Detection of target substance in sample

Abstract

To provide, in one embodiment, a novel method for detecting a target substance in a sample.SOLUTION: A method for detecting a target substance in a sample comprises contacting the sample with: an enzyme A having a portion capable of binding to the target substance; an inactive enzyme B that can be transformed from an inactive form to an active form by the action of the enzyme A; an inactive enzyme C that can be transformed from an inactive form to an active form by the action of the enzyme B, the enzyme C being capable of transforming the enzyme B from the inactive form to the active form; and a reporter substance D that is capable of emitting a signal by the action of the enzyme B and / or the enzyme C.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to detection methods, devices, reagents, and systems that involve the use of enzymatic signal amplification in the detection of target substances in a sample. [Background technology]

[0002] Techniques for detecting trace molecules in biological and environmental samples are widely required in a variety of situations, including diagnosing the presence or absence of viral infections and testing for the leakage of harmful substances and microorganisms into the environment. Specific protein detection involves the use of ELISA, which immobilizes the target protein on a substrate using antibodies and performs colorimetric quantification, and luminescent biosensors, which change the luminescence properties of chemiluminescent proteins upon antibody binding to the target protein (Non-Patent Document 1). Furthermore, a technique for detecting small molecules using protein interactions has been reported in which signal accumulation is achieved using protease activity (Non-Patent Document 2). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Alexander Grawe et al., Bioluminescence Goes Dark: Boosting the Performance of Bioluminescent Sensor Proteins Using Complementation Inhibitors. ACS Sensors 2022. [Non-patent document 2] Viktor Stein et al., Protease-based synthetic sensing and signal amplification. PNAS 2014 Summary of the Invention [Problem to be solved by the invention]

[0004] With the ELISA method, the target protein is concentrated by binding to the antibody, making it possible to detect even low concentrations of the target protein (10 pg / ml with Boster's Picokine ELISA kit). However, the test is complicated and requires multiple steps: (i) binding of the sample to the antibody, (ii) washing, and (iii) detection, making it difficult to use for self-diagnosis in the general household. In methods using luminescent biosensors, the test sample is mixed with the biosensor, a luminescent substrate is added, and changes in the intensity and spectrum of the resulting chemiluminescence are measured. This method is simpler than ELISA, and by using a commercially available smartphone to detect luminescence, it is thought that it could be applied to diagnostics in the home. However, the concentration of the luminescent biosensor in the reaction system used in the test is usually a few nM, making it difficult to apply when the target concentration is only a few pM to tens of pM, such as in the test of virus particles or blood cytokines. It has been reported that a method of controlling the activity of a protease by utilizing its binding to the test subject and accumulating the cleaved substrate can achieve high sensitivity with a detection limit of several hundred fM (Non-Patent Document 2), but this method requires approximately 10 to 15 hours and the amount of signal change is very small, making it unsuitable for use in fields that require speed, such as virus testing.

[0005] Thus, the present disclosure provides for the detection of target substances in a sample, including the use of enzymatic signal amplification. [Means for solving the problem]

[0006] In one aspect, the present disclosure relates to a method for detecting a target substance in a sample, the method comprising contacting the sample with enzyme A having a moiety capable of binding to the target substance, inactive enzyme B that can be converted from an inactive form to an active form by the action of enzyme A, inactive enzyme C that can be converted from an inactive form to an active form by the action of enzyme B and can convert enzyme B from an inactive form to an active form, and reporter substance D that can emit a signal by the action of enzyme B and / or enzyme C.

[0007] In another aspect, the present disclosure relates to a method for detecting a target substance, comprising amplifying a signal using enzyme B and enzyme C, wherein enzyme B is converted from an inactive form to an active form by enzyme C, and enzyme C is converted from an inactive form to an active form by enzyme B, and the method comprises utilizing, measuring, or detecting the enzymatic activity of enzyme B and / or enzyme C obtained by amplification through an enzymatic reaction between enzyme B and enzyme C.

[0008] In another aspect, the present disclosure relates to a device for detecting a target substance in a sample, comprising: a reagent portion; and a substrate on which the reagent portion is formed, wherein the reagent portion comprises: an enzyme A having a portion capable of binding to the target substance; an inactive enzyme B that can be converted from an inactive form to an active form by the action of enzyme A; an inactive enzyme C that can be converted from an inactive form to an active form by the action of enzyme B and can convert enzyme B from an inactive form to an active form; and a reporter substance D that can emit a signal by the action of enzyme B and / or enzyme C.

[0009] In yet another aspect, the present disclosure relates to a reagent for detecting a target substance in a sample, the reagent comprising: enzyme A having a moiety capable of binding to the target substance; inactive enzyme B that can be converted from an inactive form to an active form by the action of enzyme A; inactive enzyme C that can be converted from an inactive form to an active form by the action of enzyme B, and can convert enzyme B from an inactive form to an active form; and reporter substance D that can emit a signal by the action of enzyme B and / or enzyme C.

[0010] In still another aspect, the present disclosure relates to a system for detecting a target substance in a sample, the system including an imaging terminal that acquires a luminescence signal emitted by the target substance detection method of the present disclosure or the device of the present disclosure, and an information processing means that processes the luminescence signal data obtained by the imaging terminal, wherein the imaging terminal and the information processing means are capable of bidirectional communication via a network. [Effects of the Invention]

[0011] In one aspect, the present disclosure can provide for the detection of a target substance in a sample, which involves utilizing enzymatic signal amplification. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present inventors have conducted further research to provide a new detection method that can improve sensitivity and ease of use in detecting target substances in samples, and have discovered that signal amplification using multiple enzymes can be used.

[0013] Multiple enzymes that can be converted from inactive to active by enzymatic action are prepared. Enzyme 1 is configured to convert another enzyme (enzyme 2) from inactive to active through its enzymatic action, and enzyme 2 is configured to convert enzyme 1 from inactive to active through its enzymatic action. These enzymes repeatedly activate each other, amplifying the signal derived from the trace molecule as the number of active enzymes ( FIG. 1B ). In one or more embodiments, such signal amplification allows for exponential amplification of the signal. In one or more embodiments, the detection method of the present disclosure, which includes the above-mentioned signal amplification, is equivalent to or better than the ELISA method in terms of ease of measurement, and equivalent to or better than a biosensor using a luminescent protein in terms of detection sensitivity, and can be used for highly sensitive and rapid tests such as virus detection and measurement of blood cytokines.

[0014] One or more embodiments of the detection of a target substance of the present disclosure will be described. The detection of a target substance of the present disclosure includes signal amplification as exemplified in FIG. 1B. First, when trigger enzyme A and inactive enzyme B bind to a target substance, enzyme B is activated by trigger enzyme A (FIG. 1A). The activated enzyme B activates inactive enzyme C through its enzymatic action (reaction 1). The activated enzyme C activates inactive enzyme B through its enzymatic action (reaction 2). As these two enzymatic reactions (reactions 1 and 2) are repeated, the active enzymes B and C increase (FIG. 1B). A mathematically exponential increase is possible. The reaction signal between trigger enzyme A and enzyme B is amplified in the form of active enzymes B and C. The amplified signal can be detected by using a reporter substance D that generates a signal through the enzymatic reaction of active enzyme C (and / or active enzyme B). FIG. 1C shows an example of a reporter substance D that combines two fluorescent proteins and whose luminescent color changes upon the action of an activated enzyme C.

[0015] Method for detecting a target substance in a sample In one aspect, the present disclosure relates to a method for detecting a target substance in a sample (hereinafter also referred to as the "target substance detection method of the present disclosure"), which comprises contacting the sample with enzyme A having a moiety capable of binding to the target substance, inactive enzyme B that can be converted from an inactive form to an active form by the action of enzyme A, inactive enzyme C that can be converted from an inactive form to an active form by the action of enzyme B and can convert enzyme B from an inactive form to an active form, and reporter substance D that can emit a signal by the action of enzyme B and / or enzyme C.

[0016] [Enzyme B and Enzyme C] Enzymes B and C are enzymes that change from an inactive form to an active form through the action of an enzyme other than themselves. Such enzymes are hereinafter also referred to as enzyme-regulated enzymes. Enzyme B can be converted from an inactive form to an active form by the enzymatic action of enzyme C (active form). Enzyme C can be converted from an inactive to an active form by the enzymatic action of enzyme B (active form). By utilizing the mechanism by which these two enzyme-controlled enzymes (B and C) activate each other, the signal that begins with the trigger of activation of enzyme B (or C) can be amplified. In the present disclosure, in one or more embodiments, the term "inactive form" may include not only an enzyme in an inactive state, but also an enzyme in a state of lower activity than after activation.

[0017] Therefore, in another aspect, the method for detecting a target substance of the present disclosure relates to a method for detecting a target substance, which comprises amplifying a signal using enzyme B and enzyme C, wherein enzyme B is converted from an inactive form to an active form by enzyme C, and enzyme C is converted from an inactive form to an active form by enzyme B, and the method comprises utilizing, measuring, or detecting the enzymatic activity of enzyme B and / or enzyme C obtained by amplification through the enzymatic reaction between enzyme B and enzyme C. One or more embodiments of utilizing, measuring, or detecting the enzymatic activity of active enzyme B and / or active enzyme C is the use of reporter substance D, but may not be limited to this. Unless otherwise specified, the target substance detection method of the present disclosure may also include the above aspects.

[0018] In the method for detecting a target substance of the present disclosure, a signal is amplified by a plurality (two or more types) of enzyme-controlled enzymes. In the present disclosure, the enzyme-controlled enzymes involved in signal amplification are not limited to the two types, enzymes B and C, but may include three or more types of enzyme-controlled enzymes. Hereinafter, the term "enzyme-controlled enzyme" may include enzymes B and C unless otherwise specified.

[0019] In the present disclosure, enzyme-controlled enzymes that can be used include proteolytic enzymes (proteases), phosphorylating enzymes (kinases), dephosphorylating enzymes, glycosylation enzymes, acetylation enzymes, succinylation enzymes, propianylation enzymes, methylation enzymes, ubiquitination enzymes, nitrosylation enzymes, and lipidation enzymes. In one or more embodiments, enzyme-controlled enzymes can be obtained by obtaining variants whose activity is increased by the action of other enzymes. Furthermore, in one or more embodiments, enzymes whose enzymatic activity is increased by the action of other enzymes can be used as enzyme-controlled enzymes. In the target substance detection method of the present disclosure, these enzyme-controlled enzymes may be used in combination for signal amplification.

[0020] An example will be described in which the enzyme-controlled enzymes (enzymes B and C) are proteases. Cleavage of a substrate by a protease is irreversible, and the cleaved substrate accumulates over time. A modified protease (protease-controlled protease; PCP) whose activity increases upon cleavage by a protease can be used as an enzyme-controlled enzyme. In one or more embodiments, a PCP can be produced by inserting into a protease an amino acid sequence (hereinafter also referred to as a "cleavage sequence") that inhibits function when inserted and restores function upon cleavage. In one or more embodiments, a PCP can be produced by introducing a circular permutation sequence into a protease and inserting the cleavage sequence into the linker between the original C-terminus and N-terminus. In one or more other embodiments, a protease that exhibits activity upon cleavage of a specific portion can be produced by replacing the cleavage sequence with the cleavage sequence of another protease.

[0021] When both enzymes B and C are PCPs, the proteases are preferably specific for the amino acid sequence they cleave. That is, the cleavage sequence of enzyme B is preferably a sequence that can be specifically recognized and cleaved by enzyme C, and the cleavage sequence of enzyme C is preferably a sequence that can be specifically recognized and cleaved by enzyme B. Therefore, in one or more embodiments, enzyme B is a protease having a cleavage sequence inserted therein, the activity of which is reduced or inactivated, and the cleavage sequence of enzyme B can be cleaved by enzyme C, and cleavage of the cleavage sequence increases the activity of enzyme B. Similarly, in one or more embodiments, enzyme C is a protease having a cleavage sequence inserted therein, the activity of which is reduced or inactivated, and the cleavage sequence of enzyme C can be cleaved by enzyme B, and cleavage of the cleavage sequence increases the activity of enzyme C. That is, when both enzymes B and C are PCPs, their activity is controlled from OFF (inactive) to ON (active) by the protease activity of the other enzyme, which in one or more embodiments improves the signal amplification rate, preferably achieving exponential signal amplification.

[0022] The protease used for PCP is not particularly limited, and in one or more embodiments, examples include a protease derived from tobacco etch virus (TEV protease; TEVp) and a protease derived from tobacco vein mottling virus (TVMV protease; TVMVp). In one or more embodiments, TEVp and TVMVp can be converted into PCPs by introducing a circularly permuted sequence and inserting a cleavage sequence between the C-terminal region and the N-terminal region before the circularly permuted sequence. For example, the activity of TEVp-PCP, into which a cleavage sequence for caspase-3 has been introduced, changes by approximately 100-fold depending on the presence or absence of caspase-3 activity.

[0023] In one or more other embodiments, the protease used in PCP may be caspase-3. Caspase-3 is activated in vivo by cleavage of its regulatory site by caspase-8 and / or caspase-9. It has been reported that the activity of caspase-3 is regulated by TEVp when the regulatory site is replaced with the cleavage sequence of TEVp (Daniel C. Gray, Sami Mahrus, and James A. Wells, Cell 2010).

[0024] Therefore, in one or more embodiments, the combination of enzymes B and C includes a combination of TEVp-PCP into which a caspase-3 cleavage sequence has been introduced and caspase-3 in which the regulatory site has been replaced with the TEVp cleavage sequence.

[0025] In one or more embodiments, when enzymes B and C (referred to as PCP1 and PCP2, respectively) are produced based on TEVp, the introduction of a point mutation into TEVp can be used to change the cleavage sequence. For example, the consensus sequence of the cleavage sequence of TEVp is ENLYFQS (SEQ ID NO: 1), but when the mutations N171D and N176T are introduced, the cleavage sequence (recognition sequence) of the enzyme after the mutation is K NLYFQG (SEQ ID NO: 2), and when D148R, T173A, and N177K are introduced, the cleavage sequence (recognition sequence) of the enzyme after the mutation becomes ENLYF E G (SEQ ID NO: 3) (Figure 2). Thus, in one aspect, the present disclosure relates to a TEVp into which N171D and N176T mutations have been introduced, and in another aspect, to a TEVp into which D148R, T173A, and N177K mutations have been introduced. In addition, as a combination of PCP1 and PCP2, a circularly permuted sequence was introduced into TEVp into which the mutations N171D and N176T had been introduced, and a cleavage sequence ENLYF was inserted between the C-terminal region and the N-terminal region before the circularly permuted sequence. E G (SEQ ID NO: 3) was introduced (PCP1), and the circularly permuted TEVp into which D148R, T173A, and N177K mutations were introduced was inserted to form a cleavage sequence between the C-terminal region and the N-terminal region before the circularly permuted sequence mutation. K An example is a combination with PCP2 into which NLYFQG (SEQ ID NO: 2) has been introduced (FIG. 2). In one or more embodiments, the amino acid sequence of PCP1 is represented by SEQ ID NO:8, and the gene base sequence is represented by SEQ ID NO:9. In one or more embodiments, the amino acid sequence of PCP2 is represented by SEQ ID NO: 10, and the gene base sequence is represented by SEQ ID NO: 11. However, the enzymes B and C of the present disclosure are not limited to those shown in FIG.

[0026] In one or more embodiments, enzyme B and enzyme C may be a combination of kinase-based enzyme-controlled enzymes, or a combination of a protease-based enzyme-controlled enzyme and a kinase-based enzyme-controlled enzyme. For example, in the MAP kinase cascade, three types of enzymes, MAPKKK, MAPKK, and MAPK, activate downstream enzymes by sequentially phosphorylating them. If these enzymes are used to create two types of enzymes that phosphorylate and activate each other, they can be used as enzyme-controlled enzymes to amplify signals. In addition, the positive feedback in the thrombin cascade reaction in hemostasis reactions can also be used as an enzyme-controlled enzyme to amplify signals.

[0027] In one or more embodiments, Enzyme B and Enzyme C may be fused with a moiety that functions as an enzyme, such as a protease or a kinase, as well as other functional moieties. In one or more embodiments, examples of the functional moiety include, but are not limited to, a moiety that binds to a target substance and various peptide tags.

[0028] Signal amplification by enzyme B and enzyme C begins with activation of enzyme B (or enzyme C). In one or more embodiments, the trigger is activation of enzyme B (or enzyme C) by enzyme A ( FIG. 1A ). In one or more embodiments, to carry out the trigger reaction, enzyme B (or enzyme C) has a moiety (hereinafter simply referred to as a “binding moiety”) capable of binding to the target substance, and both enzyme A and (or enzyme C) are capable of binding to the target substance.

[0029] [Enzyme A] Enzyme A is an enzyme that has a moiety capable of binding to a target substance and can convert enzyme B (or enzyme C) from an inactive form to an active form. Below, we will explain an example in which enzyme A activates enzyme B from an inactive form. In one or more embodiments, enzyme A can activate enzyme B by binding to the target substance together with inactive enzyme B. This reaction serves as a trigger. In one or more embodiments, enzyme A and enzyme B come close to each other via the target substance, causing a trigger reaction. The mechanism of the trigger reaction (activation of enzyme B) by enzyme A may be the same as or different from that of the activation of enzyme B by enzyme C. For example, when enzymes B and C are protease-controlled proteases and enzyme A triggers the activation of enzyme B, enzyme A only needs to have protease activity that recognizes the same cleavage sequence as enzyme C. Therefore, in one or more embodiments, enzyme A may be an enzyme such as a protein-degrading enzyme (protease), a phosphorylating enzyme (kinase), a dephosphorylating enzyme, a glycosylation enzyme, an acetylation enzyme, a succinylation enzyme, a propionylation enzyme, a methylation enzyme, a ubiquitination enzyme, a nitrosylation enzyme, or a lipidation enzyme.

[0030] The binding portion of enzyme A is capable of binding to a target substance and can be appropriately selected depending on the target substance. In one or more non-limiting embodiments, the binding portion of enzyme A can be an aptamer, an antibody, an antigen-binding fragment of an antibody, a ligand, or the like. Furthermore, when the target substance has binding properties, the binding portion of enzyme A can serve as a binding partner.

[0031] From the viewpoint of detecting a target substance, in one or more embodiments, enzyme A and enzyme B are preferably capable of simultaneously binding to different portions of the target substance, each of which preferably has a binding portion that enables binding, and preferably, enzyme A is capable of activating enzyme B upon binding. Enzyme A may be capable of activating enzyme B by binding to a target substance together with enzyme B, and in one or more embodiments, enzyme B may not have a binding moiety for binding to a target substance. Enzyme A may be activated by binding to a target substance, and may then be able to activate enzyme B.

[0032] [Reporter substance D] The reporter substance D is not particularly limited as long as it can reflect the amplified signal of the activated enzyme B and / or the activated enzyme C. In one or more embodiments, the reporter substance D is a substance that can emit a signal by the action of the activated enzyme B and / or the activated enzyme C. Examples of signals from reporter substance D include luminescence, a change in luminescence color, and an electrochemical signal due to a redox reaction, but luminescence signals including luminescence and / or a change in luminescence color are preferred for ease of detection. In one or more embodiments, fluorescent proteins, chemiluminescence, and chemiluminescent proteins can be used as luminescence signals.

[0033] When enzyme B and / or enzyme C is a protease, reporter substance D, in one or more embodiments, may be in a form that emits light upon cleavage, for example, a form that includes a combination of a fluorescent protein and a quencher and emits light upon cleavage, or in a form that changes the emitted light color upon cleavage, for example, a form in which two fluorescent proteins in a FRET (Forster Resonance Energy Transfer) state change color from the acceptor side to the donor side upon cleavage. When enzyme B and / or enzyme C is a kinase, reporter substance D may, in one or more embodiments, be a protein that acquires luminescence ability when phosphorylated. When reporter substance D uses a fluorescent protein, excitation of the fluorescent protein may be performed by chemiluminescence or a chemiluminescent protein instead of fluorescence for ease of detection. This allows fluorescence emission to be used without using fluorescence.

[0034] In the present disclosure, "reporter substance D emits a signal" may include not only cases in which reporter substance D generates a directly detectable luminescent signal, such as a luminescent protein, but also cases in which reporter substance D generates an indirectly detectable signal, such as when it generates a moiety to which an aptamer, nucleic acid, antibody, etc. can bind.

[0035] [Target substance] The target substance in the detection method of the present disclosure is not particularly limited, and examples thereof include biological substances, substances contained in food, chemical substances, etc. In one or more embodiments, the target substance in the detection method of the present disclosure includes biological substances in a biological sample, allergens or harmful substances in food, pollutants or harmful substances in the natural environment such as river water or seawater, chemical substances, or pathogens, etc. In one or more embodiments, examples of biological materials include low molecular weight compounds, decomposition metabolites, nucleic acids, sugars, peptides, proteins, cells, viruses, and microorganisms in living organisms. In one or more embodiments, the sample containing the target substance is preferably a sample of an aqueous medium containing the target substance. The sample containing the target substance may be a biological sample, such as a body fluid sample such as whole blood, serum, plasma, or urine, or may be any of these samples that have been diluted and / or pretreated as necessary.

[0036] [contact] The series of reactions (FIGS. 1A to 1C) of the detection method of the present disclosure can be caused by contacting a sample with enzymes A to C and a reporter substance D. In one or more embodiments, the contact is carried out by adding the enzymes A to C and the reporter substance D to a sample containing the target substance. The addition may be done all at once, in this order, or in any order. In one or more embodiments, the contact may be performed by supplying the sample to the reagents (enzymes A to C and reporter substance D) arranged on the substrate.

[0037] The detection method of the present disclosure can improve sensitivity, and therefore can target samples containing a detection substance at a concentration of, for example, less than 1 nM. In one or more embodiments, the detection method of the present disclosure can detect a detection substance even when the concentration of the detection substance in a sample is 900 pM or less, 700 pM or less, 500 pM or less, 300 pM or less, 100 pM or less, less than 100 pM, 90 pM or less, 70 pM or less, or 50 pM or less. In one or more embodiments of the detection method of the present disclosure, the concentration of the detection substance in the sample is preferably 10 fM or more. In one or more embodiments, the concentration of the detection substance in the sample in the detection method of the present disclosure is 10 fM to 900 pM, 10 fM to 700 pM, 10 fM to 500 pM, 10 fM to 300 pM, 10 fM to 100 pM, 10 fM to less than 100 pM, 10 fM to 90 pM, 10 fM to 70 pM, or 10 fM to 50 pM.

[0038] When the concentration of the detection substance in the sample is assumed to be 10 fM or more and 900 pM or less, in one or more embodiments, the concentration of enzyme A used is, for example, 10 nM or more and 1 μM or less, the concentration of enzyme B is, for example, 100 nM or more and 10 μM or less, the concentration of enzyme C is, for example, 100 nM or more and 10 μM or less, and the concentration of reporter substance D is, for example, 10 nM or more and 10 μM or less.

[0039] [analysis] In one or more embodiments, the detection method of the present disclosure may include analyzing a signal emitted by reporter substance D. In one or more embodiments, the analysis may include calculating or determining the concentration. The analysis can be performed appropriately by those skilled in the art. From the viewpoint of improving simplicity, in one or more embodiments, the analysis preferably utilizes a luminescence signal emitted by reporter substance D. Those skilled in the art can appropriately record, capture, and process the luminescence signal. From the perspective of improving convenience, luminescence signal data can be captured using a color detector such as a color camera on a mobile device (e.g., a smartphone). This allows data from the mobile device to be transmitted and received over a network, making it easy to determine the concentration of biological substances.

[0040] device In one or more embodiments, the detection method of the present disclosure can be made more convenient by carrying out the method in the form of a device in which reporter substance D utilizes luminescence or a change in luminescence color. Thus, in one aspect, the present disclosure relates to a device for carrying out the detection method of the present disclosure. In one or more embodiments, the device of the present disclosure includes a reagent portion in which enzymes A to C and a reporter substance D are arranged, and a substrate on which the reagent portion is formed. That is, in another aspect, the present disclosure relates to a device for detecting a target substance in a sample, comprising a reagent portion and a substrate on which the reagent portion is formed, wherein the reagent portion comprises: enzyme A having a portion capable of binding to the target substance; inactive enzyme B that can be converted from an inactive form to an active form by the action of enzyme A; inactive enzyme C that can be converted from an inactive form to an active form by the action of enzyme B and can convert enzyme B from an inactive form to an active form; and reporter substance D that can emit a signal by the action of enzyme B and / or enzyme C (hereinafter also referred to as the "device of the present disclosure").

[0041] The sample, target substance, enzyme A, enzyme B, enzyme C, and reporter substance D in the device of the present disclosure are the same as those described in the detection method of the present disclosure. From the viewpoint of simplicity, it is preferable that reporter substance D emits a luminescent signal. In one or more embodiments, the reagents in the reagent portion are preferably in a dry state in order to reduce reactions with each other. The material of the substrate is not particularly limited, and in one or more embodiments, examples include fluorine-based materials such as paraffin and Teflon (registered trademark), glass, polypropylene, woven fabric, nonwoven fabric, and paper. In one or more embodiments, the substrate material is preferably hydrophobic, from the viewpoint of enabling detection of a high luminescence signal. Furthermore, the substrate is preferably one that does not interfere with imaging by imaging means such as a mobile terminal (such as a smartphone) or a digital camera.

[0042] reagent In one aspect, the present disclosure relates to a reagent for carrying out the detection method of the present disclosure or a reagent for use in manufacturing the device of the present disclosure. That is, in yet another aspect, the present disclosure relates to a reagent for detecting a target substance in a sample, the reagent comprising: enzyme A having a moiety capable of binding to the target substance; inactive enzyme B that can be converted from an inactive form to an active form by the action of enzyme A; inactive enzyme C that can be converted from an inactive form to an active form by the action of enzyme B and can convert enzyme B from an inactive form to an active form; and reporter substance D that can emit a signal by the action of enzyme B and / or enzyme C. The sample, target substance, enzyme A, enzyme B, enzyme C, and reporter substance D in the reagent of the present disclosure are the same as those described in the detection method of the present disclosure.

[0043] system In still another aspect, the present disclosure relates to a system for detecting a target substance in a sample, the system including an imaging terminal that acquires a luminescence signal emitted by the target substance detection method of the present disclosure or the device of the present disclosure, and an information processing means that processes the luminescence signal data obtained by the imaging terminal, wherein the imaging terminal and the information processing means are capable of bidirectional communication via a network. According to the system of the present disclosure, in one or more embodiments, analysis, determination, or diagnosis results by an expert can be obtained quickly and easily regardless of location, even in a remote location, without going to a special testing facility, etc. In one or more embodiments, the system of the present disclosure can be used as a remote analysis system, a remote determination system, or a remote diagnosis system.

[0044] In one or more embodiments, the imaging terminal may be a general-purpose image reading device, etc. The image reading device is not particularly limited, and in one or more embodiments, may be a mobile terminal such as a smartphone or tablet terminal, a digital camera, a CCD camera, etc. In one or more embodiments, the imaging terminal may be a mobile terminal such as a smartphone or tablet terminal, as this enables rapid measurement and reception of determination results, etc.

[0045] In one or more embodiments, the luminescence signal can be acquired by capturing an image of the luminescence signal emitted when a sample is supplied to the device of the present disclosure using an imaging terminal. In one or more embodiments, in order to improve measurement accuracy, the luminescence signal can be acquired by attaching an attachment to the camera part of the imaging terminal and placing the device of the present disclosure, to which the sample has been supplied, thereon.

[0046] In one or more embodiments, the acquired luminescence signal can be transmitted to the information processing means by the imaging terminal that acquired the luminescence signal. In one or more embodiments, the transmission to the information processing means can be performed through an application on the imaging terminal. The acquired luminescence signal may be transmitted to the information processing means by a communication terminal different from the imaging terminal. In one or more embodiments, the communication terminal may be a personal computer or the like having a communication function.

[0047] In one or more embodiments, the information processing means stores the transmitted light emitting signal data and information associated with the reference light emitting signal data, and can make a determination based on these and the light emitting signal data acquired by the imaging terminal. In one or more embodiments, the information processing means includes comparing the transmitted light emitting signal data with the reference light emitting signal data and transmitting the obtained determination result to the imaging terminal.

[0048] The present disclosure may relate to one or more of the following embodiments: [1] A method for detecting a target substance in a sample, comprising: an enzyme A having a portion capable of binding to a target substance; An inactive enzyme B that can be converted from an inactive form to an active form by the action of enzyme A; An inactive enzyme C that can be converted from an inactive form to an active form by the action of enzyme B and can convert enzyme B from an inactive form to an active form; A method comprising contacting the sample with a reporter substance D capable of emitting a signal through the action of enzyme B and / or enzyme C. [2] The method according to [1], wherein enzyme B has a portion capable of binding to the target substance, and enzymes A and B are both capable of binding to the target substance. [3] The method according to [1] or [2], wherein the enzyme A is capable of activating the enzyme B by binding to the target substance together with the inactive enzyme B. [4] A method for detecting a target substance, comprising amplifying a signal using enzyme B and enzyme C, Enzyme B is converted from an inactive form to an active form by enzyme C, Enzyme C is converted from an inactive form to an active form by enzyme B, A method comprising utilizing, measuring, or detecting the enzymatic activity of enzyme B and / or enzyme C obtained by amplification through an enzymatic reaction between enzyme B and enzyme C. [5] The method according to any one of [1] to [4], wherein Enzyme B and Enzyme C are each independently selected from the group consisting of protease, kinase, dephosphorylating enzyme, glycosylation enzyme, acetylation enzyme, succinylation enzyme, propionylation enzyme, methylation enzyme, ubiquitination enzyme, nitrosylation enzyme, and lipidation enzyme. [6] A device for detecting a target substance in a sample, comprising: A reagent portion and a substrate on which the reagent portion is formed, The reagent part comprises: an enzyme A having a portion capable of binding to a target substance; An inactive enzyme B that can be converted from an inactive form to an active form by the action of enzyme A; An inactive enzyme C that can be converted from an inactive form to an active form by the action of enzyme B and can convert enzyme B from an inactive form to an active form; and a reporter substance D capable of emitting a signal upon the action of enzyme B and / or enzyme C. [7] A reagent for detecting a target substance in a sample, comprising: an enzyme A having a portion capable of binding to a target substance; An inactive enzyme B that can be converted from an inactive form to an active form by the action of enzyme A; An inactive enzyme C that can be converted from an inactive form to an active form by the action of enzyme B and can convert enzyme B from an inactive form to an active form; a reporter substance D capable of emitting a signal by the action of enzyme B and / or enzyme C. [8] A system for detecting a target substance in a sample, comprising: An imaging terminal that acquires a luminescence signal emitted by the detection method according to any one of [1] to [5] or the device according to [6]; and an information processing means for processing the light emission signal data obtained by the imaging terminal; The system is configured such that the imaging terminal and the information processing means are capable of two-way communication via a network. [Brief explanation of the drawings]

[0049] [Figure 1] Figure 1 is a schematic diagram illustrating one embodiment of the detection of a target substance according to the present disclosure. (A): When trigger enzyme A and inactive enzyme B simultaneously bind to a target substance, enzyme B is activated by trigger enzyme A. (B): Inactive enzyme C is activated by enzyme B. Inactive enzyme B is activated by enzyme C. The signal is amplified by the repetition of these two enzyme reactions. (C): The luminescent color of reporter substance D changes due to active enzyme C. [Figure 2] FIG. 1 is a diagram illustrating an example of a TEVp-based combination of two protease-regulated proteases (enzymes B and C). [Figure 3] FIG. 3 shows the results of measuring the cleavage activity of PCP1 activated by PCP2 (Test 1) and the results of measuring the cleavage activity of PCP2 activated by PCP1 (Test 2). [Example]

[0050] The present disclosure will be described in more detail below with reference to experimental examples and examples, but these are merely illustrative and the present disclosure is not limited to these examples. All references cited in this disclosure are incorporated herein by reference.

[0051] [DNA Construction] The DNA construct for E. coli expression of TEV protease (TEVp), the source of enzyme A (trigger protease: TP) and enzymes B and C (protease-controlled proteases: PCP), was prepared by adding the T17S / N68D / I77V / I138T / S153N / T180A / S219P mutations, which contribute to improving the solubility of the recombinant protein, to the wild-type TEVp sequence, and adding a polyarginine tag, which has also been reported to improve the solubility of the recombinant. The artificial DNA sequence (eTEVp1.0) was synthesized with codons optimized for E. coli expression, and cloned into the pRSETB vector.

[0052] A DNA construct (SEQ ID NO: 5) encoding TP (enzyme A, trigger protease, SEQ ID NO: 4) was prepared by adding A169L / F217K mutations to eTEVp1.0 using the hot fusion method.

[0053] The fragments used for the DNA construct encoding PCP were amplified by PCR using the eTEVp1.0 construct as a template, with the N-terminal region sequence (11-118) and the C-terminal region sequence (119-219). The P-tag (EKLASIKEKAASAKEKLASIKEKAASAKEKLASIKEAAAKEAAAKEAAAK, SEQ ID NO: 6) or N-tag (EISALEKEASAAEKEISALEKEASAAEKEISALEKEAAAKEAAAKEAAAK, SEQ ID NO: 7) with a protease cleavage sequence added was used as the activity-regulating sequence P (P-tag plus cleavage sequence ENLYF E G (SEQ ID NO: 3)), activity regulatory sequence N (N-tag with cleavage sequence KNLYFQG (SEQ ID NO: 2) was added to the DNA sequence encoding Sumire. To improve solubility, the DNA sequence encoding Sumire was amplified by PCR. For the DNA construct (sequence number 9) encoding PCP1 (enzyme B, sequence number 8), a construct was created by incorporating the C-terminal region sequence, activity regulatory sequence P, N-terminal region sequence, and Sumire into the pRSETB vector using the hot fusion method in this order, and the N171D / N176T mutations were further added using the hot fusion method. The DNA construct (sequence number 11) encoding PCP2 (enzyme C, sequence number 10) was created by inserting the C-terminal region sequence, activity regulatory sequence N, N-terminal region sequence, and Sumire into the pRSETB vector in the same order as PCP1, and introducing the D148R / T173A / N177K mutations.

[0054] To create a luminescent indicator for protease activity, the DNA sequence of a circularly permuted mutant of Venus (YFP sequence), the DNA sequence encoding ECFP (CFP sequence), the P-tag sequence, the N-tag sequence, the DNA sequence encoding the chemiluminescent protein nanoKAZ (nanoKAZ sequence), and the sequence cleaved by PCP1 (cleavage sequence K NLYFQG (SEQ ID NO: 2)), a sequence cleaved by PCP2 (cleavage sequence ENLYF E G (SEQ ID NO: 3) was amplified using PCR. The DNA construct (SEQ ID NO: 13) of the protease activity luminescent indicator 1 (reporter substance D1, SEQ ID NO: 12) corresponding to PCP1 contains N-Tag, YFP sequence, cleavage sequence K The gene was constructed by inserting NLYFQG (SEQ ID NO: 2), the CFP sequence, and the nanoKAZ sequence, in that order, into the pRSETB vector using overlap PCR and the hot fusion method. The DNA construct (SEQ ID NO: 15) of the protease activity luminescent indicator 2 (reporter substance D2, SEQ ID NO: 14) corresponding to PCP2 also contains a P-Tag, a YFP sequence, and a cleavage sequence ENLYF. E G (SEQ ID NO: 3), the CFP sequence, and the nanoKAZ sequence were inserted into the pRSETB vector.

[0055] Protein expression and purification E. coli JM109 strain was transformed with the TP and protease activity luminescent indicator constructs and cultured in 200 ml of LB medium at 23°C for 60 hours. After centrifugation, the pellet was suspended in 10 ml of suspension buffer containing 50 mM Tris-Cl (pH 8.0) and 20 mM imidazole and disrupted using a French press. After centrifugation to remove cell debris, the soluble fraction was applied to a Ni-NTA column. The column was washed with 3 ml of suspension buffer and then eluted with 1.5 ml of elution buffer containing 50 mM Tris-Cl (pH 8.0) and 200 mM imidazole. The eluted sample was loaded onto a PD10 column, and the buffer was replaced with 50 mM Tris-Cl (pH 8.0) and 0.5 mM EDTA to prepare the experimental sample. For PCP1 and PCP2, E. coli BL21 (DE3 codon plus) was transformed with the constructs and pre-cultured overnight at 37°C in 2 ml of LB medium. The pre-culture solution was transferred to 200 ml of LB medium and cultured at 37°C. IPTG was added to a final concentration of 1 mM when the OD was between 0.4 and 0.6, and the culture was continued for an additional 3 hours at 23°C. The samples were then purified in the same manner as for the TP and protease activity luminescent indicators and used for experiments.

[0056] Tests 1 and 2 Cleavage activity was measured using PCP1 and PCP2 prepared as described above. In Test 1, samples of activated PCP1 activated by the addition of active PCP2 and inactive PCP1 without PCP2 were prepared, and the change in the indicator signal when mixed with protease activity luminescent indicator 1 was measured. In Test 2, samples of activated PCP2 activated by the addition of active PCP1 and inactive PCP2 without PCP1 were prepared, and the change in the indicator signal when mixed with protease activity luminescent indicator 2 was measured. In both tests, PCP1, PCP2, and the protease activity luminescent indicator were adjusted to a final concentration of 1 μM in the measurement sample using a buffer containing 50 mM Tris-HCl (pH 8.0), 2.5 mM EDTA, and 1 mM DTT. Measurements were performed at room temperature using a multimode plate reader, and the ratio of the YFP signal (530 nm) to the CFP signal (480 nm) was measured over time when the protease activity luminescent indicator was excited at 430 nm. In both tests, a significant difference was observed in the signal change of the luminescent protease activity indicator between the active and inactive forms, confirming that PCP1 and PCP2 can regulate each other's activity.

Claims

1. A method for detecting a target substance in a sample, comprising: an enzyme A having a portion capable of binding to a target substance; an inactive enzyme B that can be converted from an inactive form to an active form by the action of enzyme A; an inactive enzyme C that can be converted from an inactive form to an active form by the action of enzyme B and can convert enzyme B from an inactive form to an active form; A method comprising contacting the sample with a reporter substance D capable of emitting a signal by the action of enzyme B and / or enzyme C.

2. The method according to claim 1 , wherein enzyme B has a portion capable of binding to the target substance, and enzymes A and B are both capable of binding to the target substance.

3. The method according to claim 1 , wherein enzyme A is capable of activating enzyme B by binding to the target substance together with inactive enzyme B.

4. A method for detecting a target substance, comprising amplifying a signal using enzyme B and enzyme C, Enzyme B is converted from an inactive form to an active form by enzyme C, Enzyme C is converted from an inactive form to an active form by enzyme B, A method comprising utilizing, measuring, or detecting the enzymatic activity of enzyme B and / or enzyme C obtained by amplification through an enzymatic reaction between enzyme B and enzyme C.

5. The method of any one of claims 1 to 4, wherein enzyme B and enzyme C are each independently selected from the group consisting of protease, kinase, dephosphorylating enzyme, glycosylation enzyme, acetylation enzyme, succinylation enzyme, propyanylation enzyme, methylation enzyme, ubiquitination enzyme, nitrosylation enzyme, and lipidation enzyme.

6. 1. A device for detecting a target substance in a sample, comprising: A reagent portion and a substrate on which the reagent portion is formed, The reagent part comprises: an enzyme A having a portion capable of binding to a target substance; an inactive enzyme B that can be converted from an inactive form to an active form by the action of enzyme A; an inactive enzyme C that can be converted from an inactive form to an active form by the action of enzyme B and can convert enzyme B from an inactive form to an active form; a reporter substance D capable of emitting a signal upon the action of enzyme B and / or enzyme C.

7. A reagent for detecting a target substance in a sample, comprising: an enzyme A having a portion capable of binding to a target substance; an inactive enzyme B that can be converted from an inactive form to an active form by the action of enzyme A; an inactive enzyme C that can be converted from an inactive form to an active form by the action of enzyme B and can convert enzyme B from an inactive form to an active form; a reporter substance D capable of emitting a signal through the action of enzyme B and / or enzyme C.

8. 1. A system for detecting a target substance in a sample, comprising: an imaging terminal that acquires a luminescence signal emitted by the detection method according to any one of claims 1 to 4 or the device according to claim 6; and an information processing means for processing the light emission signal data obtained by the imaging terminal; The system is configured such that the imaging terminal and the information processing means are capable of two-way communication via a network.

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