Method for measuring trace substances using hydrolytic enzymes

Abstract

Provided is an electrochemical measurement method which makes it possible to easily detect or quantify a trace material.　The electrochemical measurement method uses a substrate-marker compound labeled with a marker that is used for converting a substrate into a substrate-marker compound suitable for electrochemical measurement by bonding with the substrate, and that has the property of adsorbing to an electrode.

Description

[Technical Field]

[0001] The present disclosure relates to an electrochemical method for measuring trace substances using hydrolytic enzymes. [Background technology]

[0002] When performing qualitative or quantitative measurement of a target substance by electrochemical measurement using enzymes, oxidoreductases are generally used. However, there are few cases in which enzymes other than oxidoreductases, such as hydrolases, are used. In optical techniques, measurements using hydrolases are common and are known to be used for measuring enzyme activity and for measuring endotoxins and (1→3)-β-D-glucans, which are substances derived from microorganisms (see, for example, Patent Document 1).

[0003] Endotoxin is a lipopolysaccharide that constitutes the cell walls of gram-negative bacteria. Because endotoxin is a highly stable substance, its removal or inactivation is difficult if it is introduced into pharmaceuticals, medical devices, dialysis fluids, and other products during their manufacturing or use. Endotoxin is a typical pyrogen, and even trace amounts of endotoxin can cause fever in humans or animals when administered, potentially leading to highly lethal diseases such as intravascular blood coagulation associated with sepsis. Therefore, in fields such as injectables, medical devices, biopharmaceuticals, regenerative medicine products, and dialysis agents, it is important that products are endotoxin-free (pyrogen-free). Therefore, there is a market need for technology that can rapidly and accurately measure the presence or absence of endotoxin contamination.

[0004] Currently, the Limulus test is the mainstream method for measuring endotoxin. The Limulus test is a test method based on the phenomenon in which an extract of horseshoe crab hemocytes (Limulus Amebocyte Lysate, hereinafter also referred to as LAL) reacts with endotoxin, and known methods include the gelation method, turbidimetric method, colorimetric method, and fluorometric method (see, for example, Patent Documents 1 and 2). Measurement reagents using LAL are referred to as LAL reagents, lysate reagents, Limulus reagents, etc., all of which are synonymous.

[0005] The principle of endotoxin detection using LAL is shown in the schematic diagram in Figure 1. Endotoxin converts the enzyme precursor factor C into activated factor C. Activated factor C acts as a hydrolase, converting the enzyme precursor factor B into activated factor B. Activated factor B is also a hydrolase, converting proclotting enzyme into activated clotting enzyme. The gelation method and turbidimetric method measure endotoxin by measuring the degree to which activated clotting enzyme acts as a hydrolase to convert coagulogen in the LAL reagent into clottable coagulin. On the other hand, the colorimetric method utilizes the hydrolase activity of activated clotting enzyme, which cleaves the bond between the peptide and the labeled peptide designed for endotoxin detection. The resulting color of the labeled peptide is then measured to measure endotoxin. The fluorescence method utilizes the phenomenon in which a labeled peptide designed for endotoxin measurement is acted on by a hydrolase in LAL, resulting in the production of a labeled substance from the labeled peptide, and measures the fluorescence derived from the labeled substance to measure endotoxin (see, for example, Patent Document 2).

[0006] However, because gelation and turbidimetric methods measure the coagulation that occurs in the sample, quantification can be difficult under certain conditions, such as when measuring highly turbid or colored samples. Furthermore, the amount of LAL reagent used is relatively large, and specialized measuring equipment is required, making the measurement cost relatively high. Colorimetric and fluorometric methods have the drawback of being cumbersome and requiring large amounts of reagent. Therefore, there is a need for a method that can detect microbial substances such as endotoxins using fewer reagents and simpler procedures.

[0007] Against this background, attempts have been made to develop a Limulus test using electrochemical measurement. Specifically, a method has been proposed in which a labeled peptide is synthesized using an electrochemically active label, and the labeled peptide is reacted with activated clotting enzyme, and the label generated from the labeled peptide is then electrochemically measured.

[0008] Non-Patent Document 1 proposes an electrochemical method for measuring endotoxin using a peptide bound to para-aminophenol (p-AP). Patent Document 3 proposes a method for quantifying endotoxin using a peptide bound to para-methoxyaniline, taking into account the problem that the electrochemical activity of p-AP is rapidly lost over time. Patent Document 4 discloses a proposal for modifying the electrode structure to improve the detection sensitivity in electrochemical measurement. Patent Document 5 discloses electrochemical measurement using a phenylenediamine derivative such as N,N-dimethyl-p-phenylenediamine (DMPD).

[0009] However, there is still little knowledge about techniques that utilize hydrolase activity to achieve electrochemical measurement of trace substances such as endotoxins, rather than the conventional method that utilizes oxidoreductases, and continuous technological development is required.

[0010] Trypsin is a type of serine protease, and measuring trypsin activity in blood is commonly performed in biochemical testing, such as for diagnosing pancreatic diseases. Gingipain is a type of protease produced by Porphyromonas gingivalis, a major periodontal pathogen. Measuring gingipain activity in oral samples is also used to diagnose periodontal disease in subjects. Measuring the activity of other hydrolases, such as alkaline phosphatase, esterase, and glycosidase, is commonly performed in various fields, including biochemical research and medicine, and a simple method for measuring these enzyme activities is needed. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] International Publication No. 95 / 14931 Brochure [Patent Document 2] Japanese Patent Application Laid-Open No. 2009-150903 [Patent Document 3] Japanese Patent Application Laid-Open No. 2015-187607 [Patent Document 4] Japanese Patent Application Publication No. 2018-072331 [Patent Document 5] International Publication No. 2017 / 141961 Brochure [Non-patent literature]

[0012] [Non-Patent Document 1] KY Inoue et. al, Analyst, (2013) 138, 6523-6531 Summary of the Invention [Problem to be solved by the invention]

[0013] An objective of the present disclosure is to provide a novel electrochemical method for measuring a target substance using hydrolase activity. [Means for solving the problem]

[0014] As a result of intensive research aimed at solving the above problems, the present inventors have discovered that trace substances such as endotoxins can be easily measured by electrochemically measuring a label produced by reacting a hydrolase with a substrate-labeled compound obtained by binding a label having the property of being adsorbed to an electrode with a substrate designed for measurement, and have completed the present invention, which includes this finding as one embodiment. The present inventors have also discovered that trypsin and gingipain can be easily measured, which has also been completed as one embodiment.

[0015] The present disclosure encompasses the following embodiments. [1] An electrochemical measurement method characterized by using a substrate-labeled compound labeled with a label that is used to convert a substrate into a substrate-labeled compound suitable for electrochemical measurement by binding to the substrate, and that has the property of being adsorbed to an electrode. [2] The method according to embodiment 1, wherein the label is released from the substrate-label compound by the action of a hydrolase, and the released label is measured electrochemically. [3] The method according to embodiment 1 or 2, which is for measuring the hydrolase activity of a test sample or a substance that activates a hydrolase that may be contained in the test sample. [4] The method according to any one of embodiments 1 to 3, wherein the substance that activates the hydrolase comprises endotoxin or (1→3)-β-D-glucan. [5] The method of any one of embodiments 1 to 4, wherein the hydrolase is horseshoe crab factor C, factor B, factor G, and / or proclotting enzyme, or trypsin, gingipain, or phosphatase. [6] The label has the structure of Formula I [ka] [In the formula, R 7is a straight or branched chain C optionally substituted with hydrogen or one or more X's 1-6 Alkyl, C 1-6 Alkenyl, C 1-6 Alkynyl, C 3-9 cycloalkyl, phenyl, 1-naphthyl, 2-naphthyl, anthracenyl or phenanthrenyl; R 3 , R 4 , R 5 and R 6 each independently represents a straight or branched chain C optionally substituted with hydrogen or one or more Y's; 1-6 Alkyl, C 1-6 Alkenyl, C 1-6 Alkynyl, C 1-6 alkoxy, halo, nitro, cyano, carboxy, sulfone or amino; R 8 is selected from the group consisting of phenyl, 1-naphthyl, 2-naphthyl, anthracenyl, and phenanthrenyl, optionally substituted by one or more X; wherein X is a straight or branched chain alkyl group optionally substituted with a substituent selected from the group consisting of halo, amino, cyano, carboxy, carbonyl, alkoxy, and sulfone; 1-6 Alkyl, C 1-6 Alkenyl, C 1-6 Alkynyl, C 1-6 alkoxy, anilino, phenoxy, halo, hydroxy, nitro, carboxy, cyano, sulfone or amino; Y is selected from the group consisting of halo, amino, cyano, carboxy, carbonyl, alkoxy, and sulfone. or a phenylenediamine-based compound having the formula [ka] The method according to any one of embodiments 1 to 5, wherein the aminoanthraquinone compound has the formula: [7] The phenylenediamine compound represented by formula I is [ka] [ka] [ka] and [ka] or a compound selected from the group consisting of The aminoanthraquinone compound represented by formula (II) has the following structure: [ka] 1-amino-4-hydroxyanthraquinone having the formula The following structure [ka] 2-amino-3-hydroxyanthraquinone having or a salt, anhydrate or solvate thereof.

[0016] [8] A reagent for electrochemically measuring hydrolase activity or a substance that activates a hydrolase, comprising a substrate-labeled compound, wherein the label in the substrate-labeled compound has the property of being adsorbed to an electrode. [9] A composition for measuring hydrolase activity or a substance that activates a hydrolase, comprising the reagent according to embodiment 8.

[10] An electrode comprising the reagent of embodiment 8 or the composition of embodiment 9.

[11] Electrochemical measurement system using the electrode according to embodiment 10

[12] The label has the structure of Formula I [ka] [In the formula, R 7 is a straight or branched chain C optionally substituted with hydrogen or one or more X's 1-6 Alkyl, C 1-6Alkenyl, C 1-6 Alkynyl, C 3-9 cycloalkyl, phenyl, 1-naphthyl, 2-naphthyl, anthracenyl or phenanthrenyl; R 3 , R 4 , R 5 and R 6 each independently represents a straight or branched chain C optionally substituted with hydrogen or one or more Y's; 1-6 Alkyl, C 1-6 Alkenyl, C 1-6 Alkynyl, C 1-6 alkoxy, halo, nitro, cyano, carboxy, sulfone or amino; R 8 is selected from the group consisting of phenyl, 1-naphthyl, 2-naphthyl, anthracenyl, and phenanthrenyl, optionally substituted by one or more X; wherein X is a straight or branched chain alkyl group optionally substituted with a substituent selected from the group consisting of halo, amino, cyano, carboxy, carbonyl, alkoxy, and sulfone; 1-6 Alkyl, C 1-6 Alkenyl, C 1-6 Alkynyl, C 1-6 alkoxy, anilino, phenoxy, halo, hydroxy, nitro, carboxy, cyano, sulfone or amino; Y is selected from the group consisting of halo, amino, cyano, carboxy, carbonyl, alkoxy, and sulfone. or a phenylenediamine-based compound having the formula [ka] The reagent of embodiment 8, the composition of embodiment 9, the electrode of embodiment 10, or the system of embodiment 11, wherein the aminoanthraquinone-based compound has the formula:

[12] The phenylenediamine compound represented by formula I is [ka] [ka] [ka] and [ka] or a compound selected from the group consisting of The aminoanthraquinone compound represented by formula (II) has the following structure: [ka] 1-amino-4-hydroxyanthraquinone having the formula The following structure [ka] 2-amino-3-hydroxyanthraquinone having or a salt, anhydride or solvate thereof.

[0017] This specification includes the disclosure of Japanese Patent Application No. 2020-073489, from which this application claims priority. [Effects of the Invention]

[0018] As an effect of the present disclosure, hydrolase activity or a substance that activates a hydrolase can be stably measured. [Brief explanation of the drawings]

[0019] [Figure 1] Schematic diagram of the principle of endotoxin measurement using LAL. [Figure 2] Cyclic voltammetry was performed using N-(3-isopropylphenyl) p-phenylenediamine, and the results are shown as a plot of the sweep rate and IOmax. [Figure 3]Cyclic voltammetry was performed using N-(3-tert-butylphenyl)-p-phenylenediamine, and the results are shown as a plot of the sweep rate and IOmax. [Figure 4] Cyclic voltammetry was performed using N-(3,5-di-tert-butylphenyl)-p-phenylenediamine, and the results are shown as a plot of the sweep rate and IOmax. [Figure 5] Cyclic voltammetry was performed using N-(4-aminophenyl)-N'-phenyl-p-phenylenediamine, and the results are shown as a plot of the sweep rate and IOmax. [Figure 6] Cyclic voltammetry was performed using p-aminophenol, and the results are shown as a plot of the sweep rate and IOmax. [Figure 7] Cyclic voltammetry was performed using p-aminophenol, and the results are plotted as a function of sweep rate and IOmax. The horizontal axis represents the square root of the sweep rate. [Figure 8] This shows the relationship between the endotoxin concentration in the test sample and the oxidation current value at 120 mV when cyclic voltammetry was performed using a substrate-labeled compound in which Leu-Gly-Arg (substrate) and N-(3-isopropylphenyl) p-phenylenediamine (labeled compound) were bound. [Figure 9] This shows the relationship between the endotoxin concentration in the test sample and the oxidation current value at 120 mV when cyclic voltammetry was performed using a substrate-labeled compound in which Leu-Gly-Arg (substrate) and N-(3-tert-butylphenyl)-p-phenylenediamine (labeled compound) were bound. [Figure 10] This figure shows the relationship between the endotoxin concentration in the test sample and the oxidation current value at 110 mV when cyclic voltammetry was performed using a substrate-labeled compound in which Leu-Gly-Arg (substrate) and N-(3,5-di-tert-butylphenyl)-p-phenylenediamine (labeled compound) were bound. [Figure 11]1 shows the relationship between the endotoxin concentration in a test sample and the oxidation current value when chronoamperometry was performed using a substrate-labeled compound in which labeled N-(3-isopropylphenyl)-p-phenylenediamine and the substrate Leu-Gly-Arg were bound. [Figure 12] The graph shows the results of cyclic voltammetry after reacting a substrate-labeled compound, consisting of labeled N-(3-tert-butylphenyl)-p-phenylenediamine and the substrate Leu-Gly-Arg, with trypsin solutions prepared at various concentrations. [Figure 13] The graph shows the results of cyclic voltammetry after reacting culture supernatant with a substrate-labeled compound, consisting of labeled N-(3-tert-butylphenyl)-p-phenylenediamine and the substrate Val-Pro-Arg. The culture supernatant concentration is expressed as a relative value, with the original solution set at 1. A concentration of 0 represents the negative control, which was heat-treated at 95°C for 30 minutes. DETAILED DESCRIPTION OF THE INVENTION

[0020] In certain embodiments, the present disclosure provides a label having the property of being adsorbed to an electrode. This label can bind to a substrate to form a substrate-label compound. That is, in certain embodiments, the present disclosure provides a substrate-label compound. The substrate-label compound can be suitable for electrochemical measurement. That is, in certain embodiments, the present disclosure provides an electrochemical measurement method using a substrate-label compound. In another embodiment, the present disclosure provides an electrochemical measurement method using a substrate-label compound labeled with a label having the property of being adsorbed to an electrode, the label being used to convert the substrate into a substrate-label compound suitable for electrochemical measurement by binding to the substrate.

[0021] When the bond between the substrate-labeled compound is cleaved by the action of a hydrolase or the like, a label can be generated. The generated label can be measured electrochemically. That is, in certain embodiments, the present disclosure provides an electrochemical measurement method in which a label is generated from a substrate-labeled compound by the action of a hydrolase, and the generated label is measured electrochemically. The substrate in the substrate-labeled compound can be any compound selected so that the hydrolase acts on the substrate-labeled compound, and can be, for example, but is not limited to, an amino acid, a peptide, a sugar, a nucleic acid, or the like.

[0022] In certain embodiments, the electrochemical measurement method of the present disclosure can be used to measure hydrolase activity in a test sample or a substance that activates a hydrolase that may be contained in the test sample. The substance that activates a hydrolase may include endotoxin or (1→3)-β-D-glucan.

[0023] In one embodiment, the hydrolase may be any of various enzymes classified in EC group 3. Examples include proteases such as horseshoe crab factor C, factor B, factor G, proclotting enzyme (also called clotting enzyme precursor), subtilisin, proteinase K, trypsin, chymotrypsin, thrombin, plasmin, clotting factor Xa, clotting factor VIIa, clotting factor IXa, clotting factor XIa, clotting factor XIIa, protein C, kallikrein, papain, actinidin, caspase, pepsin, renin, chymosin, coagulase, calpain, angiotensin-converting enzyme, and gingipain, as well as carboxylesterase, triacylglycerol lipase, acetonitrile, and the like. Examples of enzymes that act on carbon-nitrogen bonds other than peptides, such as asparaginase and urease, include, but are not limited to, phosphatases such as acetylcholinesterase, acetyl-CoA hydrolase, alkaline phosphatase, acid phosphatase, and protein phosphatase; esterases such as 5'-nucleotidase and 3'-nucleotidase; glycosylases such as α-amylase and α-glucosidase; ether hydrolases such as adenosylhomocysteinase and alkenylglycerophosphocholine hydrolase; enzymes that act on carbon-nitrogen bonds other than peptides, such as asparaginase and urease; and enzymes that act on acid anhydrides, such as ATPase.

[0024] In some embodiments, the label may be a phenylenediamine-based compound. In some embodiments, the phenylenediamine-based compound may be a compound of the following general formula I: [ka] [In the formula, R 7 is a straight or branched chain C optionally substituted with hydrogen or one or more X's 1-6 Alkyl, C 1-6 Alkenyl, C 1-6 Alkynyl, C 3-9 cycloalkyl, phenyl, 1-naphthyl, 2-naphthyl, anthracenyl or phenanthrenyl; R 3 , R 4 , R 5 and R6 each independently represents a straight or branched chain C optionally substituted with hydrogen or one or more Y's; 1-6 Alkyl, C 1-6 Alkenyl, C 1-6 Alkynyl, C 1-6 alkoxy, halo, nitro, cyano, carboxy, sulfone or amino; R 8 is selected from the group consisting of phenyl, 1-naphthyl, 2-naphthyl, anthracenyl, and phenanthrenyl, optionally substituted by one or more X; wherein X is a straight or branched chain alkyl group optionally substituted with a substituent selected from the group consisting of halo, amino, cyano, carboxy, carbonyl, alkoxy, and sulfone; 1-6 Alkyl, C 1-6 Alkenyl, C 1-6 Alkynyl, C 1-6 alkoxy, anilino (i.e., PhNH-group), phenoxy, halo, hydroxy, nitro, carboxy, cyano, sulfone, or amino; Y is selected from the group consisting of halo, amino, cyano, carboxy, carbonyl, alkoxy, and sulfone.

[0025] In one embodiment, the phenylenediamine compound is N-(3-isopropylphenyl)-p-phenylenediamine [ka] It could be.

[0026] In one embodiment, the phenylenediamine compound is N-(3-tert-butylphenyl)-p-phenylenediamine [ka] It could be.

[0027] In one embodiment, the phenylenediamine compound is N-(3,5-di-tert-butylphenyl)-p-phenylenediamine [ka] It could be.

[0028] In one embodiment, the phenylenediamine compound is N-(4-aminophenyl)-N'-phenyl-p-phenylenediamine [ka] It could be.

[0029] Phenylenediamine compounds can exist in redox and ionized states. In the above chemical formula, the phenylenediamine compounds are described in neutral and reduced forms. However, in addition to these forms, phenylenediamine compounds can also exist in reduced forms (diamine forms). When a free phenylenediamine compound is adsorbed onto an electrode and a potential is applied to the electrode, the phenylenediamine compound can be oxidized to become an oxidized form (diimine form).

[0030] In another embodiment, the label can be an aminoanthraquinone-based compound. In one embodiment, the aminoanthraquinone-based compound can be a compound of the following general formula II: [ka]

[0031] In some embodiments, the aminoanthraquinone compound can be 1-amino-4-hydroxyanthraquinone, which has the following structure: [ka] (CAS number 116-85-8).

[0032] In another embodiment, the aminoanthraquinone compound can be 2-amino-3-hydroxyanthraquinone, which has the following structure: [ka] (CAS No. 117-77-1). The aminoanthraquinone compound may be a salt, an anhydride, or a solvate thereof.

[0033] As used herein, alkyl refers to a straight or branched chain hydrocarbon having, for example, 6 carbon atoms. Examples of alkyl include, but are not limited to, methyl, ethyl, propyl, isopropyl, isobutyl, n-butyl, tert-butyl, isopentyl, n-pentyl, and heptyl.

[0034] As used herein, the number of atoms (such as carbon atoms) is represented, for example, as "C alkyl," which refers to an alkyl group having x to y carbon atoms. Similar notations are used for other substituents and ranges.

[0035] As used herein, the terms "optionally substituted" and "substituted or unsubstituted" refer to any substitution with one or more substituents, and include multiple degrees of substitution.

[0036] Aminoanthraquinone compounds may exist in redox and ionized states. In the above chemical formula, the aminoanthraquinone compounds are described in neutral and reduced forms. However, in addition to this form, aminoanthraquinone compounds may also be oxidized, semi-oxidized, or reduced. Furthermore, aminoanthraquinone compounds may be neutral or cationic. For convenience, when referring to aminoanthraquinone compounds, for example, aminoanthraquinone compounds represented by the above chemical formula, this includes neutral or cationic, oxidized, semi-oxidized, or reduced forms. For example, after adding a neutral and oxidized aminoanthraquinone compound to a measurement system, it may change to an oxidized and cationic compound depending on the pH of the solution or electron transfer, and such compounds are also included in the aminoanthraquinone compounds. Furthermore, when referring to aminoanthraquinone compounds, this includes their salts, acid addition salts, anhydrides, and solvates. Salts include, but are not limited to, salts of Group 1 elements and salts of Group 17 elements, such as Na salts, K salts, Cl salts, Br salts, etc. Acid addition salts include, but are not limited to, hydrochlorides, sulfates, sulfites, and nitrates.

[0037] The phenylenediamine compounds and aminoanthraquinone compounds may be artificially synthesized, obtained as natural products, or commercially available. When synthesized, organic synthesis is performed using conventional organic synthesis techniques, and the products can be confirmed by NMR, IR, mass spectrometry, etc.

[0038] Phenylenediamine compounds and aminoanthraquinone compounds can be adsorbed onto the electrode surface by contacting them with an electrode. In this case, no special procedure, such as activating the electrode surface by acid treatment, is required to achieve adsorption onto the electrode. In certain embodiments, the property of a compound (labeled compound) to adsorb onto an electrode refers to the property of the compound to physically adsorb onto the electrode, and the electrode can be made of materials such as carbon, gold, or platinum. This also includes embodiments in which the compound is adsorbed onto a primary material such as carbon powder or a carbonaceous material, and the primary material is then immobilized onto an electrode. However, this is merely a description of the properties of the compound and does not limit the method of use of the compound. That is, in certain embodiments, a method is provided in which a compound is adsorbed onto a primary material such as carbon powder or a carbonaceous material, and the primary material is then immobilized onto an electrode.

[0039] In certain embodiments, the present disclosure provides a reagent for electrochemically measuring hydrolase activity or a substance that activates a hydrolase, comprising a substrate-labeled compound. The label in the substrate-labeled compound may have the property of being adsorbed to an electrode. In certain embodiments, compounds that do not adsorb to an electrode are excluded from the labels of the present disclosure.

[0040] Whether or not a label alone or a label in a substrate-label compound adsorbs to an electrode can be confirmed using any of the electrochemical measurement methods described below. For example, whether or not a test compound adsorbs to an electrode can be confirmed by cyclic voltammetry. The sweep rate is varied, for example, from 4 mV / sec to 200 mV / sec, and the change in the maximum oxidation current (IOmax) is examined. Generally, when a label adsorbs to an electrode, the cyclic voltammetry sweep rate and IOmax are proportional. Furthermore, when a label diffuses without adsorbing to the electrode, IOmax is proportional to the 0.5 power of the sweep rate (see, for example, Electrochemical Measurement Manual: Basics (edited by the Electrochemical Society), pp. 74-94). From this relationship between IOmax and sweep rate, it is possible to determine whether a compound adsorbs to the electrode or diffuses without adsorbing.

[0041] In one embodiment, the present disclosure provides a composition for measuring hydrolase activity or a substance that activates a hydrolase, comprising the reagent for electrochemical measurement. In another embodiment, the present disclosure provides an electrode comprising the reagent for electrochemical measurement or the composition. In another embodiment, the present disclosure provides a system for electrochemical measurement using the electrode.

[0042] In one embodiment, the present disclosure provides a method for electrochemically measuring a substance that activates a hydrolase, such as a microbial substance such as endotoxin or (1→3)-β-D-glucan. Hereinafter, the electrochemical method for measuring endotoxin will be described in detail as an example of the present disclosure.

[0043] When a test subject that may contain endotoxin is mixed with an LAL reagent, factor C in the LAL reagent is activated by the endotoxin, followed by factor B, and then pro-clotting enzyme is activated to clotting enzyme. When the clotting enzyme acts on the substrate-labeled compound of the present disclosure, a labeled product is produced. The activity unit of endotoxin is expressed as EU (endotoxin unit). Unless otherwise specified, 1 EU is defined as 1 international unit (IU) of endotoxin in this specification.

[0044] In one embodiment, the present disclosure provides a method for electrochemically measuring trypsin. When trypsin acts on a substrate-labeled compound according to the present disclosure, a labeled compound is produced. One enzyme unit (U) of trypsin activity is defined as the amount of enzyme that increases the absorbance at 253 nm by 0.003 in one minute at pH 7.6 and 25°C using N-benzoyl-L-arginine ethyl ester as a substrate. In another embodiment, the present disclosure provides a method for electrochemically measuring gingipain. When gingipain acts on a substrate-labeled compound according to the present disclosure, a labeled compound is produced. One enzyme unit (U) of gingipain activity is defined as the amount of enzyme that releases 1 μmol of pNA in one hour at pH 7.6 and 37°C using N-benzoyl-L-arginine-p-nitroaniline as a substrate.

[0045] The generated labeled substance undergoes an oxidation reaction at a specific potential, making it measurable by electrochemical methods. Additionally, the labeled substance according to the present disclosure has the property of adsorbing to an electrode. Therefore, the labeled substance according to the present disclosure is highly likely to be present near or on the surface of an electrode, enabling more sensitive measurements compared to when a labeled substance that does not adsorb to an electrode is used. A correlation exists between the current value resulting from the oxidation reaction of the labeled substance and the amount of the generated labeled substance, i.e., the endotoxin concentration, and this correlation can be used to quantify the concentration of the labeled substance. In certain embodiments, the labeled substance transfers electrons to the electrode while being oxidized, without receiving electrons from other compounds such as oxidoreductases or coenzymes. In this embodiment, the labeled substance does not simply function as a mediator that mediates electron transfer from other compounds such as oxidoreductases or coenzymes to the electrode, but rather functions as a compound that releases electrons.

[0046] In addition to the generated label, uncleaved substrate-labeled compounds may also be present in the measurement system. However, because the generated label and the substrate-labeled compound have different redox potentials, it is easy to distinguish between the current caused by the label and the current caused by the substrate-labeled compound. Furthermore, the method of the present disclosure allows electrochemical measurement of even trace amounts of label, making it possible to detect endotoxin concentrations with high accuracy. Furthermore, trypsin, gingipain, and phosphatase can be detected with high accuracy.

[0047] Furthermore, in the method of the present disclosure, because the analyte is detected by electrochemical measurement and the label has the property of adsorbing to the electrode, the reaction on the electrode surface can be detected with high sensitivity, and the concentration of the substance can be measured even in trace amounts of the analyte. Or, even trace amounts of the substance contained in the subject can be measured. High-sensitivity detection means that a smaller amount of analyte is required to detect the same amount of trace substance, which also means that the amount of LAL reagent used can be reduced. It also means that trace substances can be measured more inexpensively.

[0048] Furthermore, the present disclosure utilizes the generation of a label using a substrate-label compound, and because the label has stable electrochemical activity, accurate and stable detection is possible. These properties can be advantageous, for example, for long-term measurements. Furthermore, because the method disclosed herein is not a light-based detection method like colorimetry or turbidimetry, it can also be used to measure analytes with low transparency or multi-component analytes such as interstitial fluid.

[0049] Trace substances that can be measured by the methods of the present disclosure include, but are not limited to, endotoxin, and / or (1→3)-β-D-glucan, trypsin, gingipain, and phosphatase. In one embodiment, the trace substance measured is endotoxin. In another embodiment, the trace substance measured is (1→3)-β-D-glucan. In another embodiment, the trace substance measured is trypsin. In another embodiment, the trace substance measured is gingipain. In another embodiment, the trace substance measured is phosphatase. In one embodiment, the trace substance may be derived from a microorganism.

[0050] The method for detecting endotoxin and / or (1→3)-β-D-glucan according to the present disclosure will now be described. The reaction step involves contacting a test sample, which may contain endotoxin and / or (1→3)-β-D-glucan, with an LAL reagent and a substrate-labeled compound, which then undergoes a multi-step reaction to produce a labeled product from the substrate-labeled compound.

[0051] LAL reagents may be prepared using Limulus factors such as Factor C, Factor B, Factor G, and clotting enzymes isolated and purified from biological components, or LAL equivalents prepared using recombinant Limulus factors such as Factor C produced by genetic engineering. The horseshoe crab may be any species. Examples of horseshoe crabs include, but are not limited to, Tachypleus tridentatus, Limulus polyphemus, Carcinoscorpius rotundicauda, ​​and Tachypleus gigas.

[0052] The LAL reagent may be commercially available, such as Kinetic-QCL (Lonza Walkersville, Inc.), Endospecy (Seikagaku Corporation), Pyrochrome (Associates of Cape Cod, Inc.), Pyrotel-T (Associates of Cape Cod, Inc.), Pyrotel Multitest (Associates of Cape Cod, Inc.), and Endochrome-K (Charles River Laboratories, Inc.).

[0053] Alternatively, the LAL reagent may be a reagent reconstituted to contain only any Limulus factor. In one embodiment, the reconstituted LAL reagent contains Factor C, and may or may not contain other Limulus factors (Factor B, Factor G, Proclotting Enzyme). In another embodiment, the reconstituted LAL reagent contains Factor G, and may or may not contain other Limulus factors (Factor C, Factor B, Proclotting Enzyme). Reconstituted LAL reagents can be prepared by known methods (see, for example, Nakamura T, et al., J. Biochem. 1986 Mar; 99(3): 847-57). Recombinant Limulus factor can be obtained by expressing a Limulus factor gene in a host cell transformed with a nucleic acid encoding the gene. Host cells include mammalian cells and insect cells, such as, but not limited to, Chinese hamster ovary-derived cells (CHO cells), human embryonic kidney-derived cell lines (HEK cells, e.g., HEK293 cells), and Sf9 cells (see, e.g., WO 2012 / 118226 and WO 2014 / 092079).

[0054] In certain embodiments, the substrate in the substrate-labeled compound may be an amino acid or an oligopeptide. In this case, the substrate-labeled compound may be an oligopeptide having a label attached to one end and a protecting group for the amino acid or peptide attached to the other end. Examples of such amino acids or oligopeptides include those represented by XA-labels. Here, X represents a protecting group, and A represents an amino acid or oligopeptide. The protecting group X may be a group that protects the peptide, such as, but not limited to, tert-butoxycarbonyl (Boc), benzoyl, benzyl, acetyl (Ac), or benzyloxycarbonyl (Cbz). Furthermore, the protecting group may or may not be attached to the oligopeptide. Hereinafter, compounds referred to as substrates include those with and without a protecting group attached.

[0055] Examples of amino acids include α-amino acids and β-amino acids, and are not particularly limited as long as they can be used to release a label by the action of the lysate reagent. The oligopeptide is also not particularly limited as long as it can be used to generate a label by the action of the LAL reagent. The oligopeptide may have, for example, but not limited to, 2 to 10, 2 to 5, or 3 to 4 amino acids. In certain embodiments, the oligopeptide may have Arg(R) at the C-terminus. Examples of oligopeptides include X-Asp-Pro-Arg (X-DPR), X-Val-Pro-Arg (X-VPR), X-Leu-Thr-Arg (X-LTR), X-Met-Thr-Arg (X-MTR), X-Leu-Gly-Arg (X-LGR), X-Thr-Gly-Arg (X-TGR), X-Ile-Glu-Gly-Arg (X-IEGR), and X-Ser. X-Glu-Gly-Arg (X-EGR), X-Leu-Leu-Gly-Arg (X-LLGR), and X-Met-Leu-Gly-Arg (X-MLGR). In some embodiments, X can be a peptide having 0 to 7 amino acids.

[0056] Examples of tripeptides include Leu-Gly-Arg, Thr-Gly-Arg, and Val-Pro-Arg. Also included are tripeptides having an L-amino acid represented by the general formula: R1-Gly-Arg-label, where R1 represents an N-blocked amino acid.

[0057] Also included is a tetrapeptide of the general formula: R2-A1-A2-A3-A4-labeled compound, where R2 represents hydrogen, a blocking aromatic hydrocarbon, or an acyl group, A1 represents an L-amino acid or a D-amino acid selected from Ile, Val, or Leu, A2 represents Glu or Asp, A3 represents Ala or Cys, and A4 represents Arg.

[0058] Specific examples of oligopeptides having a label attached to one end and a peptide protecting group attached to the other end include Boc-Leu-Gly-Arg-labeled product, acetate-Leu-Gly-Arg-labeled product, acetate-Ile-Glu-Ala-Arg-labeled product, Boc-Val-Pro-Arg-labeled product, and acetate-Val-Pro-Arg-labeled product.

[0059] In another embodiment, the substrate in the substrate-labeled compound may be a phosphate group. That is, in this case, the substrate-labeled compound may be a phosphate ester compound having a label at one end and a phosphate group at the other end. Examples of the compound structure are shown below.

[0060] In one embodiment, the substrate-label compound is N-(3-isopropylphenyl)-p-phenylenediamine phosphate. [ka] It could be.

[0061] In one embodiment, the substrate-label compound is N-(3-tert-butylphenyl)-p-phenylenediamine phosphate. [ka] It could be.

[0062] In one embodiment, the substrate-label compound is N-(3,5-di-tert-butylphenyl)-p-phenylenediamine phosphate. [ka] It could be.

[0063] In one embodiment, the substrate-label compound is N-(4-aminophenyl)-N'-phenyl-p-phenylenediamine phosphate [ka] It could be.

[0064] When a sample containing a trace substance is contacted with an LAL reagent and a substrate-labeled compound, a buffer may be used. The buffer can be selected depending on the hydrolase used in the measurement. For example, when an LAL reagent is used, the buffer can be one that adjusts the solution pH to 6.0 to 9.0, e.g., 7.0 to 8.5. Examples of buffers include, but are not limited to, Tris-acetate buffer, Tris-HCl buffer, phosphate buffer, HEPES buffer, PIPES buffer, etc. The buffer can also be used when measuring other hydrolases such as trypsin, gingipain, and phosphatase (including alkaline phosphatase, acid phosphatase, and protein phosphatase). Similarly A suitable solution pH (for example, pH 2 to 13) and buffer solution can be selected to allow the enzyme reaction to proceed.

[0065] In measurements using an LAL reagent, the activated clotting enzyme produced by the multistep reaction liberates the label from the peptide substrate-label compound. During the multistep reaction and liberation reaction, the solution may be heated to activate the reaction. The reaction temperature for the multistep reaction and liberation reaction may be, for example, 20°C to 50°C, 25°C to 45°C, or 30°C to 40°C, for example, approximately 37°C. In certain embodiments, the reaction time may be, for example, at least 1 minute, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 30 minutes, at least 1 hour, for example, at least 2 hours, but is not limited thereto. In other embodiments, the reaction time may be, for example, at most 2 hours, at most 1 hour, at most 30 minutes, at most 20 minutes, at most 15 minutes, at most 5 minutes, for example, at most 1 minute, but is not limited thereto.

[0066] When measuring other hydrolases such as trypsin, gingipain, and phosphatase (including alkaline phosphatase, acid phosphatase, and protein phosphatase), the label is released from the substrate-label compound by the hydrolase. To activate the hydrolysis reaction, the solution may be heated. The reaction temperature may be, for example, 20°C to 50°C, 25°C to 45°C, or 30°C to 40°C, for example, approximately 37°C. In certain embodiments, the reaction time may be, for example, but is not limited to, 1 minute or more, 5 minutes or more, 10 minutes or more, 15 minutes or more, 20 minutes or more, 30 minutes or more, 1 hour or more, for example, 2 hours or more. In other embodiments, the reaction time may be, but is not limited to, 2 hours or less, 1 hour or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, for example, 1 minute or less.

[0067] In certain embodiments, the methods of the present disclosure may measure endotoxin concentrations of 0.1 EU / L or greater, e.g., 0.2 EU / L or greater, 0.3 EU / L or greater, 0.4 EU / L or greater, 0.5 EU / L or greater, 0.6 EU / L or greater, 0.7 EU / L or greater, 0.8 EU / L or greater, 0.9 EU / L or greater, 1 EU / L or greater, 2 EU / L or greater, 3 EU / L or greater, 4 EU / L or greater, 5 EU / L or greater, 6 EU / L or greater, 7 EU / L or greater, 8 EU / L or greater, 9 EU / L or greater, e.g., 10 EU / L or greater. In certain embodiments, the methods of the present disclosure may measure endotoxin concentrations of 1000 EU / L or less, e.g., 900 EU / L or less, 800 EU / L or less, 700 EU / L or less, 600 EU / L or less, 500 EU / L or less, 400 EU / L or less, 300 EU / L or less, 200 EU / L or less, 100 EU / L or less, 90 EU / L or less, 80 EU / L or less, 70 EU / L or less, 60 EU / L or less, 50 EU / L or less, 40 EU / L or less, 30 EU / L or less, 20 EU / L or less, or 10 EU / L or less. In another embodiment, the method of the present disclosure can measure trypsin, gingipain, or phosphatase at a concentration of 0.1 U / L or more, e.g., 0.2 U / L or more, 0.5 U / L or more, 1 U / L or more, 1.5 U / L or more, 2 U / L or more, 5 U / L or more, 10 U / L or more, 50 U / L or more, 100 U / L or more, e.g., 1000 U / L or more. In another embodiment, the method of the present disclosure can measure trypsin, gingipain, or phosphatase at a concentration of 10,000 U / L or less, 5,000 U / L or less, 1000 U / L or less, 500 U / L or less, 100 U / L or less, 50 U / L or less, e.g., 10 U / L or less. Unless otherwise specified, numerical ranges in the present disclosure include both upper and lower limits (e.g., the numerical range a to b means a or more and b or less). The present disclosure also encompasses all combinations of the upper and lower limit values ​​exemplified for the numerical ranges. In one embodiment, the method of the present disclosure can measure endotoxin at a concentration of 0.1 to 1000 EU / L, for example, 1 to 1000 EU / L. In another embodiment, the method of the present disclosure can measure endotoxin at a concentration of 0.1 to 10000 U / L, for example, 1 to 10000 U / L. 1000 U / L of trypsin, gingipain, or phosphatase can be measured.

[0068] In certain embodiments, the present disclosure provides a reagent for electrochemically measuring hydrolase activity or a substance that activates a hydrolase, comprising a substrate-labeled compound, or a composition for measuring a substance comprising the reagent. These reagents or compositions may contain various substances necessary for the measurement. The concentration of the substrate-labeled compound in the reagent or composition can be selected appropriately depending on the measurement, and can be, for example, 0.01% (w / w) or more, 0.02% (w / w) or more, 0.03% (w / w) or more, 0.04% (w / w) or more, 0.05% (w / w) or more, 0.1% (w / w) or more, 0.2% (w / w) or more, 0.3% (w / w) or more, 0.4% (w / w) or more, 0.5% (w / w) or more, 1% (w / w) or more, 2% (w / w) or more, 3% (w / w) or more, 4% (w / w) or more, 5% (w / w) or more, 10% (w / w) or more, 20% (w / w) or more, 30% (w / w) or more, 40% (w / w) or more, or 50% (w / w) or more, but is not limited to these. In another embodiment, the concentration of the substrate-labeled compound in the reagent or composition can be, for example, but is not limited to, 99% (w / w) or less, 90% (w / w) or less, 50% (w / w) or less, 40% (w / w) or less, 30% (w / w) or less, 20% (w / w) or less, 10% (w / w) or less, 5% (w / w) or less, 4% (w / w) or less, 3% (w / w) or less, 2% (w / w) or less, 1% (w / w) or less, 0.5% (w / w) or less, 0.4% (w / w) or less, 0.3% (w / w) or less, 0.2% (w / w) or less, or 0.1% (w / w) or less. Note that "wt%" herein refers to % by weight on a dry weight basis.

[0069] The measurement step involves performing an electrochemical reaction on the solution after the release reaction. This allows the amount of trace substances (e.g., substances derived from microorganisms) to be quantified based on the measured current value. The solution after the reaction contains a label generated from the substrate-label compound, and the label is oxidized at a specific potential. A correlation is established between the current value resulting from this oxidation reaction and the amount of the label, i.e., the concentration of the trace substance (e.g., a substance derived from microorganisms). This correlation can be used to quantify the concentration of the label.

[0070] Measurement by electrochemical reaction can be performed by any method. Examples of measurement methods include, but are not limited to, amperometry and voltammetry. Examples of amperometry include, but are not limited to, chronoamperometry and differential pulse amperometry. Examples of voltammetry include, but are not limited to, normal pulse voltammetry, differential pulse voltammetry, and cyclic voltammetry.

[0071] In amperometry, for example, electrodes are placed in a mixed solution of the analyte, LAL reagent, and substrate-labeled compound, and measurements are performed based on the amperometry method. In one embodiment, a constant potential is applied to the working electrode, and the flowing current is measured. The potential is controlled relative to the reference electrode. The current flows between the working electrode and the counter electrode. If the current value is sufficiently small, the counter electrode may be omitted. In this case, the reference electrode can serve as the counter electrode. By plotting the voltage application time on the horizontal axis and the current value on the vertical axis, the current value after a certain time has elapsed since the start of application of the constant potential to the electrode can be graphed. A calibration curve showing the correlation between a sample with a known concentration, such as a known concentration of a trace substance, and the current value after a certain time has elapsed can be prepared in advance. This allows the concentration of the trace substance contained in a test sample, which may contain a microbial substance of unknown concentration, to be calculated from the measured current value. The applied voltage depends on the conditions and device settings, but can be, for example, -1000 mV to +1000 mV (vs. Ag / AgCl).

[0072] The electrodes are not particularly limited, and general electrodes used in electrochemical measurements can be used. For example, carbon electrodes such as glassy carbon, carbon paste, graphite, and diamond-like carbon, and electrochemically stable noble metals such as gold and platinum can be used as the working electrode. Electrochemically stable noble metals such as gold and platinum can be used as the counter electrode. An Ag / AgCl electrode or the like can be used as the reference electrode.

[0073] Furthermore, the measuring device may be a device generally used for electrochemical measurements, such as a device equipped with a potentiostat, a current amplifier, an information processing unit, etc. The information processing unit may have a CPU, memory, an external storage mechanism such as a HDD, a communication means such as a modem, a display, and input means such as a mouse and a keyboard. The information processing unit may analyze the electrical signal and calculate the concentration of the trace substance according to a program set in a predetermined area of ​​the internal memory or external storage device. The information processing device may be a general-purpose machine or a dedicated machine.

[0074] Preparation of substrate-labeled compounds The labeled compounds of the present disclosure can be linked to a substrate. In certain embodiments, the substrate-labeled compound can be a compound having a label attached to one end and a peptide protecting group attached to the other end, i.e., a protecting group-substrate-labeled compound. In another embodiment, the substrate-labeled compound can be a compound having a label attached to one end and a phosphate group attached to the other end, i.e., a phosphate-labeled compound. In certain embodiments, the substrate-labeled compound can be a compound without a protecting group attached. The substrate used can be synthesized by known synthetic methods. The structure of the substrate can be designed depending on the substrate specificity and reactivity of the hydrolase. The label can be attached to the substrate by a common method used in the field of organic synthesis. The prepared substrate-labeled compound can be separated and / or purified by thin-layer chromatography using a silica gel column or the like, and the product can be confirmed by mass spectrometry or NMR.

[0075] The practice of the present disclosure will employ conventional techniques of chemistry, organic synthesis, biochemistry, molecular biology, and electrochemistry, which are within the capabilities of one of ordinary skill in the art. Such techniques are explained in the literature. See, for example, Organic Chemistry (Jonathan Clayden (ed.), Nick Greeves (authors), Stuart Warren (authors), Peter Wothers (authors), Oxford University Press, 2000, and March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure (Michael B. Smith (authors) and Jerry March (authors)), Wiley-Interscience, 6th edition, 2007. Each of these general texts is incorporated herein by reference.

[0076] As demonstrated in the examples, endotoxin could be measured by the method of the present disclosure. Furthermore, as demonstrated in the examples, trypsin could be measured by the method of the present disclosure. Furthermore, as demonstrated in the examples, gingipain could be measured by the method of the present disclosure. These methods are characterized by using a label that adsorbs to an electrode and measuring the label by electrochemical measurement. The method of the present disclosure does not depend on a specific hydrolase, and once the label is released, the target substance can be measured electrochemically based on the same principle. [Example]

[0077] The present disclosure will be further illustrated by the following examples, but the technical scope of the present disclosure is not limited to these examples. In the examples, the following compounds may be abbreviated as follows: N,N-Diisopropylethylamine N,N-DIPEA or simply DIPEA Triethylamine Et3N Dimethyl sulfoxide DMSO Methanol MeOH Ethyl acetate AcOEt Palladium Carbon Pd / C Heating Δ Water-soluble carbodiimide hydrochloride (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) WSCl·HCl or EDC·HCl Dichloromethane DCM or CH2Cl2 Trifluoroacetic acid TFA tert-Butoxycarbonyl group Boc Benzyloxycarbonyl group Z or Cbz

[0078] [material and method] Unless otherwise specified, materials and reagents were commercially available, or were obtained or prepared according to conventional techniques or published literature. The LAL reagents used were Endospecy ES-24S Set (Seikagaku Corporation) or Limulus Color KY Single Test Wako (Fujifilm Wako Pure Chemical Industries, product code 291-53601). The endotoxin standard solution was USP Reference Standard Endotoxin (Seikagaku Corporation) diluted to the specified concentration with endotoxin-free distilled water. Trypsin derived from porcine pancreas (Fujifilm Wako Pure Chemical Industries, product code 201-19181) was used. Alkaline phosphatase is available, for example, from Fujifilm Wako Pure Chemical Industries (alkaline phosphatase, product code 018-10693). The compounds N-(3-isopropylphenyl)-p-phenylenediamine, N-(3-tert-butylphenyl)-p-phenylenediamine, N-(3,5-di-tert-butylphenyl)-p-phenylenediamine, and N-(4-aminophenyl)-N'-phenyl-p-phenylenediamine, as well as substrate-peptide compounds in which the primary amino group of the above four compounds is amide-bonded to the carboxyl group of the Arg residue in Leu-Gly-Arg or Val-Pro-Arg, were obtained from Watanabe Chemical Industry Co., Ltd. The preparation procedures for these compounds are as follows.

[0079] Using the following scheme, N-(3-isopropylphenyl)-p-phenylenediamine, N-(3-tert-butylphenyl)-p-phenylenediamine, N-(3,5-di-tert-butylphenyl)-p-phenylenediamine, and N-(4-aminophenyl)-N'-phenyl-p-phenylenediamine (represented as KIK-MO-xx in the scheme) were synthesized.

[0080] [ka]

[0081] The target compound was synthesized by adding a base to 4-fluoronitrobenzene and an arylamine corresponding to the target compound (e.g., 3-isopropylaniline, 3-tert-butylaniline, or 3,5-di-tert-butylaniline) to react with each other to synthesize a compound having a diphenylamine skeleton, and then reducing the nitro group to an amino group by catalytic reduction.

[0082] Using the following scheme, the substrates Leu-Gly-Arg or Val-Pro-Arg were conjugated to N-(3-isopropylphenyl)-p-phenylenediamine, N-(3-tert-butylphenyl)-p-phenylenediamine, N-(3,5-di-tert-butylphenyl)-p-phenylenediamine, and N-(4-aminophenyl)-N'-phenyl-p-phenylenediamine (denoted as KIK-PE-xx in the scheme). In the scheme, the labeled compounds are generally designated as "NH-Ar."

[0083] [ka] [ka]

[0084] The desired compound could be synthesized by protecting the previously synthesized KIK-MO-xx with an amino acid or oligopeptide using a protecting group such as a Z group or a Boc group, and then coupling them using a condensing agent such as a water-soluble carbodiimide.

[0085] The products were confirmed by HPLC and LC / MS. Other compounds were commercially available from Tokyo Chemical Industry Co., Ltd., Fujifilm Wako Pure Chemical Industries, Ltd., and Nacalai Tesque.

[0086] [Example 1] Confirmation of adsorption between phenylenediamine compounds and carbon electrodes Cyclic voltammetry was performed using a glassy carbon electrode with four phenylenediamine compounds (N-(3-isopropylphenyl)-p-phenylenediamine, N-(3-tert-butylphenyl)-p-phenylenediamine, N-(3,5-di-tert-butylphenyl)-p-phenylenediamine, and N-(4-aminophenyl)-N'-phenyl-p-phenylenediamine). Specifically, the measurement solution was prepared by mixing 10 μL of a 1 mM phenylenediamine compound solution, 90 μL of 100 mM potassium phosphate buffer (pH 7.0), and 100 μL of ultrapure water. Cyclic voltammetry was performed using a glassy carbon rod electrode (BAS: SGCE glassy carbon electrode 3 x 1.6 cm) as the working electrode, a platinum rod electrode (BAS: PT counter electrode 5.7 cm) as the counter electrode, and a silver / silver chloride electrode (EC Frontier: RE-11A) as the reference electrode. The sweep range was -400 mV to +800 mV. The sweep rate was varied from 4 mV / sec to 200 mV / sec to investigate how the maximum oxidation current (IOmax) changed. It is generally known that when a compound is adsorbed on the electrode, the cyclic voltammetry sweep rate and IOmax are proportional. When a compound is diffusing in solution, IOmax is proportional to the 0.5 power of the sweep rate.

[0087] Cyclic voltammetry was performed using N-(3-isopropylphenyl)-p-phenylenediamine, and the sweep rate and I0max were plotted as shown in Figure 2. As a result, it was confirmed that there was a proportional relationship between the sweep rate and I0max, indicating that N-(3-isopropylphenyl)-p-phenylenediamine was adsorbed onto the carbon electrode.

[0088] Similar tests were performed using N-(3-tert-butylphenyl)-p-phenylenediamine, N-(3,5-di-tert-butylphenyl)-p-phenylenediamine, and N-(4-aminophenyl)-N'-phenyl-p-phenylenediamine instead of N-(3-isopropylphenyl)-p-phenylenediamine, and the results are shown in Figures 3, 4, and 5. It was confirmed that there was a proportional relationship between the sweep rate and IOmax for all of the compounds, indicating that N-(3-isopropylphenyl)-p-phenylenediamine adsorbs to the carbon electrode in the same way as carbon.

[0089] Comparative Example (p-aminophenol) Similar measurements were performed using p-aminophenol, a known label, instead of the phenylenediamine compounds of the present disclosure, and the results are shown in Figures 6 and 7. It was confirmed that IOmax was proportional to the 0.5 power of the sweep rate, indicating that p-aminophenol was not adsorbed to the electrode but was diffusing.

[0090] [Example 2] Measurement of endotoxin using substrate-labeled compound Electrochemical measurement of endotoxin was performed using a compound conjugated to Leu-Gly-Arg and N-(3-isopropylphenyl)-p-phenylenediamine (hereafter referred to as LGR-N-(3-isopropylphenyl)-p-phenylenediamine). Specifically, 200 μl of the included buffer solution was added to the LAL reagent included with the Endospecy ES-24S set and mixed thoroughly to prepare an LAL solution. 10 μl of 1 mM LGR-N-(3-isopropylphenyl)-p-phenylenediamine solution, 90 μl of LAL solution, and 90 μl of endotoxin aqueous solution at a predetermined concentration (0, 1, 10, 100, 1000 EU / L) were mixed in a dry-heat sterilized test tube and allowed to stand in an incubator at 37°C for 1 hour. Cyclic voltammetry was then performed using the same electrodes and sweep range as in Example 1 under the same conditions, except for the sweep rate of 20 mV / sec.

[0091] Figure 8 shows the results of plotting endotoxin concentration on the horizontal axis and the oxidation current at 120 mV (vs. Ag / AgCl) on the vertical axis. It can be seen that the current value increases depending on the endotoxin concentration. 120 mV (vs. Ag / AgCl) is the oxidation potential of the labeled compound, N-(3-isopropylphenyl) p-phenylenediamine, and a correlation is observed between the endotoxin concentration and the oxidation current derived from the labeled compound. This demonstrates that the endotoxin concentration in a test sample can be quantified by electrochemical measurement.

[0092] Similar experiments were performed using compounds consisting of Leu-Gly-Arg and N-(3-tert-butylphenyl)-p-phenylenediamine, and Leu-Gly-Arg and N-(3,5-di-tert-butylphenyl)-p-phenylenediamine, respectively, instead of LGR-N-(3-isopropylphenyl)-p-phenylenediamine (hereafter referred to as LGR-N-(3-tert-butylphenyl)-p-phenylenediamine and LGR-N-(3,5-di-tert-butylphenyl)-p-phenylenediamine). The results are shown in Figures 9 and 10. The oxidation current values ​​shown on the vertical axis are the current at 120 mV for LGR-N-(3-tert-butylphenyl)-p-phenylenediamine and 110 mV for LGR-N-(3,5-di-tert-butylphenyl)-p-phenylenediamine. In both cases, the oxidation current value increased depending on the endotoxin concentration, and the endotoxin concentration could be measured using this method.

[0093] [Example 3] Measurement of low concentrations of endotoxin using substrate-labeled compounds Electrochemical measurement of low concentrations of endotoxin was performed using LGR-N-(3-isopropylphenyl)-p-phenylenediamine. Specifically, 200 μl of endotoxin-free distilled water was added to the LAL reagent included with the Wako Limulus Color KY Single Test and mixed thoroughly to prepare an LAL solution. 90 μl of LAL solution and 90 μl of endotoxin aqueous solution at a predetermined concentration (0, 1, 5, or 10 EU / L) were mixed in a dry-heat sterilized test tube and allowed to stand in an incubator at 37°C for 1 hour. Then, 10 μl of 4 mM LGR-N-(3-isopropyl)-p-phenylenediamine solution was added, and the mixture was allowed to stand in an incubator at 37°C for an additional 1 hour. After the reaction was completed, chronoamperometry was performed using the same electrode as in Example 1. First, a potential of 0 mV (vs. Ag / AgCl) was applied for 10 seconds, followed by a step potential of 250 mV (vs. Ag / AgCl). The current measured was the value at 1 second after the start of application of the 250 mV potential.

[0094] Figure 11 shows the results of plotting endotoxin concentration on the horizontal axis and measured current on the vertical axis. It can be seen that the current value increases depending on the endotoxin concentration. When a potential of 250 mV (vs. Ag / AgCl) is applied, the substrate-labeled LGR-N-(3-isopropyl)-p-phenylenediamine is not oxidized, and only the labeled N-(3-isopropylphenyl)p-phenylenediamine is oxidized, demonstrating a correlation between endotoxin concentration and measured current value. This demonstrates that endotoxin concentration in a test sample can be quantified by electrochemical measurement.

[0095] [Example 4] Measurement of trypsin using substrate-labeled compound Electrochemical measurements of trypsin were performed using LGR-N-(3-tert-butylphenyl)-p-phenylenediamine. Trypsin was diluted to a predetermined concentration with 0.1 mM Tris-HCl (pH 7.5). 10 μl of 1 mM LGR-N-(3-tert-butylphenyl)-p-phenylenediamine solution, 90 μl of a trypsin solution at a predetermined concentration (final concentration of 0, 630, or 6300 U / L), and 90 μl of ultrapure water were mixed in a dry-heat sterilized test tube and allowed to stand in an incubator at 37°C for 1 hour. Cyclic voltammetry was then performed under the same conditions as in Example 2.

[0096] Figure 12 shows the results of plotting trypsin concentration on the horizontal axis and the oxidation current at 120 mV (vs. Ag / AgCl) on the vertical axis. It can be seen that the current value increases in a trypsin concentration-dependent manner. 120 mV (vs. Ag / AgCl) is the oxidation potential of the labeled compound, N-(3-tert-butylphenyl)-p-phenylenediamine, and a correlation is observed between trypsin concentration and the oxidation current derived from the labeled compound. In other words, the trypsin concentration in the test sample could be quantified by electrochemical measurement.

[0097] [Example 5] Measurement of gingipain using substrate-labeled compound The P. gingivalis JCM8525 strain used for the assay was cultured as follows. A 5 mg / mL hemin stock solution was prepared by dissolving 0.05 mg of hemin (Tokyo Chemical Industry Co., Ltd., H0008) in 1 mL of 1N aqueous ammonia, adding 9 mL of distilled water, and sterilizing the solution in an autoclave. A 1 mg / mL vitamin K stock solution was prepared by dissolving 0.1 g of 2-methyl-1,4-naphthoquinone (Tokyo Chemical Industry Co., Ltd., M0373) in 100 mL of ethanol (Wako Pure Chemical Industries, Ltd.). These solutions were stored refrigerated after preparation. BHI agar medium was prepared by dissolving 5.2 g of Pearlcore Brain Heart Infusion (BHI) agar medium "Eiken" (Eiken Chemical, E-MC61) in distilled water, adding 0.1 mL of hemin stock solution (final concentration 5 μg / mL) and vitamin K stock solution (final concentration 1 μg / mL), diluting to 100 mL, and sterilizing in an autoclave. After sterilization, approximately 20 mL of the solution was spread onto sterilized petri dishes while still hot, cooled, solidified, and stored in an airtight container with Anaeropack (Mitsubishi Gas Chemical) for degassing. BHI liquid medium was prepared by adding 0.1 mL of hemin stock solution (final concentration 5 μg / mL) and vitamin K stock solution (final concentration 1 μg / mL) to 3.7 g of Pearlcore Brain Heart Infusion Broth Medium "Eiken" (Eiken Chemical, E-MC62), diluting to 100 mL, and dispensing 2 mL of the solution into test tubes with silicone stoppers. Sterilizing in an autoclave. After cooling, the mixture was stored in a sealed container containing Anaeropack (Mitsubishi Gas Chemical) and degassed. The glycerol stock of each strain was subcultured onto BHI agar medium using a platinum loop. Cultures were performed in a sealed container containing Anaeropack (Mitsubishi Gas Chemical) while degassing, and static culture was performed at 37°C for approximately one week. Once colonies were confirmed, the mixture was subcultured onto BHI liquid medium, and the cultures were subcultured in a sealed container containing Anaeropack (Mitsubishi Gas Chemical) while degassing, and static culture was performed at 37°C for approximately one week. Culture was terminated when sufficient turbidity was confirmed. The culture medium was centrifuged at 4300 × g for 20 minutes, and the supernatant was filtered using a 0.22 μm filter to remove the bacterial cells, yielding the undiluted culture supernatant. The undiluted culture supernatant was then appropriately diluted with phosphate-buffered saline (pH 7.4) (Nacalai Tesque, product code 27575-31).

[0098] Electrochemical measurements of gingipain were performed using a compound consisting of Val-Pro-Arg and N-(3-tert-butylphenyl)-p-phenylenediamine (hereafter referred to as VPR-N-(3-tert-butylphenyl)-p-phenylenediamine). Specifically, 10 μL of 1 mM VPR-N-(3-tert-butylphenyl)-p-phenylenediamine solution, 10 μL of P. gingivalis culture supernatant solution at the specified ratios (stock solution, 2-fold dilution, 4-fold dilution), and 80 μL of phosphate-buffered saline (pH 7.4) were mixed in a dry-heat sterilized test tube and allowed to stand in an incubator at 37°C for 1 hour. Cyclic voltammetry was then performed under the same conditions as in Example 2. As a negative control, a solution of P. gingivalis culture supernatant stock solution heat-treated at 95°C for 30 minutes was used, and measurements were performed in the same manner.

[0099] Figure 13 shows the results of plotting the relative concentration of the measured sample (when the undiluted culture supernatant is set to 1) on the horizontal axis and the oxidation current at 120 mV (vs. Ag / AgCl) on the vertical axis. A relative concentration of 0 represents the negative control, in which the undiluted culture supernatant was heat-treated. It can be seen that the current value increases in a concentration-dependent manner with the culture supernatant. 120 mV (vs. Ag / AgCl) is the oxidation potential of the labeled compound, N-(3-tert-butylphenyl)-p-phenylenediamine, and a correlation is observed between the culture supernatant concentration and the oxidation current derived from the labeled compound. Furthermore, the significant decrease in the oxidation current value upon heat treatment strongly suggests that this oxidation current is due to enzymatic activity. Thus, electrochemical measurements were able to quantify gingipain activity in the culture supernatant.

[0100] In the above examples, electrochemical measurements of endotoxin, trypsin, and gingipain contained in P. gingivalis culture supernatant were performed using labels based on phenylenediamine compounds. Although the clotting enzyme, trypsin, and gingipain used in endotoxin measurement are all proteases, peptidases can also be measured in the same way. Furthermore, it will be understood by those skilled in the art that the method disclosed herein is applicable to various hydrolases, such as phosphatases, e.g., alkaline phosphatases, esterases, and glycosidases.

[0101] A method for measuring alkaline phosphatase using the method of the present disclosure is described below. Electrochemical measurement of alkaline phosphatase is carried out using a compound in which phosphate is bound to a label. The phosphate substrate-labeled compound may be a compound in which phosphate is bound to N-(3-tert-butylphenyl)-p-phenylenediamine, a compound in which phosphate is bound to N-(3-isopropylphenyl)-p-phenylenediamine, or a compound in which phosphate is bound to N-(3-tert-butylphenyl)-p-phenylenediamine, or a similar compound. For example, 10 μl of a 1 mM N-(3-tert-butylphenyl)-p-phenylenediamine phosphate solution, 10 μl of an alkaline phosphatase solution of a predetermined concentration (0, 1, 2, 5, 10, or 20 U / L), and 80 μl of a 0.1 mol / L carbonate buffer solution (pH 9.8) containing 2.0 mmol / L magnesium chloride are mixed in a dry-heat sterilized test tube and allowed to stand in an incubator at 37° C. for 1 hour. Thereafter, cyclic voltammetry is performed under the same conditions as in Example 2.

[0102] The relationship between the alkaline phosphatase concentration and the oxidation current derived from the label is examined, and alkaline phosphatase is quantified by electrochemical measurement.

[0103] In the above examples, electrochemical measurements of endotoxin, trypsin, and gingipain were carried out mainly using labels based on phenylenediamine compounds. The phenylenediamine compounds used have been reported to have the property of being adsorbed to electrodes. Therefore, those skilled in the art will understand that electrochemical measurements of endotoxin, trypsin, gingipain, and phosphatase can be carried out using other compounds that have the property of being adsorbed to electrodes, such as labels based on aminoanthraquinone compounds of Formula II, instead of phenylenediamine compounds. [Industrial Applicability]

[0104] The method for measuring trace substances disclosed herein can be used in fields such as fermentation, pharmaceuticals, and biochemical research.

[0105]

[0003] Several documents, including patent applications and manufacturer's manuals, are cited herein. The disclosures of these documents are not considered relevant to the patentability of this disclosure, but are incorporated herein by reference in their entirety. More particularly, all referenced documents are incorporated herein by reference to the same extent as if each individual document were specifically and individually indicated to be incorporated by reference. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. The method is characterized by using a substrate-labeled compound labeled with a label that is used to convert the substrate into a substrate-labeled compound suitable for electrochemical measurement by binding with the substrate, and that has the property of being adsorbed to an electrode; the label is released from the substrate-label compound by the action of the hydrolase, and the released label is measured electrochemically; The present invention is intended to measure the hydrolase activity of a test sample or a substance that activates a hydrolase, which may be contained in the test sample; the substance that activates the hydrolase comprises endotoxin or (1→3)-β-D-glucan; The label has the structure of Formula I 【Chemistry 1】 [In the formula, R 7 is hydrogen, straight or branched chain C 1-6 alkyl, C 1-6 alkenyl, C 1-6 alkynyl, C 3-9 cycloalkyl, phenyl, 1-naphthyl, 2-naphthyl, anthracenyl or phenanthrenyl, optionally substituted by one or more X; R 3 , R 4 , R 5 and R 6 are each independently hydrogen, straight or branched chain C 1-6 alkyl, C 1-6 alkenyl, C 1-6 alkynyl, C 1-6 alkoxy, halo, nitro, cyano, carboxy, sulfone or amino optionally substituted by one or more Y; R 8 is selected from the group consisting of phenyl, 1-naphthyl, 2-naphthyl, anthracenyl, and phenanthrenyl, optionally substituted by one or more X; wherein X is a straight or branched chain C 1-6 alkyl, C 1-6 alkenyl, C 1-6 alkynyl, C 1-6 alkoxy, anilino, phenoxy, halo, hydroxy, nitro, carboxy, cyano, sulfone or amino, optionally substituted by a substituent selected from the group consisting of halo, amino, cyano, carboxy, carbonyl, alkoxy and sulfone; Y is selected from the group consisting of halo, amino, cyano, carboxy, carbonyl, alkoxy, and sulfone. or a phenylenediamine-based compound having the formula 【Chemistry 2】 an aminoanthraquinone-based compound having the formula Electrochemical measurement methods.

2. A label used to convert a substrate into a substrate-label compound suitable for electrochemical measurement by binding with the substrate, characterized in that a substrate-label compound labeled with a label having the property of being adsorbed to an electrode is used, the label is released from the substrate-label compound by the action of the hydrolase, and the released label is measured electrochemically; The present invention is intended to measure the hydrolase activity of a test sample or a substance that activates a hydrolase, which may be contained in the test sample; the hydrolase is trypsin, gingipain, or phosphatase; The label has the structure of Formula I 【Transformation 3】 [In the formula, R 7 is hydrogen, straight or branched chain C 1-6 alkyl, C 1-6 alkenyl, C 1-6 alkynyl, C 3-9 cycloalkyl, phenyl, 1-naphthyl, 2-naphthyl, anthracenyl or phenanthrenyl, optionally substituted by one or more X; R 3 , R 4 , R 5 and R 6 are each independently hydrogen, straight or branched chain C 1-6 alkyl, C 1-6 alkenyl, C 1-6 alkynyl, C 1-6 alkoxy, halo, nitro, cyano, carboxy, sulfone or amino optionally substituted by one or more Y; R 8 is selected from the group consisting of phenyl, 1-naphthyl, 2-naphthyl, anthracenyl, and phenanthrenyl, optionally substituted by one or more X; wherein X is a straight or branched chain C 1-6 alkyl, C 1-6 alkenyl, C 1-6 alkynyl, C 1-6 alkoxy, anilino, phenoxy, halo, hydroxy, nitro, carboxy, cyano, sulfone or amino, optionally substituted by a substituent selected from the group consisting of halo, amino, cyano, carboxy, carbonyl, alkoxy and sulfone; Y is selected from the group consisting of halo, amino, cyano, carboxy, carbonyl, alkoxy, and sulfone. or a phenylenediamine-based compound having the formula 【Chemistry 4】 an aminoanthraquinone-based compound having the formula Electrochemical measurement methods.

3. 2. The method of claim 1, wherein the hydrolase is horseshoe crab factor C, factor B, factor G, and / or proclotting enzyme.

4. The phenylenediamine compound represented by formula I is 【Transformation 5】 【Transformation 6】 【Transformation 7】 and 【Transformation 8】 or a compound selected from the group consisting of The aminoanthraquinone compound represented by formula (II) has the following structure: 【Chemistry 9】 1-amino-4-hydroxyanthraquinone having the formula The following structure 【Chemistry 10】 2-amino-3-hydroxyanthraquinone having or a salt, anhydride or solvate thereof.

5. A reagent for electrochemically measuring hydrolase activity or a substance that activates a hydrolase, comprising a substrate-labeled compound, wherein the label in the substrate-labeled compound has the property of being adsorbed to an electrode; The labeled substance is 【Chemistry 11】 【Chemistry 12】 【Chemistry 13】 and 【Chemistry 14】 or a compound selected from the group consisting of The following structure 【Chemistry 15】 1-amino-4-hydroxyanthraquinone having the formula The following structure 【Chemistry 16】 2-amino-3-hydroxyanthraquinone having or a salt, anhydride or solvate thereof, The reagent.

6. A composition for measuring hydrolase activity or a substance that activates hydrolase, comprising the reagent according to claim 5.

7. 7. An electrode comprising the reagent of claim 5 or the composition of claim 6.

8. A system for electrochemical measurements comprising the electrode according to claim 7.

9. A reagent for electrochemically measuring hydrolase activity or a substance that activates a hydrolase, comprising a substrate-labeled compound, wherein the label in the substrate-labeled compound has the property of being adsorbed to an electrode, the reagent is for measuring the hydrolase activity of a test sample or a substance that activates the hydrolase, which may be contained in the test sample; the substance that activates the hydrolase comprises endotoxin or (1→3)-β-D-glucan; The label has the structure of Formula I 【Chemistry 17】 [In the formula, R 7 is a straight or branched chain C optionally substituted with hydrogen or one or more X's 1-6 Alkyl, C 1-6 Alkenyl, C 1-6 Alkynyl, C 3-9 cycloalkyl, phenyl, 1-naphthyl, 2-naphthyl, anthracenyl or phenanthrenyl; R 3 , R 4 , R 5 and R 6 each independently represents a straight or branched chain C optionally substituted with hydrogen or one or more Y's; 1-6 Alkyl, C 1-6 Alkenyl, C 1-6 Alkynyl, C 1-6 alkoxy, halo, nitro, cyano, carboxy, sulfone or amino; R 8 is selected from the group consisting of phenyl, 1-naphthyl, 2-naphthyl, anthracenyl, and phenanthrenyl, optionally substituted by one or more X; wherein X is a straight or branched chain alkyl group optionally substituted with a substituent selected from the group consisting of halo, amino, cyano, carboxy, carbonyl, alkoxy, and sulfone; 1-6 Alkyl, C 1-6 Alkenyl, C 1-6 Alkynyl, C 1-6 alkoxy, anilino, phenoxy, halo, hydroxy, nitro, carboxy, cyano, sulfone or amino; Y is selected from the group consisting of halo, amino, cyano, carboxy, carbonyl, alkoxy, and sulfone. or a phenylenediamine-based compound having the formula [Chemistry 18] an aminoanthraquinone-based compound having the formula The reagent.

10. A reagent for electrochemically measuring hydrolase activity or a substance that activates a hydrolase, comprising a substrate-labeled compound, wherein the label in the substrate-labeled compound has the property of being adsorbed to an electrode, the reagent is for measuring the hydrolase activity of a test sample or a substance that activates the hydrolase, which may be contained in the test sample; the hydrolase is trypsin, gingipain, or phosphatase; The label has the structure of Formula I 【Chemistry 19】 [In the formula, R 7 is a straight or branched chain C optionally substituted with hydrogen or one or more X's 1-6 Alkyl, C 1-6 Alkenyl, C 1-6 Alkynyl, C 3-9 cycloalkyl, phenyl, 1-naphthyl, 2-naphthyl, anthracenyl or phenanthrenyl; R 3 , R 4 , R 5 and R 6 each independently represents a straight or branched chain C optionally substituted with hydrogen or one or more Y's; 1-6 Alkyl, C 1-6 Alkenyl, C 1-6 Alkynyl, C 1-6 alkoxy, halo, nitro, cyano, carboxy, sulfone or amino; R 8 is selected from the group consisting of phenyl, 1-naphthyl, 2-naphthyl, anthracenyl, and phenanthrenyl, optionally substituted by one or more X; wherein X is a straight or branched chain alkyl group optionally substituted with a substituent selected from the group consisting of halo, amino, cyano, carboxy, carbonyl, alkoxy, and sulfone; 1-6 Alkyl, C 1-6 Alkenyl, C 1-6 Alkynyl, C 1-6 alkoxy, anilino, phenoxy, halo, hydroxy, nitro, carboxy, cyano, sulfone or amino; Y is selected from the group consisting of halo, amino, cyano, carboxy, carbonyl, alkoxy, and sulfone. or a phenylenediamine-based compound having the formula 【Chemistry 20】 an aminoanthraquinone-based compound having the formula The reagent.

11. A composition for measuring hydrolase activity or a substance that activates a hydrolase, comprising the reagent described in claim 9 or 10.

12. An electrode comprising the reagent described in claim 9 or 10 or the composition described in claim 11.

13. A system for electrochemical measurements comprising the electrode described in claim 12.

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