Apparatus and method for measuring enzyme activity using substrate-immobilized carrier with non-constant substrate immobilization density

Abstract

To provide a simpler method for measuring enzyme activity.SOLUTION: A method for estimating enzyme activity using a substrate having spots formed thereon where substrates are immobilized comprises: (a) allowing an enzyme to act on the substrate in the spots to obtain an enzyme product; (b) defining one or more compartments in the spots and obtaining the density of the substrate in each compartment; (c) measuring the density of the enzyme product in each compartment; and (d) estimating the enzyme activity of the enzyme based on the relationship between the density of the substrate in each compartment obtained in step (b) and the density of the enzyme product in each compartment obtained in step (c).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The embodiments disclosed in the present specification and drawings relate to an apparatus and method for measuring enzyme activity using a substrate-immobilized support in which the density of immobilized substrate is not constant. [Background technology]

[0002] The catalytic action of an enzyme on a substrate, i.e., the magnitude of enzyme activity, is evaluated by the rate v at which a reactant (enzyme product) is produced from the substrate in the presence of the enzyme. The production rate v varies depending on the substrate concentration, and reaches a maximum when the substrate concentration is sufficiently high. Therefore, the enzymatic activity of an enzyme on a substrate is generally evaluated based on two indicators, the maximum production rate Vmax and the affinity constant (Michaelis constant: Km), based on the Michaelis-Menten equation.

[0003] A common method for determining indicators of enzyme activity (e.g., Km, Vmax) of a specific enzyme against a substrate is to add a fixed concentration of the enzyme to multiple substrate solutions containing different substrate concentrations, calculate the rate of enzyme product formation (enzyme product concentration / reaction time), and fit this to the Michaelis-Menten equation to determine Km and the maximum rate of enzyme product formation, v, which is then equal to Vmax to determine enzyme activity. This method is effective when the product has luminescence, fluorescence, or absorbance properties and can be measured directly. However, when direct measurement of the product is difficult and labeling procedures are required, there is a problem that such procedures make B / F separation difficult.

[0004] One method for measuring enzyme activity involves immobilizing a substrate on a carrier beforehand and then reacting it with the enzyme. This method has the advantage that, when a labeling procedure is required to detect the enzyme product from an enzymatic reaction, it is easy to perform B / F separation of the labeled substance. Immobilization of the substrate is generally performed by contacting the substrate solution with the solid phase. One example is a method in which a spot of immobilized substrate is created on a carrier and then reacted with the enzyme.

[0005] Since the enzyme reaction occurs at the solid-liquid interface in the immobilized substrate, (i) the reaction between the immobilized substrate and the enzyme requires that the immobilized area for each spot be the same to maintain a constant enzyme supply rate for each spot, and (ii) that spots be formed with different amounts of immobilized substrate.

[0006] To satisfy (i), conventional methods have been employed to make the immobilization area of ​​each spot the same, but in the method of immobilizing a substrate by contacting the substrate with the substrate, the viscosity and surface tension of the liquid vary depending on the amount of substrate contained in the substrate, making it difficult to make the immobilization area of ​​each spot the same, especially on a continuous support such as a glass slide. To make the immobilization area of ​​each spot the same, the substrate is usually patterned (e.g., by forming a hydrophilic / hydrophobic surface, a pattern with or without a linker substance, etc.) before immobilization, but this requires a lot of time and effort.

[0007] To satisfy (ii), a method has traditionally been used in which a known amount of substrate solution with a known concentration is brought into contact with a support. However, because the amount of substrate immobilized on the support is affected by variations in multiple factors (e.g., concentration of the substrate solution, amount of spotting solution, density of the linker substance on the support, binding efficiency of the substrate to the support, etc.), the amount of immobilized substrate is not necessarily the same as the value expected at the time of spotting, and spots with the expected amount of substrate may not be obtained. Furthermore, because it is necessary to prepare multiple immobilized spots with different amounts of substrate on a solid support with a finite area, the number of spots that can be made is limited, and there are also problems such as the need to prepare substrate solutions of different concentrations to create spots. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Japanese Patent Application Laid-Open No. 2000-325086 Summary of the Invention [Problem to be solved by the invention]

[0009] One of the problems to be solved by the embodiments disclosed in this specification and the drawings is to enable simple measurement of enzyme activity. However, the problems to be solved by the embodiments disclosed in this specification and the drawings are not limited to the above problem. Problems corresponding to the effects of each configuration shown in the embodiments described below can also be positioned as other problems. [Means for solving the problem]

[0010] An embodiment of the method for measuring enzyme activity is a method for estimating enzyme activity using a substrate having spots formed thereon where a substrate is immobilized. This method includes the steps of: (a) allowing an enzyme to act on the substrate in the spot to obtain an enzyme product; (b) defining one or more compartments in the spot and obtaining the density of the substrate in each compartment; (c) measuring the density of the enzyme product in each compartment; and (d) estimating the enzyme activity of the enzyme based on the relationship between the density of the substrate in each compartment obtained in step (b) and the density of the enzyme product in each compartment obtained in step (c).

[0011] An enzyme activity measuring device according to an embodiment is an information processing device that estimates enzyme activity using a substrate having spots formed thereon where substrates are immobilized. The device comprises: means for causing an enzyme to act on the substrate in the spots to obtain an enzyme product; means for setting one or more compartments in the spots and acquiring the density of the substrate in each compartment; means for measuring the density of the enzyme product in each compartment; and means for estimating the enzyme activity of the enzyme based on the relationship between the density of the substrate in each compartment and the density of the enzyme product in each compartment. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 shows an example of a fluorescent image of spots on a protein array plate where substrates are immobilized at non-uniform immobilization densities. [Figure 2]FIG. 2 is a diagram showing an example of the flow of the enzyme activity measurement method according to the first embodiment. [Figure 3] Figure 3 shows a schematic diagram of the procedure for phosphorylating and labeling multiple compartments (spatially independent groups of spots) containing different amounts of solid-phase substrate (Src) for 2 hours, as well as fluorescent images of the compartments after the procedure. [Figure 4] Figure 4 shows a graph plotting the fluorescence images of multiple compartments (spatially independent spots) containing different amounts of immobilized substrate (Src) after 2 hours of phosphorylation and labeling, and the density values ​​of the enzyme product (I785nm) in each compartment as a function of the density values ​​of the substrate (I670nm) in each compartment. [Figure 5] Figure 5 shows fluorescent images of multiple compartments (virtual compartments set up so that each compartment is one pixel (10 × 10 μm) within one spot) containing different amounts of solid-phase substrate (Src) after 2 hours of phosphorylation and labeling, and plots the density value of the enzyme product (I785 nm) in one compartment as a function of the density value of the substrate (I670 nm) in that compartment. [Figure 6] Figure 6 shows a graph plotting the density value (I785nm) of the enzyme product in each compartment (virtual compartments set up so that each compartment is one pixel (10 × 10 μm) within one spot) containing different amounts of solid-phase substrate (Src) after 2 hours of phosphorylation and labeling as a function of the density value (I670nm) of the substrate in each compartment. [Figure 7] Figure 7 shows the results of applying smoothing (mean filter) to the fluorescent image of a spot where each section was set to one pixel (10 × 10 μm). [Figure 8]Figure 8 shows a graph plotting the density value of the enzyme product (I785nm) in each compartment as a function of the density value of the substrate (I670nm) in each compartment after applying smoothing (Mean filter) to the fluorescent image of the compartments after 2 hours of phosphorylation reaction and labeling for multiple compartments (virtual compartments set to one pixel (10 x 10 μm) within one spot) containing different amounts of solid-phase substrate (Src). [Figure 9] Figure 9 shows a graph plotting the density value (I785nm) of the enzyme product in each compartment (virtual compartments set up so that each compartment is 1 pixel (10 x 10 μm) within one spot) containing different amounts of solid-phase substrate (HCK) after 2 hours of phosphorylation and labeling as a function of the density value (I670nm) of the substrate in each compartment. [Figure 10] Figure 10 shows a graph plotting the density value (I785nm) of the enzyme product in each compartment after 2 hours of phosphorylation and labeling as a function of the density value (I670nm) of the substrate in each compartment (virtual compartments set up so that each compartment is 1 pixel (10 x 10 μm) within one spot) containing different amounts of solid-phase substrate (ABL1). DETAILED DESCRIPTION OF THE INVENTION

[0013] Hereinafter, embodiments of an enzyme activity measuring device and an enzyme activity measuring method will be described in detail with reference to the drawings.

[0014] (First embodiment) The enzyme activity measurement method according to the first embodiment is a method for estimating enzyme activity using a substrate having spots formed thereon where a substrate is immobilized. This method includes the steps of: (a) allowing an enzyme to act on the substrate in the spots to obtain an enzyme product; (b) defining one or more compartments in the spots and obtaining the density of the substrate in each compartment; (c) measuring the density of the enzyme product in each compartment; and (d) estimating the enzyme activity of the enzyme based on the relationship between the density of the substrate in each compartment obtained in step (b) and the density of the enzyme product in each compartment obtained in step (c).

[0015] The enzyme activity measurement method according to the first embodiment is a method for estimating enzyme activity using a substrate on which spots of immobilized substrates are formed.

[0016] The term "substrate" in the enzyme activity measurement method according to the first embodiment refers to a carrier for immobilizing a substrate, and includes, but is not limited to, a two-dimensional plate, a polymer gel, a fiber or fiber sheet, beads, a rod, and the like. The surface of the "substrate" may be smooth, or may have a micro / nano structure such as a porous structure or a fiber. The "substrate" is preferably a two-dimensional plate, and examples of two-dimensional plates include plate-like substrates such as glass slides and cover glasses, and well substrates such as array plates. The "substrate" is more preferably a well substrate such as an array plate. An array plate has spots containing a substrate and is used for comprehensive analysis of samples; it is also called a microchip, microarray, protein chip, DNA chip, and the like.

[0017] The "substrate" in the enzyme activity measurement method according to the first embodiment is not particularly limited as long as it can be physically or chemically immobilized on a substrate and there is a means for measuring the amount of the "substrate," and specific examples include proteins, peptides, nucleic acids, sugar chains, and glycoproteins. The "substrate" may be immobilized on the substrate by being directly bound thereto, by being bound via a linker substance, or by being incorporated into a gel substance.

[0018] In the enzyme activity measurement method according to the first embodiment, a "spot" refers to a certain enclosed area on a substrate where a substrate is immobilized. The shape of the "spot" is not particularly limited and may be, for example, a square, rectangle, circle, ellipse, etc., but is preferably a circle. A "spot" may be continuous or discontinuous in a two-dimensional plane. Furthermore, in a "spot," the substrate may be immobilized two-dimensionally or three-dimensionally depending on the substrate. Preferably, one type of substrate is immobilized in a "spot." However, if two or more types of substrates and their respective enzyme products can be distinguishably measured, two or more types of substrates may be immobilized in the same area. According to a preferred aspect of the first embodiment, a "spot" is a circular area on a two-dimensional plate where one type of substrate is immobilized.

[0019] The method for measuring enzyme activity according to the first embodiment comprises the step of (a) allowing an enzyme to act on a substrate on a spot to obtain an enzyme product.

[0020] The "enzyme product" in the enzyme activity measurement method according to the first embodiment refers to a substance obtained as a result of the reaction of an enzyme with a substrate. Such enzyme products include those produced by an addition reaction of a substrate, those produced by a substitution reaction of a substrate, or those in which a portion of the substrate has been cleaved by a cleavage reaction (the cleavage site has disappeared), or combinations thereof. The "enzyme" in the enzyme activity measurement method according to the first embodiment is not particularly limited and includes all substances that catalyze addition reactions, substitution reactions, cleavage reactions, or combinations thereof. Examples of the "enzyme" in the enzyme activity measurement method according to the first embodiment include proteases, kinases, oxidases, nucleases, etc., and preferably kinases.

[0021] The method for measuring enzyme activity according to the first embodiment comprises the step of (b) setting one or more compartments on the spot and acquiring the density of the substrate in each compartment.

[0022] In the "spot" of the enzyme activity measuring method according to the first embodiment, the substrate may be uniformly or non-uniformly immobilized. Here, "uniformly immobilized" of the substrate means that the substrate is substantially uniform, and does not mean that regions with non-uniform densities are not permitted. Furthermore, in the "spot" of the enzyme activity measuring method according to the first embodiment, the substrate may be uniformly immobilized in some regions, and non-uniformly immobilized in some regions of the same spot.

[0023] In the enzyme activity measurement method according to the first embodiment, a single closed region in which the density of immobilized substrates is uniform is defined as a "compartment." Here, "uniform density of immobilized substrates" means that the density is substantially uniform, and does not mean that it is not permitted to include regions in which the density is not the same. A "compartment" may have the same or different density of immobilized substrates as other "compartments." Furthermore, a "compartment" may be continuous or discontinuous with other "compartments" in a two-dimensional plane. The shape of a "compartment" is not particularly limited, and may be, for example, a square, rectangle, circle, ellipse, etc.

[0024] In the method for measuring enzyme activity according to the first embodiment, the uniform density of the immobilized substrate may be the average density in one compartment.

[0025] According to a first preferred embodiment, the compartment is selected from spatially independent spots. In this case, the spatially independent spots are used as a unit of the compartment, and this corresponds to, for example, spatially independent spots having a uniform density of immobilized substrates.

[0026] According to another preferred first embodiment, the section is smaller than a single spot and is selected from a single closed area in which the density of the immobilized substrate is uniform, and multiple sections can be set within a single spot. Here, "uniform density of the immobilized substrate" means that it is substantially uniform, and does not mean that it is not permitted to include areas with different densities. Such a single closed area is referred to as a "virtual section," and a "virtual section" is considered to be one unit of the section. The size and shape of the "virtual section" may be determined arbitrarily. Similar to a "section," a "virtual section" may have the same or different density of the immobilized substrate from other "virtual sections." Furthermore, a "virtual section" may be continuous or discontinuous with other "virtual sections" in a two-dimensional plane. The shape of the "virtual section" is not particularly limited, and may be, for example, a square, rectangle, circle, ellipse, etc. A "virtual section" may be, for example, a section of several hundred μm in size, including areas with different densities of the immobilized substrate. 2 In a spot of this size, a section set in the form of a square of about 1 pixel (10 μm×10 μm) can be exemplified.

[0027] In the method for measuring enzyme activity according to the first embodiment, the density of the uniform immobilized substrate may be the average density in one virtual compartment.

[0028] In the enzyme activity measurement method according to the first embodiment, the method for producing spots with non-uniform densities of immobilized substrate is not particularly limited, and they can be produced according to known methods, such as tilting the substrate, using the coffee ring phenomenon, changing the temperature (convection control), changing the immobilization density of the substrate linker substance, pinning the liquid using a contact method, spotting in multiple stages, or spotting substrate solutions of different concentrations at different points.

[0029] In the enzyme activity measurement method according to the first embodiment, the density of the substrate in a compartment may be determined by measuring the amount of substrate in the compartment and dividing it by the area of ​​the compartment, or by obtaining this value if it is already known. The amount of substrate in a compartment can be measured by known methods, such as quantification using surface plasmon resonance (SPR), quantification based on electrochemical properties (e.g., potential, current, impedance, capacitance, etc.), quantification based on element distribution obtained by X-ray spectroscopy, quantification using AFM, and quantification based on signal information derived from a labeling substance introduced into the substrate. The labeling substance is not particularly limited, but is preferably optically detectable. Examples of such labeling substances include fluorescent substances, light-absorbing substances, light-emitting substances, scattering substances, polarizing substances, and redox substances.

[0030] The method for measuring enzyme activity according to the first embodiment comprises the step (c) of measuring the density of the enzyme product in each compartment.

[0031] In the enzyme activity measurement method according to the first embodiment, the density of the enzyme product in a compartment can be determined by measuring the amount of enzyme product in the compartment and dividing it by the area of ​​the compartment. The amount of enzyme product in a compartment can be measured by known methods, including, for example, quantification using surface plasmon resonance (SPR), quantification based on electrochemical properties (e.g., potential, current, impedance, capacitance, etc.), quantification based on element distribution obtained by X-ray spectroscopy, quantification using AFM, and quantification based on signal information derived from a labeling substance introduced into the enzyme product. The labeling substance is not particularly limited, but is preferably optically detectable. Examples of such labeling substances include fluorescent substances, light-absorbing substances, light-emitting substances, scattering substances, polarizing substances, and redox substances.

[0032] In the method for measuring enzyme activity according to the first embodiment, the density of the enzyme product in each compartment may be the average density in one compartment.

[0033] The enzyme activity measurement method according to the first embodiment comprises (d) a step of estimating the enzyme activity of the enzyme based on the relationship between the density of the substrate in each compartment obtained by step (b) and the density of the enzyme product in each compartment obtained by step (c).

[0034] In the enzyme activity measurement method according to the first embodiment, the enzyme activity is estimated by first calculating the maximum production rate (Vmax) of the enzyme product based on the relationship between the density of the substrate and the density of the enzyme product in each compartment, and then estimating the enzyme activity based on the obtained maximum production rate of the enzyme product. This estimation of the enzyme activity may be performed by further taking into account the value of the affinity constant (Michaelis constant: Km), and may also take into account other parameters required for estimation, if any.

[0035] As described above, in the enzyme activity measurement method according to the first embodiment, the enzyme activity is estimated by first calculating the value of the maximum production rate of the enzyme product based on the relationship between the density of the substrate and the density of the enzyme product in each compartment, and then estimating the enzyme activity based on the obtained value of the maximum production rate of the enzyme product. Therefore, the enzyme activity measurement method according to the first embodiment may also be used as a method for measuring the maximum production rate of the enzyme product.

[0036] This method for measuring the maximum production rate of an enzyme uses a substrate having spots formed thereon on which substrates are immobilized, and comprises the steps of: (a) allowing an enzyme to act on the substrate in the spots to obtain an enzyme product; (b) defining one or more compartments in the spots and obtaining the density of the substrate in each compartment; (c) measuring the density of the enzyme product in each compartment; and (d) calculating the maximum production rate of the enzyme based on the relationship between the density of the substrate in each compartment obtained in step (b) and the density of the enzyme product in each compartment obtained in step (c).

[0037] The value of the maximum production rate can be calculated, for example, based on the results of plotting the density value of the enzyme product in each compartment or the value obtained by dividing said value by the reaction time on a graph as a function of the density value of the substrate in each compartment. In this case, the relationship between these values ​​may be fitted using the Michaelis-Menten equation. Therefore, estimation of enzyme activity and calculation of the value of the maximum production rate preferably include a step of fitting using the Michaelis-Menten equation. When fitting using the Michaelis-Menten equation is performed, the affinity constant of the substrate for the enzyme may also be calculated. Note that instead of fitting using the Michaelis-Menten equation, an equation obtained by taking the reciprocal of the Michaelis-Menten equation (Lineweaver-Burk equation) may also be used.

[0038] When the decrease in reaction rate due to a decrease in the amount of unreacted substrate during the enzymatic reaction is not taken into consideration, fitting using the Michaelis-Menten equation can be performed, for example, by performing an enzymatic reaction for a certain reaction time, determining the density of the substrate and the density of the enzyme product in each compartment, and then dividing the density of the enzyme product by the reaction time to convert it into the production rate. This can then be fitted using the Michaelis-Menten equation to calculate the maximum production rate. In this case, the density of the substrate at which the maximum production rate is 1 / 2 can be calculated as the affinity constant.

[0039] On the other hand, when taking into consideration the decrease in reaction rate due to the decrease in the amount of unreacted substrate during the enzyme reaction, this can be done, for example, by (i) assuming a certain affinity constant and maximum production rate, and calculating the production rate of the enzyme product at the start of the reaction (t = 0) for the substrate density in each compartment using the Michaelis-Menten equation; (ii) calculating the amount of enzyme product produced and the decrease in the amount of unreacted substrate after a unit time has elapsed, and also calculating the production rate of the enzyme product at t = unit time for the unreacted substrate density; (iii) repeating (i) and (ii) to obtain an evaluation result of the relationship between the substrate density and the density of the enzyme product after a specified reaction time has elapsed; and (iv) converging the maximum production rate and affinity constant so as to minimize the error between the evaluation result obtained in (iii) and the actual measurement result.

[0040] In the enzyme activity measurement method according to the first embodiment, the "specimen" containing the enzyme to be measured can be determined appropriately by the person performing the method depending on the purpose, and anything containing an enzyme can be the "specimen." The "specimen" includes biologically derived substances, extracts from biological organisms, blood, blood-derived substances, food, food-derived substances, natural products, substances derived from natural products, and culture medium-derived substances. The "specimen" may be pretreated as appropriate depending on the purpose and procedure, or a reagent may be added beforehand. The "specimen" may be in the form of a gas, solid, or liquid, but may be diluted, suspended, or extracted in water, physiological saline, buffer solution, or other solution as appropriate and used in liquid form. The specimen may contain preservatives and other additives. Furthermore, a reagent may be added to the "specimen" depending on the purpose.

[0041] 2 is a diagram showing an example of the flow of the enzyme activity measurement method according to the first embodiment. The enzyme activity measurement method shown in FIG. 2 includes the steps of: setting multiple compartments on a spot and acquiring the density of the substrate in each compartment; contacting a sample with the spot to react the substrate with the enzyme and acquire the enzyme product; measuring the density of the enzyme product in each compartment; plotting the density value of the enzyme product in each compartment or the value obtained by dividing said value by the reaction time on a graph as a function of the density value of the substrate in each compartment; determining whether there is a region in which the density value of the enzyme product in each compartment or the value obtained by dividing said value by the reaction time is zero-order with respect to the density value of the substrate in each compartment; and calculating the enzyme activity of the enzyme based on the function if there is a zero-order region; and determining that calculation of the enzyme activity of the enzyme is impossible if there is no zero-order region. In this method, the enzyme activity is calculated using a substrate on which a spot of immobilized substrate is formed. Furthermore, in this method, the substrate density in each compartment may be obtained at any timing before the step of plotting the density value of the enzyme product in each compartment or the value obtained by dividing said value by the reaction time on a graph as a function of the substrate density value in each compartment, and determining whether there is a region in which the density value of the enzyme product in each compartment or the value obtained by dividing said value by the reaction time is zero-order with respect to the substrate density value in each compartment.

[0042] The enzyme activity measurement method shown in Figure 2 includes the steps of setting multiple compartments on a spot and obtaining the substrate density in each compartment. The substrate density may be obtained by measurement, or if the value is known, it may be used.

[0043] The method for measuring enzyme activity shown in FIG. 2 includes the steps of contacting a spot with a sample to allow the substrate to react with the enzyme, and obtaining the enzyme product.

[0044] The method for measuring enzyme activity shown in FIG. 2 includes a step of measuring the density of the enzyme product in each compartment.

[0045] The method for measuring enzyme activity shown in Figure 2 includes the steps of plotting the density value of the enzyme product in each compartment or the value obtained by dividing said value by the reaction time on a graph as a function of the density value of the substrate in each compartment, and determining whether there is a region in which the density value of the enzyme product in each compartment or the value obtained by dividing said value by the reaction time is zero-order with respect to the density value of the substrate in each compartment.

[0046] The zero-order region refers to a region on the graph where the enzyme product value (or the value obtained by dividing said value by the reaction time) remains constant even when the substrate value is increased. Normally, in measurements, the increase in the enzyme product relative to the increase in the substrate amount (or the increase in the value obtained by dividing that value by the reaction time) (slope) gradually decreases and finally becomes zero-order. Therefore, the zero-order region includes "a region where the increase in the enzyme product amount (or the value obtained by dividing that value by the reaction time) relative to the increase in the substrate amount has a smaller slope than the slope of the region on the graph where the increase in the enzyme product amount (or the value obtained by dividing that value by the reaction time) relative to the increase in the substrate amount occurs linearly (seen in the region with a low substrate amount)."

[0047] The absence of a zero-order region refers to the case where there is a constant slope between the substrate mass and the amount of enzyme product (or the value obtained by dividing that value by the reaction time) within the range of the measured substrate mass, and no change is observed in the slope of the increase in the amount of enzyme product (or the value obtained by dividing that value by the reaction time) with an increase in the substrate mass.

[0048] The method for measuring enzyme activity shown in Figure 2 includes a step of calculating the value of the enzyme activity of the enzyme based on the function if there is a zero-order region, and determining that the enzyme activity of the enzyme cannot be calculated if there is no zero-order region.

[0049] In the enzyme activity measurement method shown in Figure 2, the maximum production rate (Vmax) of the enzyme product is first calculated based on the function, and then the enzyme activity value is calculated based on the obtained maximum production rate of the enzyme product. This enzyme activity value may be calculated further taking into account the affinity constant (Michaelis constant: Km), and may also be calculated taking into account other parameters necessary for calculation, if any.

[0050] As described above, the enzyme activity value in the enzyme activity measurement method shown in Figure 2 is calculated based on the maximum production rate of the enzyme product, which is calculated based on the calculated maximum production rate of the enzyme product. Therefore, the enzyme activity measurement method shown in Figure 2 may also be used as a method for measuring the maximum production rate of the enzyme product.

[0051] This method for measuring the maximum production rate includes the steps of: setting multiple zones on a spot and acquiring the density of the substrate in each zone; contacting a sample with the spot to react the substrate with the enzyme and acquire the enzyme product; measuring the density of the enzyme product in each zone; plotting the density of the enzyme product in each zone or the value obtained by dividing said value by the reaction time on a graph as a function of the density of the substrate in each zone; determining whether there is a region where the density of the enzyme product in each zone or the value obtained by dividing said value by the reaction time is zero-order with respect to the density of the substrate in each zone; and calculating the value of the maximum production rate of the enzyme based on the function if there is a zero-order region; and determining that calculation of the maximum production rate of the enzyme is impossible if there is no zero-order region. In this method, the maximum production rate is calculated using a substrate on which a spot of immobilized substrate is formed. In this method, the substrate density in each compartment may be obtained at any timing before the step of plotting the density value of the enzyme product in each compartment or the value obtained by dividing said value by the reaction time on a graph as a function of the substrate density value in each compartment, and determining whether there is a region in which the density value of the enzyme product in each compartment or the value obtained by dividing said value by the reaction time is zero-order with respect to the substrate density value in each compartment.

[0052] (Second embodiment) The enzyme activity measuring device according to the second embodiment is an information processing device that estimates enzyme activity using a substrate having spots formed thereon where substrates are immobilized. This device comprises: means for making an enzyme react with the substrate in the spot to obtain an enzyme product; means for defining one or more compartments in the spot and acquiring the density of the substrate in each compartment; means for measuring the density of the enzyme product in each compartment; and means for estimating the enzyme activity of the enzyme based on the relationship between the density of the substrate in each compartment and the density of the enzyme product in each compartment.

[0053] The meanings and references of the terms "substrate," "spot," "substrate," "compartment," etc. used to describe the second embodiment are the same as those used to describe the first embodiment.

[0054] The enzyme activity measuring device according to the second embodiment may further include a means for changing the measurement point. The means for changing the measurement point is not particularly limited, but includes a scanning method, a method of integrating the measurement means, and the like. Examples of the scanning method include moving the spot side and / or the measurement means side. Examples of the method of integrating the measurement means include optical sensors such as PMT, CMOS, CCD, and SPAD, and electrochemical sensors such as integrated FET.

[0055] The amount of substrate and enzyme product can be measured, for example, by labeling the common site of the substrate (a site whose structure does not change before and after the enzyme reaction, which serves as an indicator of the amount of substrate) and the enzyme product-specific site (a structural site that exists only after the enzyme reaction, which serves as an indicator of the enzyme product) with different fluorescent substances, and then acquiring the fluorescence intensity at each point using a confocal unit and scanning this at each point within the spot.

[0056] According to at least one of the embodiments described above, enzyme activity can be measured simply and easily.

[0057] Although several embodiments have been described, these embodiments are presented as examples and are not intended to limit the scope of the invention. These embodiments can be implemented in various other forms, and various omissions, substitutions, modifications, and combinations of embodiments can be made without departing from the spirit of the invention. These embodiments and their modifications are included within the scope and spirit of the invention, as well as within the scope of the invention and its equivalents as defined in the claims. [Example]

[0058] The present invention will be described in detail based on the following examples, but the present invention is not limited to these examples. Contents are expressed in mass % unless otherwise specified.

[0059] Estimation of the maximum phosphorylation rate of kinases on immobilized Src In this example, various kinases contained in a cell extract of the human lung adenocarcinoma cell line PC9 were selected as the kinases to be measured, and an example is described in which the maximum phosphorylation reaction rate when these kinases phosphorylate immobilized Src is estimated.

[0060] An array plate with solid-phase spots of Src (approximately 100 μm in diameter) was obtained by dropping three spots of GST (glutathione-S-transferase) tagged Src fusion solution onto a glutathione (GSH)-coated glass slide prepared using the method described in a non-patent document (Tadashi Manabe et al., "IGF2 Autocrine-Mediated IGF1R Activation Is a Clinically Relevant Mechanism of Osimertinib Resistance in Lung Cancer", Mol Cancer Res. 2020 Apr;18(4):549-559.) so that the Src concentration ratios were 1, 2, 4, and 8. In addition, cell extracts from the human lung adenocarcinoma cell line PC9 were mixed with a kinase reaction solution (25 mM Tris-HCl, 5 mM β-glycerophosphate, 0.1 mM Na3VO4, 10 mM MgCl2, 1 mM ATP, and 2 mM DTT) and contacted with the Src-immobilized spots. The mixture was then incubated at 30°C for 2 hours to induce phosphorylation on the spots. The reaction was then stopped by contacting the immobilized spots with a reaction stop solution (50 mM EDTA, 10 mM HEPES-NaOH [pH 7.4], 150 mM NaCl, and 0.05% [v / v] Tween 20) and incubating at 30°C for 5 minutes. After washing the array plate with TBST, a primary antibody reaction solution (a cocktail of mouse anti-phosphotyrosine and rabbit anti-GST antibodies) was added and the plate was incubated at 30°C for 1 hour.After washing the array plate with TBST, a secondary antibody solution (a cocktail of goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 790 (Invitrogen) and goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 680 (Invitrogen)) was added and incubated at 30°C for 1 hour. The phosphorylated tyrosine in the immobilized Src spots was labeled with Alexa Fluor 790, and the GST tag in Src was labeled with Alexa Fluor 680. Fluorescence images of the labeled array plate were captured using a microarray scanner with confocal optics. Alexa Fluor 790 was measured using a 785 nm excitation laser (the fluorescence signal at this time is denoted as I785nm), and Alexa Fluor 680 was measured using a 670 nm excitation laser (the fluorescence signal at this time is denoted as I670nm) (Figure 3). From the acquired fluorescence images, the maximum phosphorylation rate of Src was obtained using two methods.

[0061] In the first method, spatially independent spots were set on the plate as zones for determining the density of Src and the density of the enzyme product. The density value (I785nm) of the enzyme product (phosphorylated tyrosine) in each spot was plotted as a function of the density value (I670nm) of the substrate in each spot (Figure 4). The plots were made by adding the same concentration of Src solution to three spots, and the average I785nm and I670nm values ​​and their standard deviations were plotted. Each spot was identified as an area where the I670nm intensity exceeded a certain threshold. The plots were calculated using the Michaelis-Menten equation, which takes into account the presence of background (Bg).

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[0062] In the second method, a virtual area of ​​1 pixel (10 × 10 μm), smaller than a single spot, was defined to measure the density of Src and the density of the enzyme product. The density of the enzyme product (phosphotyrosine) (I785 nm) in one virtual area (1 pixel) within a single spot (the rightmost spot in Figure 3) was plotted as a function of the density of the substrate (I670 nm) in that area (Figure 5). This procedure was repeated throughout the entire spot to obtain a series of plots of substrate and enzyme product density. Fitting of the resulting plots using Equation (1) yielded the maximum phosphorylation reaction rate at 2 h, Vmax( / 2 h) = 27512, and the Michaelis constant (affinity constant) Km = 6637 and Bg = 5105 (Figure 6). The slight difference in values ​​between the first and second methods was thought to be due to the fact that the distribution of substrate density (I670nm) used for fitting was somewhat narrower in the first method.

[0063] In the second method, local variations in substrate density within a single spot are used as the distribution of substrate densities between virtual compartments, allowing for the acquisition of information on the density of enzyme products over a wider range of substrate density distributions. Furthermore, although the Vmax and Km values ​​obtained here are in arbitrary units based on fluorescence intensity, they can also be converted into units of substrate density and enzyme product density by creating a calibration curve from the fluorescence intensity obtained for compartments with known substrate and enzyme product densities.

[0064] In both the first and second methods, it is possible to apply a filter to each acquired fluorescence image. Figure 7 shows the results of using a median filter. This allows localized noise components to be canceled out while maintaining the distribution information of the substrate and enzyme product amounts in the fluorescence image (Figure 7). After determining the substrate density and enzyme product density for each section, it is also possible to sort the sections in descending order of substrate density and take a moving average. Figure 8 shows the results of sorting the substrate density and enzyme product density distribution for each section (1 pixel (10 × 10 μm)) obtained using the second method, sorting the sections in descending order of substrate density, and then taking a moving average for every 10 sections. This operation makes it easier to grasp the tendency for enzyme products to change with substrate density, facilitating fitting operations (Figure 8).

[0065] Estimation of the maximum phosphorylation rate of kinases on immobilized HCK The procedure was the same as for Src. The results are shown in Figure 9. Note that, in calculating the maximum phosphorylation reaction rate Vmax, fitting based on the Michaelis-Menten equation is not necessarily required; Vmax may be calculated based on the density of the enzyme product in a region where the density of the enzyme product is constant relative to the density of the substrate.

[0066] Estimation of the maximum phosphorylation rate of kinases on immobilized ABL1 The procedure was the same as for Src. The results are shown in Figure 10. Note that, in calculating the maximum phosphorylation reaction rate Vmax, fitting based on the Michaelis-Menten equation is not necessarily required; Vmax may be calculated based on the density of the enzyme product in a region where the density of the enzyme product is constant relative to the density of the substrate.

[0067] Estimation of the maximum reaction rate of each enzyme when multiple enzymes act on multiple substrates The reaction rate of the enzyme was estimated based on the maximum reaction rate obtained with the immobilized substrate.

[0068] When one type of enzyme x1 converts one type of substrate Y1 into enzyme product Y1', what is the maximum reaction rate obtained for substrate Y1?

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[0069] One type of enzyme x1 reacts with multiple types of substrate Y j (j=1, 2, …, n) is the enzyme product Y j ' (j=1, 2, ..., n), the substrate Y j The maximum reaction rate obtained for

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[0070] Multiple enzymes x i (i=1, 2, ..., m) is one or more types of substrate Y j (j=1, 2, …, n) when acting on the substrate Y j The maximum reaction rate obtained for

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[0071] The simultaneous equations (4) have m×n unknowns and n equations, and cannot be solved except in the case of m=1 mentioned above.

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[0072] As mentioned above, one type of enzyme x1 has multiple types of substrates Y j Maximum reaction rate for (j=1, 2, …, n)

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[0073] Enzyme x i (i=1, 2, ..., m) is one or more types of substrate Y j The ratio of the maximum reaction rates when acting on (j=1, 2, ..., n) is

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[0074] Separation of substrate-quantity-limiting / enzyme-reaction-limiting regions within a spot If we plot the substrate density and enzyme product density in each subregion within a single spot, we can see that the density of the enzyme product increases with increasing substrate density, and that the density of the enzyme product is constant (zero-order) with respect to the substrate density. The former is considered the substrate mass-limited region, and the latter the enzyme reaction rate-limited region. In such a spot, the enzyme reaction occurs at its maximum reaction rate in the enzyme reaction rate-limited region, where the density of the enzyme product is proportional to the maximum reaction rate of the enzyme. On the other hand, the presence of a substrate mass-limited region is necessary for estimating the affinity constant Km. For a given substrate, if either the substrate mass-limited or enzyme reaction rate-limited region is extremely low (e.g., less than 1%), the maximum reaction rate and affinity constant are estimated based on the substrate density and enzyme product density in the small region, which can be easily affected by noise and can result in large estimation errors.

[0075] Based on the above, in applications where enzyme activity is compared and analyzed based on the density of enzyme products, it is possible to separate the regions within a spot into substrate mass-limiting / enzyme reaction-limiting regions and present them to the user. Possible methods of presentation include overlaying each region on an image of the spot, or presenting the percentage of each region for each spot, substrate type, or plate. Alternatively, the device can determine whether the substrate mass-limiting / enzyme reaction-limiting region has a sufficient area after separation, and if not, present the user with an error message informing them that the estimated enzyme activity index may be unreliable.

Claims

1. A method for estimating enzyme activity using a substrate on which spots of immobilized substrates are formed, comprising: (a) allowing an enzyme to act on the substrate on the spot to obtain an enzyme product; (b) setting one or more zones in the spot and acquiring the density of the substrate in each zone; (c) measuring the density of the enzyme product in each of the compartments; and (d) estimating the enzymatic activity of the enzyme based on the relationship between the density of the substrate in each of the compartments obtained in step (b) and the density of the enzyme product in each of the compartments obtained in step (c); A method comprising:

2. The method of claim 1 , wherein the section is a single spot that is spatially independently arranged.

3. The method according to claim 1 , wherein the section is smaller than one spot and is selected from a plurality of areas that can be set within one spot.

4. The method according to any one of claims 1 to 3, wherein the enzyme activity is the maximum production rate (Vmax) of the enzyme product.

5. The method according to claim 4 , wherein the calculation of the value of the maximum production rate includes a step of fitting using the Michaelis-Menten equation.

6. 6. The method of claim 5, further comprising calculating the affinity constant (Km) of the substrate for the enzyme.

7. An information processing device for estimating enzyme activity using a substrate on which spots of immobilized substrates are formed, comprising: a means for allowing an enzyme to act on the substrate in the spot to obtain an enzyme product; a means for setting one or more sections in the spot and acquiring the density of the substrate in each section; a means for measuring the density of the enzyme product in each of the compartments; a means for estimating the enzymatic activity of the enzyme based on the relationship between the density of the substrate in each compartment and the density of the enzyme product in each compartment; An information processing device comprising:

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