Method for determining reaction kinetic parameters

Abstract

According to the present invention, a method is provided for determining kinetic parameters of a reaction between an analyte and a ligand attached to a test surface of a flow cell, the method comprising the steps of: (a) flowing a first volume of sample fluid (V1) containing the analyte through the test surface between a first time point (t1) and a second time point (t2); (b) flowing a first volume of buffer fluid (Vb1) without the analyte through the test surface between a third time point (t3) and a fourth time point (t4); (c) flowing a sample fluid (V2) containing at least a second volume of the analyte through the test surface between a fifth time point (t5) and a sixth time point (t6); (d) flowing a buffer fluid (Vb2) without the analyte through the test surface between a seventh time point (t7) and an eighth time point (t8); (e) measuring the binding of the analyte to the ligand on the test surface using a sensor to obtain a binding curve; and (f) using only a portion of the binding curve within a predefined time interval and excluding other portions of the binding curve to determine the kinetic parameters.

Description

Technical Field

[0001] The present invention relates to a method for determining the kinetic parameters of the reaction between an analyte and a ligand attached to a test surface in a flow cell; and particularly to a method for determining kinetic parameters by using a significantly smaller portion of the conjugation curve representing the association between the analyte and the ligand, and instead primarily using the portion of the conjugation curve representing the dissociation of the analyte and the ligand. Background Technology

[0002] In existing methods for determining the kinetic parameters of the reaction between an analyte and a ligand attached to a test surface in a flow cell, a volume of sample fluid containing the analyte continuously flows over the test surface. As the volume of sample fluid containing the analyte continuously flows over the test surface, a sensor is used to measure the amount of analyte bound to the ligand on the test surface; the sensor outputs a curve representing the amount of analyte bound to the ligand on the test surface, called the binding curve. The binding curve will include an association phase and a dissociation phase. The association phase occurs as a volume of sample fluid passes over the test surface and the analyte in the sample fluid binds to the ligand; the binding curve will show an increase in the amount of analyte bound to the ligand on the test surface during the association phase. The dissociation phase occurs after a volume of sample fluid has passed over the test surface and before the next volume of sample fluid flows over the test surface; the binding curve will show a decrease in the amount of analyte bound to the ligand on the test surface during the dissociation phase, and previously bound analytes become dissociated (or detached) from the ligand.

[0003] The kinetic parameters of the reaction between the analyte and the ligand on the test surface are then determined using the entire conjugation curve (i.e., all the association and dissociation phases); typically, this is done by identifying a predefined model with known kinetic parameters that fits best to the conjugation curve; the kinetic parameters of the model that fits best to the conjugation curve are then considered as the kinetic parameters of the reaction.

[0004] However, the problem with using the entire binding curve to determine the kinetic parameters of the reaction between the analyte and the ligand on the test surface is that it can lead to inaccurate determination of the reaction kinetic parameters: existing sensors used to measure the amount of analyte bound to the test surface and output the binding curve are sensitive to changes in the refractive index of the sample fluid volume; when a certain volume of sample fluid containing the analyte flows across the test surface, the change in the refractive index of that volume of sample fluid can introduce artifacts into the binding curve; these artifacts exist in the correlation phase of the binding curve; because the entire binding curve (i.e., all correlation and dissociation phases) is then used to determine the kinetic parameters of the reaction between the analyte and the ligand on the test surface, these artifacts, in turn, lead to inaccurate determination of the reaction kinetic parameters.

[0005] The object of this invention is to eliminate or mitigate at least some of the drawbacks associated with existing methods in the art. In particular, the object of this invention is to provide an improved method for determining the kinetic parameters of a reaction that is less susceptible to artifacts in the bonding curve created by changes in refractive index, which occur when a volume of sample fluid flows over the test surface of a flow cell. Summary of the Invention

[0006] According to the invention, this objective is achieved by a method having at least the steps described in claim 1. The dependent claims describe advantageous, optional steps that can be performed in various embodiments of the invention.

[0007] Advantageously, in the method of the present invention, the dissociation phase of the binding curve (i.e., the portion of the binding curve corresponding to the period after a certain volume of sample fluid has flowed over the test surface and before the next volume of sample fluid flows over the test surface), rather than the association phase of the binding curve (i.e., the portion of the binding curve corresponding to when a certain volume of sample fluid is flowing over the test surface and the analyte in the sample fluid is binding with the ligand), is primarily used to determine the kinetic parameters of the reaction.

[0008] Specifically, in the first embodiment, only the dissociation phase of the joint curve is used, without using the association phase, to determine the kinetic parameters; and in the second embodiment, only a very small portion of both the dissociation phase and the association phase of the joint curve are used to determine the kinetic parameters. In the second embodiment, the small portion of the association phase of the joint curve can be so small that artifacts in those portions of the joint curve have a negligible effect on the determination of the kinetic parameters. In either embodiment, the amount of association phase of the joint curve used to determine the kinetic parameters is reduced compared to the prior art.

[0009] Therefore, in this invention, the artifacts in the bonding curve created by the refractive index change that occurs when a certain volume of sample fluid flows through the test surface of the flow cell will have a relatively small impact on the determination of kinetic parameters. Attached Figure Description

[0010] Exemplary embodiments of the present invention are disclosed in the specification and illustrated in the accompanying drawings, wherein:

[0011] Figure 1 It is a complete bonding curve, which includes six association phases and six dissociation phases, generated by continuously flowing six corresponding volumes of sample fluid containing the analyte over the test surface of the flow cell.

[0012] Figure 2 It shows Figure 1 The portion of the joining curve within multiple predefined time intervals;

[0013] Figure 3 Normalized concentration curves used to determine the plurality of predefined time intervals are shown. Detailed Implementation

[0014] According to the present invention, a method is provided for determining the kinetic parameters of the reaction between an analyte and a ligand attached to a test surface of a flow cell. The method includes the following steps:

[0015] (a) Between a first time point (t1) and a second time point (t2), a sample fluid (V1) containing a first volume of the analyte is passed over the test surface.

[0016] (b) Between a third time point (t3) and a fourth time point (t4) (the third time point (t3) may be equal to the second time point (t2)), a first volume of buffer fluid (Vb1) (which does not contain analytes) is allowed to flow over the test surface; most preferably, between the third time point (t3) and the fourth time point (t4), at least some of the analytes that have bound to the ligands on the test surface are dissociated from the first volume of sample fluid (V1). In one embodiment, the buffer fluid is configured to facilitate the dissociation of the bound analytes from the ligands.

[0017] (c) Between the fifth time point (t5) and the sixth time point (t6) (the fifth time point (t5) may be equal to the fourth time point (t4)), a sample fluid (V2) containing at least a second volume of the analyte is passed over the test surface;

[0018] (d) Between a seventh time point (t7) and an eighth time point (t8) (the seventh time point (t7) may be equal to the sixth time point (t6)), a second volume of buffer fluid (Vb2) (which does not contain analytes) is allowed to flow over the test surface; most preferably, between the seventh time point (t7) and the eighth time point (t8), at least some of the analytes that have bound to the ligands on the test surface are dissociated from the second volume of sample fluid (V2). In one embodiment, the buffer fluid is configured to facilitate the dissociation of the bound analytes from the ligands.

[0019] (e) Use a sensor to measure the binding of the analyte with the ligand on the test surface to obtain a binding curve.

[0020] (f) Use only the portion of the joint curve within a predefined time interval, and not the other portion of the joint curve, to determine the dynamic parameters.

[0021] To determine the kinetic parameters of the bonding curve only for a portion of the predefined time interval, the following steps are performed: A normalized concentration curve (c(t)) is established, which describes the concentration of the analyte at the test surface over time; the kinetic parameters Rmax and k are estimated. a k d The value of , where Rmax is a predefined theoretical maximum binding curve value corresponding to ligand saturation, such as, for example, when all binding sites of the ligand are occupied by the analyte bound to the ligand, k a It is the correlation rate constant, and k d It is the dissociation rate constant; using the normalized concentration curve (c(t)) and kinetic parameters Rmax, k a k d The estimated value is used to solve the differential reaction equation:

[0022]

[0023] In order to obtain the simulated bonding curve sbc = R(t).

[0024] Once the differential reaction equation is obtained, the predefined time interval is extracted ( The portion of the simulated joint curve in the ) is used to provide the corresponding partial simulated joint curve (sbcj).

[0025] Then, the partial chi-square of the simulated bonding curve is established according to the following equation ( ):

[0026]

[0027] Where dmbj is the junction curve in the j-th predefined interval time period ( The part in ), where sbcj is the j-th partial simulated bonding curve, and N dmbj It is the joining curve in the j-th predefined interval time period ( The number of data points in the part of ).

[0028] In the already determined simulated joint curve, the partial chi-square ( After that, then minimize the determined partial chi-square ( ), where minimizing the determined partial chi-square ( The dynamic parameters Rmax and k of the above are... a k d The value defines the kinetic parameter of the reaction between the analyte and the ligand attached to the test surface of the flow cell.

[0029] In a preferred embodiment, the dynamic parameters Rmax and k are estimated. a k d The steps involved in finding the value of the partial chi-square () include using the Levenberg-Marquard algorithm to find the value of the partial chi-square () Minimize the dynamic parameters.

[0030] In another preferred embodiment, the dynamic parameters Rmax and k are estimated. a k d The steps involved in finding the value of the partial chi-square () include using an estimator to find the value of the partial chi-square () Minimize the dynamic parameters.

[0031] In a preferred embodiment, the method includes the following steps: continuously measuring the binding of the analyte to the ligand on the test surface using a sensor from a first time point (t1) to a ninth time point (t9), and then extracting a portion of the binding curve within the predefined time interval; and using only the extracted portion of the binding curve to determine kinetic parameters.

[0032] In another embodiment, the method includes the following steps: continuously measuring the binding of the analyte to the ligand on the test surface using a sensor from a first time point (t1) to a ninth time point (t9); zeroing the portion of the binding curve outside the predefined time interval; and then using only the non-zeroed portion of the binding curve to determine kinetic parameters.

[0033] Exemplary Example 1:

[0034] In the first embodiment, the predefined interval time period is the first and second interval time periods ( , ); where the first interval time period ( ) occurs between the second time point (t2) and the fifth time point (t5); and the second interval time period ( The dissociation occurs between the sixth time point (t6) and the ninth time point (t9), where the ninth time point (t9) occurs sometime after the eighth time point (t8) (or, in another embodiment, the ninth time point (t9) is equal to the eighth time point (t8)). Advantageously, in this embodiment, only the portion of the engagement curve corresponding to the dissociation phase is used to determine the kinetic parameters.

[0035] In one embodiment, the first interval time period ( ) is a portion of the duration between the second time point (t2) and the fifth time point (t5); and the second interval period ( The interval (t9) is a portion of the duration between the sixth time point (t6) and the ninth time point (t9), where the ninth time point (t9) occurs sometime after the eighth time point (t8) (or, in another embodiment, the ninth time point (t9) is equal to the eighth time point (t8)). For example, the first interval time period (t9) The second interval can be within a predefined time period after the second time point (t2), up to the fifth time point (t5), or up to the fifth time point (t5); and the second interval time period ( The interval can be between a predefined time period after the sixth time point (t6), up to the eighth time point (t8), or a predefined time period before the eighth time point (t8). In another embodiment, the first interval time period ( The second time interval is defined by the entire time interval between the second time point (t2) and the fifth time point (t5); and the second interval time period ( The time interval is defined by the entire time interval between the sixth time point (t6) and the ninth time point (t9), where the ninth time point (t9) occurs sometime after the eighth time point (t8) (or, in another embodiment, the ninth time point (t9) is equal to the eighth time point (t8)).

[0036] The first embodiment includes the following steps:

[0037] (a) Between a first time point (t1) and a second time point (t2), a sample fluid (V1) containing a first volume of the analyte is passed over the test surface.

[0038] (b) Between a third time point (t3) and a fourth time point (t4) (the third time point (t3) may be equal to the second time point (t2)), a first volume of buffer fluid (Vb1) (which does not contain analytes) is allowed to flow over the test surface; most preferably, between the third time point (t3) and the fourth time point (t4), at least some of the analytes that have bound to the ligands on the test surface are dissociated from the first volume of sample fluid (V1). In one embodiment, the buffer fluid is configured to facilitate the dissociation of the bound analytes from the ligands.

[0039] (c) Between the fifth time point (t5) and the sixth time point (t6) (the fifth time point (t5) may be equal to the fourth time point (t4)), a sample fluid (V2) containing at least a second volume of the analyte is passed over the test surface;

[0040] (d) Between a seventh time point (t7) and an eighth time point (t8) (the seventh time point (t7) may be equal to the sixth time point (t6)), a second volume of buffer fluid (Vb2) (which does not contain analytes) is allowed to flow over the test surface; most preferably, between the seventh time point (t7) and the eighth time point (t8), at least some of the analytes that have bound to the ligands on the test surface are dissociated from the second volume of sample fluid (V2). In one embodiment, the buffer fluid is configured to facilitate the dissociation of the bound analytes from the ligands.

[0041] (e) at least during the first and second intervals ( , During this period, sensors are used to measure the binding of the analyte to ligands on the test surface, wherein the first interval time period ( ) occurs between the second time point (t2) and the fifth time point (t5), and the second interval period ( The interval occurs between the sixth time point (t6) and the ninth time point (t9), where the ninth time point (t9) occurs sometime after the eighth time point (t8) (or, in another embodiment, the ninth time point (t9) equals the eighth time point (t8)), to obtain the joining curve. In one embodiment, the first interval time period ( ) is a portion of the duration between the second time point (t2) and the fifth time point (t5); and the second interval period ( The interval (t9) is a portion of the duration between the sixth time point (t6) and the ninth time point (t9), where the ninth time point (t9) occurs sometime after the eighth time point (t8) (or, in another embodiment, the ninth time point (t9) is equal to the eighth time point (t8)). For example, the first interval time period (t9) The second interval can be within a predefined time period after the second time point (t2), up to the fifth time point (t5), or up to the fifth time point (t5); and the second interval time period ( The interval can be between a predefined time period after the sixth time point (t6), up to the eighth time point (t8), or a predefined time period before the eighth time point (t8). In another embodiment, the first interval time period ( The second time interval is defined by the entire time interval between the second time point (t2) and the fifth time point (t5); and the second interval time period ( The time interval is defined by the entire time interval between the sixth time point (t6) and the ninth time point (t9), where the ninth time point (t9) occurs sometime after the eighth time point (t8) (or, in another embodiment, the ninth time point (t9) is equal to the eighth time point (t8)).

[0042] (f) Using the joining curve during the first and second interval time periods ( , The part in ) without using the joining curve at the first time point (t1) and the second time point (t2) 2a The dynamic parameters are determined between the time points t5 and t6, and between the fifth time point (t5) and the sixth time point (t6).

[0043] In this first embodiment, there are essentially no binding curves between the first time point (t1) and the second time point (t2), or between the fifth time point (t5) and the sixth time point (t6), used to determine the kinetic parameters. Therefore, artifacts in the binding curves created by the refractive index change that occurs when a certain volume of sample fluid flows through the test surface of the flow cell will have a relatively small impact on the determination of the kinetic parameters.

[0044] In one embodiment, the method includes the steps of: continuously measuring the binding of the analyte to a ligand on a test surface using a sensor from a first time point (t1) to a ninth time point (t9), and then extracting the binding curve during the first and second time intervals (t1 and t9). , The portion of the curve; and the portion extracted from the joint curve used only to determine the kinetic parameters.

[0045] In another embodiment, the method includes the steps of: continuously measuring the binding of the analyte to the ligand on the test surface using a sensor from a first time point (t1) to a ninth time point (t9); and plotting the binding curves at the first and second time intervals (t1, t2, and t9). , The portion outside of the zeroed-out portion of the engagement curve is zeroed out; and then only the portion of the engagement curve that is not zeroed out is used to determine the dynamic parameters.

[0046] The joining curve was used during the first and second interval time periods ( , The step of determining the kinetic parameters in the portion of the joint curve between the first time point (t1) and the second time point (t2), and between the fifth time point (t5) and the sixth time point (t6), can be carried out using any known technique for determining kinetic parameters using a joint curve; in this invention, those exact same techniques can be used to determine the kinetic parameters, the difference being that the technique is applied to the joint curve during the first and second time intervals (t1 and t2). , The portion of the curve to be joined between the first time point (t1) and the second time point (t2), and between the fifth time point (t5) and the sixth time point (t6), shall not be applied.

[0047] In the most advantageous embodiment, a bonding curve is used during the first and second time intervals ( , The step of determining the kinetic parameters using a portion of the curve between the first time point (t1) and the second time point (t2), and between the fifth time point (t5) and the sixth time point (t6), preferably includes the following steps: establishing a normalized concentration curve (c(t)) that describes the concentration of the analyte at the test surface over time (preferably, the concentration curve is established first and then normalized to provide the normalized concentration curve (c(t))); estimating the kinetic parameters Rmax, k a k d The value of , where Rmax is the predefined theoretical maximum binding curve value corresponding to ligand saturation, such as, for example, when all binding sites of the ligand are occupied by the analyte bound to the ligand, k a It is the correlation rate constant, and k d It is the dissociation rate constant; using the normalized concentration curve (c(t)) and kinetic parameters Rmax, k a k d The estimated value is used to solve the differential reaction equation:

[0048]

[0049] In order to obtain the simulated bonding curve sbc = R(t).

[0050] Once the differential reaction equation is obtained, the simulated bonding curve is extracted during the first time interval ( A portion of the simulated joint curve (sbc1) is used to provide a first partial simulated joint curve; and the simulated joint curve is extracted during the second interval time period ( The portion of ) is used to provide a second partial simulated bonding curve (sbc2); and the partial chi-square of the simulated bonding curve is determined according to the following equation ( ):

[0051]

[0052] Where dmb1 is the junction curve during the first interval time period ( The portion of the curve in the second interval ( ), and dmb2 is the junction curve in the second interval time period ( ). The part in ), where sbc1 is the first partial simulated bonding curve, and sbc2 is the second partial simulated bonding curve, and where N dmb1 The joining curve is in the first interval time period ( The number of data points in N, and N dmb2 It is the number of data points of the joining curve in the second time interval (t2).

[0053] In the already determined simulated joint curve, the partial chi-square ( After that, then minimize the determined partial chi-square ( ), where minimizing the determined partial chi-square ( The dynamic parameters Rmax and k of the above are... a k d The value defines the kinetic parameter of the reaction between the analyte and the ligands attached to the test surface of the flow cell.

[0054] In a preferred embodiment, the dynamic parameters Rmax and k are estimated. a k d The steps involved in finding the value of the partial chi-square () include using the Levenberg-Marquard algorithm to find the value of the partial chi-square () Minimize the dynamic parameters.

[0055] In another preferred embodiment, the dynamic parameters Rmax and k are estimated. a k d The steps involved in finding the value of the partial chi-square () include using an estimator to find the value of the partial chi-square () Minimize the dynamic parameters.

[0056] Exemplary Example 2:

[0057] In the first embodiment described above, the predefined interval time period includes the first and second interval time periods ( , ); where the first interval time period ( ) occurs between the second time point (t2) and the fifth time point (t5); and the second interval time period ( The time interval occurs between the sixth time point (t6) and the ninth time point (t9), where the ninth time point (t9) occurs sometime after the eighth time point (t8) (or, in another embodiment, the ninth time point (t9) equals the eighth time point (t8)). However, in this second embodiment, a predefined interval time period is determined based on the concentration curve (or based on the normalized concentration curve). The second embodiment includes the following steps:

[0058] (a) Between a first time point (t1) and a second time point (t2), a sample fluid (V1) containing a first volume of the analyte is passed over the test surface.

[0059] (b) Between a third time point (t3) and a fourth time point (t4) (the third time point (t3) may be equal to the second time point (t2)), a first volume of buffer fluid (Vb1) (which does not contain analytes) is allowed to flow over the test surface; most preferably, between the third time point (t3) and the fourth time point (t4), at least some of the analytes that have bound to the ligands on the test surface are dissociated from the first volume of sample fluid (V1). In one embodiment, the buffer fluid is configured to facilitate the dissociation of the bound analytes from the ligands.

[0060] (c) Between the fifth time point (t5) and the sixth time point (t6) (the fifth time point (t5) may be equal to the fourth time point (t4)), a sample fluid (V2) containing at least a second volume of the analyte is passed over the test surface;

[0061] (d) Between a seventh time point (t7) and an eighth time point (t8) (the seventh time point (t7) may be equal to the sixth time point (t6)), a second volume of buffer fluid (Vb2) (which does not contain analytes) is allowed to flow over the test surface; most preferably, between the seventh time point (t7) and the eighth time point (t8), at least some of the analytes that have bound to the ligands on the test surface are dissociated from the second volume of sample fluid (V2). In one embodiment, the buffer fluid is configured to facilitate the dissociation of the bound analytes from the ligands.

[0062] (e) Use a sensor to measure the binding of the analyte with the ligand on the test surface to obtain a binding curve.

[0063] (f) Using the joining curve during a predefined first interval time period ( ) and the second interval time period ( The dynamic parameters are determined by the part in ) .

[0064] In this second embodiment, a significantly smaller portion of the bonding curve between the first time point (t1) and the second time point (t2), and between the fifth time point (t5) and the sixth time point (t6), is used to determine the kinetic parameters. Therefore, artifacts in the bonding curve created by the refractive index change that occurs when a certain volume of sample fluid flows through the test surface of the flow cell will have a smaller impact on the determination of the kinetic parameters.

[0065] In one embodiment, the method includes the steps of: continuously measuring the binding of the analyte to a ligand on a test surface using a sensor from a first time point (t1) to a ninth time point (t9), and then extracting the binding curve during the first and second time intervals (t1 and t9). , The portion of the curve; and the portion extracted from the joint curve used only to determine the kinetic parameters.

[0066] In another embodiment, the method includes the steps of: continuously measuring the binding of the analyte to the ligand on the test surface using a sensor from a first time point (t1) to a ninth time point (t9); and plotting the binding curves at the first and second time intervals (t1, t2, and t9). , The portion outside of the first and second intervals is zeroed out; and then only the portion outside of the first and second intervals is used. , The dynamic parameters are determined by the portion of the engagement curve (i.e., the portion of the engagement curve that is not zero) in the equation.

[0067] The second embodiment further includes a calibration step, which may be performed before steps (a)-(f) or after steps (a)-(e) (performed before step (f)), to determine the first and second interval time periods ( , ).

[0068] In one embodiment, the calibration step includes: establishing a concentration curve describing the concentration of the analyte at the test surface over time, and then normalizing the concentration curve to provide a normalized concentration curve (c(t)); and then using the normalized concentration curve (c(t)) to determine the first and second time intervals ( , In another embodiment, the calibration step includes: establishing a concentration profile describing the concentration of the analyte at the test surface over time; and then using the concentration profile to determine the first and second time intervals. , It should also be noted that, in addition to determining the first and second time intervals from the concentration curve (c(t)), , The same steps were performed to determine the first and second time intervals from the normalized concentration curve (c(t)). , In both variations, the normalized concentration curve (c(t)) is used to determine the kinetic parameters.

[0069] In this exemplary second embodiment, the establishment of a concentration profile describing the concentration of the analyte at the test surface over time will be described, and the concentration profile will then be normalized to provide a normalized concentration profile (c(t)); and the normalized concentration profile (c(t)) will then be used to determine the first and second time intervals. , ):

[0070] In this exemplary second embodiment, sample fluids (V1, V2) of first and second volumes flow through the test surface of the flow cell to establish a bonding curve, and a first volume of refractive index standard fluid (V1') containing reference molecules of known concentration flows through the test surface between a first reference time point (t'1) and a second reference time point (t'2).

[0071] Importantly, the rate (i.e. flow rate) of the first volume of refractive index standard fluid (V1') flowing through the test surface is equal to the rate of the first volume of sample fluid (V1) flowing through the test surface; the duration between the first reference time point (t'1) and the second reference time point (t'2) is also equal to the duration between the first time point (t1) and the second time point (t2).

[0072] After a first volume of refractive index standard fluid (V1') containing reference molecules of known concentration has flowed over the test surface, between the third reference time point (t'3) and the fourth reference time point (t'4) (the third reference time point (t'3) may be equal to the second reference time point (t'2)), a first volume of buffer fluid (Vb1') (which does not contain reference molecules) flows over the test surface.

[0073] Then, between the fifth reference time point (t'5) and the sixth reference time point (t'6), a second volume of refractive index standard fluid (V2') containing reference molecules of known concentration flows over the test surface.

[0074] Importantly, the flow rate (i.e., the flow rate) of the second volume of refractive index standard fluid (V2') across the test surface is equal to the flow rate of the second volume of sample fluid (V2) across the test surface; the duration between the fifth reference time point (t'5) and the sixth reference time point (t'6) is also equal to the duration between the fifth time point (t5) and the sixth time point (t6); the ratio of the concentration of the second volume of refractive index standard fluid (V2') to the concentration of the first volume of refractive index standard fluid (V1') is also equal to the ratio of the concentration of the second volume of sample fluid (V2) to the concentration of the first volume of sample fluid (V1); and the duration between the second reference time point (t'2) and the fifth reference time point (t'5) is also equal to the duration between the second time point (t2) and the fifth reference time point (t'5).

[0075] After a second volume of refractive index standard fluid (V2') containing reference molecules of known concentration flows over the test surface, between the seventh reference time point (t7) and the eighth reference time point (t8) (the seventh time point (t7) may be equal to the sixth reference time point (t'6)), a second volume of buffer fluid (Vb2') (which does not contain reference molecules) flows over the test surface.

[0076] The concentration of reference molecules at the test surface is continuously measured during the period from the first reference time point (t'1) to the ninth reference time point (t'9), wherein the ninth reference time point (t'9) occurs at some time after the eighth reference time point (t'8) (or, in another embodiment, the ninth reference time point (t'9) is equal to the eighth reference time point (t'8)) in order to obtain a concentration profile (cmc).

[0077] Then, the concentration curve (cmc) is normalized by dividing the concentration curve (cmc) by the maximum value of the concentration curve (max(cmc)) and multiplying the result by the known maximum concentration (cmax) of the analyte in the first and second sample fluid volumes (V1, V2), as illustrated in the equation below:

[0078]

[0079] To obtain the normalized concentration curve (c(t)).

[0080] In this example, the analyte concentration (c(V1)) in the first volume of sample fluid (V1) is equal to the analyte concentration (c(V2)) in the second volume of sample fluid (V2) (i.e., the first and second volumes of sample fluid (V1, V2) have the same analyte concentration); however, if one volume of sample fluid (V1, V2) contains a higher concentration of analyte than the other, then the higher concentration value will define c. max (Right now It should be understood that any suitable technique known in the art can be used to determine the concentration of an analyte in a given volume of sample fluid.

[0081] In order to determine the first and second time intervals using the normalized concentration curve (c(t)) , Select a threshold concentration. Most preferably, the threshold concentration is determined as a predefined percentage of the maximum value of the normalized concentration curve (c(t)); for example, if the maximum value of the normalized concentration curve (c(t)) is "200", then the threshold concentration can be selected as 5% of the maximum value, which in this example would be 5% * 200 = 10; therefore, the threshold concentration in this example would be "10"; in another example, the threshold concentration could be 2% of the maximum value, which in this example would be 2% * 200 = 4, therefore, the threshold concentration in this example would be "4".

[0082] The earliest time interval on the normalized concentration curve (c(t)) – from the time when the normalized concentration curve (c(t)) drops below the threshold concentration until the time when the normalized concentration curve (c(t)) equals the threshold concentration – defines the first interval period. The next time interval on the normalized concentration curve (c(t)) (which occurs during the first interval) The second interval period is defined as the time from when the normalized concentration curve (c(t)) drops below the threshold concentration until the ninth reference time point (t'9). ).

[0083] It should be understood that, in a variation of this embodiment, the concentration profile (unnormalized) can instead be used to determine the first and second time intervals. , Similarly, in this variant of the embodiment, a threshold concentration is selected. Most preferably, the threshold concentration is determined as a predefined percentage of the maximum value of the concentration curve; for example, if the maximum value of the concentration curve is "2000", the threshold concentration can be selected as 5% of the maximum value, which in this example would be 5% * 2000 = 100; therefore, the threshold concentration in this example would be "100"; in another example, the threshold concentration could be 2% of the maximum value, which in this example would be 2% * 2000 = 40, therefore, the threshold concentration in this example would be "40". The earliest time interval on the concentration curve—from the time when the concentration curve drops below the threshold concentration until the time when the concentration curve equals the threshold concentration—defines the first interval time period. The next time interval on the concentration curve (which occurs during the first interval) A second interval period is defined as the time from when the concentration curve drops below the threshold concentration until the ninth reference time point (t'9). ).

[0084] In the above example, most preferably, the refractive index standard fluid preferably comprises a buffer fluid containing a reference molecule of known concentration; and the reference molecule (which exists in the refractive index standard fluid at a known concentration) may include, for example, DMSO or glucose. Most preferably, the concentration of the reference molecule in the refractive index standard fluid (and therefore the concentration of the reference molecule in the first volume refractive index standard fluid (V1'), and also the concentration of the reference molecule in the second volume refractive index standard fluid (V2')) is preferably 0.1% v / v, or 0.5% v / v, or 1% v / v, so that the refractive index of the refractive index standard fluid is different from the refractive index of the first and second volume buffer fluids (Vb1', Vb2') without reference molecules.

[0085] The joining curve was used during the first and second interval time periods ( , The step (f) in determining the kinetic parameters in the part of the joint curve can be carried out using any known technique for determining kinetic parameters using the joint curve; in this invention, the exact same technique can be used to determine the kinetic parameters, the difference being that the technique is applied to the joint curve during the first and second time intervals ( , Part of ).

[0086] In the most advantageous embodiment, a bonding curve is used during the first and second time intervals ( , The step of determining the kinetic parameters in the part of the application preferably includes the following steps: establishing a normalized concentration curve (c(t)) that describes the concentration of the analyte at the test surface over time (if a normalized concentration curve (c(t)) has not yet been established; for example, a normalized concentration curve (c(t)) established in the calibration step can be used) (using any steps described in this application for obtaining a normalized concentration curve (c(t))); estimating the kinetic parameters Rmax, k a k d The value of , where Rmax is a predefined theoretical maximum binding curve value corresponding to ligand saturation, such as, for example, when all binding sites of the ligand are occupied by the analyte bound to the ligand, k a It is the correlation rate constant, and k d It is the dissociation rate constant; using the normalized concentration curve (c(t)) and kinetic parameters Rmax and k determined in the calibration step. a k dUsing the estimated value, solve the differential reaction equation:

[0087]

[0088] In order to obtain the simulated bonding curve sbc = R(t).

[0089] Once the differential reaction equation is obtained, the simulated bonding curve is extracted during the first time interval ( A portion of the simulated joint curve (sbc1) is used to provide a first partial simulated joint curve; and the simulated joint curve is extracted during the second interval time period ( The portion of ) is used to provide a second partial simulated bonding curve (sbc2); and the partial chi-square of the simulated bonding curve is determined according to the following equation ( ):

[0090]

[0091] Where dmb1 is the junction curve during the first interval time period ( The portion of the curve in the second interval ( ), and dmb2 is the junction curve in the second interval time period ( ). The part in ), where sbc1 is the first partial simulated bonding curve, and sbc2 is the second partial simulated bonding curve, and where N dmb1 The joining curve is in the first interval time period ( The number of data points in N, and N dmb2 The joining curve is in the second interval time period ( The number of data points in ).

[0092] In the already determined simulated joint curve, the partial chi-square ( After that, then minimize the determined partial chi-square ( ), where minimizing the determined partial chi-square ( The dynamic parameters Rmax and k of the above are... a k d The value defines the kinetic parameter of the reaction between the analyte and the ligand attached to the test surface of the flow cell.

[0093] In a preferred embodiment, the dynamic parameters Rmax and k are estimated. a k d The steps involved in finding the value of the partial chi-square () include using the Levenberg-Marquard algorithm to find the value of the partial chi-square () Minimize the dynamic parameters.

[0094] In another preferred embodiment, the dynamic parameters Rmax and k are estimated. a k dThe steps involved in finding the value of the partial chi-square () include using an estimator to find the value of the partial chi-square () Minimize the dynamic parameters.

[0095] Establish normalized concentration curves:

[0096] Each embodiment of the present invention involves the step of establishing a concentration profile that describes the concentration of the analyte at the test surface over time. It should be noted that the concentration profile can be established using any suitable known method in the art.

[0097] In this invention, the step of establishing the concentration curve can be accomplished using a certain volume of a refractive index standard fluid containing reference molecules of known concentration, as described in the second embodiment above.

[0098] In another embodiment, the concentration curve describing the concentration of the analyte at the test surface over time can be established using mathematical models of convection and diffusion fluid dynamics transport phenomena.

[0099] In yet another embodiment, the concentration curve describing the concentration of the analyte at the test surface over time can be established using a simplified model that does not consider transport phenomena: in this embodiment, the concentration curve is established by the time period before the first time point (t1), the time period after the first interval (t2), and the concentration curve after the first interval (t3). During the period, and in the second interval ( During this period, the concentration curve is set to a "zero" value to form a concentration curve; the concentration curve is set to a constant value of c1 between the first time point (t1) and the second time point (t2), where c1 is the analyte concentration at the test surface between the first time point (t1) and the second time point (t2) when the first volume of sample fluid (V1) flows over the test surface (i.e., c1 is the analyte concentration in the first volume of sample fluid); the concentration curve is set to a constant value of c2 between the fifth time point (t5) and the sixth time point (t6), where c2 is the average concentration of the analyte at the test surface between the fifth time point (t5) and the sixth time point (t6) when the second volume of sample fluid (V2) flows over the test surface (i.e., c2 is the analyte concentration in the first volume of sample fluid).

[0100] Regardless of the steps taken to establish the concentration profile, in each embodiment of the invention, after the concentration profile has been established, it is then normalized to provide a normalized concentration profile (c(t)); and this normalized concentration profile (c(t)) is then used to determine the kinetic parameters in the same manner as described in the above embodiments.

[0101] It should be understood that in some embodiments (e.g., the second embodiment), the concentration profile or normalized concentration profile can be further used to determine a predefined time interval on the bonding curve, which is used to determine kinetic parameters.

[0102] An exemplary embodiment of a sample fluid with multiple volumes:

[0103] Importantly, the examples described above (particularly the first and second embodiments) depict the flow of first and second volumes of sample fluid (V1, V2) containing the analyte through the test surface of the flow cell. However, it should be understood that any number of volumes of sample fluid can flow through the test surface of the flow cell (with corresponding volumes of buffer fluid flowing through the test surface between the volumes of sample fluid); the same principles of the invention apply when multiple volumes of sample fluid (e.g., more than two) flow through the test surface of the flow cell. Regarding the second embodiment, during the calibration step, the number of volumes of refractive index standard fluid flowing through the test surface of the flow cell corresponds to the number of volumes of sample fluid containing the analyte that have already flowed or will flow through the test surface of the flow cell.

[0104] Therefore, the method of the present invention may include the following steps: continuously flowing multiple volumes of sample fluid over a test surface, each volume of sample fluid containing the analyte, wherein there is an interval time period between each corresponding volume of sample fluid flowing over the test surface; and during each corresponding interval time period, flowing a corresponding volume of buffer fluid (which does not contain the analyte) over the test surface; measuring the binding of the analyte to a ligand on the test surface using a sensor to obtain a binding curve; and using only a portion of the binding curve within multiple predefined interval time periods to determine kinetic parameters.

[0105] Multiple predefined time intervals can be determined in the manner described in the first and second embodiments.

[0106] As in the first and second embodiments, the method may include the steps of: continuously measuring the binding of the analyte to a ligand on the test surface using a sensor between the time when a first volume of sample fluid flows over the test surface and the time when a final volume of buffer fluid flows over the test surface; extracting portions of the binding curve within the plurality of predefined time intervals; and using only the extracted portions of the binding curve to determine kinetic parameters. In another embodiment, the method includes extracting the middle portion of the binding curve within the plurality of predefined time intervals and the portion of the binding curve after the final volume of sample fluid flows over the test surface until the binding level drops to a predefined threshold level; and using only the extracted portions of the binding curve to determine kinetic parameters.

[0107] Alternatively, the method may include the steps of: continuously measuring the binding of the analyte to the ligand on the test surface using a sensor between the time when a first volume of sample fluid flows over the test surface and the time when a final volume of buffer fluid flows over the test surface; zeroing out the portion of the binding curve that is not within the plurality of predefined time intervals; and using only the non-zeroed portion of the binding curve to determine kinetic parameters. Optionally, the portion of the binding curve after the final volume of sample fluid has flowed over the test surface until the binding level drops to a predefined threshold level may not be zeroed out.

[0108] As in the first and second embodiments, preferably, the buffer fluid has components that enable the buffer to facilitate the dissociation of the analyte from the ligand, which binds to the ligand on the test surface. Most preferably, each volume of sample fluid preferably has a buffer fluid composition containing the analyte; and each volume of buffer fluid preferably has the same buffer fluid composition as the respective volumes of sample fluid, but without the analyte.

[0109] The step of determining the kinetic parameters using only a portion of the bonding curve within the plurality of predefined time intervals (and optionally, a portion of the bonding curve after the last volume of sample fluid has flowed across the test surface until the bonding level drops to a predefined threshold level) can be performed in the same manner as the first or second embodiment described above, the only difference being that in the first and second embodiments, only two volumes of sample fluid (first and second volumes of sample fluid (V1, V2)) and only two time intervals (first time interval (V1, V2)) are used. ) and the second interval time period ( In the second embodiment, the concept is extended to multiple volumes of sample fluid and multiple predefined time intervals.

[0110] Any known technique for determining kinetic parameters using a bonding curve can be used to perform the step of determining kinetic parameters using only a portion of the bonding curve within the plurality of predefined time intervals (and optionally, a portion of the bonding curve after the last volume of sample fluid has flowed over the test surface until the bonding level drops to a predefined threshold level); in this invention, the exact same technique can be used to determine kinetic parameters, the difference being that the technique is exclusively applied only to a portion of the bonding curve within the plurality of predefined time intervals (and optionally, a portion of the bonding curve after the last volume of sample fluid has flowed over the test surface until the bonding level drops to a predefined threshold level).

[0111] Most preferably, in order to determine only the portion of the bonding curve within the plurality of predefined time intervals (and optionally, the portion of the bonding curve after the last volume of sample fluid has flowed over the test surface until the bonding level drops to a predefined threshold level), the following steps are performed: establishing a normalized concentration curve (c(t)) describing the concentration of the analyte at the test surface over time (using any techniques described in this application); estimating the kinetic parameters Rmax, k a k d The value of , where Rmax is a predefined theoretical maximum binding curve value corresponding to ligand saturation, such as, for example, when all binding sites of the ligand are occupied by the analyte bound to the ligand, k a It is the correlation rate constant, and k d It is the dissociation rate constant; using the normalized concentration curve (c(t)) and kinetic parameters Rmax and k determined in the calibration step. a k d Using the estimated value, solve the differential reaction equation:

[0112]

[0113] In order to obtain a simulated bonding curve.

[0114] Once the differential reaction equation is obtained, the simulated bonding curves are extracted over the multiple predefined time intervals ( The part in ) to provide the corresponding partial simulated joint curve (sbcj).

[0115] Then, the partial chi-square of the simulated bonding curve is established according to the following equation ( ):

[0116]

[0117] Where dmbj is the junction curve in the j-th interval ( The part in ), where sbcj is the j-th partial simulated bonding curve, and N dmbj It is the joining curve in the j-th predefined interval time period ( The number of data points in the part of ).

[0118] In the already determined simulated joint curve, the partial chi-square ( After that, then minimize the determined partial chi-square ( ), where minimizing the determined partial chi-square ( The dynamic parameters Rmax and k of the above are... a k d The value defines the kinetic parameter of the reaction between the analyte and the ligands attached to the test surface of the flow cell.

[0119] In a preferred embodiment, the dynamic parameters Rmax and k are estimated. a k d The steps involved in finding the value of the partial chi-square () include using the Levenberg-Marquard algorithm to find the value of the partial chi-square () Minimize the dynamic parameters.

[0120] In another preferred embodiment, the dynamic parameters Rmax and k are estimated. a k d The steps involved in finding the value of the partial chi-square () include using an estimator to find the value of the partial chi-square () Minimize the dynamic parameters.

[0121] Figure 1-3 Description:

[0122] Figure 1-3 This can be used to demonstrate the principles outlined in the second embodiment above when multiple quantities of sample fluid flow through the test surface of a flow cell.

[0123] Figure 1An example of a complete binding profile is shown, obtained when six volumes of sample fluid (V1-V6) flow continuously through the test surface of a flow cell. The complete binding profile shows the amount of analyte binding to the ligand on the test surface as follows: when a first volume of sample fluid (V1) containing the analyte flows through the test surface (T1-T2); after the first volume of sample fluid containing the analyte flows through the test surface and before the second volume of sample fluid flows through the test surface (T2-T3); during the time period T2-T3, a first volume of buffer fluid (V'1) (which does not contain the analyte) flows through the test surface; when a second volume of sample fluid (V2) containing the analyte flows through the test surface (T3-T4); when the second volume of sample fluid containing the analyte... After the sample fluid containing the analyte flows across the test surface and before the third volume of sample fluid flows across the test surface (T4-T5), during the time period T4-T5, the second volume of buffer fluid (V'2) (which does not contain the analyte) flows across the test surface; when the third volume of sample fluid containing the analyte (V3) flows across the test surface (T5-T6); after the third volume of sample fluid containing the analyte flows across the test surface and before the fourth volume of sample fluid flows across the test surface (T6-T7), during the time period T6-T7, the third volume of buffer fluid (V'3) (which does not contain the analyte) flows across the test surface; when the third volume of sample fluid containing the analyte ...2) flows across the test surface; when the third volume of sample fluid containing the analyte flows across the test surface (T5-T6); after the third volume of sample fluid containing the analyte flows across the test surface and before the fourth volume of sample fluid flows across the test surface (T6-T7), the second volume of buffer fluid (V'2) (which does not contain the analyte) flows across the test surface; when the third volume of sample fluid containing the analyte flows across the test surface (T5-T6); after the third volume of sample fluid containing the analyte flows across the test surface and before the fourth volume of sample fluid When the fourth volume of sample fluid containing the analyte (V4) flows across the test surface (T7-T8); after the fourth volume of sample fluid containing the analyte flows across the test surface and before the fifth volume of sample fluid flows across the test surface (T8-T9), during the time period T8-T9, the fourth volume of buffer fluid (V'4) (which does not contain the analyte) flows across the test surface; when the fifth volume of sample fluid containing the analyte (V5) flows across the test surface (T9-T10); after the fifth volume of sample fluid containing the analyte flows across the test surface and before the sixth volume of sample fluid flows across the test surface (T1... During the time period T10-T11, a fifth volume of buffer fluid (V'5) (which does not contain analyte) flows over the test surface; while a sixth volume of sample fluid (V6) containing analyte flows over the test surface (T11-T12); after the sixth volume of sample fluid containing analyte flows over the test surface, the sixth volume of buffer fluid (V'6) (which does not contain analyte) flows over the test surface until the amount of analyte bound to the ligand decreases to a predefined threshold level, or until a predefined time period has elapsed (T12-T13), preferably during the time period T12-T13.

[0124] Preferably, each volume of buffer fluid (V'1-V'6) may preferably have components that allow the buffer fluid to facilitate the dissociation of the analyte from the ligand bound to the test surface. Most preferably, each volume of sample fluid (V'1-V'6) consists of buffer fluid mixed with the analyte; each volume of buffer fluid (V'1-V'6) consists only of buffer fluid (i.e., without the analyte), and most preferably, the buffer fluid in each volume of buffer fluid (V'1-V'6) is the same as the buffer fluid in that volume of sample fluid (V'1-V'6).

[0125] Each of the six sample fluid volumes (V1-V6) contains a known concentration of analyte. The analyte concentration in each volume can be determined using any method known in the art. Therefore, the analyte concentration in each of the six sample fluid volumes (V1-V6) is known. The sample fluid volume (V1-V6) with the highest analyte concentration is the volume with the highest concentration (c). max In this example, each of the six volumes of sample fluid (V1-V6) happens to have the same analyte concentration (therefore each volume of sample fluid has a concentration equal to the maximum concentration (c)). max (The analyte concentration); however, in another embodiment, the six volumes of sample fluid (V1-V6) can have different analyte concentrations, in which case the maximum concentration (c) is... max ) is the analyte concentration in the sample fluid (V1-V6) with the highest concentration.

[0126] Figure 2 The extracted Figure 1 The joining curve has multiple predefined time intervals. It should be understood that... Figure 2 It can be alternatively considered to show Figure 1 The bonding curve is defined by multiple predefined time intervals, where other parts of the bonding curve have been "zeroed out". Regardless of the method, in this invention, Figure 2 The illustration shows a portion of the engagement curve, which is used exclusively in this invention to determine kinetic parameters (e.g., in step (e) of the first embodiment).

[0127] It should be understood that the predefined interval time period is preferably determined from the concentration curve or from the normalized concentration curve. Figure 3 Normalized concentration curves are shown for determining predefined interval time periods; these predefined interval time periods need to be determined to know the extraction... Figure 1 Which parts of the joining curve are used to form Figure 2 The curve shown.

[0128] In this configuration, six volumes of sample fluid (V1-V6) flow continuously through the test surface of the flow cell to establish the bonding profile. Figure 1 Therefore, six volumes of refractive index standard fluid (containing reference molecules of known concentration) flow through the test surface to obtain... Figure 3 The normalized concentration curve in the figure.

[0129] Figure 3The normalized concentration curves were obtained as follows: After the first volume of refractive index standard fluid (V”1) flows over the test surface; after the first volume of refractive index standard fluid flows over the test surface and before the second volume of refractive index standard fluid flows over the test surface, during this time period, the first volume of buffer fluid (V’1) (which does not contain reference molecules) flows over the test surface; when the second volume of refractive index standard fluid (V”2) flows over the test surface; after the second volume of refractive index standard fluid flows over the test surface and before the third volume of refractive index standard fluid flows over the test surface, during this time period, the second volume of buffer fluid (V’2) (which does not contain reference molecules) flows over the test surface; when the third volume of refractive index standard fluid (V”3) flows over the test surface; after the third volume of refractive index standard fluid flows over the test surface and before the fourth volume of refractive index standard fluid flows over the test surface, during this time period, the third volume of buffer fluid (V’3) (which does not contain reference molecules) flows over the test surface; when the fourth volume of refractive index standard fluid flows over the test surface... When a volume of refractive index standard fluid (V”4) flows over the test surface; after the fourth volume of refractive index standard fluid flows over the test surface and before the fifth volume of refractive index standard fluid flows over the test surface, during this time period, a fourth volume of buffer fluid (V’4) (which does not contain reference molecules) flows over the test surface; when the fifth volume of refractive index standard fluid (V”5) flows over the test surface; after the fifth volume of refractive index standard fluid flows over the test surface and before the sixth volume of refractive index standard fluid flows over the test surface, during this time period, a fifth volume of buffer fluid (V’5) (which does not contain reference molecules) flows over the test surface; when the sixth volume of refractive index standard fluid (V”6) flows over the test surface; after the sixth volume of refractive index standard fluid flows over the test surface until the concentration of reference molecules at the test surface decreases to a predefined threshold level or until a predefined time period has elapsed, preferably, during this time period, a sixth volume of buffer fluid (V’6) (which does not contain reference molecules) flows over the test surface. While performing each of the above steps, the concentration of reference molecules at the test surface is measured (preferably before the first volume of sample fluid (V1) flows over the test surface, until the last step is completed, at which point the sixth volume of buffer fluid (V'6) flows over the test surface, until the concentration of reference molecules at the test surface decreases to a predefined threshold level, or until a predefined time period has elapsed, generating a concentration curve (cmc).

[0130] Importantly, the flow rate (i.e., the rate at which the corresponding volume of refractive index standard fluid flows across the test surface is equal to the flow rate of the corresponding six volumes of sample fluid (V1-V6) across the test surface. The concentration ratio of each volume of refractive index standard fluid (V1”-V”6) is also equal to the concentration ratio of each volume of sample fluid (V1-V6).

[0131] Then, by dividing the concentration curve (cmc) by the maximum value of the concentration curve (max(cmc)) and multiplying the result by the known maximum concentration (c) of the analyte in the corresponding six volumes of sample fluid (V1-V6), the concentration of the analyte was determined. max The concentration curve (cmc) is normalized using the following equation:

[0132]

[0133] To provide in Figure 3 The normalized concentration curve (c(t)) is shown in the figure.

[0134] In order to Figure 3 The normalized concentration curve (c(t)) shown defines each of several predefined time intervals. A threshold concentration is selected; in this example, the threshold concentration is 5% of the maximum value of the normalized concentration curve (c(t)); since the maximum value of the normalized concentration curve (c(t)) is "200", the threshold concentration in this example is "10" (i.e., 5% * 200 = 10). Several predefined time intervals are defined by the time interval in which the normalized concentration curve (c(t)) drops below "10" until it rises again to be equal to or higher than "10". Reference Figure 3 The normalized concentration curve (c(t)) shown is as follows: at time T'2, the normalized concentration curve (c(t)) drops below the level of "10", and at time T'3, the normalized concentration curve (c(t)) rises to "10", thus T'2-T'3 defines a predefined interval time period; at time T'4, the normalized concentration curve (c(t)) drops below the level of "10", and at time T'5, the normalized concentration curve (c(t)) rises to "10", thus T'4-T'5 defines another predefined interval time period. Interval time periods; at time T'6, the normalized concentration curve (c(t)) drops below the level of "10", and at time T'7, the normalized concentration curve (c(t)) rises to "10", therefore T'6-T'7 defines another predefined interval time period; at time T'8, the normalized concentration curve (c(t)) drops below the level of "10", and at time T'9, the normalized concentration curve (c(t)) rises to "10", therefore T'8-T'9 defines another predefined interval time period; at time T' 10 The normalized concentration curve (c(t)) drops below 10 at time T' 11 The normalized concentration curve (c(t)) rises to "10", therefore T' 10 -T' 11 Another predefined time interval is defined; at time T' 12The normalized concentration curve (c(t)) drops below 10 at time T' 13 The predefined time period has passed, therefore T' 12 -T' 13 The last predefined interval time period was defined.

[0135] In the corresponding predefined interval time periods T'2-T'3, T'4-T'5, T'6-T'7, T'8-T'9, T' 10 -T' 11 T' 12 -T' 13 The time that happened Figure 1 The portion of the joining curve shown is then extracted to provide Figure 2 The engagement curves shown are used to determine the kinetic parameters Rmax and k in the manner described above in each embodiment of the invention. a k d .

[0136] Various modifications and variations to the embodiments described herein will be apparent to those skilled in the art without departing from the scope of the invention as defined by the appended claims. Although the invention has been described in conjunction with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments.

Claims

1. A method for determining kinetic parameters of the reaction between an analyte and a ligand attached to a test surface of a flow cell, the method comprising the steps of: (a) Between a first time point (t1) and a second time point (t2), a sample fluid (V1) containing a first volume of the analyte is allowed to flow over the test surface; (b) Between the third time point (t3) and the fourth time point (t4), a first volume of buffer fluid (Vb1) without analyte is allowed to flow over the test surface; (c) Between the fifth time point (t5) and the sixth time point (t6), a sample fluid (V2) containing at least a second volume of the analyte is passed over the test surface; (d) Between the seventh time point (t7) and the eighth time point (t8), at least a second volume of buffer fluid (Vb2) without analyte is passed over the test surface; (e) Use sensors to measure the binding of the analyte to the ligands on the test surface to obtain binding profiles; (f) Determining kinetic parameters using only a portion of the engagement curve within a predefined time interval, without using other portions of the engagement curve; wherein the predefined time interval includes first and second time intervals. , ); where the first interval time period ( ) occurs between the second time point (t2) and the fifth time point (t5); and the second interval time period ( The event occurs between the sixth time point (t6) and the ninth time point (t9), where the ninth time point (t9) is after the eighth time point (t8), or the ninth time point (t9) is equal to the eighth time point (t8), where each predefined interval corresponds to the dissociation phase of the bonding curve.

2. The method according to claim 1, wherein, First interval time period ( ) is a portion of the duration between the second time point (t2) and the fifth time point (t5); and the second interval period ( ) is a portion of the duration between the sixth time point (t6) and the ninth time point (t9); or The first interval time period ( The second time interval is defined by the entire time interval between the second time point (t2) and the fifth time point (t5); and the second interval time period ( The time interval between the sixth time point (t6) and the ninth time point (t9) is defined.

3. The method of claim 1, further comprising a calibration step to determine a predefined interval time period; The calibration steps include establishing a concentration curve that describes the concentration of the analyte at the test surface over time; and using the concentration curve to determine a predefined time interval.

4. The method according to claim 3, wherein, The steps for using the concentration curve to determine the predefined time interval include: Select the threshold concentration; Mark the moment when the concentration curve equals the threshold concentration; Each corresponding predefined interval is defined by the time interval between the moment when the concentration curve is at a threshold concentration and the next moment when the concentration curve is at the threshold concentration, wherein the concentration curve shows a decrease in concentration before the moment and an increase in concentration before the next moment.

5. The method according to claim 4, wherein, The steps for selecting the threshold concentration include, The maximum value of the concentration curve is indicated; Select a percentage value, where the threshold concentration is defined by multiplying the maximum value of the concentration curve by the percentage value.

6. The method of claim 1, further comprising a calibration step to determine a predefined interval time period; The calibration steps include: establishing a concentration curve describing the concentration of the analyte at the test surface over time; normalizing the concentration curve to provide a normalized concentration curve (c(t)); and then using the normalized concentration curve (c(t)) to determine a predefined time interval.

7. The method according to claim 6, wherein, The steps for determining a predefined time interval using the normalized concentration curve include: Select the threshold concentration; This indicates the moment when the normalized concentration curve equals the threshold concentration. Each corresponding predefined interval is defined as the time interval between the moment when the normalized concentration curve is at the threshold concentration and the next moment when the normalized concentration curve is at the threshold concentration, wherein the normalized concentration curve shows a decrease in concentration before the moment and an increase in concentration before the next moment.

8. The method according to claim 7, wherein, The steps for selecting the threshold concentration include, Identify the maximum value of the normalized concentration curve; Choose a percentage value, where the threshold concentration is defined by multiplying the maximum value of the normalized concentration curve by the percentage value.

9. The method according to claim 1, comprising: The binding of the analyte to the ligand on the test surface is continuously measured using a sensor from a first time point (t1) or before the first time point (t1) up to an eighth time point (t8) or after the eighth time point (t8); a portion of the binding curve is extracted within the predefined time interval to provide the extracted portion of the binding curve; The kinetic parameters are determined using only the extracted portion of the bonding curve.

10. The method of claim 1, comprising: The binding of the analyte to the ligand on the test surface is continuously measured using a sensor from the first time point (t1) or before the first time point (t1) up to the eighth time point (t8) or after the eighth time point (t8); the portion of the binding curve outside the predefined time interval is zeroed out; The dynamic parameters are determined using only the portion of the engagement curve that has not returned to zero.

11. The method according to claim 1, comprising the following steps: Multiple volumes of sample fluid are continuously flowed over a test surface, each volume of sample fluid containing the analyte, wherein there is an interval time between each corresponding volume of sample fluid flowing over the test surface; During each corresponding time interval, a corresponding volume of buffer fluid without analyte is allowed to flow over the test surface; Sensors are used to measure the binding of the analyte to the ligands on the test surface to obtain binding curves; The dynamic parameters are determined using only a portion of the engagement curve within a number of predefined time intervals.

12. The method according to claim 1, wherein, The steps for determining kinetic parameters using only a portion of the engagement curve within a predefined time interval, without using other portions of the engagement curve, include the following steps: A normalized concentration curve (c(t)) is established, which describes the concentration of the analyte at the test surface over time; Estimate the dynamic parameters Rmax, k a k d The value of , where Rmax is the predefined maximum response value corresponding to ligand saturation on the test surface, k a It is the correlation rate constant, and k d It is the dissociation rate constant; Using the normalized concentration curve (c(t)) and kinetic parameters Rmax, k a k d Given the estimated value, solve the differential reaction equation: In order to obtain a simulated joint curve; Extracting simulated bonding curves over multiple predefined time intervals ( The part in ) to provide the corresponding partial simulated joint curve (sbcj); The partial chi-square of the simulated bonding curve is determined according to the following equation ( ): Where dmbj is the junction curve in the j-th predefined interval time period ( The part in ), where sbcj is the j-th partial simulated bonding curve, and N dmbj It is the joining curve in the j-th predefined interval time period ( The number of data points in the portion of ); Minimize the determined partial chi-square ( ), where minimizing the determined partial chi-square ( The dynamic parameters Rmax and k of the above are... a k d The value defines the kinetic parameter of the reaction between the analyte and the ligand attached to the test surface of the flow cell.

13. The method according to claim 12, wherein, The steps to establish a normalized concentration curve (c(t)) include using the Levenberg-Marquard algorithm to find the partial chi-square value. The kinetic parameters are minimized, and the normalized concentration curve (c(t)) describes the concentration of the analyte at the test surface over time.

14. The method according to claim 12, wherein, Estimate the dynamic parameters Rmax, k a k d The steps involved in finding the value of the partial chi-square () include using an estimator to find the value of the partial chi-square () Minimize the dynamic parameters.

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