WO2025077984A1

WO2025077984A1 - A method of determining a binding parameter between a first particle and a second particle

Info

Publication number: WO2025077984A1
Application number: PCT/DK2024/050245
Authority: WO
Inventors: Henrik Jensen
Original assignee: Fida Biosystems Aps
Current assignee: Fida Biosystems Aps
Priority date: 2023-10-13
Filing date: 2024-10-10
Publication date: 2025-04-17
Anticipated expiration: 2026-04-13

Abstract

A method of determining a binding parameter between a first particle (L) and a second particle (A) capable of noncovalent interacting with the first particle. The method comprises obtaining N sets of raw data, wherein each set of raw data is obtained by performing a Taylor dispersion assay, and processing each of the at least one set of raw data, wherein each Taylor dispersion assay for obtaining each respective set of raw data comprises i) feeding liquid portions comprising a sample portion comprising said second particle (A) and at least one supplementing portion into a microfluidic channel such that the sample portion and the at least one supplementing portion form an interfacial contact in the microfluidic channel, wherein at least one of the liquid portions comprises a concentration of the first particle (L); ii) providing the liquid portions to a flow in the microfluidic channel and iii) obtaining the set of raw data comprising reading signal intensity as a function of time s(t) of the marker. Each set of raw data comprises at least one out-of-equilibrium reading and the processing of each of the N sets of raw data comprises determining at least one of the apparent diffusivity D and the hydrodynamic radius Rh and from this determining TD and fitting the TD determined for each of the N sets of raw data to a fitting equation.

Description

P81680PC01 1 A METHOD OF DETERMINING A PARAMETER BETWEEN A FIRST PARTICLE AND A SECOND PARTICLE TECHNICAL FIELD The invention relates to a method determining a binding parameter between a first particle, and a second particle, wherein the first and the second particle are capable of non-covalent binding to form a complex. The method of the invention is in particularly suitable for determining at least one kinetic binding parameter between the first and the second particle. BACKGROUND ART Fully or partly characterization of particle association in particular where at least one of the first and the second particles is a biomolecule is of fundamental importance for many technologies, such as within various chemical processes, e.g. within pharmacy, foods and diagnostic for example for understanding, developing and administration of drugs, for understanding and controlling chemical processing of food, for understanding biological systems and/or for performing diagnostic investigation and optionally for identifying biochemical disorders. In 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 is described determination of the equilibrium constant K_D for a protein Puf4 binding to its cognate RNA sequence wherein Puf4 was mixed over a series of concentrations, with a trace amount of the labeled RNA and at different temperatures and at different incubation times, where after the fraction of bound RNA was determined by non-denaturing gel electrophoresis. It were concluded that to reach equilibrium an incubation time of more than 4.5 hours was required and that an incubation for only 30 min would give an apparent K_D value that is seven times higher than after a 24 hours incubation. It was discussed that many of the K_D values available may be incorrect since the time of equilibrium may have been too short. Further it is shown that the temperature may highly influence the KD value. The article “Study of Binding between Protein A and lmmunoglobulin G Using a Surface Tension Probe” by Yang et al. Biophysical Journal Volume 84 January 2003 509—522, is disclosed a method of determining molecular interaction by surface tension measurement. It is described that the surface tension measurement method can quantify molecular binding ratios (or capacities) between the interacting species. P81680PC01 2 The article “Time, the Forgotten of Ligand Binding Teaching” by Javier orzo, BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION. Vol.34, No.6, pp.413–416, 2006 discuss the importance of the kinetic parameter constants kon and koff and the determination of individual constants, kon and koff by surface plasmon resonance. There is still a need for a new and reliable method for determining a binding parameter between a first particle (A) and a second particle capable of non-covalent interacting. DISCLOSURE OF INVENTION An objective of the present invention is to provide a relatively fast and reliable method for determining a binding parameter between a first particle and a second particle capable of interact with the first particle. In an embodiment, it is an objective to provide relatively fast and reliable method for determining a binding parameter between a first molecule and a second molecule capable of non-covalently interacting. In an embodiment, it is an objective to provide relatively fast and reliable method for determining a binding parameter between a first molecule and a second molecule capable of non-covalently interacting, wherein the binding parameter comprises at least one kinetic binding parameter, preferably comprising at least one of k_off (Rate of disassociation); k_on (Rate of association); k_obs (Rate constant for reaching equilibrium); KD (Equilibrium dissociation constant); RAL:(Hydrodynamic radius of AL); RA (Hydrodynamic radius of A); DA (Diffusitivity of unbound A); DAL (Diffusitivity of AL) and/or any binding parameter derived from one or more of the mentioned binding parameters. These and other objects have been solved by the inventions or embodiments thereof as defined in the claims and as described herein below. It has been found that the inventions or embodiments thereof have a number of additional advantages, which will be clear to the skilled person from the following description. Abbreviations and signs as applied herein: KD or Kd: Equilibrium dissociation constant P81680PC01 3 k_off: Rate of dissociation kon: Rate of association kobs: Rate constant for reaching equilibrium RA: Hydrodynamic radius of A R_AL: Hydrodynamic radius of AL R_h: Apparent Hydrodynamic radius of A + AL t½: Reaction half life (association) k(t): Time dependent dispersion coefficient t: time D(t): Time dependent diffusitivity constant D: Apparent diffusitivity of the second particle A (Free A and Bound A+L complex) ^_^: Diffusitivity of unbound A ^_^^: Diffusitivity of AL Rc: Capillary radius or if non circular = 2* cross-sectional area/perimeter t_R: Peak appearance time ^_^: Convoluted diffusivity L: First particle A: Second particle S(t): Signal intensity as a function of time a, p and q: Fitting constants σ²: Signal peak varians ^(^): ½ peak width as a function of time η: Viscosity ^_^: Linear flow rate P81680PC01 4 T: Temperature in Kelvin Af(t): Temporal dilution factor ^_^: Average dilution factor Pr: Run pressure Pi: Injection pressure C_A: Base concentration of A (Concentration of A in sample portion) C_L: Base concentration of L (Concentration of L in sample portion) SL: Base concentration of L (Concentration of L in supplementing portion portion) ^_^: Total A concentration in the sample/ dispersion zone with no dilution ~ CA L_T: Total L concentration in the sample/dispersion zone with no dilution ~ C_L ^_^: Average diluted concentration of total A (Average concentration of A in dispersion zone) ^_^: Average diluted concentration of total L (Average concentration of L in dispersion zone) ^_^ = ^_^^ ∙ (^_^ + ^_^) + ^_^^^

[AL] in the sample (dispersion zone) at the time zero [L]: Concentration of unbound L [A]: Concentration of unbound A [LA] or [AL]: Concentration of the complex AL/LA C(x,t): Particle concentration (solute concentration), position x at time t. x: Axial position of dispersion zone front TDA: Taylor dispersion assay The phrase “molecular interaction” means any non-covalent interactions between the first particle and the second particle, wherein the particles independent of each other may be a molecule a cluster of molecules or a complex of molecules. P81680PC01 5 The term “particle” is herein used to any portion of matter comprising at least one molecule, such as an organic molecule or an inorganic molecule. The particle may for example comprise an aggregate, a cluster, a complex or any combinations comprising one or more of these. The terms “first particle L”, “first particle” and the abbreviation “L” are used interchangeable. The terms “second particle A”, “second particle” and the abbreviation “A” are used interchangeable. The phrase “concentration of the first particle L” means herein the total concentration of the first particle in bound and unbound condition unless otherwise specified or clear from the context. The phrase “concentration of the second particle L” means herein the total concentration of the first particle in bound and unbound condition unless otherwise specified or clear from the context. All concentrations are molar concentrations unless otherwise specified or clear from the context. The term “binding partner” is herein used to mean any molecule or group of molecules, capable of non-covalent interacting with the particle. The microfluidic channel is also referred to as a “capillary”. The terms, “marker” or “label” are used interchangeable and are herein used to mean any intrinsic or extrinsic marker/label capable of being detected by a reader arrangement. In an embodiment, the marker /label comprises an element, group of elements, moieties and/or any combination comprising one or more of these, where the marker is capable of being detected by a reader arrangement directly and/or after being influenced from an external and/or internal source. In the same way the terms “marked” and “labelled” are used interchangeable. The term ”reader arrangement” means any detector or detector system capable of detection a signal associated with the binding partner and/or particle, such as an optical signal and/or an electrochemical signal. P81680PC01 6 The term “buffer” means an aqueous which is resistant to changes in pH value in the context where the buffer is used. The buffer advantageously comprises an aqueous solution of either a weak acid and its salt or a weak base and its salt. It should be emphasized that the term “comprises/comprising” when used herein is to be interpreted as an open term, i.e. it should be taken to specify the presence of specifically stated feature(s), such as element(s), unit(s), integer(s), step(s) component(s) and combination(s) thereof, but does not preclude the presence or addition of one or more other stated features. Reference made to “some embodiments” or “an embodiment” means that a particular feature(s), structure(s), or characteristic(s) described in connection with such embodiment(s) is included in at least one embodiment of the subject matter disclosed. Thus, the appearance of the phrases “in some embodiments” or “in an embodiment” in various places throughout the specification is not necessarily referring to the same embodiment(s). Further, the skilled person will understand that particular features, structures, or characteristics may be combined in any suitable manner within the scope of the invention as defined by the claims. Throughout the description or claims, the singular encompasses the plural unless otherwise specified or required by the context. All features of the invention and embodiments of the invention as described herein, including ranges and preferred ranges, may be combined in various ways within the scope of the invention, unless there are specific reasons not to combine such features. The inventor of the present invention has realized that the one or preferably more binding parameters associated to a noncovalent interaction between a first particle L and a second particle A may be found in an alternative and very effective way by applying a combination of Taylor dispersion assay technology data processing of one or more obtained raw data. Taylor dispersion assay technology is well known in the art for the determination of the diffusion coefﬁcient of a particle species and, thus, the hydrodynamic radius of a particle species. Taylor dispersion assay technology is based on the dispersion of a sample plug containing the particle species e.g. between buffer plugs in a narrow channel under a laminar Poiseuille ﬂow (pressure induced flow). The dispersion is due to the combined action of the dispersive parabolic velocity proﬁle and the molecular diffusion of the particle P81680PC01 7 species that redistributes the molecules the cross-section of the channel. The Taylor dispersion signal profile may be recorded as a function of time S(t). The TDA is for example described in US9,310,359, in wo22237946, in Conditions under which dispersion of a solute in a stream of solvent can be used to measure molecular diffusion .By SIR Geoffrey Taylor (1954) https://royalsocietypublishing.org/ on 27 February 2023 or in “Microfluidics and the quantification of biomolecular interactions”, by Otzen et al. Current Opinion in Structural Biology 2021, 70:8—15. https:l/doi.orgl10.1016lj.sbi.2021.02.006. The method of the invention is directed to determining at least one binding parameter between a first particle (L) and a second particle (A) capable of noncovalent interacting with the first particle, wherein the second particle comprises a selected marker. The marker may conveniently be or comprise an intrinsic marker and/or where desired an extrinsic marker. The method comprises obtaining N sets of raw data, wherein each set of raw data is obtained by performing a Taylor dispersion assay, and processing each of the at least one set of raw data. Each Taylor dispersion assay for obtaining each respective set of raw data comprises i. feeding liquid portions comprising a sample portion comprising the second particle (A) and at least one supplementing portion into a microfluidic channel such that the sample portion and the at least one supplementing portion form an interfacial contact in the microfluidic channel, wherein at least one of the liquid portions comprises a concentration of the first particle (L); ii. providing the liquid portions to a flow in the microfluidic channel; iii. obtaining the set of raw data comprising reading signal intensity as a function of time s(t) of the marker. The term “feeding” and “injecting” are used interchangeable. The formation of the interfacial contact in the microfluidic channel between the sample portion and the at least one supplementing portion may provide a modification of the sample portion to a modified sample portion and may introduce a concentration jump of at least one on the first particle L and the second particle A in the modified sample portion. Thus the concentration of the second particle A and the at least one supplementing portion may be different from each other and advantageously the supplementing portion should be free of the second particle A or the concentration of the second particle should conveniently be very low such as at P81680PC01 8 most 50 %, such as at most 10 %, such at most 1 % of the concentration of the second particle A in the sample portion.. The concentration jump may induce an association reaction or a dissociation reaction providing that the first particle L and the second particle A and the interaction there between will be out-of-equilibrium. Each set of raw data comprises a plurality of intensity readings of the marker of the second particle A, wherein each set of raw data comprises at least one and preferably a plurality out-of-equilibrium reading (s). The processing of each of the N sets of raw data comprises determining at least one of the apparent diffusivity D and the hydrodynamic radius Rh, determining ^_^ from the apparent diffusivity D by applying the equation 12a ^_^ = ½ ∙ ^^{^} = ^{^^^∙^^} ^^^ (12) and/or

determining ^_^ from the hydrodynamic radius Rh by applying the equation 23 ^_^ = ^{^∙^^∙^∙^^} ^_{∙^∙^^^∙^^} (23) and fitting the ^_^

^_^ = ^{^} ^ ∙ ^^_^ + ^{^} ^ ∙ ^^ ^ ^{^^^} ^_^^∙^^∙^^^^ (21) wherein

one binding parameter from one or more of the fitting parameters, wherein the binding parameter preferably comprises at least one of a kinetic parameter or an affinity parameter. The inventor has found that the fitting parameters may described as equations, wherein the equation to be applied depend on whether the concentration jump induces an association reaction or a dissociation reaction. Further the equation to be applied may depend on whether the concentration of respectively the first particle L and the second particle A in the dispersion zone may be considered constant or not. In an embodiment, wherein at least one of the N raw data sets is obtained from Taylor dispersion assay wherein the concentration jump induces an association P81680PC01 9 reaction and the method comprises the at least one binding parameter from one or more of the fitting parameters a, p and q by applying one or more of the equations E1, E2 and/or E3 ^ = ^_^ + (^_^^ − ^_^) ∙ ^{^^} ^_^^^^^^^ (E1) , wherein LX

considered constant and Ld when the concentration of L in the dispersion zone may not be considered constant and wherein A_X is 0 when the concentration of A in the dispersion zone may be considered constant and A_d when the concentration of A in the dispersion zone may not be considered constant. The equations E1, E2 and E3 are also called a set of equations E1, E2 and E3. In an embodiment, wherein at least one of the N raw data sets is obtained from Taylor dispersion assay wherein the concentration jump induces an dissociation reaction and the method comprises determining the at least one binding parameter from one or more of the fitting parameters a, p and q by applying one or more of the equations ^ = (^_^^ − ^_^) ∙ ^{^^^∙^^} ^_^ + ^_^ (50)

The first particle is herein 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 independently of each other be a molecule, a cluster, an aggregate or a complex. Advantageously, one of or both of the first particle L and the second particle A is a molecule. The equations 50, 51 and 52 are also called a set of equations 50, 51 and 52. P81680PC01 10 A Taylor dispersion assay may also be as a dispersion assay wherein Taylor conditions apply. An out-of-equilibrium reading is a reading where the interaction between the first particle and the second particle is out of equilibrium. Reference to the set of raw data means herein at least one of the N set of raw data. The processing of each respective set of raw data of the N sets of raw data to determine ^_^ may be identical or different as further described below and/or as illustrated in the examples. The apparent diffusivity D may be determined from the set of raw data using any method, such as the method known in the art, e.g. as described in the article “Flow induced dispersion analysis rapidly quantifies proteins in human plasma samples” Poulsen et al, DOI: 10.1039/C5AN00697J. Analyst, 2015, 140, 4365-4369, in the article “Flow Induced Dispersion Analysis Quantifies Noncovalent Interactions in Nanoliter Samples, Jensen et al. Journal of the American Chemical Society 132(12):4070-1 March 2010. DOI:10.1021/ja100484d and/or as described in US9310359 The apparent diffusivity D may preferably be determined by a method comprising fitting the set of raw data to the equation ^(^) = ^_^ + ^_^ ∙ ^^^ ^− ^{(^^^^)^∙^^^} ^_^^∙^^ ^ (12b) Here ^_^ is a detector/background offset and ^_^ 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, during the Taylor dispersion assay, and by fitting the raw data to equation 12b, the parameters ^_^, D, ^_^ and ^_^ are determined. The hydrodynamic radius Rh may be determined from the raw data using any method, such as describe in the above identified articles and/or as described in WO21180289. In an embodiment, the hydrodynamic radius Rh is determined based on the apparent diffusivity D by applying the equation 22 ^_^ = ^{^^∙^} ^_∙^∙^∙^ (22)

P81680PC01 11 The processing of the respective N raw data may comprise a preprocessing step of filtering the raw data, for example comprising removing outlies, signal noise etc. The liquid portions are advantageously comprises a buffer as basic liquid, wherein the mentioned particle or particles may be dispersed, wherein the at least one supplementing portion in an embodiment may be pure buffer. The buffer is advantageously selected in dependence of the first particle L and the second particle A to provide a desired pH level for the interaction between the particles. After large effort and thorough analysis the inventor of the present invention has reach the method of determining the one or more binding properties in a relative simple, fast and reliable way. Especially it has been an aim to determine the kinetic binding parameters in particular comprising k_off (Rate of dissociation); k_on (Rate of association) and k_obs (Rate constant for reaching equilibrium) which in prior art methods have been cumbersome and difficult to determining with adequate accuracy. However, it is also very beneficial that other binding parameters such as the kinetic parameters KD: (Equilibrium dissociation constant); RAL :( Hydrodynamic radius of AL); R_A (Hydrodynamic radius of A); D_A (Diffusitivity of unbound A); D_AL (Diffusitivity of AL) may be determined with a relatively high accuracy by applying an embodiment of the method of the invention. Advantageously, the at least one binding parameter comprises one or more of k_off (Rate of disassociation); k_on (Rate of association); k_obs (Rate constant for reaching equilibrium); RAL:(Hydrodynamic radius of AL); RA (Hydrodynamic radius of A); DA (Diffusitivity of unbound A); DAL (Diffusitivity of AL) and/or any derivative of one or more of the mentioned binding parameters. The rationale behind the present invention and the effects thereof may be explained on the following theoretical framework. It is assumed that binding kinetics may be assessed by realizing conditions where equilibrium is not established. It is here assumed that a simple concentration change of one of the equilibrium species may be applied. It has been found that the Taylor dispersion assay may be applied for providing a rapid changing of a particle concentration in a microfluidic capillary flow system. P81680PC01 12 This may be provided as describes wherein the liquid portions comprising a sample portion comprising the second particle (A) and at least one supplementing portion and optionally an affecting portion are fed into and provided to a laminar flow in the microfluidic channel, wherein at least one of the liquid portions comprises a concentration of the first particle (L) Whereas there are many variations of the content of A and L in the sample portion and in the one or more (normally two - a leading supplementing portion and a trailing supplementing portion) and the optional affecting portion, there are mainly four ground types of Taylor dispersion assay for inducing the out- of equilibrium condition. Type 1, referred to as “capmix” wherein the sample portion comprises a concentration of unbound second particle A and free of the first particle L and the supplementing portion(s) comprises a concentration of the first particle L and is essentially free of the second particle A. Type 2, referred to as “premix” wherein the sample portion comprises a concentration of the second particle A and a concentration of the first particle in equilibrium and wherein the supplementing portion(s) comprises a concentration of the first particle L and is free of the second particle A. Type 3, referred to as “dismix” wherein the sample portion comprises a concentration of the second particle A and a concentration of the first particle in equilibrium and wherein the supplementing portion(s) are essentially pure buffer free of both A and L. Type 4, referred to as “cap-dis-mix” wherein the liquid portions further comprises an affecting portion feed into the microfluidic channel to form an interfacial contact with the sample portion, wherein the sample portion comprises a concentration of the second particle A and is essentially free of the first particle L, the affecting portion comprises a concentration of the first particle L and is essentially free of the second particle A and wherein the supplementing portion(s) is/are essentially free of both the first particle L and the second particle A In the premix assay, the first particle L and the second particle A are mixed prior to the performing of the Taylor dispersion assay to ensure that equilibrium is established. Further L is introduced in the supplementing portion(s). Where the concentration of L in the supplementing portion(s) is different from the concentration of L in the sample portion the concentration of L in the modified sample concentration will rapidly be as in the supplementing portion and thereby a P81680PC01 13 concentration jump has been induced modified sample portion inducing the out- of equilibrium condition especially where A initially was in surplus in the sample portion. Where A is in surplus in the sample portion and the concentration of L is larger in the supplementing portion(s) than in the sample portion, the concentration jump induces an association reaction. Where A is in surplus in the sample portion and the concentration of L is smaller in the supplementing portion(s) than in the sample portion, the concentration jump induces an dissociation reaction. In the capmix assay, there is no binding between A and L at time zero, i.e. prior to feeding the liquid portions to the microfluidic channel, and as the sample passes through the capillary (microfluidic channel), mixing and binding between A and L takes place – i.e. the concentration jump in a capmix assay induces an association reaction. Ideally, the assay conditions may be chosen so that the injected sample is only a very small part of the capillary i.e. the volume of the sample portion is much smaller than the total volume of the supplementing portion(s). Under such conditions, L is mixed into the modified sample portion rapidly and it can be assumed that L is uniformly distributed in the capillary at time greater than zero. Assuming that the apparent diffusion coefficient describing the dispersion, is a time dependent weighted average of the diffusivities of bound and non-bound A. It has been found that under such conditions a closed form solution of the dispersion dependence on kon and koff may be obtained. When the binding kinetics is sufficiently slow to perform out-of-equilibrium readings, it has been found that the data can be analyzed in such a way that kinetic information may be extracted. LA is an in-solution complex composed of L and A. LA= L + A , K_d = [L][A]/[LA] in equilibrium (1) koff LA->LA + A (2) kon L + A -> LA (3) Kd = koff/kon (4) P81680PC01 14 The different limiting cases may be on the binding kinetics. It is useful to define a relaxation kinetic constant as: k_obs = k_off + k_on*[A] (5) In terms of kinetics, a convenient estimate of when a system can be studied is the reaction half-life defined as: t½ = ln2/kobs (6) The Taylor dispersion assay also called flow induced dispersion analysis (FIDA) may conveniently be performed in microfluidic channels in the form of thin capillaries with peak appearance times as low as 10 seconds or even lower. Depending on the size (or marker e.g. fluorescence) change upon binding a rough estimate of accessible binding kinetics would be conditions corresponding to t_½ of 1-5 seconds and longer. On the other hand, using very low flow rates / long capillaries it is possible to reach peak appearance times in the order of 1 hour (3600 seconds) and possibly even longer. In the following referred to as a first case wherein L is in excess of A, a first theoretical framework for a capmix type assay is elucidated. It is assumed that t_½ is between 1 sec and about 3600 seconds (60 min). In the following equations used to fit to a binding curve representing apparent Rh as a function of concentration of L (L_T), wherein the concentration of L in the supplementing portion(s) is in excess of the concentration of A in the sample portion. Due to the fast mixing (low volume of sample portion) it can be assumed that the concentration of L is constant in the dispersion zone (the zone of the microfluidic channel comprising the modified sample). Under these conditions the kinetics may be considered independent of the exact A concentration. Furthermore, it is assumed that the Taylor conditions apply. Taylor conditions means that the mass transport problem is significantly simplified. It means that the signal shape can be described by the following differential equation: ^^{^} _^ ⁽ _^,^ ⁾ = ^ ^{^^(^,^)} ^_^ ^{^} (^) ∙ ^^ (7)

P81680PC01 15

In the limiting case of a constant diffusivity k(t) (and D(t)) are simply constants. However, for the present situation, the diffusivity is modeled as a weighted average of bound and unbound A. The apparent diffusivity is thus time dependent (governed by the kinetics of complex formation), but decoupled from the spatial diffusion. It is convenient to define a convoluted diffusivity, ^_^, as: ^_^ = _∫ ^{^^} ^ ^(^)^^ = ^^{^} ∙ _∫ ^{^^ ^} ^_^(^)^^ (10)

By change of variables, equation 7 can then be rewritten as: ^^{^}^(^,^_^) ^_^ ^{^} = ^{^^(^,^^)} ^{^^} _^ (11)

that used under Taylor´s conditions and using the boundary condition corresponding to pulse injection. From the general Gaussian solution, ^_^ is related to the peak variance (at tR) as: ^_^ = ½ ∙ ^^{^} = ^{^^^∙^^} ^^^ (12) Where D is the apparent diffusivity obtained from fitting the raw data (S(t), fluorescence vs time). Under conditions where Taylor conditions apply, the raw data may be fitted to the following equation: (^) = ^_^ + ^_^ ∙ ^^^ ^− ^{(^^ )^} ^ ^{^^ ∙^^^} ^_^^∙^^ ^ (12b) P81680PC01 16 Here ^_^ is a detector/background offset ^_^ 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 from the raw data to equation 12b, the parameters ^_^, D, ^_^ and ^_^ are determined. Then, ^_^ may be obtained from equation 12. The apparent diffusivity changes according to the fraction bound of A. Initially, A is 0% bound to L, but during the experiments, the fraction bound increases as described by homogeneous binding kinetics. Under these conditions the time dependent diffusivity, D(t), is linked to fraction bound according to: D(t) = x(t) ^D_I + (1-x(t)) ^D_AL (13) The rate equation (corresponding to equations 2 and 3) can be solved for AL, and for the boundary condition where [AL] is zero at time zero, the solution is: [^^](^) = ^{^^∙^^ ^^^^^ ^^ ∙^ ^∙^} ^{^} _^ ^{^^} _^ ∙ _^1 − ^ ^{^ ^^ ^} _^ (14)

concentration. It then follow: 1 − ^(^) = ^{^^ ^^^^^^^^^ ∙^ ^∙^} ^{^} _^ ^{^^} _^ ∙ ^1 − ^ ^{^ ^} ^ (15)

^⁽^⁾ = ^_^ + ⁽^_^^ − ^_^ ⁾ ∙ ^{^^} ^_^^^^ + (^_^ − ^_^^) ∙ ^{^^} ^_^^^^ ∙ ^^{^^^^^^^^^^∙^^^∙^} (16)

Note that the two first (time independent) terms correspond to case 1 and the normal 1-1 equilibrium binding model used to obtain Kd´s in Fida experiments. Equation 16 may be simplified using the following parameters, which may then be applied as fitting parameters as described below: ^ = ^_^ + (^_^^ − ^_^) ∙ ^{^^} ^_^^^^ (17)

P81680PC01 17

concentration of L and A in the dispersion zone may be considered constant. The equations 17, 18 and 19 are also called a set of equations 17, 18, 19. It then follow that ^_^ evaluated at tR (equation 10) is given by: ^_^ = _∫ ^{^^} ^ ^(^)^^ = ^^{^} ∙ _∫ ^{^^ ^} ^_^(^)^^ = ^^{^} ∙ _∫ ^{^^ ^} ^_^^^∙^^∙^ ^^ (20)

^_^ = ^{^} ^ ∙ ^^_^ + ^{^} ^ ∙ ^^ ^ ^{^^^} ^_^^∙^^∙^^^^ (21)

Through this set of equations ^_^ is linked to DA, DAL, Kd and the kinetic parameters (koff and kon). Therefore, varying LT in a titration measuring ^_^ from equation 12 and then fitting it to equation 21 would in principle give access to all the above parameters. In practice, even better accuracy may be obtained doing 2 or more experiments comprising a premix giving DA, DAL and KD, and a capmix where the remaining unknown kinetic parameters may be fitted. Alternatively or in addition, several capmix experiments can be performed at different tR values. In many cases it is more convenient to use hydrodynamic radius (Rh) rather than diffusivities. Diffusivity can be converted into hydrodynamic radius using the Stokes – Einstein equation (equation 22). ^_^ = ^{^^∙^} ^_∙^∙^∙^ (22)

P81680PC01 18 ^_^ = ^{^∙^^∙^} ^^{∙^^} ^_{∙^∙^^∙^^} Therefore ^ is link

^ ed to RA, RAL, Kd above using appropriate fitting procedures Rh obtained from raw data can be used to obtain hydrodynamic radius of A (RA), hydrodynamic radius of AL (RAL), K_d and the kinetics (k_off and k_on). An example of the implementation may comprise determining apparent Rh from sets of raw data from raw data at different L_T values (i.e. concentration of L in the supplementing portion, which is assumed to be the same in the modified sample/dispersion zone). Determining and fitting the respective ^_^ to obtain kinetic parameters pertaining to the binding from one or more of the fitting parameters a, p and q. In the following second case, a second theoretical framework for a capmix type assay is elucidated. Also here it is assumed that t½ is between 1 sec and about 3600 seconds (60 min). In the following equations used to fit to a binding curve representing apparent Rh as a function of concentration of L (LT), wherein the concentration of L in the supplementing portion(s) is not in excess of the concentration of A in the sample portion. Although the second case is similar to the first case, some differences may be considered. When the A concentration is high it cannot in general be assumed that the concentration of free L, [L], can be approximated by LT. The reason is that a significant portion of L is bound to A. LT = [L]+[AL] (24) The amount of L bound to A may be determined by the Kd of the interaction as well as the concentration of A. In order to treat the second the concentration of A is estimated. At time zero, the total concentration of A is CA, and it may be estimated based on the readable marker of the sample portion prior to injection into the capillary. During the capmix assay A (unbound A and AL) are diluted due to the dispersion process (Taylor dispersion) in the capillary. The concentration of A in the dispersion zone is thus not constant and in order to describe the second case it is convent to P81680PC01 19 describe the dilution process occurring the experiment. It has been found that the raw data in itself may be a good measure of how the sample is diluted during the experiment as the Gaussian signal is effectively a temporal concentration profile. The spatial width of the initial dispersion (zone of A) ( ^i) may be obtained by the linear flow rate during sample injection (u_i) times the injection time (t_i): ^_^ = ^_^ ∙ ^_^ (25) During the experiment, the sample zone is dispersed as a Gaussian distribution as described by the Taylor theory. The temporal dispersion is described by the peak width, Z ^ ^(t), where Z is a scaling factor which may be assumed to be 2, and may thus be used to calculate the spatial width of the dispersion zone at tR ( ^s): ^_^ = ^_^ ∙ 2 ∙ ^(^) (26) Where ^_^ is the linear flow rate during the actual experiment and: ^⁽ _^ ⁾ ₌ ^{^ ^^^} ^^^ ∙ _√^ (27)

In equation 27 it is assumed that D is an average diffusion coefficient during the experiment which can be obtained from the raw data as described above. The temporal dilution factor, A_f(t), is then given by: ^_^(^) = ^{^^ ^^∙^^ ^^∙^^} ^_^^^^ = _{^^∙^^^^^∙^∙^(^)} = _{^^∙^^^^^∙^∙^(^)} (28)

Where Pi is the injection pressure and Pr is the run pressure in the experiment. Here we take advantage of the fact that according to the Hagen – Poiseuilles equation, u and P are directly proportional. P81680PC01 20 Equations 28 and 27 can be combined rewritten as: ^_^(^) = ^{^} ^_^^∙√^ (29) Where b is given by: ^ = 2 ∙ ^{^ ^} _^^ ^^∙^ ∙ ^ ^{^} _^ ^_^ ^{∙^} _^ ^ (30)

are specific (and known) to the experiment or directly obtainable from raw data. The dilution factor is time dependent as shown in equation 29. It may also be noted that t_R is proportional to P_r. Therefore Af(t) is proportional to sqrt(t), meaning that the dilution factor increased (less dilution) at high t values. The reason is that diffusion over time will counteract the flow induced dispersion. In the following it is assumed that the dilution effect of A is well described by an average dilution factor (A_f) during the experiment vide infra. ^_^ = ^{^} ^_^^^∫ ^{^^} ^ ^_^(^)^^ = ^{^} ^_^ ∙ _∫ ^{^^ ^} ^_^^^∙√^ ^^ (31)

The integral in equation 31 can be solved analytically. The result is: ^_^ = ^{^} ^_∙√^^ − ^{^} ^_^∙^^ ∙ ln (^ ∙ _√^_^ + 1) (32)

It can be noted that ^_^ may be calculated directly from raw data (D and ^_^) and known experimental parameters. Using the result of equation 32, the total indicator concentration during the experiment is thus simply given by: ^_^ = ^_^ ∙ ^_^ = ^_^ (33)

P81680PC01 21 Where C_A is the total concentration of the sample portion. The rate equations pertaining to second case may then be solved in a way similar to simple homogeneous kinetics. The solution is: [^^](^) = ^{^^∙^^ ^^^^^ ^^ ∙(^ ^^ )^∙^} ^{^} _^ ^{^^} _^ ^{^^} _^ ∙ ^1 − ^ ^{^ ^^ ^ ^} ^ (34)

slightly different definitions of a, p and q of the first case: ^ = ^_{^^^ ^^^^} = − ^^_^^^ + ^_^^ ⁽^_^ + ^_^ ⁾^ (35)

concentration of L in the dispersion zone may be considered constant and the concentration of A is considered not constant. The equations 36, 37 and 35 are also called a set of equations 36, 37 and 35. In a third case and a fourth case respectively a third theoretical framework and a fourth theoretical framework for the dismix type assays are discussed. Also here it is assumed that t_½ is between 1 sec and about 3600 seconds (60 min). In the third case, the concentration of L in the sample portion is in excess of the concentration of A in the sample portion. And in the fourth case, the concentration of L in the sample portion may not be in excess of the concentration of A in the sample portion. Dilution of both unbound A and/or unbound L in the dispersion zone during the Taylor dispersion assay are treated as average dilutions that are independent of kinetics and vice versa. However, Kinetics is indirectly taken into account since dilution may be obtained from the fraction bound. It means that the concentration of unbound A and unbound L may be assumed to be adequately described by average P81680PC01 22 concentrations when Taylor conditions This conveniently simplifies the solution of the mass transport equations describing the distribution of particles. It is thus possible to obtain a general solution, albeit using different boundary conditions than the solution pertaining to capmix as applied in the first and the second cases. In general: [^^^] = ^ ∙ ^^{^^^∙^} + ^{^^^∙^^∙^^} ^{^} _^ (38)

Where ^_^ and ^_^ are the average diluted concentrations of total A and L respectively. In the third case it may be assumed that LT = [L] (that is L is in excess). At t=0 the concentration is determined by the Kd: [_^^ ^] ₌ ^{[^]∙[^] (^^^[^^])∙^^ ^^∙^^ [^^]∙^} _^ ^_^ = ^{^} _^ = ^{^} _^ − ^{^} _^ (40)

s^{econd particle in the sample portion at the time of injecting the sample.} [_{^^] ∙ (^^ + ^^) = ^^ ∙ ^^ (41)}

^ = ^ − ^{^^^∙^^∙^^ ^^∙^^ ^^^∙^^∙^^} ^{^} _^ = _(^^^^^) − _^^ (44)

P81680PC01 23

t=0: [^^] = ^{[^]∙[^]} = ^{(^^^[^^])∙(^^^[^^])} ^{^} _^ ^{^^}

Of which the physically meaningful solution is: ^ = ½ ∙ _^(^_^ + ^_^ + ^_^) + _^(^_^ + ^_^ + ^_^)^{^} − 4 ∙ ^_^ ∙ ^_^^

P81680PC01 24

from the fitting parameters a, q and p for the third case and the fourth case Taylor dispersion signal assay(s) may be determined. The average concentrations (Ad) are obtained as described before for A. When L is in excess of A, Ld can be obtained in an analogue procedure. ^_^(^) ∙ ^_^ = ^_^^ (53) Where Ld1 refers to the limiting case and Af(L) is obtained from eq 30 and 31 using the diffusivity of L in the calculation of the constant b. For the limiting case where L is not in excess of A: L_T ^ [AL], Ld may be found using the dilution factor of A. ^_^ ⁽^^⁾ ∙ ^_^ = ^_^^ (55) For the general case, the dilution factor of L may be found as a weighted average of the two limiting cases. ^_^ = ^_^ ^{^} _^ ^{^} ∙ ^_^^ + ^_^ ^{^^} ∙ ^_^^ (56) Where ^_^ ^{^} _^ ^{^} is the average fraction bound of L and ^_^ ^{^^} is the average fraction free of L.

P81680PC01 25

^_^^ ⁽^⁾ = 1 − ^_^ ⁽^⁾ (58) Using numerical algorithms the equation above may be conveniently solved. However, it is also accurate to use average concentrations which simplifies the approach significantly. Using average concentrations we get: ^_^ ^{^^} = ^{^^^[^^]^^} ^{^} _^ (59) ^_^ ^{^} _^ ^{^} = 1 − ^_^ ^{^^} (60)

W^{here [^^]^^ is the average AL concentration during the experiment.} [_{^^]^^ can be found from the experimental measurement of fraction bound of A} d^{uring the experiment:} [_{^^] = ^ ^} _{^^ ^ ∙ ^^^^^^ (61)} Inserting in equations 59 and 60 gives: ^_^ ^{^} ^ ^^{^^} = ^{^} ^{^} _^ ∙ ^{^} _^ ^{^^} _^^ (62)

Where D is the measured apparent diffusivity. Combining equations 56-62 gives: ^_^ = ^{^^} ^_^ ∙ ^{^ ^} ^_^^^^^ ∙ ^_^^ + _^1 − ^{^^} ^_^ ∙ ^{^ ^} ^_^^^^^^ ∙ ^_^^ = ^{^^ ^ ^} ^_^ ∙_^^^^^^ ∙ (^_^^ − ^_^^) + ^_^^ (63)

Rearranging gives the following 2. order equation: ^^{^} _^ − ^_^ ∙ ^_^^ + ^_^ ∙ ^{^ ^} ^_^^^^^ ∙ (^_^^ − ^_^^) = 0 (64)

P81680PC01 26

Of which only equation 66 is the physical meaningful solution: ^_^ = ½ ∙ ^_^^ + ½ ∙ _^^^{^} _^^ − 2 ∙ ^_^ ∙ ^{^} _^ ^_^ ^{^^} _^^ ∙ (^_^^ − ^_^^) (66)

It is recognized that the hydrodynamic radii of L and AL may also be used in the calculations of the dilution factors as hydrodynamic radius is related to diffusivity according to the Stokes Einstein equation (equation 22). The type 4 assay “cap-dis-mix as defined above may be said to be a mixture of the capmix and the dismix type, where the concentration jump induces an association reaction and where the concentration of both A and L in the dispersion zone are not considered to be constant. The total volume of the sample portion and the affecting portion is advantageously relatively small, preferably less than 5 %, such as less than 2% and ideally 1 % or less of the total volume of the liquid portions Under these conditions, which herein is also referred to as the fifth case, the above formulas for Ad and Ld holds and the boundary condition at time zero is similar to case 1 and 2. The fitting parameters a, p and q may therefore be as the following equations 57, 58, 59 ^ = ^_{^^^ ^^^^} = − _^^_^^^ + ^_^^(^_^ + ^_^)_^ (67)

P81680PC01 27 In a corresponding way, equations for the desired binding parameters from the fitting parameters a, q and p for the fifth case Taylor dispersion signal assay(s) may be determined. The equations 67, 68 and 69 are also called a set of equations 67, 68, 69. In an embodiment, the at least one binding parameter comprises one or more of koff (Rate of disassociation); k_on (Rate of association); k_obs (Rate constant for reaching equilibrium); R_AL:(Hydrodynamic radius of AL); R_A (Hydrodynamic radius of A); D_A (Diffusitivity of unbound A); DAL (Diffusitivity of AL) and/or any derivative of one or more of the mentioned binding parameters. The preferred binding parameter to be determined by embodiments of the invention may be as described above. Advantageously, formation of the interfacial contact between the sample portion and the at least one supplementing portion in the microfluidic channel, provides a modification of the sample portion to a modified sample portion comprising dispersion between the sample portion and the at least one supplementing portion, to thereby induce a concentration jump of at least one on the first particle L and the second particle A in the modified sample portion, wherein the concentration jump provides the first particle and the second particles to be out of equilibrium in the modified sample portion, wherein the concentration jump induces an association reaction or a dissociation reaction. The zone along the microfluidic channel comprising the second particle which initially was in the sample portion is referred to as the dispersion zone. The association reaction induces first particle L and the second particle A in the sample portion to bind to form bound first and second particles (AL). Where bound first and second particles (AL) are already present in the modified sample portion the association reaction induces the concentration of bound first and second particles (AL) to increase. The dissociation reaction induces bound first and second particles (AL) to dissociate to the first particle L and the second particle A to reduce the concentration of bound first and second particles (AL). The modified sample portion is defined by the second particles A, which carries the selected marker (intrinsic or extrinsic), which are read during the Taylor dispersion P81680PC01 28 assay. The modified sample portion in microfluidic channel is also referred to as the dispersion zone. In an embodiment, at least one of the N raw data sets is obtained from a Taylor dispersion assay wherein the second particle (A) of the sample portion is dispersing to form a dispersion zone in the microfluidic channel and wherein the molar concentration of the second particle is equal to or lower than the molar concentration of the first particle in the dispersion zone and the method comprises determining the at least one binding parameter from one or more of the fitting parameters a, p and q by applying one or more of the equations 17, 18 and/or 19 ^ = ^_^ + (^_^^ − ^_^) ∙ ^{^^} ^_^^^^ (17) , preferably at least half of

Taylor dispersion assay wherein the second particle (A) of the sample portion is dispersing to form the dispersion zone in the microfluidic channel and wherein the molar concentration of the second particle is equal to or lower than the molar concentration of the first particle in the dispersion zone. In an embodiment, at least one of the N raw data sets is obtained from a Taylor dispersion assay wherein the second particle (A) of the sample portion is dispersing to form a dispersion zone in the microfluidic sample and wherein the molar concentration of the second particle is equal to or larger than the molar concentration of the first particle in the dispersion zone and wherein the method comprises determining the at least one binding parameter from one or more of the fitting parameters a, p and q by applying one or more of the equations 35, 36 and/or 37 ^ = − ^^_^^^ + ^_^^(^_^ + ^_^)^ (35) ,

P81680PC01 29 preferably at least half of or all or the N data sets are each obtained from a Taylor dispersion assay wherein the second particle (A) of the sample portion is dispersing to form a dispersion zone in the microfluidic sample and wherein the molar concentration of the second particle is equal to or larger than the molar concentration of the first particle in the dispersion zone. In an embodiment, the at least one binding parameter is determined from one or more of the fitting parameters by applying one or more of the equations 17-19 and the at least one binding parameter is determined from one or more of the fitting parameters by applying one or more of the equations 35-37, where after average values of the at least one binding parameter may be determined, to thereby determining the at least one binding parameter with an even higher accuracy. This may in particular be desired where the N sets of raw data comprises at least one set of raw data obtained from a Taylor dispersion assay wherein the molar concentration of the second particle is equal to or lower than the molar concentration of the first particle in the dispersion zone and at least one set of raw data obtained from a Taylor dispersion assay wherein the molar concentration of the second particle is equal to or higher than the molar concentration of the first particle in the dispersion zone. The N raw data sets may comprise one or more data sets obtained from respective one or more Taylor dispersion assay of the capmix type, one or more data sets obtained from respective one or more Taylor dispersion assay of the dismix type, one or more data sets obtained from respective one or more Taylor dispersion assay of the cap-dis-mix type and/or one or more data sets obtained from respective one or more Taylor dispersion assay of the premix type. The interfacial contact between the sample portion and the at least one supplementing portion in the microfluidic channel, provides a modification of the sample portion to a modified sample portion comprising dispersion between the sample portion and the at least one supplementing portion. Thereby a concentration jump of at least one on the first particle L and the second particle A in the sample portion may be induced. The concentration jump provides the first particle and the second particles to be out of equilibrium in the modified sample portion, wherein the concentration jump induces an association reaction or wherein the concentration jump induces a dissociation reaction. To ensure a desired large concentration jump, it is desired that sample portion has a volume which is 5 % or less than the total volume of the sample portion and the at P81680PC01 30 least one supplementing portion, the sample portion at the time of feeding the sample portion has a volume which is 1 % or less, such as 0.5 % or less, such as 0.1 % or less, such as 0.05 % or less than the total volume of the sample portion and the at least one supplementing portion. In practice the sample portion may be a very few nanoliter (nL), since the microfluidic channel usually may be rather narrow to ensure laminar flow and to establish the desired Taylor conditions. The microfluidic channel may preferably have a cross- sectional dimension of about 1 mm or less, such as of about 0.5 mm or less, such as of about 0.1 mm or less, such as of about 75 pm or even less, The microfluidic unit may conveniently be shaped as a tube with equal diameter in its entire length. Such tube is also referred to as a capillary tube or simply a capillary. Examples of suitable apparatus for performing the Taylor dispersion assay are disclosed in WO21180289 and/or marketed by FIDA Biosystems, e.g. the apparatus sold under the tradename FIDA 1. The microfluidic channel may conveniently have a length of 10 cm or longer, such as from 20 cm to several meters, depending on the required run time for an assay. Thus for assays with relatively slow reaction kinetic (such as a long t½ times), the microfluidic channel may advantageously be relative long such as up to 5 m, such as up to 2 m, such as up to 1 m. In an embodiment, the total molar amount of the second particle in the total volume of the sample portion and the at least one supplementing 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 supplementing portion, preferably the total molar amount of the second particle in the total volume of the sample portion and the at least one supplementing portion is less than 5 %, such as less than 1 %, such as 0.5 % or less of the total molar amount of the first particle in the total volume of the sample portion and the at least one supplementing portion. Since the second particle A, is the particle hat is read, this embodiment may provide a reading that may be simpler to process. In an embodiment, there is one supplementing portion only and the single supplementing portion may be fed to the microfluidic channel before or after the sample portion. However, it is generally preferred that the at least one supplementing portion comprise at least two supplementing portions. P81680PC01 31 In an embodiment, the at least one portion comprising a leading supplementing portion and a trailing supplementing portion, wherein the feeding of the liquid portions onto the microfluidic channel comprises providing the leading supplementing portion and the trailing supplementing portion on either sides of the sample portion. The leading supplementing portion comprises a supplementing portion adapted for leading the flow in the microfluidic channel i.e. in front of the dispersion zone and the trailing supplementing portion comprises a supplementing portion adapted for trailing the flow in the microfluidic channel i.e. after the dispersion zone. The leading supplementing portion and the trailing supplementing portion may be equal or different in volume and/or content. Advantageously, the leading supplementing portion and the trailing supplementing portion has identical content at the time of feeding to the microfluidic channel. In an embodiment, at least one of the N raw data sets is obtained from a Taylor dispersion assay, wherein the sample portion comprises a base concentration of the second particle at the time of feeding the sample portion to the microfluidic channel and wherein the at least one of the supplementing portion is free of or has a lower concentration than the base concentration of the second particle at time of feeding the at least one supplementing portion to the microfluidic channel, and wherein the formation of the interfacial contact between the sample portion and the at least one supplementing portion in the microfluidic channel, provides a modification of the sample portion to a modified sample portion comprising dispersion between the sample portion and the at least one supplementing portion, to thereby induce a concentration jump of at least the first particle L in the modified sample portion, wherein the concentration jump provides the first particle and the second particles to be out of equilibrium in the modified sample portion, wherein the concentration jump induces an association reaction. The Taylor dispersion assay in this embodiment may be a capmix type assay, wherein the at least one supplementing portion is free of the second particle A or a capmix variation type assay, wherein the capmix variation type assay means that the at least one of the supplementing portion is not free of the second particle A, but has a lower concentration than the base concentration of the second particle at time of feeding the at least one supplementing portion to the microfluidic channel P81680PC01 32 The concentration jump may provide a of the sample portion to the modified sample portion forming the dispersion zone, and wherein the dispersion zone comprises a concentration of the first particle dispersed from the at least one supplementing portion, to thereby induce a concentration jump of at least the first particle in the sample portion. Where a data set is obtained from a capmix type assay or a capmix variation type assay the ^_^ derived from the set of da may be very beneficial for deriving fitting parameters based on which kon may be determined with a desired very high accuracy. Due to the small volume of the sample portion relative to the at least one supplementing portion the molar concentration of the first particle (L) in the modified sample portion will very fast reach the a molar concentration of the first particle (L) which may considered to be identical to the molar concentration of the first particle (L) of the at least one supplementing portion. Where the supplementing portion comprises the second particle, but in a lower concentration than the concentration of the second particle in the sample portion, it is desired that the concentration of the second particle in the at least one supplementing portion is less than 50 % of the base concentration of the second particle in the sample portion, such as less than 10 %, such as less than 5 %, such as less than 1 % of the base concentration of the second particle in the sample portion. The signal of the second particles initially present in the at least one supplementing portion may be withdrawn as background noise in the processing of the raw data, e.g. in a preprocessing step of filtering the raw data. In an embodiment, wherein the Taylor dispersion assay is a capmix type assay or a capmix variation type assay, the at least one supplementing portion is free of the second particle or wherein the second particle of the supplementing portion is free of the selected marker and/or wherein the concentration of the second particle is less than 10 % of the base concentration of the second particle at the time of feeding the sample portion to the microfluidic channel, such as less than 1 % of the base concentration of the second particle at the time of feeding the sample portion to the microfluidic channel. In a preferred embodiment, at least one of the N raw data sets is obtained from a Taylor dispersion assay is a capmix type assay, wherein the sample portion comprises a base concentration of the second particle A and is free of the first particle L at the time of feeding the sample portion to the microfluidic channel and P81680PC01 33 wherein the at least one supplementing is free of the second particle and has a concentration of the first particle L at the time of feeding the at least one supplementing portion to the microfluidic channel. The formation of the interfacial contact between the sample portion and the at least one supplementing portion in the microfluidic channel, provides a modification of the sample portion to a modified sample portion comprising dispersion between the sample portion and the at least one supplementing portion, to thereby induce a concentration jump of at least the first particle L in the modified sample portion. The concentration jump provides the first particle and the second particles to be out of equilibrium in the modified sample portion, wherein the concentration jump induces an association reaction. In an embodiment, at least one of the N raw data sets is obtained from a Taylor dispersion assay, wherein the sample portion comprises a base concentration of the second particle (A) and a base concentration of the first particle (L) at the time of feeding the sample portion to the microfluidic channel and wherein the at least one supplementing portion is free of the first particle (in which case the Taylor dispersion assay is a dismix type assay) or has a different concentration of L, such as a higher concentration than the base concentration of the first particles at the time of feeding the at least one supplementing portion to the microfluidic channel (in which case the Taylor dispersion assay is a premix type assay) or has a different concentration of A, such as a lower concentration than the base concentration of the second particles at the time of feeding the at least one supplementing portion to the microfluidic channel (in which case the Taylor dispersion assay is a premix variation type assay), and wherein the formation of the interfacial contact between the sample portion and the at least one supplementing portion in the microfluidic channel, provides a modification of the sample portion to a modified sample portion comprising dispersion between the sample portion and the at least one supplementing portion, to thereby induce a concentration jump of at least one on the first particle L and the second particle A in the modified sample portion, wherein the concentration jump provides the first particle and the second particles to be out of equilibrium in the modified sample portion, wherein the concentration jump induces an association reaction or a dissociation reaction depending on the concentration of particles in the supplementing portion(s) relative to the sample portion. Advantageously, the at least one supplementing portion wherein the at least one supplementing portion has a different concentration of the first particle, such as a higher concentration of the first particles at the time of feeding the sample portion to P81680PC01 34 the microfluidic channel, which differs 50 %, such as at least 90 % from the base concentration of the sample portion of the first particle L. In a preferred embodiment, at least one of the N raw data sets is obtained from a Taylor dispersion assay, which is a dismix type assay, wherein the sample portion comprises a base concentration of the second particle (A) and a base concentration of the first particle (L) at the time of feeding the sample portion to the microfluidic channel and wherein the at least one supplementing portion is free of the first particle and free of the second particle at the time of feeding the at least one supplementing portion to the microfluidic channel, and wherein the formation of the interfacial contact between the sample portion and the at least one supplementing portion in the microfluidic channel, provides a modification of the sample portion to a modified sample portion comprising dispersion between the sample portion and the at least one supplementing portion, to thereby induce a concentration jump of at least one on the first particle L and the second particle A in the modified sample portion, wherein the concentration jump provides the first particle and the second particles to be out of equilibrium in the modified sample portion, wherein the concentration jump induces a dissociation reaction. Where a data set is obtained from a dismix type assay, the ^_^ derived from the set of da may be very beneficial for deriving fitting parameters based on which k_off may be determined with a desired very high accuracy. In the dismix type assay it is preferred that the first particle and the second particle of the sample portion are in equilibrium at the time of feeding the sample portion to the microfluidic channel. In an embodiment, wherein at least one of the supplementing portion comprises the first particle L and/or the second particle A it is desired that the leading supplementing portion has a lead portion concentration of the first particle and the trailing supplementing portion has a trail portion concentration of the first particle, wherein the lead portion concentration of the first particle and the trail portion concentration of the first particle is equal. In a variation thereof the a lead portion concentration may differ from the a trail portion concentration, wherein preferably the lead portion concentration of the first particle and the trail portion concentration of the first particle is equal or differs with less than 10 % from the average concentration, such as less than 1 % of the average of the trail portion concentration of the first particle and the lead portion concentration of the first particle. P81680PC01 35 In an embodiment, wherein the leading portion is free of or has a lead portion concentration of the second particle and the trailing supplementing portion is free of or has a trail portion concentration of the second particle, wherein the leading supplementing portion concentration of the second particle and the trail portion concentration of the second particle is equal or differs, preferably the lead portion concentration of the second particle and the trail portion concentration of the second particle is equal or differs with less than 10 % from the average concentration, such as less than 1 % of the average of the trail portion concentration of the second particle and the lead portion concentration of the second particle. The leading supplementing portion has a lead portion volume and the trailing supplementing portion has a trail portion volume, wherein the lead portion volume and the trail portion volume may be identical or may differ. Generally it is desired that preferably each of the lead portion volume and the trail portion volume is larger than the sample volume, such as larger than 5 times the sample volume, such as larger than 10 times the sample volume, such as larger than 20 times the sample volume. In an embodiment wherein at least one of the N raw data sets is obtained from a type 3 (dismix) Taylor dispersion assay, the sample portion comprises a base concentration of the second particle (A) and a base concentration of the first particle (L) at the time of feeding the sample portion to the microfluidic channel and wherein the at least one supplementing portion is free of the first particle and free of the second particle at the time of feeding the at least one supplementing portion to the microfluidic channel, and wherein the formation of the interfacial contact between the sample portion and the at least one supplementing portion in the microfluidic channel, provides a modification of said sample portion to a modified sample portion comprising dispersion between the sample portion and the at least one supplementing portion, to thereby induce a concentration jump of at least one on the first particle L and the second particle A in said modified sample portion, wherein the concentration jump provides the first particle and the second particles to be out of equilibrium in the modified sample portion, wherein the concentration jump induces a dissociation reaction. In the embodiment wherein at least one of the N raw data sets is obtained from a type 3 (dismix) Taylor dispersion assay, the method may advantageously comprise determining the at least one binding parameter from one or more of the fitting parameters a, p and q by applying one or more of the equations 50, 51 and/or 52 P81680PC01 36

sets are each obtained from a type 2 (premix) or from a type 3 (dismix) Taylor dispersion assay. In an embodiment, at least one of the N raw data sets is obtained from a type 4 (cap- dis-mix) Taylor dispersion assay wherein the liquid portions further comprises an affecting portion feed into the microfluidic channel to form an interfacial contact with the sample portion, wherein the sample portion comprises a base concentration of the second particle A and is essentially free of the first particle L at the time of feeding the sample portion to the microfluidic channel, the affecting portion comprises a base concentration of the first particle L and is essentially free of the second particle A at the time of feeding the affecting portion to the microfluidic channel and wherein the supplementing portion(s) is/are essentially free of both the first particle L and the second particle A at the time of feeding the at least one supplementing portion to the microfluidic channel, and wherein the formation of the interfacial contact between the sample portion and the affecting portion and/or between the sample portion and the at least one supplementing portion in the microfluidic channel, provides a modification of said sample portion to a modified sample portion comprising dispersion between the sample portion, the affecting portion and the at least one supplementing portion, to thereby induce a concentration jump of at least the first particle L in said modified sample portion, wherein the concentration jump provides the first particle and the second particles to be out of equilibrium in the modified sample portion, wherein the concentration jump induces an association reaction. In the embodiment wherein at least one of the N raw data sets is obtained from a type 4 (cap-dis-mix) Taylor dispersion assay, the method advantageously comprises determining the at least one binding parameter from one or more of the fitting parameters a, p and q by applying one or more of the equations 57, 58 and/or 59 ^ = − ^^_^^^ + ^_^^(^_^ + ^_^)^ (67)

P81680PC01 37

preferably at least half of or all or the N raw data sets, such as all of the N data raw sets are each obtained from a type 4 (cap-dis-mix) Taylor dispersion assay. In an embodiment, at least one of the set of equations17, 18, 19, the set of equations 36, 37 and 35, the set of equations 50, 51 and 52 and the set of equations 67, 68 and 69 is/are applied in the determination of the at least one binding parameter from one or more of the fitting parameters a, p and q. In an embodiment two, three or all four of the equations set of equations 36, 37 and 35, the set of equations 50, 51 and 52 and the set of equations 67, 68 and 69 is/are applied in the determination of the at least one binding parameter from one or more of the fitting parameters a, p and q. The number of readings to generate each respective set of data, is advantageously sufficient to generate a Gaussian representation, such as a Taylorgram. In an embodiment, the obtaining of each set of raw data comprises performing a plurality of readings of the signal intensity at a read out section of the microfluidic channel, wherein the plurality of readings preferably comprises at least 5 intensity readings, comprising at least one intensity reading at the peak appearance time (tR), at least one intensity reading before the peak appearance time (tR) and at least one intensity reading after the peak appearance time (t_R), preferably the obtaining of each set of raw data comprises recording the intensity with a recording rate of at least 1 Hz, such as 5 to 12 Hz or more, such as to obtain a full Gaussian reading profile (i.e. a Taylorgram) The method may conveniently comprise obtaining the N of sets of raw data wherein the N respective sets of raw data are obtained from N respective Taylor dispersion assays, wherein N is an integer of at least 1. N may advantageously be from 1 to 50 such as from 2 to 20, such as 3 to 10. In an embodiment, at least of the binding parameters KD (Equilibrium dissociation constant), DA (Diffusitivity of unbound A) and/or; DAL (Diffusitivity of AL) is a known binding parameter and wherein the one or more known parameter is applied for P81680PC01 38 determining at least one other binding from one or more of the fitting parameters p, q and a. In this embodiment, in particular wherein at least one of the N data sets is based on a dismix type assay the required number of data set may be relatively low. For example, N may be 5 or less such as 1, 2 or 3. Preferably the at least one of the N sets of raw data is obtained from a Taylor dispersion assay wherein the sample portion comprises a base concentration of the first particle L and a base concentration of the second particle A in equilibrium. In a preferred embodiment, N is two or more and the N sets of raw data comprises at least one set of raw data obtained from a Taylor dispersion assay wherein the concentration jump induces an association reaction and at least one set of raw data obtained from a Taylor dispersion assay wherein the concentration jump induces a dissociation reaction. It has been found that this combination may provide determination of the one or more binding parameters with remarkable high accuracy. In an embodiment, N is two or more and the N sets of raw data comprises at least one set of raw data obtained from a Taylor dispersion assay wherein the second particle (A) of the sample portion is dispersing to form a dispersion zone in the microfluidic channel and wherein the molar concentration of the second particle is equal to or lower than the molar concentration of the first particle in the dispersion zone and at least one set of raw data obtained from a Taylor dispersion assay wherein the second particle (A) of the sample portion is dispersing to form a dispersion zone in the microfluidic sample and wherein the molar concentration of the second particle is equal to or larger than the molar concentration of the first particle in the dispersion In an embodiment, wherein N is two or more and wherein the N sets of raw data comprises a plurality of data sets obtained from respective Taylor dispersion assay wherein the base concentration of the second particle A in the sample portion differs. In an embodiment, wherein N is two or more and wherein the N sets of raw data comprises a plurality of data sets obtained from respective Taylor dispersion assay wherein a base concentration of the first particle in the at least one supplementing portion in the sample portion differs. The selected marker may in principle be any kind of readable marker. The selected marker comprises an intrinsic marker, such as tryptophan and/or an extrinsic marker P81680PC01 39 capable of being detected, preferably are optically readable markers, such as florescence markers and/or light absorbing markers. In an embodiment, a conformational change of the first particle, preferably the selected marker changes signal in dependence of conformation of the first particle and changes thereof, such as in dependence of change in binding/dissociation and/or in structure. The selected marker is advantageously an optically readable marker, such as a light absorbing marker and/or a fluorescent marker, preferably operating in the UV/Vis wavelength range preferably from about 190 nm to about 700 nm. In an embodiment, the selected marker is an electrochemically readable marker, such as an electroactive marker. The feeding of the liquid portions into the microfluidic device is conveniently performed at an injection pressure Pi and the provision of the liquid portions to the flow in the microfluidic device is performed at a run pressure Pr ensuring a laminar flow in the microfluidic channel. To ensure a high control of the assay it may be desired that the run pressure Pr is larger than the injection pressure Pi. Advantageously, the run pressure Pr is adjustable, e.g. such that the run pressure may be adjusted to ensure that the dispersed zone reaches a reading section of the microfluidic channel at a desired time to ensure that at least one out-of-equilibrium reading may be performed. The application of the run pressure may ensure forcing the liquid portions to pass lengthwise in the channel, more preferably the flow velocity of the sample portion along the length if the microfluidic channel may be adjusted by adjusting the run pressure, e.g. to provide the sample portion at a selected location of the microfluidic channel at a selected time frame to performing a reading of the selected marker at the selected time frame. Advantageously, each reading to obtain a reading result of the set of reading results comprises reading marker signals of the concentration of marked second particles along a length section of the microfluidic channel containing the sample portion preferably comprising the dispersion zone. The reading of the marker signals of the concentration of marked second particles along the length section of the microfluidic channel containing the dispersion zone is from a leading edge to a trailing edge of the dispersion zone comprises reading the intensity of the marker signals as a P81680PC01 40 function of time as the dispersion zone a reading section of the microfluidic channel. Advantageously, each reading to obtain a reading result of the set of reading results comprises obtaining the reading while the liquid sample is in laminar flow. Advantageously, each reading to obtain a reading result of the set of reading results comprises reading marker signals of the concentration of marked second particles along a length section of the microfluidic channel containing the sample portion, wherein each reading is performed within a time frame not exceeding the time it takes the sample portion to pass a reading section at the microfluidic channel. Advantageously, the at least one reading result of an out-of-equilibrium reading is obtained by reading marker signals of the concentration of marked second particles before the interaction between the first particle and the second particle has reached equilibrium. Preferably the method comprises performing a plurality of out-of- equilibrium readings, optionally all of the reading results of the set of reading results are reading results of out-of-equilibrium readings. In an embodiment, the set of reading results of readings comprises a reading result of an in-equilibrium reading, preferably obtained by reading marker signals of the concentration of marked second particles wherein the interaction between the first particle and the second particle are in-equilibrium. The first particle L and the second particle A may independently of each other be any type of particle capable of being dispersed in the microfluidic channel. Wherein the first particle L and the second particle A are capable on non-covalent interaction with each other. The first particle and the second particle may conveniently differ from each other. Preferably the first particle and the second particle differs in type of particle, molecular weight, dispersion properties or any combinations thereof. Advantageously, at least one of the first particle and the second particle comprises a drug or a drug candidate. As examples of first particle L and second particle A it may be mentioned that at least one of the first particle and the second particle maybe selected from a biomolecule; a protein, such as an antibody (monoclonal or polyclonal), a nanobody, an antigen, an enzyme and/or a hormone; a nucleotide; a nucleoside; a nucleic acid, P81680PC01 41 such a RNA, DNA, PNA or any thereof and/or any combinations comprising at least one of these. The binding parameter may advantageously comprise a kinetic parameter comprising one or more of an association rate constant k_on, a dissociation rate constant k_{off or} a derivative thereof, such as residence time (1/ K_off), t½ (0.693/V K_off) or any other binding parameter as discussed above. BRIEF DESCRIPTION OF THE EXAMPLES AND DRAWING The invention is being illustrated further below in connection with examples and embodiments and with reference to the figures. The figures are schematic and may not be drawn to scale. The examples and embodiments are merely given to illustrate the invention and should not be interpreted to limit the scope of the invention Figures 1a, 1b and 1c schematically illustrates a Taylor dispersion assay in the form of a capmix type assay. Figures 1a’, 1b’ and 1c’ schematically illustrates a Taylor dispersion assay in the form of a cap-dis-mix type assay. Figures 2a, 2b and 2c schematically illustrates a Taylor dispersion assay in the form of a dismix type assay. Figure 3 is a flow diagram illustrating an embodiment of the method of the invention. Figure 4 is a flow diagram illustrating another embodiment of the method of the invention. Figure 5 is a flow diagram illustrating a further embodiment of the method of the invention. Figure 6 is a flow diagram illustrating a further embodiment of the method of the invention. Figure 7 is a flow diagram illustrating a further embodiment of the method of the invention. P81680PC01 42 The Taylor dispersion assay in the form a capmix type assay illustrated in figures 1a, 1b and 1c show a length section of a capillary at the times t0 (figure 1a), t1 (figure 1b) and t2 (figure 1c), The arrow above the figures illustrates that the capillary may be very long as described above. At the time t0 as illustrated in figure 1a, a leading supplementing portion 2’, an affecting portion 1a, a sample portion 1’ and a trailing supplementing portion 3’ have been injected into the capillary at an injection pressure Pi, which advantageously may be relatively low to maintain the sample portion 1 substantially undispersed between the leading supplementing portion 2 and the trailing supplementing portion 3. As illustrated in figure 1a, the sample portion may be maintained substantially undispersed at the time t0. The sample portion 1 comprises a base concentration of the second particle each of the leading supplementing portion 2 and the trailing supplementing 3 comprises a concentration of the first particle L. In practice the concentration jump may already have been initiated, depending on the injection pressure applied. In figure 1b a run pressure Pr has been applied, the liquid portions have been induced to be in laminar flow and it can be seen that a concentration jump in the sample portion has taken place. Due to the very small sample portion 1 relative to the supplementing portions 2 and 3, the sample portion will immediately be modified to have a concentration of first particle L essentially identical to the concentration of the first particle L in the leading supplementing portion 2 and the trailing supplementing portion 3. Thereby an association reaction has been induces and the first particle L and the second particle A will interact, first it will be out-of-equilibrium, but as time passes an equilibrium will eventually be reached. At the time t1 the modified sample portion forms a parabolic shape in the capillary with a parabolic top face towards the leading supplementing portion 2 and a parabolic valley face towards the trailing supplementing portion 3. It can be seen that the modified sample portions forms a dispersion zone DZ, which may grow lengthwise as the modified sample passes along the capillary. I figure 1c, at the time t2 the dispersion zone has broadened and this may continue as the dispersion zone DZ passes further downstream to a reading section of the P81680PC01 43 capillary, wherein the signals of the of the second particle A are read as the dispersion zone passes the reading section of the capillary. The Taylor dispersion assay in the form of a cap-dis-mix type assay illustrated in figures 1a’, 1b’ and 1c’ show a length section of a capillary at the times t0 (figure 1a’), t1 (figure 1b’) and t2 (figure 1c’), At the time t0 as illustrated in figure 1a’, a leading supplementing portion 2’, a sample portion 1 and a trailing supplementing portion 3 have been injected into the capillary at an injection pressure Pi, which advantageously may be relatively low to maintain the affecting portion 1a and the sample portion 1’ substantially unmixed and undispersed between the leading supplementing portion 2’ and the trailing supplementing portion 3’. As illustrated in figure 1a’, the affecting portion 1a and the sample portion 1’ may be maintained substantially unmixed and undispersed at the time t0. The sample portion 1’ comprises a base concentration of the second particle, the affecting portion 1a comprises a concentration of the first particle L and each of the leading supplementing portion 2’ and the trailing supplementing 3’ are buffer without first particle L and the second particle A. In practice the concentration jump may already have been initiated, depending on the injection pressure applied. In figure 1b’ a run pressure Pr has been applied, the liquid portions have been induced to be in laminar flow and it can be seen that a concentration jump in the sample portion 1’ has taken place. Due to the very small sample portion 1’ and the very small affecting portion 1a, these two portions will relative fast coalesce to form a combined diffusion zone DZ, wherein the diffusion zone DZ comprises the sample portion and the affecting portion only very slightly diluted by buffer from the supplementing portions. Thereby an association reaction has been induces and the first particle L and the second particle A will interact. First it will be out-of-equilibrium, but as time passes an equilibrium will eventually be reached. At the time t1 the modified sample portion, now combined with the affecting portion 1a, forms a parabolic shape in the capillary with a parabolic top face towards the leading supplementing portion 2’ and a parabolic valley face towards the trailing supplementing portion 3’. It can be seen that the modified sample portion, now comprising the affecting portion forms the dispersion zone DZ, which may grow lengthwise as the modified sample portion passes along the capillary. P81680PC01 44 I figure 1c’, at the time t2 the dispersion DZ has broadened and this may continue as the dispersion zone DZ passes further downstream to a reading section of the capillary, wherein the signal of the marker of the second particle A are read as the dispersion zone DZ passes the reading section of the capillary. The Taylor dispersion assay in the form of a dismix type assay illustrated in figures 2a, 2b and 2c show a length section of a capillary at the times t0 (figure 2a), t1 (figure 2b) and t2 (figure 2c), At the time t0 as illustrated in figure 2a, a leading supplementing portion 12, a sample portion 11 and a trailing supplementing portion 13 have been injected into the capillary at an injection pressure Pi. Prior to the injection into the capillary the particles in the sample portions have reached equilibrium so that they are in equilibrium at the time of injection. The injection pressure may advantageously be relatively low to maintain the sample portion 11 substantially undispersed between the leading supplementing portion 12 and the trailing supplementing portion 13. As illustrated in figure 2a, the sample portion may be maintained substantially undispersed at the time t0. The sample portion 11 comprises a base concentration of first particle L and a base concentration of the second particle A and each of the leading supplementing portion 2 and the trailing supplementing portion 3 do not contain any of the first particle L or the second particle A but are buffer. In figure 2b, at the time t1, a run pressure Pr has been applied, the liquid portions have been induced to be in laminar flow and it can be seen that a concentration jump in the sample portion has taken place comprising that the sample portion has been diluted by buffer. At the time t1 the modified sample portion forms a parabolic shape in the capillary with a parabolic top face towards the leading supplementing portion 2 and a parabolic valley face towards the trailing supplementing portion 3. It can be seen that the modified sample portions forms a dispersion zone DZ, which may grow lengthwise as the modified sample passes along the capillary. Where the particles A and L are in equilibrium a relatively large concentration of the complex AL may be present and since the complex AL must be assumed to be relatively large relative to unbound A and unbound B the dispersion may occur relatively fast leading to a relatively fast broadening of the dispersion zone DZ and thereby a relatively fast dilution the first particle L and the second particle A in the dispersion zone. The concentration jump induces a dissociation reaction. P81680PC01 45 In figure 2c, at the time t2 the has broadened even further and this may continue as the dispersion zone DZ passes further downstream to a reading section of the capillary, wherein the signals of the marker of the second particle A are read as the dispersion zone passes the reading section of the capillary. The flow diagram shown in figure 3 illustrates an embodiment of the method of the invention wherein each of the N data sets are based on Taylor dispersion assays of the capmix type assay. In step 31a, a capmix type assay is performed e.g. as described above, The capmix type assay is performed where the sample portion comprises a base concentration X1 of the second particle A and wherein each of the leading supplementing portion and the trailing supplementing portion comprises a concentration Y1 of the first particle L. In step 31b the raw data set 1 is derived from the Taylor dispersion assay of step 31a. In step 31c at least one of Rh and apparent D is/are derived from the raw data set 1. In step 31d ^_^ is determined from the apparent Rh and/or the apparent D. In step 32a, a capmix type assay i assay is performed where the sample portion comprises a base concentration X2 of the second particle A and wherein each of the leading supplementing portion and the trailing supplementing portion comprises a concentration Y2 of the first particle L. In step 32b the raw data set 1 is derived from the Taylor dispersion assay of step 32a. In step 32c at least one of Rh and apparent D is/are derived from the raw data set 1. In step 32d ^_^ is determined from the apparent Rh and/or the apparent D. In step 33a, a capmix type assay i assay is performed where the sample portion comprises a base concentration X3 of the second particle A and wherein each of the leading supplementing portion and the trailing supplementing portion comprises a concentration Y3 of the first particle L. In step 33b the raw data set 1 is derived from the Taylor dispersion assay of step 33a. In step 33c at least one of Rh and apparent D is/are derived from the raw data set 1. In step 33d ^_^ is determined from the apparent Rh and/or the apparent D. In step 34a, a capmix type assay i assay is performed where the sample portion comprises a base concentration X4 of the second particle A and wherein each of the leading supplementing portion and the trailing supplementing portion comprises a concentration Y4 of the first particle L. In step 34b the raw data set 1 is derived from P81680PC01 46 the Taylor dispersion assay of step step 34c at least one of Rh and apparent D is/are derived from the raw data set 1. In step 34d ^_^ is determined from the apparent Rh and/or the apparent D. In this example N is 4. In variations thereof it may be more or less e.g. as described above. From each N data sets a ^_^ has now been determined. In step 35, the determined ^_^ are fitted to the equation 21 and the fitting parameters p, q and a are derived from the fitting. In step 36, one or more of the binding parameters is /are determined from the equations 18, 19 and 20 and/or from the equations 35, 36 and 37. Where one or more of the data sets has been obtained from a Taylor dispersion assay where the first particle L has been in excess of the second particle A in the dispersion zone, the equations 18, 19 and 20 may advantageously be applied for the determination og one or more of the binding parameters. Where one or more of the data sets has been obtained from a Taylor dispersion assay where the first particle L has not been in excess of the second particle A in the dispersion zone, the equations 35, 36 and 3720 may advantageously be applied for the determination og one or more of the binding parameters. In many cases both equations 18, 19 and 20 and the equations 35, 36 and 37 may be applied and beneficially an average determination based on respectively the equations 18, 19 and 20 and the equations 35, 36 and 37 may provide an even higher accuracy. In the above embodiment the base concentrations of A: X1, X2, X3 and X4, may advantageously be identical and the concentrations of L in the supplementing portions: Y1, Y2, Y3 and Y4 may advantageously differ. In a variation thereof the base concentrations of A: X1, X2, X3 and X4 differ and the concentrations of L in the supplementing portions: Y1, Y2, Y3 and Y4 are identical. The flow diagram shown in figure 4 illustrates an embodiment of the method of the invention, wherein each of the N data sets are based on Taylor dispersion assays of the dismix type assay. P81680PC01 47 In step 41a, a dismix type assay is e.g. as described above, The dismix type assay is performed where the sample portion comprises a base concentration X1 of the second particle A and a base concentration Y1 of the first particle L wherein each of the leading supplementing portion and the trailing supplementing portion are buffer free of the first particle L and the second particle A. In step 41b the raw data set 1 is derived from the Taylor dispersion assay of step 41a. In step 41c at least one of Rh and apparent D is/are derived from the raw data set 1. In step 41d ^_^ is determined from the apparent Rh and/or the apparent D. In step 42a, a dismix type assay i assay is performed where the sample portion comprises a base concentration X2 of the second particle A and a base concentration Y2 of the first particle L wherein each of the leading supplementing portion and the trailing supplementing portion are buffer free of the first particle L and the second particle A. In step 42b the raw data set 1 is derived from the Taylor dispersion assay of step 42a. In step 42c at least one of Rh and apparent D is/are derived from the raw data set 1. In step 42d ^_^ is determined from the apparent Rh and/or the apparent D. In step 43a, a dismix type assay i assay is performed where the sample portion comprises a base concentration X3 of the second particle A and a base concentration Y3 of the first particle L wherein each of the leading supplementing portion and the trailing supplementing portion are buffer free of the first particle L and the second particle A. In step 43b the raw data set 1 is derived from the Taylor dispersion assay of step 43a. In step 43c at least one of Rh and apparent D is/are derived from the raw data set 1. In step 43d ^_^ is determined from the apparent Rh and/or the apparent D. In step 44a, a dismix type assay i assay is performed where the sample portion comprises a base concentration X4 of the second particle A and a base concentration Y4 of the first particle L wherein each of the leading supplementing portion and the trailing supplementing portion are buffer free of the first particle L and the second particle A. In step 44b the raw data set 1 is derived from the Taylor dispersion assay of step 44a. In step 44c at least one of Rh and apparent D is/are derived from the raw data set 1. In step 44d ^_^ is determined from the apparent Rh and/or the apparent D. P81680PC01 48 In the above embodiment the base of A in the sample portion: X1, X2, X3 and X4, may advantageously be identical and the concentrations of L in the sample portion: Y1, Y2, Y3 and Y4 may advantageously differ. In a variation thereof the base concentrations of A in the sample portion: X1, X2, X3 and X4 differ and the concentrations of L in the sample portion: Y1, Y2, Y3 and Y4 are identical. In this example N is 4. In variations thereof it may be more or less e.g. as described above. From each N data sets a ^_^ value has now been determined. In step 45, the determined ^_^ are fitted to the equation 21 and the fitting parameters p, q and a are derived from the fitting. In step 46, one or more of the binding parameters is /are determined from the equations 18, 19 and 20 and/or from the equations 35, 36 and 37. Where one or more of the data sets has been obtained from a Taylor dispersion assay where the first particle L has been in excess of the second particle A in the dispersion zone, the equations 18, 19 and 20 may advantageously be applied for the determination og one or more of the binding parameters. Where one or more of the data sets has been obtained from a Taylor dispersion assay where the first particle L has not been in excess of the second particle A in the dispersion zone, the equations 35, 36 and 37 may advantageously be applied for the determination og one or more of the binding parameters. In many cases both equations 18, 19 and 20 and the equations 35, 36 and 37 may be applied and beneficially an average determination based on respectively the equations 18, 19 and 20 and the equations 35, 36 and 37 may provide an even higher accuracy. The flow diagram shown in figure 5 illustrates an embodiment of the method of the invention, which combines the embodiment illustrated in figures 3 and 4. In step 51a, a capmix type assay is performed e.g. as described above, The capmix type assay is performed where the sample portion comprises a base concentration X1 of the second particle A and wherein each of the leading supplementing portion and the trailing supplementing portion comprises a concentration Y1 of the first particle L. In step 51b the raw data set 1 is derived from the Taylor dispersion assay P81680PC01 49 of step 51a. In step 51c at least one of and apparent D is/are derived from the raw data set 1. In step 51d ^_^ is determined from the apparent Rh and/or the apparent D. In step 52a, a capmix type assay i assay is performed where the sample portion comprises a base concentration X2 of the second particle A and wherein each of the leading supplementing portion and the trailing supplementing portion comprises a concentration Y2 of the first particle L. In step 52b the raw data set 1 is derived from the Taylor dispersion assay of step 52a. In step 52c at least one of Rh and apparent D is/are derived from the raw data set 1. In step 52d ^_^ is determined from the apparent Rh and/or the apparent D. In step 53a, a dismix type assay i assay is performed where the sample portion comprises a base concentration X3 of the second particle A and a base concentration Y3 of the first particle L wherein each of the leading supplementing portion and the trailing supplementing portion are buffer free of the first particle L and the second particle A. In step 53b the raw data set 1 is derived from the Taylor dispersion assay of step 53a. In step 53c at least one of Rh and apparent D is/are derived from the raw data set 1. In step 53d ^_^ is determined from the apparent Rh and/or the apparent D. In step 54a, a dismix type assay i assay is performed where the sample portion comprises a base concentration X4 of the second particle A and a base concentration Y4 of the first particle L wherein each of the leading supplementing portion and the trailing supplementing portion are buffer free of the first particle L and the second particle A. In step 54b the raw data set 1 is derived from the Taylor dispersion assay of step 54a. In step 54c at least one of Rh and apparent D is/are derived from the raw data set 1. In step 54d ^_^ is determined from the apparent Rh and/or the apparent D. In step 55, the determined ^_^ are fitted to the equation 21 and the fitting parameters p, q and a are derived from the fitting. In step 56, one or more of the binding parameters is /are determined from the equations 18, 19 and 20 and/or from the equations 35, 36 and 37 e.g. as described above. P81680PC01 50 The flow diagram shown in figure 6 an embodiment of the method of the invention, wherein each of the N data sets are based on Taylor dispersion assays of the cap-dis-mix type assay (On the figure called “capdismix”). In step 61a, a cap-dis-mix type assay is performed e.g. as described above, The dismix type assay is performed where the sample portion comprises a base concentration X1 of the second particle A and the affecting portion comprises a concentration of the first particle L AFL which is Y1 and wherein each of the leading supplementing portion and the trailing supplementing portion are buffer free of the first particle L and the second particle A. In step 61b the raw data set 1 is derived from the Taylor dispersion assay of step 61a. In step 61c at least one of Rh and apparent D is/are derived from the raw data set 1. In step 61d ^_^ is determined from the apparent Rh and/or the apparent D. In step 62a, a cap-dis-mix type assay i assay is performed where the sample portion comprises a base concentration X2 of the second particle A and the affecting portion comprises a concentration of the first particle L AF_L which is Y2 and wherein each of the leading supplementing portion and the trailing supplementing portion are buffer free of the first particle L and the second particle A. In step 62b the raw data set 1 is derived from the Taylor dispersion assay of step 62a. In step 62c at least one of Rh and apparent D is/are derived from the raw data set 1. In step 62d ^_^ is determined from the apparent Rh and/or the apparent D. In step 63a, a cap-dis-mix type assay i assay is performed where the sample portion comprises a base concentration X3 of the second particle A and the affecting portion comprises a concentration of the first particle L AFL which is Y3 and wherein each of the leading supplementing portion and the trailing supplementing portion are buffer free of the first particle L and the second particle A. In step 63b the raw data set 1 is derived from the Taylor dispersion assay of step 63a. In step 63c at least one of Rh and apparent D is/are derived from the raw data set 1. In step 63d ^_^ is determined from the apparent Rh and/or the apparent D. In step 64a, a cap-dis-mix type assay i assay is performed where the sample portion comprises a base concentration X4 of the second particle A and the affecting portion comprises a concentration of the first particle L AFL which is Y4 and wherein each of the leading supplementing portion and the trailing supplementing portion are buffer free of the first particle L and the second particle A. In step 64b the raw data set 1 is derived from the Taylor dispersion assay of step 64a. In step 64c at least one of Rh P81680PC01 51 and apparent D is/are derived from the data set 1. In step 64d ^_^ is determined from the apparent Rh and/or the apparent D. In the above embodiment the base concentrations of A in the sample portion: X1, X2, X3 and X4, may advantageously be identical and the concentrations of L in the affecting portion: Y1, Y2, Y3 and Y4 may advantageously differ. In a variation thereof the base concentrations of A in the sample portion: X1, X2, X3 and X4 differ and the concentrations of L in the affecting portion: Y1, Y2, Y3 and Y4 are identical. In this example N is 4. In variations thereof, N may be more or less e.g. as described above. From each N data sets a ^_^ value has now been determined. In step 65, the determined ^_^ are fitted to the equation 21 and the fitting parameters p, q and a are derived from the fitting. In step 66, one or more of the binding parameters is /are determined from the set of equations 18, 19 and 20 and/or from the set of equations 35, 36 and 37 and/or from the set of equations 50, 51 and 52 and/or the set of equations 67, 68 and 69. In this example it is preferred to apply the set of equations 67, 68 and 69 optionally in combination with one or more of the other sets of equations. Where one or more of the data sets has been obtained from a Taylor dispersion assay where the first particle L has been in excess of the second particle A in the dispersion zone, the equations 18, 19 and 20 may advantageously be applied for the determination og one or more of the binding parameters. Where one or more of the data sets has been obtained from a Taylor dispersion assay where the first particle L has not been in excess of the second particle A in the dispersion zone, the equations 35, 36 and 37 may advantageously be applied for the determination og one or more of the binding parameters. In many cases both equations 18, 19 and 20 and the equations 35, 36 and 37 may be applied and beneficially an average determination based on respectively the equations 18, 19 and 20 and the equations 35, 36 and 37 may provide an even higher accuracy. Alternatively, the set of equations 50, 51 and 52 may be combined with one or more of the set of equations 18, 19 and 20 and/or the set of equations 35, 36 and 37 P81680PC01 52 The flow diagram shown in figure 7 an embodiment of the method of the invention, which combines the embodiment illustrated in figures 3 and 6. In step 71a, a cap-dis-mix type assay is performed e.g. as described above, The cap-dis-mix type assay is performed where the sample portion comprises a base concentration X1 of the second particle A and the affecting portion comprises a concentration of the first particle L AFL which is Y1 and wherein each of the leading supplementing portion and the trailing supplementing portion are buffer free of the first particle L and the second particle A. In step 71b the raw data set 1 is derived from the Taylor dispersion assay of step 71a. In step 71c at least one of Rh and apparent D is/are derived from the raw data set 1. In step 71d ^_^ is determined from the apparent Rh and/or the apparent D. In step 72a, a cap-dis-mix type assay i assay is performed where the sample portion comprises a base concentration X2 of the second particle A and the affecting portion comprises a concentration of the first particle L AF_L which is Y2 and wherein each of the leading supplementing portion and the trailing supplementing portion are buffer free of the first particle L and the second particle A. In step 72b the raw data set 1 is derived from the Taylor dispersion assay of step 72a. In step 72c at least one of Rh and apparent D is/are derived from the raw data set 1. In step 72d ^_^ is determined from the apparent Rh and/or the apparent D. In step 73a, a dismix type assay i assay is performed where the sample portion comprises a base concentration X3 of the second particle A and a base concentration Y3 of the first particle L wherein each of the leading supplementing portion and the trailing supplementing portion are buffer free of the first particle L and the second particle A. In step 73b the raw data set 1 is derived from the Taylor dispersion assay of step 73a. In step 73c at least one of Rh and apparent D is/are derived from the raw data set 1. In step 73d ^_^ is determined from the apparent Rh and/or the apparent D. In step 74a, a dismix type assay i assay is performed where the sample portion comprises a base concentration X4 of the second particle A and a base concentration Y4 of the first particle L wherein each of the leading supplementing portion and the trailing supplementing portion are buffer free of the first particle L and the second particle A. In step 74b the raw data set 1 is derived from the Taylor dispersion assay of step 74a. In step 74c at least one of Rh and apparent D is/are P81680PC01 53 derived from the raw data set 1. In step ^_^ is determined from the apparent Rh and/or the apparent D. In step 75, the determined ^_^ are fitted to the equation 21 and the fitting parameters p, q and a are derived from the fitting. In step 76, one or more of the binding parameters is /are determined from the set of equations 18, 19 and 20 and/or from the set of equations 35, 36 and 37 and/or from the set of equations 50, 51 and 52 and/or the set of equations 67, 68 and 69. In this example it is preferred to apply the set of equations 67, 68 and 69 optionally in combination with one or more of the other sets of equations.

Claims

P81680PC01 54 PATENT CLAIMS 1. A method of determining a binding parameter between a first particle (L) and a second particle (A) capable of noncovalent interacting with the first particle, wherein the second particle comprises a selected marker, wherein the method comprises obtaining N sets of raw data, wherein each set of raw data is obtained by performing a Taylor dispersion assay, and processing each of the at least one set of raw data, wherein each Taylor dispersion assay for obtaining each respective set of raw data comprises i. feeding liquid portions comprising a sample portion comprising said second particle (A) and at least one supplementing portion into a microfluidic channel such that the sample portion and the at least one supplementing portion form an interfacial contact in the microfluidic channel, wherein at least one of the liquid portions comprises a concentration of the first particle (L); ii. providing the liquid portions to a flow in the microfluidic channel; iii. obtaining the set of raw data comprising reading signal intensity as a function of time s(t) of the marker; wherein each set of raw data comprises at least one out-of-equilibrium reading and wherein the processing of each of the N sets of raw data comprises determining at least one of the apparent diffusivity D and the hydrodynamic radius Rh, determining ^_^ from the apparent diffusivity D by applying the equation 12a ^_^ = ½ ∙ ^^{^} = ^{^^^∙^^} ^^^ (12) and/or

determining ^_^ from the hydrodynamic radius Rh by applying the equation 23 ^_^ = ^{^^∙^∙^^} ^_∙^∙^^ _^∙^^ (23) and fitting the ^_^

^_^ = ^{^} ^ ∙ ^^_^ + ^{^} ^ ∙ ^^ ^ ^{^^^} ^_^^∙^^∙^^^^ (21) wherein

one binding parameter from one or more of the fitting parameters, wherein the P81680PC01 55 binding parameter preferably 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 k_off (Rate of disassociation); k_on (Rate of association); k_obs (Rate constant for reaching equilibrium); R_AL:(Hydrodynamic radius of AL); R_A (Hydrodynamic radius of A); DA (Diffusitivity of unbound A); DAL (Diffusitivity of AL) and/or any derivative of one or more of the mentioned binding parameters. 3. The method of claim 1 or claim 2, wherein the formation of the interfacial contact between the sample portion and the at least one supplementing portion in the microfluidic channel, provides a modification of said sample portion to a modified sample portion comprising dispersion between the sample portion and the at least one supplementing portion, to thereby induce a concentration jump of at least one on the first particle L and the second particle A in said modified sample portion, wherein the concentration jump provides the first particle and the second particles to be out of equilibrium in the modified sample portion, wherein the concentration jump induces an association reaction or a dissociation reaction. 4. The method of any one of the preceding claims, wherein at least one of the N raw data sets is obtained from Taylor dispersion assay wherein the concentration jump induces an association reaction and the method comprises determining the at least one binding parameter from one or more of the fitting parameters a, p and q by applying one or more of the equations E1, E2 and/or E3 ^ = ^_^ + (^_^^ − ^_^) ∙ ^{^^} ^_^^^^^^^ (E1) , wherein LX is LT when

considered constant and Ld when the concentration of L in the dispersion zone may not be considered constant and wherein AX is 0 when the concentration of A in the dispersion zone may be considered constant and A_d when the concentration of A in the dispersion zone may not be considered constant, or P81680PC01 56 wherein at least one of the N raw data is obtained from Taylor dispersion assay wherein the concentration jump induces an dissociation reaction and the method comprises determining the at least one binding parameter from one or more of the fitting parameters a, p and q by applying one or more of the equations ^ = (^_^^ − ^_^) ∙ ^{^^^∙^^} ^_^ + ^_^ (50) 5. The

of the N raw data sets is obtained from a type 1 (capmix) Taylor dispersion assay wherein the second particle (A) of the sample portion is dispersing to form a dispersion zone in said microfluidic channel and wherein the molar concentration of the second particle is equal to or lower than the molar concentration of said first particle in the dispersion zone and the method comprises determining the at least one binding parameter from one or more of the fitting parameters a, p and q by applying one or more of the equations 17, 18 and/or 19 ^ = ^_^ + (^_^^ − ^_^) ∙ ^{^^} ^_^^^^ (17) , preferably at least half of

sets are each obtained from a type 1 (capmix) Taylor dispersion assay wherein the second particle (A) of the sample portion is dispersing to form a dispersion zone in said microfluidic channel and wherein the molar concentration of the second particle is equal to or lower than the molar concentration of said first particle in the dispersion zone. 6. The method of the preceding claims, wherein at least one of the N raw data sets is obtained from a type 1 (capmix) Taylor dispersion assay wherein the second particle (A) of the sample portion is dispersing to form a dispersion zone in said microfluidic channel and wherein the molar concentration of the second particle is equal to or larger than the molar concentration of said first particle in the dispersion zone and wherein the method comprises determining the at least one P81680PC01 57 binding parameter from one or more of fitting parameters a, p and q by applying one or more of the equations 35, 36 and/or 37 ^ = − ^^_^^^ + ^_^^(^_^ + ^_^)^ (35) ^ = ^_^ + (^_^^ − ^_^) ∙ ^{^^} ^_^^^^^^^ (36) , preferably at least half of

Taylor dispersion assay wherein the second particle (A) of the sample portion is dispersing to form a dispersion zone in said microfluidic channel and wherein the molar concentration of the second particle is equal to or larger than the molar concentration of said first particle in the dispersion zone. 7. The method of any one of claims 4-7, wherein the interfacial contact between the sample portion and the at least one supplementing portion in the microfluidic channel, provides a modification of said sample portion to a modified sample portion comprising dispersion between the sample portion and the at least one supplementing portion, to thereby induce a concentration jump of at least one on the first particle L and the second particle A in said sample portion, wherein the concentration jump provides the first particle and the second particles to be out of equilibrium in the modified sample portion, wherein the concentration jump induces an association reaction. 8. The method of any one of the preceding claims, wherein the sample portion at the time of feeding (time zero) has a volume which is 5 % or less than the total volume of the liquid portions, preferably the sample portion at the time of feeding the sample portion has a volume which is 1 % or less, such as 0.5 % or less, such as 0.1 % or less, such as 0.05 % or less than the total volume of the liquid portions comprising the sample portion and the at least one supplementing portion. 9. The method of 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 supplementing 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 supplementing portion, preferably the total molar amount of the second particle in the total volume of P81680PC01 58 the sample portion and the at least one portion is less than 5 %, such as less than 1 %, such as 0.5 % or less of the total molar amount of the first particle in the total volume of the sample portion and the at least one supplementing portion. 10. The method of any one of the preceding claims, wherein the at least one supplementing portion comprising a leading supplementing portion and a trailing supplementing portion and wherein the feeding of the liquid portions onto the microfluidic channel comprises providing the leading supplementing portion and the trailing supplementing portion on either sides of the sample portion. 11. The method of any one of the preceding claims, wherein at least one of the N raw data sets is obtained from a type 1 (capmix) Taylor dispersion assay, wherein the sample portion comprises a base concentration of the second particle at the time of feeding the sample portion to the microfluidic channel and wherein the at least one of the supplementing portion is free of or has a lower concentration than the base concentration of the second particle optionally bound with the first particle at time of feeding the at least one supplementing portion to the microfluidic channel, and wherein the formation of the interfacial contact between the sample portion and the at least one supplementing portion in the microfluidic channel, provides a modification of said sample portion to a modified sample portion comprising dispersion between the sample portion and the at least one supplementing portion, to thereby induce a concentration jump of at least the first particle L in said modified sample portion, wherein the concentration jump provides the first particle and the second particles to be out of equilibrium in the modified sample portion, wherein the concentration jump induces an association reaction. 12. The method of claim 11, wherein the at least one supplementing portion is free of the second particle or wherein the second particle of the supplementing portion is free of said selected marker and/or wherein the concentration of the second particle is less than 10 % of the base concentration of the second particle at the time of feeding the sample portion to the microfluidic channel, such as less than 1 % of the base concentration of the second particle at the time of feeding the sample portion to the microfluidic channel. 13. The method of any one of the preceding claims, wherein at least one of the N raw data sets is obtained from a type 1 (capmix) Taylor dispersion assay, wherein the sample portion comprises a base concentration of the second particle and is free of the first particle at the time of feeding the sample portion to the P81680PC01 59 microfluidic channel and wherein the at one supplementing portion is free of the second particle and has a concentration of the first particle L at the time of feeding the at least one supplementing portion to the microfluidic channel, and wherein the formation of the interfacial contact between the sample portion and the at least one supplementing portion in the microfluidic channel, provides a modification of said sample portion to a modified sample portion comprising dispersion between the sample portion and the at least one supplementing portion, to thereby induce a concentration jump of at least the first particle L in said modified sample portion, wherein the concentration jump provides the first particle and the second particles to be out of equilibrium in the modified sample portion, wherein the concentration jump induces an association reaction. 14. The method of any one of the preceding claims, wherein at least one of the N raw data sets is obtained from a type 2 (premix) Taylor dispersion assay, wherein the sample portion comprises a base concentration of the second particle (A) and a base concentration of the first particle (L) at the time of feeding the sample portion to the microfluidic channel and wherein the at least one supplementing portion has a concentration of the first particle, such as a concentration that is equal to or different from the base concentration of the first particles at the time of feeding the at least one supplementing portion to the microfluidic channel, and wherein the formation of the interfacial contact between the sample portion and the at least one supplementing portion in the microfluidic channel, provides a modification of said sample portion to a modified sample portion comprising dispersion between the sample portion and the at least one supplementing portion, to thereby induce a concentration jump of at least one on the first particle L and the second particle A in said modified sample portion, wherein the concentration jump provides the first particle and the second particles to be out of equilibrium in the modified sample portion, wherein the concentration jump induces an association reaction or a dissociation reaction. 15. The method of claim 14 wherein the at least one supplementing portion has a different concentration of the first particle, such as a higher concentration of the first particles at the time of feeding the sample portion to the microfluidic channel, which differs at least 50 %, such as at least 90 % from the base concentration of the sample portion of the first particle L. P81680PC01 60 16. The method of any one preceding claims, wherein at least one of the N raw data sets is obtained from a type 3 (dismix) Taylor dispersion assay, wherein the sample portion comprises a base concentration of the second particle (A) and a base concentration of the first particle (L) at the time of feeding the sample portion to the microfluidic channel and wherein the at least one supplementing portion is free of the first particle and free of the second particle at the time of feeding the at least one supplementing portion to the microfluidic channel, and wherein the formation of the interfacial contact between the sample portion and the at least one supplementing portion in the microfluidic channel, provides a modification of said sample portion to a modified sample portion comprising dispersion between the sample portion and the at least one supplementing portion, to thereby induce a concentration jump of at least one on the first particle L and the second particle A in said modified sample portion, wherein the concentration jump provides the first particle and the second particles to be out of equilibrium in the modified sample portion, wherein the concentration jump induces a dissociation reaction. 17. The method of any one of claims 14-16, wherein the first particle and the second particle of the sample portion are in equilibrium at the time of feeding the sample portion to the microfluidic channel. 18. The method of any one of claims 14-17, wherein the method comprises determining the at least one binding parameter from one or more of the fitting parameters a, p and q by applying one or more of the equations 50, 51 and/or 52 ^ = (^_^^ − ^_^) ∙ ^{^^^∙^^} ^_^ + ^_^ (50) preferably at least half

sets are each obtained from a type 2 (premix) or from a type 3 (dismix) Taylor dispersion assay. 19. The method of any one of the preceding claims, wherein at least one of the N raw data sets is obtained from a type 4 (cap-dis-mix) Taylor dispersion assay wherein the liquid portions further comprises an affecting portion feed into the microfluidic channel to form an interfacial contact with the sample portion, wherein the sample portion comprises a base concentration of the second particle A and is P81680PC01 61 essentially free of the first particle L at time of feeding the sample portion to the microfluidic channel, the affecting portion comprises a base concentration of the first particle L and is essentially free of the second particle A at the time of feeding the affecting portion to the microfluidic channel and wherein the supplementing portion(s) is/are essentially free of both the first particle L and the second particle A at the time of feeding the at least one supplementing portion to the microfluidic channel, and wherein the formation of the interfacial contact between the sample portion and the affecting portion and/or between the sample portion and the at least one supplementing portion in the microfluidic channel, provides a modification of said sample portion to a modified sample portion comprising dispersion between the sample portion, the affecting portion and the at least one supplementing portion, to thereby induce a concentration jump of at least the first particle L in said modified sample portion, wherein the concentration jump provides the first particle and the second particles to be out of equilibrium in the modified sample portion, wherein the concentration jump induces an association reaction. 20. The method of claim 19, wherein the sample portion and the affecting portion in total has a volume which is 5 % or less than the total volume of the liquid portions at the time of feeding the sample portion to the microfluidic channel, preferably the sample portion and the affecting portion together has a volume which is 1 % or less, such as 0.5 % or less, such as 0.1 % or less, such as 0.05 % or less than the total volume of the liquid portions. 21. The method of claim 19 or claim 20, wherein the method comprises determining the at least one binding parameter from one or more of the fitting parameters a, p and q by applying one or more of the equations 57, 58 and/or 59 ^ = − ^^_^^^ + ^_^^(^_^ + ^_^)^ (67)

^ = (^_^ − ^_^^) ∙ ^{^^} ^_^^^^^^^ (69) preferably at least half of or all or the N raw data sets, such as all of the N data raw sets are each obtained from a type 4 (cap-dis-mix) Taylor dispersion assay. P81680PC01 62 22. The method of any one of claims 10 -15 and 17, wherein the leading supplementing portion has a lead portion concentration of the first particle and the trailing supplementing portion has a trail portion concentration of the first particle, wherein the lead portion concentration of the first particle and the trail portion concentration of the first particle is equal or differs, preferably the lead portion concentration of the first particle and the trail portion concentration of the first particle is equal or differs with less than 10 % from the average concentration, such as less than 1 % of the average of the trail portion concentration of the first particle and the lead portion concentration of the first particle. 23. The method of any one of claims 10 -15 and 17, wherein the leading supplementing portion is free of or has a lead portion concentration of the second particle and the trailing supplementing portion is free of or has a trail portion concentration of the second particle, wherein the leading supplementing portion concentration of the second particle and the trail portion concentration of the second particle is equal or differs, preferably the lead portion concentration of the second particle and the trail portion concentration of the second particle is equal or differs with less than 10 % from the average concentration, such as less than 1 % of the average of the trail portion concentration of the second particle and the lead portion concentration of the second particle. 24. The method of any one of claims 9-23, wherein the leading supplementing portion has a lead portion volume and the trailing supplementing portion has a trail portion volume, wherein the lead portion volume and the trail portion volume may be identical or may differ, preferably each of the lead portion volume and the trail portion volume is larger than the sample volume, such as larger than 5 times the sample volume, such as larger than 10 times the sample volume, such as larger than 20 times the sample volume. 25. The method of any one of the preceding claims, wherein the obtaining of each set of raw data comprises performing a plurality of readings of the signal intensity at a read out section of the microfluidic channel, wherein the plurality of readings preferably comprises at least 5 intensity readings, comprising at least one intensity reading at the peak appearance time (tR), at least one intensity reading before the peak appearance time (tR) and at least one intensity reading after the peak appearance time (t_R), preferably the obtaining of each set of raw data P81680PC01 63 comprises recording the intensity with a rate of at least 1 Hz, such as 5 to 12 Hz or more. 26. The method of any one of the preceding claims, wherein the method comprises obtaining the N of sets of raw data wherein the N respective sets of raw data are obtained from N respective Taylor dispersion assays, wherein N is an integer of at least 1, preferably N is from 1 to 50 such as from 2 to 20, such as from 3 to 10. 27. The method of any one of the preceding claims, wherein at least of the binding parameters KD (Equilibrium dissociation constant), DA (Diffusitivity of unbound A) and/or; DAL (Diffusitivity of AL) is a known binding parameter and wherein the one or more known parameter is applied for determining at least one other binding parameter from one or more of the fitting parameters p, q and a, wherein N preferably is 5 or less such as 1, 2 or 3 and wherein at least one of the N sets of raw data preferably is obtained from a Taylor dispersion assay wherein the sample portion comprises a base concentration of the first particle L and a base concentration of the second particle A in equilibrium. 28. The method of any one of the preceding claims, wherein N is two or more and wherein the N sets of raw data comprises at least one set of raw data obtained from a Taylor dispersion assay wherein the concentration jump induces an association reaction 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 of any one of the preceding claims, wherein N is two or more and wherein the N sets of raw data comprises at least one set of raw data obtained from a Taylor dispersion assay wherein the second particle (A) of the sample portion is dispersing to form a dispersion zone in said microfluidic channel and wherein the molar concentration of the second particle is equal to or lower than the molar concentration of said first particle in the dispersion zone and at least one set of raw data obtained from a Taylor dispersion assay wherein the second particle (A) of the sample portion is dispersing to form a dispersion zone in said microfluidic channel and wherein the molar concentration of the second particle is equal to or larger than the molar concentration of said first particle in the dispersion zone. 30. The method of any one of the preceding claims, wherein N is two or more and wherein the N sets of raw data comprises a plurality of data sets obtained P81680PC01 64 from respective Taylor dispersion the base concentration of the second particle A in the sample portion differs. 31. The method of any one of the preceding claims, wherein N is two or more and wherein the N sets of raw data comprises a plurality of data sets obtained from respective Taylor dispersion assay wherein a base concentration of the first particle in the at least one supplementing portion in the sample portion differs. 32. The method of any one of the preceding claims, wherein the selected marker comprises an intrinsic marker and/or an extrinsic marker capable of being detected, preferably the markers are optically readable markers, such as florescence markers and/or light absorbing markers. 33. The method of any one of the preceding claims, wherein the selected marker is sensitive to the molecular interaction, such a sensitive to a conformational change of the first particle, preferably the selected marker changes signal in dependence of conformation of the first particle and changes thereof, such as in dependence of change in binding/dissociation and/or in structure. 34. The method of any one of the preceding claims, wherein the feeding of the liquid portions into the microfluidic channel is performed at an injection pressure Pi and wherein the provision of the liquid portions to the flow in the microfluidic channel is performed at a run pressure Pr ensuring a laminar flow in the microfluidic channel, wherein the run pressure Pr is larger than the injection pressure Pi, preferably the run pressure Pr is adjustable. 35. The method of any one of the preceding claims, wherein each reading to obtain a reading result of the set of reading results comprises reading marker signals of the concentration of marked second particles along a length section of the microfluidic channel containing the sample portion preferably comprising the dispersion zone, preferably the reading of the marker signals of the concentration of marked second particles along the length section of the microfluidic channel containing the dispersion zone is from a leading edge to a trailing edge of the dispersion zone comprises reading the intensity of the marker signals as a function of time as the dispersion zone is passing a reading section of the microfluidic channel. 36. The method of any one of the preceding claims, wherein the at least one reading result of an out-of-equilibrium reading is obtained by reading marker P81680PC01 65 signals of the concentration of marked particles before said interaction between said first particle and said second particle has reached equilibrium, preferably the method comprises performing a plurality of out-of-equilibrium readings, optionally all of the reading results of the set of reading results are reading results of out-of-equilibrium readings. 37. The method of any one of the preceding claims, wherein the set of reading results of readings comprises a reading result of an in-equilibrium reading, preferably obtained by reading marker signals of the concentration of marked second particles wherein said interaction between said first particle and said second particle are in-equilibrium. 38. The method of any one of the preceding claims, wherein said first particle and said second particle differs from each other, preferably said first particle and said second particle differs in type of particle, molecular weight, dispersion properties or any combinations thereof. 39. The method of any one of the preceding claims, wherein at least one of said first particle and said second particle comprises a drug or a drug candidate. 40. The method of any one of the preceding claims, wherein at least one of the first particle and the second particle comprises a biomolecule; a protein, such as an antibody (monoclonal or polyclonal), a nanobody, an antigen, an enzyme and/or a hormone; a nucleotide; a nucleoside; a nucleic acid, such a RNA, DNA, PNA or any fragments thereof and/or any combinations comprising at least one of these.