic channel, and wherein the molar concentration of the second particles in the dispersion region is equal to or less than the molar concentration of the first particles; and at least one set of raw data obtained from a Taylor dispersion assay, wherein the second particles (A) of the sample portion are dispersed to form a dispersion region in a microfluidic sample, and wherein the molar concentration of the second particles in the dispersion region is equal to or greater than the molar concentration of the first particles.

[0372] In one embodiment, N is 2 or greater, and N sets of raw data comprise multiple datasets obtained from corresponding Taylor dispersion determinations, wherein the baseline concentration of the second particle A differs in the sample portions.

[0373] In one embodiment, N is 2 or greater, and N sets of raw data comprise multiple datasets obtained from corresponding Taylor dispersion determinations, wherein the base concentration of the first particle differs in at least one supplementary portion of the sample portion.

[0374] The selected marker can, in principle, be any type of readable marker. The selected marker includes detectable intrinsic markers (such as tryptophan) and / or extrinsic markers, and preferably, the marker is an optically readable marker (such as a fluorescent marker and / or a light-absorbing marker).

[0375] In one embodiment, a conformational change of the first particle (preferably a selected marker) alters the signal based on the conformation of the first particle and its changes, such as based on binding / dissociation and / or structural changes.

[0376] The selected markings are advantageously optically readable markings, such as light-absorbing markings and / or fluorescent markings, preferably operating in the UV / Vis wavelength range, preferably from about 190 nm to about 700 nm.

[0377] In one embodiment, the selected label is an electrochemically readable label, such as an electroactive label.

[0378] A liquid portion is conveniently supplied to the microfluidic device at an injection pressure Pi, and a liquid portion is supplied to the flow within the microfluidic device at an operating pressure Pr to ensure laminar flow in the microfluidic channel. To ensure height control of the measurement, it is desirable that the operating pressure Pr be greater than the injection pressure Pi. Advantageously, the operating pressure Pr is adjustable, for example, so that the operating pressure can be adjusted to ensure that the dispersion region reaches the reading section of the microfluidic channel at the desired time, thereby ensuring that at least one imbalance reading can be performed.

[0379] The application of operating pressure ensures that the liquid portion is forced to pass through the channel along its length. More preferably, if the microfluidic channel can be adjusted by regulating the operating pressure, the flow rate of the sample portion along its length can be ensured, for example, that the sample portion is provided at a selected location in the microfluidic channel within a selected time range to perform the reading of a selected mark within the selected time range.

[0380] Advantageously, each read, the purpose of which is to acquire one of the reads in the set, includes reading the label signal of the concentration of the second particle of the label along a length cross-section of the microfluidic channel including the sample portion (preferably including the dispersion region). Reading the label signal of the concentration of the second particle of the label along the length cross-section of the microfluidic channel including the dispersion region is from the leading edge of the dispersion region to the following edge of the dispersion region, and includes reading the intensity of the label signal as a function of time as the dispersion region passes through the read section of the microfluidic channel.

[0381] Advantageously, each read aimed at obtaining one of the reads in the set includes obtaining the read while the liquid sample is in laminar flow.

[0382] Advantageously, each read, the purpose of which is to obtain one of the reads in the set, involves reading a label signal indicating the concentration of a second particle of the label along a cross-section of the length of the microfluidic channel that includes the sample portion, wherein each read is performed within a time range not exceeding the time taken for the sample portion to pass through the read cross-section of the microfluidic channel.

[0383] Advantageously, at least one read result of the imbalance read is obtained by reading a labeled signal of the concentration of the labeled second particle before the interaction between the first and second particles reaches equilibrium. Preferably, the method includes performing multiple imbalance reads, optionally all read results of this set of reads being the results of an imbalance read.

[0384] In one embodiment, the set of reads includes balanced reads, preferably obtained by reading the marker signal of the concentration of the second marker particle, wherein the interaction between the first and second particles is balanced.

[0385] The first particle L and the second particle A can be any type of particle that can be dispersed in a microfluidic channel, independently of each other. The first particle L and the second particle A can interact with each other non-covalently.

[0386] The first particle and the second particle can conveniently be different from each other. Preferably, the first particle and the second particle differ in particle type, molecular weight, dispersion properties, or any combination thereof.

[0387] Advantageously, at least one of the first and second particles includes a drug or a drug candidate.

[0388] As an example of the first particle L and the second particle A, it may be mentioned that at least one of the first particle and the second particle may be selected from: biomolecules, proteins (such as antibodies (monoclonal or polyclonal antibodies), nanobodies, antigens, enzymes and / or hormones), nucleotides, nucleosides, nucleic acids (such as RNA, DNA, PNA or any fragment thereof) and / or any combination including at least one of these.

[0389] The parameters can advantageously include kinetic parameters, such as the association rate constant k. on Dissociation rate constant k off Or its derived parameters, such as dwell time (1 / K) off ),t ½ (0.693 / VK off (or one or more of the other combination parameters as described above.) Attached Figure Description

[0390] The invention will be further described below with reference to examples and embodiments and the accompanying drawings. The drawings are schematic and may not be drawn to scale. The examples and embodiments given are merely illustrative of the invention and should not be construed as limiting the scope of the invention.

[0391] Figure 1a , 1b Figure 1c schematically illustrates the Taylor dispersion assay in the form of a capmix assay.

[0392] Figure 1a' , 1b’ Figure 1c' schematically illustrates a Taylor dispersion assay in the form of a cap-dis-mix assay.

[0393] Figure 2a , 2b Figure 2c schematically illustrates a Taylor dispersion assay in the form of a dismix assay.

[0394] Figure 3 This is a flowchart illustrating one embodiment of the method of the present invention.

[0395] Figure 4 This is a flowchart illustrating another embodiment of the method of the present invention.

[0396] Figure 5 This is a flowchart illustrating another embodiment of the method of the present invention.

[0397] Figure 6 This is a flowchart illustrating another embodiment of the method of the present invention.

[0398] Figure 7This is a flowchart illustrating another embodiment of the method of the present invention. Detailed Implementation

[0399] Figure 1a , 1b The Taylor dispersion measurement in the form of a capmix type measurement, shown in 1c, illustrates the time t0 ( Figure 1a ), t1 ( Figure 1b ) and t2 ( Figure 1c The length and cross-section of the capillary at that time.

[0400] The arrow at the top of the attached diagram shows that capillaries can be very long, as described above.

[0401] like Figure 1a As shown, at time t0, the leading supplement portion 2', the influencing portion 1a, the sample portion 1', and the following supplement portion 3' have been injected into the capillary at an injection pressure Pi. This injection pressure Pi can advantageously be relatively low to maintain the sample portion 1 substantially undispersed between the leading supplement portion 2 and the following supplement portion 3. Figure 1a As shown, at time t0, the sample portions remain essentially undispersed. Sample portion 1 contains a base concentration of second particles, while each of the leading supplement portion 2 and the following supplement portion 3 contains a certain concentration of first particles L. In reality, a concentration jump may have already begun, depending on the applied injection pressure.

[0402] exist Figure 1b In this process, operating pressure Pr has been applied, the liquid portion is induced to be in laminar flow, and a concentration jump can be observed in the sample portion. Since sample portion 1 is very small relative to supplementary portions 2 and 3, the sample portion will be immediately modified to have a concentration of the first particle L that is substantially the same as that in the leading supplementary portion 2 and the following supplementary portion 3. This induces an association reaction, and the first particle L and the second particle A will interact. Initially, there will be an imbalance, but it will eventually reach equilibrium over time.

[0403] At time t1, the modified sample portion forms a parabolic shape in the capillary, the parabolic shape having a parabolic top surface facing the leading supplementary portion 2 and a parabolic valley surface facing the following supplementary portion 3. It can be seen that the modified sample portion forms a dispersion region DZ, which can grow along the length direction as the modified sample passes through the capillary.

[0404] exist Figure 1c At time t2, the dispersion region has widened, and this may continue as the dispersion region DZ moves further downstream through the capillary reading section, where the signal of the marker of the second particle A is read as the dispersion region passes through the capillary reading section.

[0405] Figure 1a' , 1b’ The Taylor dispersion measurement in the form of a cap-dis-mix type measurement, shown in 1c', illustrates the time t0 ( Figure 1a' ), t1 ( Figure 1b' ) and t2 ( Figure 1c' The length and cross-section of the capillary at that time.

[0406] At time t0, such as Figure 1a' As shown, the leading supplement portion 2', sample portion 1, and follower supplement portion 3 have been injected into the capillary at an injection pressure Pi, which is advantageously relatively low to maintain the influencing portion 1a and sample portion 1' substantially unmixed and undispersed between the leading supplement portion 2' and the follower supplement portion 3'. Figure 1a' As shown, at time t0, the influencing portion 1a and the sample portion 1' remain substantially unmixed and undispersed. Sample portion 1' contains a base concentration of second particles, the influencing portion 1a contains a certain concentration of first particles L, and each of the leading supplement portion 2' and the following supplement portion 3' is a buffer without first particles L and second particles A. In reality, a concentration jump may have already begun, depending on the applied injection pressure.

[0407] exist Figure 1b' In this process, operating pressure Pr has been applied, the liquid portion has been induced into laminar flow, and a concentration jump has been observed in sample portion 1'. Because sample portion 1' is very small, and the influence portion 1a is very small, these two portions will merge relatively quickly to form a combined diffusion region DZ, which includes the sample portion and the influence portion, which is only very slightly diluted by the buffer from the supplementary portion. This induces an association reaction, and the first particle L and the second particle A will interact. Initially, they will be out of equilibrium, but will eventually reach equilibrium over time.

[0408] At time t1, the modified sample portion now combined with the influencing portion 1a forms a parabolic shape in the capillary. This parabolic shape has a parabolic top surface facing the leading supplementary portion 2' and a parabolic valley surface facing the following supplementary portion 3'. It can be seen that the modified sample portion now including the influencing portion forms a dispersion region DZ, which can grow along its length as the modified sample portion passes through the capillary.

[0409] exist Figure 1c' At time t2, the dispersion region DZ has widened, and this may continue as the dispersion region DZ moves further downstream through the reading section of the capillary, where the signal of the marker of the second particle A is read as the dispersion region DZ passes through the reading section of the capillary.

[0410] Figure 2a , 2b The Taylor dispersion measurement in the form of a dismix type measurement, shown in 2c, illustrates the time t0 ( Figure 2a ), t1 ( Figure 2b ) and t2 ( Figure 2c The length and cross-section of the capillary at that time.

[0411] At time t0, such as Figure 2a As shown, the leading supplement portion 12, the sample portion 11, and the following supplement portion 13 have been injected into the capillary at an injection pressure Pi. Prior to injection into the capillary, the particles in the sample portion have reached equilibrium, ensuring they are in equilibrium upon injection. The injection pressure can advantageously be relatively low to maintain the sample portion 11 substantially undispersed between the leading supplement portion 12 and the following supplement portion 13. Figure 2a As shown, at time t0, the sample portion remains substantially undispersed. Sample portion 11 includes a base concentration of first particles L and a base concentration of second particles A, and each of the leading supplement portion 2 and the following supplement portion 3 does not include any first particles L or second particles A, but rather serves as a buffer.

[0412] exist Figure 2b At time t1, the operating pressure Pr has been applied, the liquid portion is induced to be in laminar flow, and it can be seen that a concentration jump (including dilution of the sample portion by the buffer) has occurred in the sample portion. At time t1, the modified sample portion forms a parabolic shape in the capillary, which has a parabolic apex facing the leading supplementary portion 2 and a parabolic valley facing the following supplementary portion 3. It can be seen that the modified sample portion forms a dispersion region DZ, which can grow along the length direction as the modified sample passes through the capillary.

[0413] When particles A and L are in equilibrium, there may be a relatively high concentration of the complex AL. Since the complex AL must be assumed to be relatively large relative to unbound A and unbound B, dispersion may occur relatively quickly, leading to a relatively rapid widening of the dispersion region DZ and a relatively rapid dilution of the first particle L and the second particle A within the dispersion region. This concentration jump induces a dissociation reaction.

[0414] exist Figure 2c At time t2, the dispersion region has widened further, and this may continue as the dispersion region DZ moves further downstream through the capillary reading section, where the signal of the marker of the second particle A is read as the dispersion region passes through the capillary reading section.

[0415] Figure 3The flowchart shown illustrates an embodiment of the method of the present invention, wherein each of the N datasets is based on a Taylor dispersion determination of a capmix type determination.

[0416] In step 31a, a capmix assay is performed, for example as described above. The capmix assay is performed when the sample portion includes a second particle A at a base concentration of X1, and each of the leading supplement and the following supplement includes a first particle L at a concentration of Y1. In step 31b, the raw dataset 1 is obtained from the Taylor dispersion assay of step 31a. In step 31c, R is obtained from the raw dataset 1. h and at least one of appearance D. In step 31d, by appearance R h and / or apparent D determination .

[0417] In step 32a, the capmix-type assay is performed when the sample portion includes a second particle A at a baseline concentration of X2, and each of the leading supplement and the following supplement includes a first particle L at a certain concentration of Y2. In step 32b, the raw dataset 1 is obtained from the Taylor dispersion assay of step 32a. In step 32c, R is obtained from the raw dataset 1. h and at least one of appearance D. In step 32d, by appearance R h and / or apparent D determination .

[0418] In step 33a, the capmix-type assay is performed when the sample portion includes a second particle A of a base concentration X3, and each of the leading supplement and the following supplement includes a first particle L of a certain concentration Y3. In step 33b, the raw dataset 1 is obtained from the Taylor dispersion assay of step 33a. In step 33c, R is obtained from the raw dataset 1. h and at least one of appearance D. In step 33d, by appearance R h and / or apparent D determination .

[0419] In step 34a, the capmix-type assay is performed when the sample portion includes a second particle A at a base concentration of X4, and each of the leading supplement and the following supplement includes a first particle L at a certain concentration of Y4. In step 34b, the raw dataset 1 is obtained from the Taylor dispersion assay of step 34a. In step 34c, R is obtained from the raw dataset 1. h and at least one of appearance D. In step 34d, by appearance R h and / or apparent D determination .

[0420] In this example, N is 4. In its variations, it can be larger or smaller, as described above. This is now determined from each N dataset. .

[0421] In step 35, the determined Fit to Equation 21 and obtain the fitting parameters p, q and a from the fit.

[0422] In step 36, one or more binding parameters are determined according to equations 18, 19 and 20 and / or according to equations 35, 36 and 37.

[0423] When one or more datasets have been obtained from Taylor dispersion determination, where the first particle L has surpassed the second particle A in the dispersion region, equations 18, 19 and 20 can be advantageously applied to determine one or more binding parameters.

[0424] When one or more datasets have been obtained from Taylor dispersion determination, where the first particle L does not exceed the second particle A in the dispersion region, equations 35, 36 and 37 can be advantageously applied to determine one or more binding parameters.

[0425] In many cases, both equations 18, 19 and 20 and equations 35, 36 and 37 can be applied, and advantageously, the average determination based on equations 18, 19 and 20 and equations 35, 36 and 37 can provide even higher accuracy.

[0426] In the above embodiments, the base concentrations of A: X1, X2, X3 and X4 can advantageously be the same, and the concentrations of L in the supplementary portion: Y1, Y2, Y3 and Y4 can advantageously be different.

[0427] In its variants, the base concentrations of A: X1, X2, X3, and X4 are different, while the concentrations of L in the supplementary parts: Y1, Y2, Y3, and Y4 are the same.

[0428] Figure 4 The flowchart shown illustrates an embodiment of the method of the present invention, wherein each of the N datasets is based on a Taylor dispersion determination of a dismix type determination.

[0429] In step 41a, a dismix assay is performed, for example as described above. The dismix assay is performed when the sample portion includes a second particle A at a baseline concentration X1 and a first particle L at a baseline concentration Y1, wherein each of the leading supplement and the following supplement is a buffer free of the first particle L and the second particle A. In step 41b, raw dataset 1 is obtained from the Taylor dispersion assay of step 41a. In step 41c, R is obtained from raw dataset 1. hand at least one of appearance D. In step 41d, by appearance R h and / or apparent D determination .

[0430] In step 42a, the dismix-type assay is performed when the sample portion includes a second particle A at a baseline concentration of X2 and a first particle L at a baseline concentration of Y2, wherein each of the leading supplement and the following supplement is a buffer free of the first particle L and the second particle A. In step 42b, the raw dataset 1 is obtained from the Taylor dispersion assay of step 42a. In step 42c, R is obtained from the raw dataset 1. h and at least one of appearance D. In step 42d, by appearance R h and / or apparent D determination .

[0431] In step 43a, the dismix-type assay is performed when the sample portion includes a second particle A at a baseline concentration of X3 and a first particle L at a baseline concentration of Y3, wherein each of the leading supplement and the following supplement is a buffer free of the first particle L and the second particle A. In step 43b, the raw dataset 1 is obtained from the Taylor dispersion assay of step 43a. In step 43c, R is obtained from the raw dataset 1. h and at least one of appearance D. In step 43d, by appearance R h and / or apparent D determination .

[0432] In step 44a, the dismix-type assay is performed when the sample portion includes a second particle A at a baseline concentration of X4 and a first particle L at a baseline concentration of Y4, wherein each of the leading supplement and the following supplement is a buffer free of the first particle L and the second particle A. In step 44b, the raw dataset 1 is obtained from the Taylor dispersion assay of step 44a. In step 44c, R is obtained from the raw dataset 1. h and at least one of appearance D. In step 44d, by appearance R h and / or apparent D determination .

[0433] In the above embodiments, the base concentrations of A in the sample portions: X1, X2, X3 and X4 can advantageously be the same, and the concentrations of L in the sample portions: Y1, Y2, Y3 and Y4 can advantageously be different.

[0434] In its variants, the base concentrations of A in the sample portions are different: X1, X2, X3, and X4, while the concentrations of L in the sample portions are the same: Y1, Y2, Y3, and Y4.

[0435] In this example, N is 4. In its variations, it can be larger or smaller, as described above. This is now determined from each N dataset. value.

[0436] In step 45, the determined Fit to Equation 21 and obtain the fitting parameters p, q and a from the fit.

[0437] In step 46, one or more binding parameters are determined according to equations 18, 19 and 20 and / or according to equations 35, 36 and 37.

[0438] When one or more datasets have been obtained from Taylor dispersion determination, where the first particle L has surpassed the second particle A in the dispersion region, equations 18, 19 and 20 can be advantageously applied to determine one or more binding parameters.

[0439] When one or more datasets have been obtained from Taylor dispersion determination, where the first particle L does not exceed the second particle A in the dispersion region, equations 35, 36 and 37 can be advantageously applied to determine one or more binding parameters.

[0440] In many cases, both equations 18, 19, and 20 and equations 35, 36, and 37 can be applied, and advantageously, the determination based on the average of equations 18, 19, and 20 and equations 35, 36, and 37 can provide greater accuracy.

[0441] Figure 5 The flowchart shown illustrates one embodiment of the method of the present invention, which combines... Figure 3 and Figure 4 The example shown.

[0442] In step 51a, a capmix-type assay is performed, for example as described above. The capmix-type assay is performed when the sample portion includes a second particle A at a baseline concentration of X1, and each of the leading supplement and the following supplement includes a first particle L at a concentration of Y1. In step 51b, the raw dataset 1 is obtained from the Taylor dispersion assay of step 51a. In step 51c, R is obtained from the raw dataset 1. h and at least one of appearance D. In step 51d, by appearance R h and / or apparent D determination .

[0443] In step 52a, the capmix-type assay is performed when the sample portion includes a second particle A at a baseline concentration of X2, and each of the leading supplement and the following supplement includes a first particle L at a certain concentration of Y2. In step 52b, the raw dataset 1 is obtained from the Taylor dispersion assay of step 52a. In step 52c, R is obtained from the raw dataset 1. h and at least one of appearance D. In step 52d, by appearance R h and / or apparent d determination .

[0444] In step 53a, the dismix-type assay is performed when the sample portion includes a second particle A at a baseline concentration of X3 and a first particle L at a baseline concentration of Y3, wherein each of the leading supplement and the following supplement is a buffer free of the first particle L and the second particle A. In step 53b, the raw dataset 1 is obtained from the Taylor dispersion assay of step 53a. In step 53c, R is obtained from the raw dataset 1. h and at least one of appearance D. In step 53d, by appearance R h and / or apparent D determination .

[0445] In step 54a, the dismix-type assay is performed when the sample portion includes a second particle A at a baseline concentration of X4 and a first particle L at a baseline concentration of Y4, wherein each of the leading supplement and the following supplement is a buffer free of the first particle L and the second particle A. In step 54b, the raw dataset 1 is obtained from the Taylor dispersion assay of step 54a. In step 54c, R is obtained from the raw dataset 1. h and at least one of appearance D. In step 54d, by appearance R h and / or apparent D determination .

[0446] In step 55, the determined Fit to Equation 21 and obtain the fitting parameters p, q and a from the fit.

[0447] In step 56, for example as described above, one or more binding parameters are determined according to equations 18, 19 and 20 and / or according to equations 35, 36 and 37.

[0448] Figure 6 The flowchart shown illustrates an embodiment of the method of the present invention, wherein each of the N datasets is based on a Taylor dispersion determination of the cap-dis-mix type determination (referred to as "capdismix" in the figure).

[0449] In step 61a, for example as described above, a cap-dis-mix assay is performed. The dismix assay includes a second particle A at a baseline concentration of X1 in the sample portion and an influence portion including a certain concentration of AF. L The process is performed when the first particle L (of which is Y1) is used, and each of the leading supplement and the following supplement is a buffer containing neither the first particle L nor the second particle A. In step 61b, the original dataset 1 is obtained from the Taylor dispersion determination in step 61a. In step 61c, R is obtained from the original dataset 1. h and at least one of appearance D. In step 61d, by appearance R h and / or apparent D determination .

[0450] In step 62a, the cap-dis-mix type assay i measures the second particle A, which includes a base concentration of X2, in the sample portion and the influence portion, which includes a certain concentration of AF. L The first particle L (of which is Y2) is executed, and each of the leading supplement and the following supplement is a buffer containing neither the first particle L nor the second particle A. In step 62b, the original dataset 1 is obtained from the Taylor dispersion determination in step 62a. In step 62c, R is obtained from the original dataset 1. h and at least one of appearance D. In step 62d, by appearance R h and / or apparent D determination .

[0451] In step 63a, the cap-dis-mix type assay measures the second particle A, which includes a base concentration of X3, in the sample portion and the affected portion, which includes a certain concentration of AF. L The first particle L (of which is Y3) is executed, and each of the leading supplement and the following supplement is a buffer containing neither the first particle L nor the second particle A. In step 63b, the original dataset 1 is obtained from the Taylor dispersion determination in step 63a. In step 63c, R is obtained from the original dataset 1. h and at least one of appearance D. In step 63d, by appearance R h and / or apparent D determination .

[0452] In step 64a, the cap-dis-mix type assay measures the second particle A, which includes a base concentration of X4, in the sample portion and the affected portion, which includes a certain concentration of AF. LThe first particle L (of which is Y4) is executed, and each of the leading supplement and the following supplement is a buffer containing neither the first particle L nor the second particle A. In step 64b, the original dataset 1 is obtained from the Taylor dispersion determination in step 64a. In step 64c, R is obtained from the original dataset 1. h and at least one of appearance D. In step 64d, by appearance R h and / or apparent D determination .

[0453] In the above embodiments, the base concentrations of A in the sample portion: X1, X2, X3 and X4 can advantageously be the same, and the concentrations of L in the affected portion: Y1, Y2, Y3 and Y4 can advantageously be different.

[0454] In its variants, the baseline concentrations in sample portion A (X1, X2, X3, and X4) are different, while the concentrations of L in the affected portion (Y1, Y2, Y3, and Y4) are the same.

[0455] In this example, N is 4. In its variations, N can be larger or smaller, as described above. This has now been determined from each N dataset. value.

[0456] In step 65, the determined Fit to Equation 21 and obtain the fitting parameters p, q and a from the fit.

[0457] In step 66, one or more combination parameters are determined based on a set of equations 18, 19, and 20 and / or a set of equations 35, 36, and 37 and / or a set of equations 50, 51, and 52 and / or a set of equations 67, 68, and 69. In this example, a set of equations 67, 68, and 69 is preferably applied, and alternatively, one or more other sets of equations may be combined.

[0458] When one or more datasets have been obtained from Taylor dispersion determination, where the first particle L has surpassed the second particle A in the dispersion region, equations 18, 19 and 20 can be advantageously applied to determine one or more binding parameters.

[0459] When one or more datasets have been obtained from Taylor dispersion determination, where the first particle L does not exceed the second particle A in the dispersion region, equations 35, 36 and 37 can be advantageously applied to determine one or more binding parameters.

[0460] In many cases, both equations 18, 19, and 20 and equations 35, 36, and 37 can be applied, and advantageously, the determination based on the average of equations 18, 19, and 20 and equations 35, 36, and 37 can provide greater accuracy.

[0461] Alternatively, a set of equations 50, 51 and 52 may be combined with one or more of a set of equations 18, 19 and 20 and / or a set of equations 35, 36 and 37.

[0462] Figure 7 The flowchart shown illustrates one embodiment of the method of the present invention, which combines... Figure 3 and Figure 6 The example shown.

[0463] In step 71a, for example as described above, a cap-dis-mix assay is performed. The cap-dis-mix assay includes a second particle A at a baseline concentration of X1 in the sample portion and an influence portion including a certain concentration of AF. L The process is performed when the first particle A (of which is Y1) is used, and each of the leading supplement and the following supplement is a buffer containing neither the first particle L nor the second particle A. In step 71b, the original dataset 1 is obtained from the Taylor dispersion determination in step 71a. In step 71c, R is obtained from the original dataset 1. h and at least one of appearance D. In step 71d, by appearance R h and / or apparent D determination .

[0464] In step 72a, the cap-dis-mix type assay i measures the second particle A, which includes a base concentration of X2, in the sample portion and the influence portion, which includes a certain concentration of AF. L The first particle L (of which is Y2) is executed, and each of the leading supplement and the following supplement is a buffer containing neither the first particle L nor the second particle A. In step 72b, the original dataset 1 is obtained from the Taylor dispersion determination in step 72a. In step 72c, R is obtained from the original dataset 1. h and at least one of appearance D. In step 72d, by appearance R h and / or apparent D determination .

[0465] In step 73a, the dismix-type assay is performed when the sample portion includes a second particle A at a baseline concentration of X3 and a first particle L at a baseline concentration of Y3, wherein each of the leading supplement and the following supplement is a buffer free of the first particle L and the second particle A. In step 73b, the raw dataset 1 is obtained from the Taylor dispersion assay of step 73a. In step 73c, R is obtained from the raw dataset 1. h and at least one of appearance D. In step 73d, by appearance R h and / or apparent D determination .

[0466] In step 74a, the dismix-type assay is performed when the sample portion includes a second particle A at a baseline concentration of X4 and a first particle L at a baseline concentration of Y4, wherein each of the leading supplement and the following supplement is a buffer free of the first particle L and the second particle A. In step 74b, the raw dataset 1 is obtained from the Taylor dispersion assay of step 74a. In step 74c, R is obtained from the raw dataset 1. h and at least one of appearance D. In step 74d, by appearance R h and / or apparent d determination .

[0467] In step 75, the determined Fit to Equation 21 and obtain the fitting parameters p, q and a from the fit.

[0468] In step 76, one or more combination parameters are determined based on a set of equations 18, 19, and 20 and / or a set of equations 35, 36, and 37 and / or a set of equations 50, 51, and 52 and / or a set of equations 67, 68, and 69. In this example, a set of equations 67, 68, and 69 is preferably applied, optionally combined with one or more other sets of equations.

Claims

1. A method for determining binding parameters between a first particle (L) and a second particle (A) capable of non-covalently interacting with the first particle, wherein the second particle includes a selected marker, wherein the method includes N sets of raw data are acquired, each set of raw data is obtained by performing a Taylor dispersion determination, and each of the at least one set of raw data is processed, wherein each Taylor dispersion determination for acquiring the corresponding set of raw data includes... i. A liquid portion comprising a sample portion containing the second particle (A) and at least one supplementary portion is supplied into a microfluidic channel such that the sample portion and the at least one supplementary portion form an interfacial contact in the microfluidic channel, wherein at least one of the liquid portions comprises a certain concentration of the first particle (L). ii. Providing the liquid portion to the flow in the microfluidic channel; iii. Acquire the set of raw data, which includes reading the signal strength s(t) of the marker as a function of time; Each set of raw data includes at least one unbalanced read, and The processing of each of the N sets of raw data includes determining the apparent diffusivity D and the hydrodynamic radius R. h At least one of them, The apparent diffusivity D is determined by applying Equation 12a. (12) and / or By applying Equation 23, the hydrodynamic radius R... h Sure (23) And will be determined for each of the N sets of original data. Fit to Equation 21 (21) One or more of a, p, and q are fitting parameters, and at least one binding parameter is determined by one or more of the fitting parameters, wherein the binding parameter preferably includes at least one of a kinetic parameter or an affinity parameter.

2. The method of claim 1, wherein the at least one binding parameter comprises one or more of the following: k off (Dissociation rate), k on (Association rate), k obs (The rate constant at which equilibrium is reached), R AL (hydrodynamic radius of AL), R A (The hydrodynamic radius of A), D A (Diffusion rate of unbound A), D AL (AL diffusion rate) and / or any derived parameter of one or more of the mentioned binding parameters.

3. The method according to claim 1 or claim 2, wherein the formation of the interfacial contact between the sample portion and the at least one supplementary portion in the microfluidic channel provides modification of the sample portion to a dispersed modified sample portion comprising the sample portion and the at least one supplementary portion, thereby inducing a concentration jump of at least one of the first particle L and the second particle A in the modified sample portion, wherein the concentration jump causes the first particle and the second particle to become unbalanced in the modified sample portion, wherein the concentration jump induces an association reaction or a dissociation reaction.

4. The method according to any one of the preceding claims, wherein at least one of the N original datasets is obtained from a Taylor dispersion assay, wherein the concentration jump induces an association reaction, and the method comprises determining the at least one binding parameter by applying one or more of the fitting parameters a, p, and q using equations E1, E2, and / or E3. (E1) (E2) (E3)， Where the concentration of L in the dispersion region can be considered constant, L X It is L T And when the concentration of L in the dispersion region can be considered not constant, L X It is L d And wherein, when the concentration of A in the dispersion region can be considered constant, A X It is 0, and when the concentration of A in the dispersion region can be considered not constant, A X It is A d , or At least one of the N original datasets is obtained from a Taylor dispersion assay, wherein the concentration jump induces a dissociation reaction, and the method includes determining the at least one binding parameter by applying one or more of the following equations from one or more of the fitting parameters a, p, and q. (50) (51) (52)。 5. The method according to any one of the preceding claims, wherein at least one of the N original datasets is obtained from a type 1 (capmix) Taylor dispersion assay, wherein the second particle (A) of the sample portion is dispersed to form a dispersion region in the microfluidic channel, and wherein the molar concentration of the second particle in the dispersion region is equal to or less than the molar concentration of the first particle, and the method comprises determining the at least one binding parameter by applying one or more of the fitting parameters a, p, and q. (17) (18) (19)， Preferably, at least half or all or all of the N original datasets, such as all of the N original datasets, each is obtained from a type I (capmix) Taylor dispersion assay, wherein the second particles (A) of the sample portion are dispersed to form a dispersion region in the microfluidic channel, and wherein the molar concentration of the second particles in the dispersion region is equal to or less than the molar concentration of the first particles.

6. The method according to the preceding claim, wherein at least one of the N original datasets is obtained from a type 1 (capmix) Taylor dispersion assay, wherein the second particle (A) of the sample portion is dispersed to form a dispersion region in the microfluidic channel, and wherein the molar concentration of the second particle in the dispersion region is equal to or greater than the molar concentration of the first particle, and wherein the method comprises determining the at least one binding parameter by applying one or more of the fitting parameters a, p, and q. (35) (36) (37)， Preferably, at least half or all or each of the N original datasets is obtained from a Taylor dispersion assay, wherein the second particles (A) of the sample portion are dispersed to form a dispersion region in the microfluidic channel, and wherein the molar concentration of the second particles in the dispersion region is equal to or greater than the molar concentration of the first particles.

7. The method according to any one of claims 4-7, wherein the interfacial contact between the sample portion and the at least one supplementary portion in the microfluidic channel provides modification of the sample portion to a modified sample portion comprising a dispersion between the sample portion and the at least one supplementary portion, thereby inducing a concentration jump of at least one of the first particle L and the second particle A in the sample portion, wherein the concentration jump causes the first particle and the second particle to become unbalanced in the modified sample portion, wherein the concentration jump induces an association reaction.

8. The method according to any one of the preceding claims, wherein at the time of supply (time 0), the volume of the sample portion is 5% or less of the total volume of the liquid portion including the sample portion and the at least one supplementary portion, preferably, at the time of supplying the sample portion, the volume of the sample portion is 1% or less of the total volume of the liquid portion, such as 0.5% or less, such as 0.1% or less, such as 0.05% or less.

9. The method according to any one of the preceding claims, wherein the total molar amount of the second particle in the total volume of the sample portion and the at least one supplementary portion is less than the total molar amount of the first particle in the total volume of the sample portion and the at least one supplementary portion, preferably, the total molar amount of the second particle in the total volume of the sample portion and the at least one supplementary portion is less than 5%, such as less than 1%, such as 0.5% or less.

10. The method according to any one of the preceding claims, wherein the at least one replenishment portion comprises a leading replenishment portion and a following replenishment portion, and wherein supplying the liquid portion to the microfluidic channel comprises providing the leading replenishment portion and the following replenishment portion on either side of the sample portion.

11. The method according to any one of the preceding claims, wherein at least one of the N original datasets is obtained from a type I (capmix) Taylor dispersion assay, wherein when the sample portion is supplied to the microfluidic channel, the sample portion comprises a baseline concentration of the second particles, and wherein when the at least one supplementary portion is supplied to the microfluidic channel, the at least one supplementary portion does not contain the second particles or has a concentration lower than the baseline concentration of the second particles, optionally, the second particles are bound to the first particles, and wherein the formation of the interfacial contact between the sample portion and the at least one supplementary portion in the microfluidic channel modifies the sample portion to provide a modified sample portion comprising a dispersion between the sample portion and the at least one supplementary portion, thereby inducing a concentration jump of at least the first particles L in the modified sample portion, wherein the concentration jump causes an imbalance between the first particles and the second particles in the modified sample portion, wherein the concentration jump induces an association reaction.

12. The method of claim 11, wherein the at least one supplementary portion does not contain the second particle, or wherein the second particle of the supplementary portion does not contain the selected marker, and / or wherein when the sample portion is supplied to the microfluidic channel, the concentration of the second particle is less than 10% of the base concentration of the second particle, such as less than 1% of the base concentration of the second particle when the sample portion is supplied to the microfluidic channel.

13. The method according to any one of the preceding claims, wherein at least one of the N original datasets is obtained from a type I (capmix) Taylor dispersion assay, wherein when the sample portion is supplied to the microfluidic channel, the sample portion includes a base concentration of the second particles and contains no first particles, and wherein when the at least one supplementary portion is supplied to the microfluidic channel, the at least one supplementary portion contains no second particles and has a certain concentration of the first particles L, and wherein the formation of an interfacial contact between the sample portion and the at least one supplementary portion in the microfluidic channel modifies the sample portion to provide a modified sample portion comprising a dispersion between the sample portion and the at least one supplementary portion, thereby inducing a concentration jump of at least the first particles L in the modified sample portion, wherein the concentration jump causes an imbalance between the first particles and the second particles in the modified sample portion, wherein the concentration jump induces an association reaction.

14. The method according to any one of the preceding claims, wherein at least one of the N original datasets is obtained from a type 2 (premix) Taylor dispersion assay, wherein when the sample portion is supplied to the microfluidic channel, the sample portion comprises a baseline concentration of the second particle (A) and a baseline concentration of the first particle (L), and wherein when the at least one supplementary portion is supplied to the microfluidic channel, the at least one supplementary portion has a concentration of the first particle, such as a concentration equal to or different from the baseline concentration of the first particle, and wherein the formation of the interfacial contact between the sample portion and the at least one supplementary portion in the microfluidic channel modifies the sample portion to provide a modified sample portion comprising a dispersion between the sample portion and the at least one supplementary portion, thereby inducing a concentration jump of at least one of the first particle L and the second particle A in the modified sample portion, wherein the concentration jump causes the first particle and the second particle to become unbalanced in the modified sample portion, wherein the concentration jump induces an association reaction or a dissociation reaction.

15. The method of claim 14, wherein when the sample portion is supplied to the microfluidic channel, the at least one supplementary portion has a different concentration of the first particles, such as a higher concentration of the first particles, which differs from the baseline concentration of the first particles L in the sample portion by at least 50%, such as at least 90%.

16. The method according to any one of the preceding claims, wherein at least one of the N original datasets is obtained from a type 3 (dismix) Taylor dispersion assay, wherein when the sample portion is supplied to the microfluidic channel, the sample portion comprises a baseline concentration of the second particle (A) and a baseline concentration of the first particle (L), and wherein when the at least one supplementary portion is supplied to the microfluidic channel, the at least one supplementary portion contains neither the first particle nor the second particle, and wherein the formation of an interfacial contact between the sample portion and the at least one supplementary portion in the microfluidic channel provides modification of the sample portion to a modified sample portion comprising a dispersion between the sample portion and the at least one supplementary portion, thereby inducing a concentration jump of at least one of the first particle L and the second particle A in the modified sample portion, wherein the concentration jump causes the first particle and the second particle to become unbalanced in the modified sample portion, and wherein the concentration jump induces a dissociation reaction.

17. The method according to any one of claims 14-16, wherein when the sample portion is supplied to the microfluidic channel, the first particles and the second particles of the sample portion are in equilibrium.

18. The method according to any one of claims 14-17, wherein the method comprises determining the at least one binding parameter by applying one or more of the fitting parameters a, p, and q. (50) (51) (52)， Preferably, at least half or all of the N original datasets, such as all of the N original datasets, are obtained from type 2 (premix) or type 3 (dismix) Taylor dispersion determinations.

19. The method according to any one of the preceding claims, wherein at least one of the N original datasets is obtained from a type 4 (cap-dis-mix) Taylor dispersion assay, wherein the liquid portion further includes an influence portion supplied to the microfluidic channel to form an interface contact with the sample portion, wherein when the sample portion is supplied to the microfluidic channel, the sample portion comprises a baseline concentration of the second particle A and substantially contains no first particle L, and when the influence portion is supplied to the microfluidic channel, the influence portion comprises a baseline concentration of the first particle L and substantially contains no second particle A, and wherein when the at least one supplementary portion is supplied... When supplied to the microfluidic channel, the supplementary portion is substantially free of both the first particle L and the second particle A, and the formation of the interfacial contact between the sample portion and the influencing portion and / or between the sample portion and the at least one supplementary portion in the microfluidic channel provides modification of the sample portion to a dispersed modified sample portion comprising the sample portion, the influencing portion and the at least one supplementary portion, thereby inducing a concentration jump of at least the first particle L in the modified sample portion, wherein the concentration jump causes an imbalance between the first particle and the second particle in the modified sample portion, and wherein the concentration jump induces an association reaction.

20. The method of claim 19, wherein when the sample portion is supplied to the microfluidic channel, the total volume of the sample portion and the influencing portion is 5% or less of the total volume of the liquid portion, preferably 1% or less of the total volume of the liquid portion, such as 0.5% or less, such as 0.1% or less, such as 0.05% or less.

21. The method of claim 19 or claim 20, wherein the method comprises determining the at least one binding parameter by applying one or more of equations 57, 58 and / or 59 from one or more of the fitting parameters a, p and q. (67) (68) (69)， Preferably, at least half or all of the N original datasets, such as all of the N original datasets, are obtained from a type 4 (cap-dis-mix) Taylor dispersion determination.

22. The method according to any one of claims 10-15 and 17, wherein the leading supplement portion has a leading portion concentration of the first particles, and the following supplement portion has a following portion concentration of the first particles, wherein the leading portion concentration of the first particles and the following portion concentration of the first particles are equal or different, preferably, the leading portion concentration of the first particles and the following portion concentration of the first particles are equal to or differ from the average concentration of the following portion concentration of the first particles and the leading portion concentration of the first particles by less than 10%, such as differing from the average concentration by less than 1%.

23. The method according to any one of claims 10-15 and 17, wherein the leading supplement portion does not contain the second particle or has a leading portion concentration of the second particle, and the following supplement portion does not contain the second particle or has a following portion concentration of the second particle, wherein the leading supplement portion concentration of the second particle and the following portion concentration of the second particle are equal or different, preferably, the leading portion concentration of the second particle and the following portion concentration of the second particle are equal or differ from the average concentration of the following portion concentration of the second particle and the leading portion concentration of the second particle by less than 10%, such as differing from the average concentration by less than 1%.

24. The method according to any one of claims 9-23, wherein the leading supplement portion has a leading portion volume and the following supplement portion has a following portion volume, wherein the leading portion volume and the following portion volume may be the same or different, preferably, each of the leading portion volume and the following portion volume is larger than the sample volume, such as being 5 times larger than the sample volume, such as being 10 times larger than the sample volume, such as being 20 times larger than the sample volume.

25. The method according to any one of the preceding claims, wherein acquiring each set of raw data comprises performing multiple reads of signal intensity at the readout section of the microfluidic channel, wherein the multiple reads preferably comprise at least 5 intensity reads, including at the peak occurrence time (t... R At least one intensity reading at the time of peak occurrence (t) R At least one intensity reading prior to the peak occurrence time (t) R At least one intensity read after that, preferably, acquiring each set of raw data includes recording intensity at a recording rate of at least 1 Hz, such as 5 to 12 Hz or higher.

26. The method according to any one of the preceding claims, wherein the method comprises acquiring the N sets of raw data, wherein the corresponding N sets of raw data are acquired from corresponding N Taylor dispersion measurements, wherein N is an integer of at least 1, and N is preferably from 1 to 50, such as from 2 to 20, such as from 3 to 10.

27. The method according to any one of the preceding claims, wherein at least parameter K is incorporated. D (Equilibrium dissociation constant), D A (Diffusion rate of unbound A) and / or D AL (Diffusion rate of AL) is a known binding parameter, and one or more of the known parameters are applied to determine at least one other binding parameter by one or more of the fitting parameters p, q and a, wherein N is preferably 5 or less, such as 1, 2 or 3, and wherein preferably, at least one of the N sets of raw data is obtained from a Taylor dispersion determination, wherein the sample portion comprises the first particle L at a baseline concentration in equilibrium and the second particle A at a baseline concentration.

28. The method according to any one of the preceding claims, wherein N is 2 or greater, and wherein said N sets of raw data comprise: At least one set of raw data obtained from Taylor dispersion assays, wherein the concentration jump induces association reactions, and At least one set of raw data obtained from a Taylor dispersion assay, wherein the concentration jump induces a dissociation reaction.

29. The method according to any one of the preceding claims, wherein N is 2 or greater, and wherein said N sets of raw data comprise: At least one set of raw data obtained from a Taylor dispersion assay, wherein the second particle (A) of the sample portion is dispersed to form a dispersion region in the microfluidic channel, and wherein the molar concentration of the second particle in the dispersion region is equal to or less than the molar concentration of the first particle, and At least one set of raw data obtained from a Taylor dispersion assay, wherein the second particle (A) of the sample portion is dispersed to form a dispersion region in the microfluidic channel, and wherein the molar concentration of the second particle in the dispersion region is equal to or greater than the molar concentration of the first particle.

30. The method according to any one of the preceding claims, wherein N is 2 or greater, and wherein the N sets of raw data comprise multiple datasets obtained from corresponding Taylor dispersion determinations, wherein the baseline concentration of the second particle A in the sample portions is different.

31. The method according to any one of the preceding claims, wherein N is 2 or greater, and wherein the N sets of raw data comprise multiple datasets obtained from corresponding Taylor dispersion determinations, wherein the base concentration of the first particle in the at least one supplementary portion of the sample portion is different.

32. The method according to any one of the preceding claims, wherein the selected mark comprises a detectable intrinsic mark and / or extrinsic mark, preferably, the mark is an optically readable mark, such as a fluorescent mark and / or a light-absorbing mark.

33. The method according to any one of the preceding claims, wherein the selected marker is sensitive to the molecular interactions, such as to conformational changes of the first particle, preferably, the selected marker changes the signal according to the conformation of the first particle and its changes, such as changing the signal according to binding / dissociation and / or structural changes.

34. The method according to any one of the preceding claims, wherein the liquid portion is supplied to the microfluidic channel at an injection pressure Pi, and wherein the liquid portion is supplied to the flow in the microfluidic channel at an operating pressure Pr to ensure laminar flow in the microfluidic channel, wherein the operating pressure Pr is greater than the injection pressure Pi, preferably, the operating pressure Pr is adjustable.

35. The method according to any one of the preceding claims, wherein each reading of the set of readings comprises reading a marker signal of the concentration of a second particle along a length cross-section of the microfluidic channel including the sample portion, preferably including the dispersion region, preferably reading the marker signal of the concentration of the second particle along the length cross-section of the microfluidic channel including the dispersion region from the leading edge of the dispersion region to the following edge of the dispersion region, comprising reading the intensity of the marker signal as a function of time as the dispersion region passes through the reading cross-section of the microfluidic channel.

36. The method according to any one of the preceding claims, wherein at least one read result of the imbalance read is obtained by reading a labeled signal of the concentration of a labeled second particle before the interaction between the first particle and the second particle reaches equilibrium, preferably, the method includes performing a plurality of imbalance reads, optionally, all read results in the set of read results are read results of imbalance reads.

37. The method according to any one of the preceding claims, wherein the set of reads includes reads of balanced readings, preferably obtained by reading a marker signal of the concentration of a second marked particle, wherein the interaction between the first particle and the second particle is balanced.

38. The method according to any one of the preceding claims, wherein the first particle and the second particle are different from each other, preferably, the first particle and the second particle are different in terms of particle type, molecular weight, dispersion properties or any combination thereof.

39. The method according to any one of the preceding claims, wherein at least one of the first particle and the second particle comprises a drug or a drug candidate.

40. The method according to any one of the preceding claims, wherein at least one of the first particle and the second particle comprises: 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 including at least one of these.

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