Electrochemical measurement using phenylenediamine derivative

Abstract

The present invention addresses the problem of providing a means for making highly sensitive and stable measurements possible using electrochemical measurement methods. This problem is resolved by providing: a labeling substance represented by general formula (1) and used to label a substrate; a measurement substrate that is formed by being labeled using the labeling substance; a method of measuring using electrochemical measurement methods that use the measurement substrate; and a reagent kit that includes as components the labeling substance and/or the measurement substrate. ##STR00001##
(In general formula (1), R.sub.1 is either H or an alkyl group (C.sub.mH.sub.2m+1), m is an integer of 1-4, R.sub.2 is an alkyl group (C.sub.nH.sub.2n+1), and n is an integer of 1-4.)

Claims

1. A method of measuring an endotoxin and/or (1.fwdarw.3)--D-glucan in a sample, comprising: (1) contacting the sample with a measuring substrate represented by formula (2): ##STR00009## wherein R.sub.1 is H, R.sub.2 is methyl, and X.sub.1 is a peptide with an Arg (R) residue at the C-terminus which is coupled to the NH group of the measuring substrate by an amide bond and an enzyme that degrades the measuring substrate when the endotoxin and/or (1.fwdarw.3)--D-glucan is present in the sample, and (2) measuring the cleavage of the peptide from the measuring substrate by the enzyme by an electrochemical measurement method.

2. The method according to claim 1, wherein the enzyme is a Limulus Factor C, B, or G and/or a pro-clotting enzyme.

3. The method according to claim 1, wherein the electrochemical measurement method is a voltammetric method or an amperometric method.

4. The method according to claim 1, wherein the peptide having the Arg group at the C-terminus is a peptide represented by any one of the following formulas (a) to (g):
Y-Asp-Pro-Arg (Y-DPR)(a),
Y-Val-Pro-Arg (Y-VPR)(b),
Y-Leu-Thr-Arg (Y-LTR)(c),
Y-Met-Thr-Arg (Y-MTR)(d),
Y-Leu-Gly-Arg (Y-LGR)(e),
Y-Ile-Glu-Gly-Arg (Y-IEGR) (SEQ ID No. 1)(f), and
Y-Glu-Gly-Arg (Y-EGR)(g); wherein Y may be present or may not be present, and when Y is present, Y is a protective group of an amino group of amino acid at the N-terminus of the peptide.

5. The method according to claim 4, wherein the protective group is Cbz (benzyl oxycarbonyl group) or Boc (tert-butoxycarbonyl group).

Description

EXAMPLES

(1) Hereinafter, the present invention is described in detail based on examples, however, a technical scope of the present invention is not limited to only these examples.

<Reference Example 1> Acquisition of Labeling Substance and Substrate for Measurement

(2) P-aminophenol (pAP), N, N-dimethyl-p-phenylenediamine (DMPD), and N-methyl-p-phenylenediamine (MPDD) being labeling substances were purchased from Wako Pure Chemical Corporation. Cbz-LGR-pAP, Cbz-LGR-DMPD, and Cbz-LGR-MPDD being substrates for measurement obtained by amide-bonding the labeling substances and Cbz-Leu-Gly-Arg (Cbz-LGR) were acquired by contracting out synthesis to WATANABE CHEMICAL INDUSTRIES, LTD. In the examples of the application concerned, a substrate for measurement prepared as TFA (trifluoroacetate) salt was used.

(3) Hereinafter, Cbz-LGR-pAP, Cbz-LGR-DMPD, and Cbz-LGR-MPDD are respectively abbreviated to LGR-pAP, LGR-DMPD, and LGR-MPDD.

<Reference Example 2> Evaluation of Quantitative Performance

(4) As a result of performing a cyclic voltammetric measurement in which the mixture ratio of a substrate and free molecule (labeling substance) was varied in stages, in a voltammogram of a mixed solution of LGR-pAP and pAP, two oxidation peaks at 0.15 V and 0.49 V were confirmed. From individual voltammograms of a substrate molecule and a free molecule, the peak near 0.49 V is considered to be derived from oxidation of LGR-pAP, and the peak near 0.15 V is considered to be derived from oxidation of pAP. Similarly, from a voltammogram of a mixed solution of LGR-DMPD and DMPD, an oxidation peak derived from LGR-DMPD was confirmed near 0.45V, and an oxidation peak derived from DMPD was confirmed near 0.19 V. Further, from a voltammogram of a mixed solution of LGR-MPDD and MPDD, an oxidation peak derived from LGR-MPDD was confirmed near 0.46 V, and an oxidation peak derived from MPDD was confirmed near 0.14 V. Further, when using any substrate, an oxidation peak current value of free molecules increased as the percentage of a free molecule increased. From these facts, it was considered that a concentration of free molecules in each mixed solution could be amperometrically quantified at a potential of 0.3 V vs. Ag/AgCl. Next, in each mixed solution, chronoamperometry was performed. Based on a temporal change in current value just after (0 seconds) a potential of a working electrode was changed to 0.3 V vs. Ag/AgCl, a current value at 20 seconds with respect to a concentration of a free molecule in each mixed solution was plotted, and as a result, determination coefficients were R.sub.2=0.9973 for pAP, R.sub.2=0.9986 for DMPD, and R.sub.2=0.9972 for MPDD, and high linearity was confirmed.

<Example 1> Evaluation of Stability of Labeling Substances and Substrates for Measurement

(5) Stabilities of compounds of the labeling substances and substrates for measurement acquired in <Reference Example 1> were evaluated.

(6) The labeling substances (pAP, MPDD, and DMPD) and the substrates for measurement (LGR-pAP, LGR-MPDD, LGR-DMPD) were dissolved into 0.1 M HEPES (pH 7.8) and prepared to be 1 mM, and were left to stand for 1 to 24 hours in an incubator at 37 C. Thereafter, a test was performed according to the following procedures.

(7) <Electrochemical Measurement Method>

(8) As an electrochemical measurement method, cyclic voltammetry and a potential step chronoamperometric method were used. As a measuring device, a working electrode, a reference electrode, and a counter electrode, those described in <Measurement Conditions> below were used. In each measurement, the working electrode, the reference electrode, and the counter electrode were inserted in a solution, and respectively connected to connectors of a potentiostat for a working electrode, a reference electrode, and a counter electrode. After each measurement, the electrode surface of the working electrode was polished with 0.05 m polishing alumina, and cleaned with ultrapure water. In the cyclic voltammetric measurement, potential scanning was performed by setting an initial potential and a finish potential to 0 V, a first turnover potential to 0.4 V (0.6 V for LGR-pAP and LGR-DMPD, 0.7 V for LGR-MPDD), and a second turnover potential to 0.2 V, and the current was monitored. In the potential step chronoamperometric measurement, after 0.0 V was applied to the working electrode for 10 seconds, the potential was momentarily changed to 0.3 V, and the current was monitored for 35 seconds.

(9) <Measurement Conditions>

(10) Measuring device: Potentiostat (CompactStat manufactured by IVIUM)

(11) Working electrode: Glassy carbon disk electrode, 1 mm in diameter (manufactured by BAS)

(12) Reference electrode: Silver-silver chloride electrode

(13) Counter electrode: Platinum electrode

(14) <Confirmation on Stabilities of Substrates and Free Molecules>

(15) 50 ml of test solutions (1.0 mM) of the respective substrates (LGR-pAP, LGR-MPDD, LGR-DMPD) and free molecules (pAP, MPDD, DMPD) were prepared by using 0.1 M HEPES (pH=7.8), put in a disposable 50 ml centrifuge tube (manufactured by IWAKI), and left to stand at 37 C. under light-shielded conditions. When a predetermined time (0.5 h, 1 h, 2 h, 4 h, and 24 h) elapsed, 5 ml was sampled from the test solution and subjected to cyclic voltammetric measurement. MPDD was measured when 6 hours and 8 hours elapsed as well. By using a test solution just after preparation, a measurement at a predetermined time of 0 hours was performed.

(16) <Quantitative Evaluation of Free Molecules>

(17) A 1.0 mM LGR-pAP solution and a 1.0 mM pAP solution were prepared by using 0.1M HEPES (pH=7.8), and these were mixed at a ratio of 100:0, 75:25, 50:50, 25:75, or 0:100 to prepare mixed solutions whose pAP concentrations were varied in stages. Mixed solutions of the LGR-DMPD solution and the DMPD solution and mixed solutions of the LGR-MPDD solution and the MPDD solution were also prepared in a similar manner. These mixed solutions were subjected to a cyclic voltammetric measurement.

(18) As a result of the cyclic voltammetric measurements of each synthetic substrate and each free molecule left to stand at 37 C. under light-shielded conditions, from the results of the measurements of the synthetic substrates, it was found that the forms of voltammograms of the synthetic substrates did not greatly change in 24 hours, and were stable. It is considered that in a reaction time of 1 hour in actual endotoxin detection, the synthetic substrates do not deteriorate. On the other hand, from the results of the measurements of the free molecules, it was found that the forms of voltammograms of the free molecules gradually changed in 24 hours, and were lower in stability than the synthetic substrates. These results are shown in Table 1.

(19) TABLE-US-00001 TABLE 1 Time pAP DMPD MPDD 0 100.0 100.0 100.0 0.5 94.2 95.3 102.4 1 95.0 94.3 102.3 2 83.0 84.5 101.0

(20) The numerical values shown in Table 1 are relative values of heights of peaks in the voltammograms of the free molecules after predetermined times elapse, where the oxidation peak current value just after preparation (0 hours) is set as 100%, in voltammograms of the respective free molecules. Because detection of an endotoxin is usually finished in 1 hour, the oxidation peak current values in voltammograms of pAP, DMPD, and MPDD in one hour after preparation were compared with values just after solution preparation as references, resulting in 95.0%, 94.3%, and 102.3%, respectively.

(21) As shown in Table 1, among the labeling substances that are substances to be freed from the substrate for measurement by Limulus reaction, detected values of pAP and DMPD showed a tendency to successively decrease in 2 hours after preparation. However, no decrease in detected value of MPDD was found in 2 hours after preparation.

(22) It was shown above that MPDD is more stable than pAP, and is accordingly a labeling substance that enables a stable measurement.

<Example 2> Limulus Test (1)

(23) Limulus tests using the substrate for measurement acquired in <Reference Example 1> described above were performed, and endotoxin measurements were performed by an amperometric method. As a Limulus reagent, Endospecy ES-24S (manufactured by SEIKAGAKU CORPORATION) was used. In the procedures described hereinafter, Endospecy ES-24S is abbreviated to ES-24S. The procedures of the test are described below.

(24) <Limulus Reaction>

(25) According to a method prescribed in Japanese Pharmacopoeia, a standard endotoxin sample (USP-RSE) was dissolved into an injection solvent (manufactured by Otsuka Pharmaceutical Co., Ltd.) to prepare endotoxin solutions of 20,000 EU/L, 2,000 EU/L, 200 EU/L, 20 EU/L, 2 EU/L, and 0 EU/L. In addition, a 1.0 mM LGR-pAP solution was prepared by using a buffer solution supplied with ES-24S, and by using this solution, the Limulus reagent ES-24S was dissolved. Each endotoxin solution at the respective concentration and the Limulus reagent containing LGR-pAP were mixed at a volume ratio of 1:1 and reacted for 1 hour at 37 C., and thereafter, subjected to a chronoamperometric measurement. For LGR-DMPD and LGR-MPDD, the tests were also performed using the same procedures. The solution of 20,000 EU/L was measured only once, and for the solutions of 2,000 EU/L, 200 EU/L, 20 EU/L, 2 EU/L, and 0 EU/L, the test from the solution preparation to the measurement was performed for three times on different measurement dates.

(26) <Measurement Conditions>

(27) The conditions are the same as those described in <Measurement Conditions> of <Example 1> described above.

(28) The results are shown in Table 2.

(29) TABLE-US-00002 TABLE 2 EU/L LGR-pAP LGR-DMPD LGR-MPDD 0 0.023 0.005 0.008 0.001 0.010 0.000 1 0.033 0.011 0.015 0.002 0.012 0.001 10 0.075 0.032 0.021 0.002 0.022 0.002 50 0.275 0.006 0.039 0.013 0.061 0.001 100 0.313 0.027 0.075 0.011 0.087 0.007 1,000 0.276 0.013 0.130 0.013 0.235 0.003

(30) The numerical values shown in Table 2 are averages of measured values (current values/PA after 20 seconds in amperograms) and standard deviations obtained when the Limulus test was performed 3 times by using the respective substrates. In the present example, measurable ranges of endotoxin concentration when using each substrate were evaluated by using a value calculated by adding a value three times the standard deviation to an average of the measured values of the sample of 0 EU/L as a detection limit value.

(31) From Table 2, when a conventional substrate for measurement (LGR-pAP) is used, a measured value of the sample of 1 EU/L was not more than the detection limit value. In addition, a measured value of the sample of 100 EU/L was higher than a measured value of the sample of 1,000 EU/L. That is, it was shown that when the conventional substrate for measurement (LGR-pAP) was used, a quantitative range of endotoxin concentration was 1.0 to 100 EU/L.

(32) On the other hand, when the substrate for measurement (LGR-MPDD, LGR-DMPD) of the present invention was used, a measured value of the sample of 1 EU/L exceeded the detection limit value, and the measured value increased in proportion to an endotoxin concentration in an endotoxin concentration range of 1 to 1,000 EU/L. That is, it was shown that when the substrate for measurement (LGR-MPDD, LGR-DMPD) of the present invention was used, a quantitative range of endotoxin concentration was 1 to 1,000 EU/L, and this shows that a wider range of endotoxin concentration can be quantified as compared with a case where a conventional substrate for measurement (LGR-pAP) is used.

(33) Values of standard deviations when measurements were performed by using the substrates for measurement (LGR-MPDD, LGR-DMPD) of the present invention were found to become smaller than a value of a standard deviation when a measurement was made by using a conventional substrate for measurement (LGR-pAP). That is, it was shown that, by using MPDD or DMPD as a labeling molecule, stable measurements with reduced daily errors were enabled. Further, it was found that, when measurements were performed by using the substrates for measurement (LGR-MPDD, LGR-DMPD) of the present invention, measured values of the sample of 0 EU/L were smaller than in the case where a measurement was performed by using LGR-pAP, showing the background of the measured value can be kept low.

<Example 3> Limulus Test (2)

(34) Limulus tests using the substrates for measurement acquired in <Reference Example 1> described above were performed, and measurements of endotoxin were performed by a voltammetric method. The test procedures are shown below.

(35) <Limulus Reaction>

(36) The same conditions as those described in <Limulus Reaction> of <Example 2> described above were applied except that a cyclic voltammetric measurement was performed instead of the chronoamperometric measurement.

(37) <Measurement Conditions>

(38) The same conditions as described in <Measurement Conditions> of <Example 1> above were applied.

(39) As a result of measurements according to a voltammetric method, when using any of LGR-pAP, LGR-DMPD, and LGR-MPDD as a substrate, only an oxidation peak of the substrate was observed in the absence of endotoxin (0 EU/L). On the other hand, it was confirmed that in the presence of endotoxin, as the endotoxin concentration increase, an oxidation peak derived from a free molecule became higher. When using a conventional substrate for measurement (LGR-pAP) as a substrate, an oxidation peak derived from a free molecule when the sample of 1,000 EU/L was set as a measurement target was smaller than that in the case where the sample of 100 EU/L was set as a measurement target. On the other hand, when the substrate for measurement (LGR-MPDD, LGR-DMPD) of the present invention was used, in the endotoxin concentration range of 1 to 1,000 EU/L, an oxidation peak derived from a free molecule increased in proportion to the endotoxin concentration. That is, it was shown that the endotoxin concentration could be measured in the case using a voltammetric method as an electrochemical measurement method in a similar manner as in the case using an amperometric method.

(40) In a voltammogram at this time, a current value of an oxidation peak derived from a free molecule in the case where the sample of 1,000 EU/L was set as a measurement target was 0.31 A when LGR-DMPD was used as a substrate, and 0.60 A when LGR-MPDD was used as a substrate. On the other hand, a voltammogram of LGR-pMA being a substrate labeled by para methoxyaniline (pMA), is disclosed in FIG. 9 of Patent Literature 1, and a current value of an oxidation peak caused by a free molecule when the sample of 1,000 EU/L is set as a measurement target is approximately 0.3 A. That is, it was shown that by using the labeling substance (MPDD or DMPD) of the present invention, a current value equal to or higher than that in the case where the conventional labeling substance (pMA) was used was obtained. In particular, it was clarified that when using MPDD as a labeling substance, a current value was obtained approximately twice as high as a current value when pMA was used, and this shows that a highly sensitive measurement of endotoxin can be available.

(41) Oxidation peak current values caused by 0.5 mM DMPD and MPDD, shown by the results shown in <Reference Example 2>, were approximately 0.9 A and approximately 1.1 A, respectively. On the other hand, FIG. 4 of Patent Literature 1 shows that an oxidation peak current value caused by 1 mM pMA is approximately 3.0 A, so that an oxidation peak current value caused by 0.5 mM pMA is approximately 1.5 A. That is, from the results of measurement of a free molecule itself, both of MPDD and DMPD being labeling substances to be used for preparation of the substrate for measurement of the present invention were presumed to show lower current values as compared with that of pMA being a conventional labeling substance. However, although FIG. 8 of Patent Literature 1 shows that a maximum value (upper limit) of an oxidation peak current value obtained when bonded to the substrate (LGR) and used for a Limulus test is approximately 0.28 A when using LGR-pMA, maximum values when using LGR-DMPD and LGR-MPDD being the substrates for measurement of the present invention were 0.31 A and 0.60 A. These values are shown in Table 3.

(42) TABLE-US-00003 TABLE 3 Labeling Measured Measured Measured value B/ reagent value A value B Measured value A (%) MPDD 1.1 A 0.60 A 55% DMPD 0.9 A 0.31 A 34% pMA 1.5 A 0.28 A 19%

(43) In Table 3, the measured value A shows an oxidation peak current value when each of 0.5 mM labeling substances itself is measured by a voltammetric method, and the measured value B shows a maximum value of an oxidation peak current value obtained by using, for a Limulus test, a substrate for measurement (0.5 mM) obtained by bonding each labeling substance to a peptide (LGR).

(44) That is, when using any of the labeling substances, a maximum value (upper limit) of an oxidation peak current value obtained after the labeling substance was bonded to the peptide and used for a Limulus test showed a tendency to be lower than an oxidation peak current value estimated from a molarity, however, when using MPDD and DMPD being the labeling substances of the present invention, the degree of decrease was reduced as compared with the case using pMA being a conventional labeling substance, and these were found to be efficiently sensed in an electrochemical measurement method. Based on a measured value of a labeling substance alone, while a person skilled in the art expects that pMA is more efficiently sensed in an electrochemical measurement method when used as a substrate for measurement, the result that MPDD and DMPD are more efficiently sensed is unexpected. This result is particularly noticeable in MPDD.

INDUSTRIAL APPLICABILITY

(45) According to the present invention, a highly sensitive and stable measurement using an electrochemical measurement method can be performed. Therefore, the present invention is extremely useful in both clinical practice and research practice.

(46) The disclosure of Japanese Patent Application No. 2016-026925 (date of filing: Feb. 16, 2016) is incorporated herein by reference in its entirety. All literatures, patent applications, and technical standards described herein are incorporated herein by reference to the same extent as if individual literatures, patent applications, and technical standards are specifically and individually indicated to be incorporated by reference.