Method for quantifying reducing sugar and kit for measuring reducing sugar amount

Abstract

In this method for quantifying reducing sugar, a tetrazolium salt that generates water-soluble formazan by being reduced is added to a reducing sugar-containing sample containing reducing sugar, water-soluble formazan is generated by directly reducing the tetrazolium salt under a strong alkaline condition by means of the reducibility of the reducing sugar in the reducing sugar-containing sample, and the reducing sugar amount in the sample is measured by optically quantifying the generated water-soluble formazan.

Description

Method for quantifying reducing sugars and kit for measuring the amount of reducing sugars

[0001] This disclosure relates to a method for quantifying reducing sugars and a kit for measuring the amount of reducing sugars.

[0002] Methods for quantifying reducing sugars are used, for example, to measure the decomposition activity of polysaccharides such as biomass and cellulose. Known methods for measuring polysaccharide decomposition activity include, for example, measuring glucose, which has been decomposed to monosaccharides, by liquid chromatography. In contrast, using a method for quantifying reducing sugars allows for a simpler and more accurate measurement of polysaccharide decomposition activity by including the reducing sugars produced during the decomposition process from polysaccharides to monosaccharides.

[0003] Conventionally, the Somogyi-Nelson method, which utilizes color development using a copper reagent, has been commonly used as a method for quantifying reducing sugars (e.g., Non-Patent Documents 1 and 2). The tetrazolium blue method, which quantifies the resulting blue formazan by measuring its absorbance after reducing tetrazolium blue with a reducing sugar, is also known (e.g., Non-Patent Document 3).

[0004] Somogyi, M., J. Biol. Chem., 125, 399 (1938)Nelson, N., J. Biol. Chem., 153, 375 (1944)Chong K. Jue & Peter N. Lipke, Journal of Biochemical and Biophysical Methods, 11,109-115 (1985)

[0005] Of the conventional methods for quantifying reducing sugars, the Somogyi-Nelson method described above had problems: insufficient sensitivity, the use of toxic substances such as arsenic, and a complicated measurement procedure. The tetrazolium blue method has a wider detectable range and higher sensitivity compared to the Somogyi-Nelson method, does not use toxic substances such as arsenic, and is relatively simple to use. However, the stability of the measured values ​​when optically measuring the resulting blue formazan had not been sufficiently studied. Furthermore, because the reaction to reduce tetrazolium blue needs to be carried out at a relatively high temperature of 100°C, the equipment configuration and process became complicated, and safety could be insufficient in some cases. Therefore, there was a need for a simpler detection process that could perform measurements more stably and safely. In addition, further improvements in detection performance were desired.

[0006] This disclosure can be realized in the following forms: (1) According to one embodiment of this disclosure, a method for quantifying reducing sugars is provided. This method for quantifying reducing sugars involves adding a tetrazolium salt that produces water-soluble formazan upon reduction to a reducing sugar-containing sample containing reducing sugars, and under strongly alkaline conditions, directly reducing the tetrazolium salt due to the reducing properties of the reducing sugars in the reducing sugar-containing sample to produce water-soluble formazan, and then optically quantifying the resulting water-soluble formazan to measure the amount of reducing sugar in the sample. According to this embodiment of the method for quantifying reducing sugars, since a tetrazolium salt that produces water-soluble formazan upon reduction is used to quantify the reducing sugars, the stability of the measurement value when optically measuring the resulting formazan can be increased, enabling more sensitive measurements. At this time, the reaction in which the tetrazolium salt is directly reduced by the reducing properties of the reducing sugars in the reducing sugar-containing sample, i.e., NAD ＋Because this method utilizes a reaction that directly reduces tetrazolium salts using the reducing properties of the aldehyde group present in reducing sugars, without the need for coenzymes such as NADH or electron carriers (electron mediators), the composition of the reaction solution and the process for quantification can be simplified. Furthermore, the reduction of tetrazolium salts can be carried out at relatively low temperatures, simplifying the apparatus configuration and process, and improving safety. In addition, it is possible to broaden the concentration range of reducing sugars that can be quantified and to improve the sensitivity of reducing sugar measurement. (2) In the above-described method for quantifying reducing sugars, the tetrazolium salt may be any of the following: WST-1 (2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt), WST-8 (1-(2-Methoxy-4-nitrophenyl)-3-(2,4-disulfophenyl)-5-(4-nitrophenyl) formazan, disodium salt hydrate), MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt), and XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium, inner salt, monosodium salt hydrate). With this configuration, it becomes possible to set a lower reaction temperature when performing the quantitative determination method for reducing sugars, thereby simplifying the equipment configuration used for measurement and improving the safety of the measurement operation.(3) In the above-described method for quantifying reducing sugars, the tetrazolium salt may be WST-1 (2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) or WST-8 (1-(2-Methoxy-4-nitrophenyl)-3-(2,4-disulfophenyl)-5-(4-nitrophenyl) formazan, disodium salt hydrate). With such a configuration, it is possible to set the reaction temperature when carrying out the method for quantifying reducing sugars at a lower temperature, thereby simplifying the equipment configuration used for measurement and improving the safety of the measurement operation. (4) In the above-described method for quantifying reducing sugars, the tetrazolium salt is WST-1 (2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) or XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium, inner salt, monosodium salt, hydrate), and the step of directly reducing the tetrazolium salt by the reducing properties of the reducing sugar to produce a water-soluble formazan may be carried out in a temperature range of 4°C to 100°C. With such a configuration, the reaction temperature when carrying out the method for quantifying reducing sugars can be set in a wide temperature range of 4°C to 100°C, thereby improving the convenience of performing the operation to quantify reducing sugars.(5) In the above-described method for quantifying reducing sugars, the tetrazolium salt is one of WST-1 (2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt), WST-8 (1-(2-Methoxy-4-nitrophenyl)-3-(2,4-disulfophenyl)-5-(4-nitrophenyl) formazan, disodium salt hydrate), and XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium, inner salt, monosodium salt hydrate), and the step of directly reducing the tetrazolium salt by the reducing properties of the reducing sugar to produce water-soluble formazan may be carried out in a temperature range of 4°C to 60°C. With this configuration, the reaction temperature when performing the method for quantifying reducing sugars is set in a relatively low temperature range of 4°C to 60°C, thereby simplifying the equipment configuration used for measurement and increasing the safety of the measurement operation. (6) In the method for quantifying reducing sugars according to the above embodiment, the step of directly reducing the tetrazolium salt by the reducing properties of the reducing sugar to produce water-soluble formazan may be performed in a temperature range of 4°C to room temperature. With this configuration, the reaction temperature when performing the method for quantifying reducing sugars is set in a relatively low temperature range of 4°C to room temperature, so there is no need to use special heating means when performing the reaction to reduce the tetrazolium salt in order to quantify the reducing sugar, thereby simplifying the equipment configuration used for measurement and increasing the safety of the measurement operation. (7) In the method for quantifying reducing sugars according to the above embodiment, the step of optically quantifying the water-soluble formazan may be performed using at least one of the wavelengths of 400 to 440 nm and 540 to 650 nm as the measurement wavelength. With this configuration, the amount of reducing sugar in a reducing sugar-containing sample can be measured with high accuracy.(8) In the above-described method for quantifying reducing sugars, the step of directly reducing the tetrazolium salt by the reducing properties of the reducing sugar to produce water-soluble formazan may be performed in a temperature range of 4°C to 30°C, and the step of optically quantifying the water-soluble formazan may be performed with a measurement wavelength of 540 to 650 nm. With such a configuration, the amount of reducing sugar in a reducing sugar-containing sample can be measured with high accuracy. (9) According to another embodiment of the present disclosure, a reducing sugar amount measurement kit is provided for measuring the amount of reducing sugar in a reducing sugar-containing sample containing a reducing sugar. This reducing sugar amount measurement kit comprises a tetrazolium salt that is reduced to produce water-soluble formazan, and a strongly alkaline solution for preparing a reaction solution with strongly alkaline conditions as a reaction solution for reacting the reducing sugar-containing sample with the tetrazolium salt. According to this type of reducing sugar content measurement kit, a tetrazolium salt that generates water-soluble formazan upon reduction is added to a reducing sugar-containing sample containing reducing sugars. Under strongly alkaline conditions, the reducing properties of the reducing sugars in the reducing sugar-containing sample directly reduce the tetrazolium salt to generate water-soluble formazan. The amount of reducing sugar in the sample can then be easily measured by optically quantifying the resulting water-soluble formazan. This disclosure can be implemented in various forms other than those described above, for example, in a method for measuring the decomposition activity of polysaccharides such as real biomass and cellulose.

[0007] A flowchart illustrating the method for quantifying reducing sugars. An explanatory diagram showing the relationship between glucose concentration and absorbance in TB. An explanatory diagram showing the results of deriving the regression line from Figure 2. An explanatory diagram showing the results of comparing the measurement values ​​immediately after cooling with the measurement values ​​15 minutes later. An explanatory diagram showing the measurement results for WST-1. An explanatory diagram showing the measurement results for MMT. An explanatory diagram showing the measurement results for NTB. An explanatory diagram showing the measurement results for INT. An explanatory diagram showing an image of the assay plate after reaction cessation. An explanatory diagram showing the results of absorbance measurement at wavelengths of 320 nm to 760 nm. An explanatory diagram showing the results of absorbance measurement at wavelengths of 460 nm to 700 nm. An explanatory diagram showing the absorbance when the final concentration of WST-1 is 0.05%. An explanatory diagram showing the measurement values ​​immediately after reaction cessation and 10 minutes after reaction cessation superimposed. An explanatory diagram showing the absorbance when the reaction conditions are heated at 60°C for 5 minutes. An explanatory diagram showing the measurement values ​​immediately after reaction cessation and 10 minutes after reaction cessation superimposed. An explanatory diagram showing the absorbance measurement results when the reaction temperature is set to room temperature. An explanatory diagram showing the regression line derived from the measured values. An explanatory diagram showing the absorbance measurement results at a measurement wavelength of 420 nm. An explanatory diagram showing the absorbance measurement results at a measurement wavelength of 600 nm. An explanatory diagram showing the regression line derived from the measured values. An explanatory diagram showing the regression line derived from the measured values. An explanatory diagram showing the regression line derived from the measured values. An explanatory diagram showing the regression line derived from the measured values. An explanatory diagram showing the regression line derived from the measured values. An explanatory diagram showing the results of direct reduction with reducing sugars. An explanatory diagram showing an image of the assay plate after reaction cessation. An explanatory diagram showing the results of measuring absorbance after changing the pH of the reaction solution.

[0008] A. First Embodiment: Figure 1 is a flowchart showing a method for quantifying reducing sugars as an embodiment of the present disclosure. When quantifying reducing sugars, first, a reducing sugar-containing sample is prepared as the object of measurement (step T100). The reducing sugar-containing sample is not particularly limited as long as it is a sample that contains the reducing sugar to be quantified. For example, it can be a sample obtained by decomposing actual biomass or polysaccharides such as cellulose, and in such cases, the method for quantifying reducing sugars of this embodiment can be used to measure the decomposition activity of polysaccharides.

[0009] After step T100, a tetrazolium salt is added to the reducing sugar-containing sample prepared in step T100 to prepare a strongly alkaline reaction solution (step T110). Then, the tetrazolium salt is directly reduced by the reducing sugar to produce water-soluble formazan (step T120). Subsequently, the water-soluble formazan is optically quantified (step T130) to quantify the amount of reducing sugar in the reducing sugar-containing sample. In this embodiment of the method for quantifying reducing sugars, by performing steps T110 and T120, under strongly alkaline conditions, the reducing properties of the reducing sugar in the reducing sugar-containing sample directly reduce the tetrazolium salt to produce water-soluble formazan from the tetrazolium salt. Then, in step T130, the amount of water-soluble formazan produced is measured by optical measurement using an appropriate wavelength corresponding to the water-soluble formazan produced.

[0010] The above-mentioned "strongly alkaline conditions" refer to a reaction solution with a pH of 11 or higher when direct reduction is carried out with reducing sugars, preferably 11.5 or higher, and possibly 12 or higher. Such a reaction solution can be prepared by adding a strong alkali such as sodium hydroxide or potassium hydroxide to a final concentration of 0.01 to 1.0 M. Furthermore, it is desirable that the above-mentioned strongly alkaline reaction solution contains sodium potassium tartrate to a final concentration of 0.1 to 1.0 M.

[0011] Furthermore, "directly reducing tetrazolium salts" means reducing tetrazolium salts using the reducing properties of reducing sugars, and NAD ＋ This refers to the direct reduction of a tetrazolium salt by the reducing properties of the aldehyde group of a reducing sugar, without the need for coenzymes such as NADH or electron carriers (electron mediators).

[0012] The tetrazolium salt used is not particularly limited, as long as it can be directly reduced by a reducing sugar under strongly alkaline conditions to produce a water-soluble formazan. Such tetrazolium salts can be, for example, any of the following: WST-1 (2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt), WST-8 (1-(2-Methoxy-4-nitrophenyl)-3-(2,4-disulfophenyl)-5-(4-nitrophenyl) formazan, disodium salt hydrate), MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt), and XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium, inner salt, monosodium salt hydrate). Among these, WST-1 (2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) is preferred.

[0013] The reaction in step T120 to produce water-soluble formazan from the tetrazolium salt can be carried out in a temperature range of, for example, 4°C to 100°C, or in a temperature range of 4°C to 60°C, or in a temperature range of 4°C to room temperature. Furthermore, the water-soluble formazan produced from the tetrazolium salt in step T120 can be measured using, for example, at least one of the wavelengths between 400 and 440 nm and 540 and 650 nm as the measurement wavelength.

[0014] Among the tetrazolium salts, WST-1, WST-8, MTS, and XXT, as previously described, can be directly reduced by reducing sugars in a relatively low temperature range of 4°C to 30°C. Therefore, the reaction temperature for directly reducing tetrazolium salts can be set in a relatively low temperature range of 4°C to 30°C, for example, to room temperature. As described above, tetrazolium salts that can be directly reduced by reducing sugars over a temperature range of 4°C to 30°C are also called "low-temperature reactive tetrazolium salts." In particular, WST-1 and WST-8 are preferred because they exhibit particularly excellent low-temperature reactivity, being directly reduced by reducing sugars in a relatively low temperature range such as 4°C to 25°C, and can show higher values ​​when their absorbance is measured after the completion of the reduction reaction.

[0015] Furthermore, among the tetrazolium salts, WST-1, WST-8, and XTT, as previously described, can be directly reduced by reducing sugars over a wider temperature range, including a relatively low temperature range of 4°C to 60°C. Therefore, the reaction temperature for directly reducing tetrazolium salts can be set to a temperature range of 4°C to 60°C or 4°C to 50°C. In addition, among the tetrazolium salts, WST-1 and XTT, as previously described, can be directly reduced by reducing sugars over a wider temperature range, from a relatively low temperature range of 4°C to 100°C to a relatively high temperature range, and can be suitably used for the quantitative determination of reducing sugars.

[0016] When measuring the amount of reducing sugar in a reducing sugar-containing sample, the result can be generalized by, for example, converting it to glucose equivalent. In such cases, the measurement wavelength for the resulting water-soluble formazan can be set as the wavelength at which the absorbance peak is observed, or a wavelength near the peak (a wavelength with a difference of 20 nm or less from the peak wavelength), by performing the reducing sugar quantification method shown in Figure 1 using a glucose solution as the reducing sugar-containing sample, and measuring the absorbance while varying the wavelength. Of the low-temperature reactive tetrazolium salts mentioned above, WST-1 shows absorbance peaks at 420 nm and 590 nm, WST-8 shows absorbance peaks at 440 nm and 640 nm, MTS shows an absorbance peak at 540 nm, and XTT shows absorbance peaks at 420 nm and 600 nm.

[0017] The tetrazolium salt used in the method for quantifying reducing sugars in this embodiment can be any tetrazolium salt other than those described above, as long as it can be directly reduced by the reducing properties of the reducing sugar. For example, it is sufficient if it has the property of being able to quantify the amount of reducing sugar in terms of glucose. From the viewpoint of quantifying the amount of reducing sugar in terms of glucose, it is desirable that the tetrazolium salt used in the method for quantifying reducing sugars in this embodiment has the following properties.

[0018] Specifically, 195 μL of a tetrazolium salt solution containing a final concentration of 0.01–0.1% tetrazolium salt, 0.5 M potassium sodium tartrate, and 0.05 M sodium hydroxide is mixed with 5 μL of a 0–20 mM glucose solution. The reaction solution is heated at 100°C for 5 minutes or at 25°C for 60 minutes, then cooled, and the absorbance is measured at a wavelength corresponding to the tetrazolium salt. A regression line (linear approximation line) is then created by plotting the glucose concentration in the glucose solution added to the tetrazolium salt solution (hereinafter simply referred to as "glucose concentration") on the x-axis and the measured absorbance on the y-axis. The coefficient of determination R of the regression line thus created is calculated. ２ It is desirable to use a tetrazolium salt that yields a regression line of 0.99 or higher. Furthermore, the coefficient of determination R ２Within the glucose concentration range where a regression line of 0.99 or higher can be obtained, the regression line can be suitably used as a calibration curve (standard curve) for quantifying reducing sugars in glucose equivalent. ２ The coefficient of determination (R) is equal to the squared value of the correlation coefficient (r), and is also called the rate of explanation of variance (%). It represents the proportion of the variance of the dependent variable (y) explained by the explanatory variable (x). ２ A value of 0.99 or higher indicates sufficient linearity in the relationship between glucose concentration and absorbance. Although the properties of tetrazolium salts, such as oxidation-reduction potential, change depending on the structure of the side chain, any tetrazolium salt that satisfies the above properties can be used effectively in the quantitative determination method of reducing sugars in this embodiment. In the following description, when the concentration in solution is expressed as a percentage, it will be expressed as mass percentage.

[0019] The "measurement wavelength corresponding to the tetrazolium salt" mentioned above can be identified as follows, provided that the tetrazolium salt has some activity that allows it to be directly reduced by reducing sugars. Specifically, by using a glucose solution as a reducing sugar-containing sample and performing the quantitative analysis method for reducing sugars shown in Figure 1, and measuring the absorbance while varying the wavelength, the wavelength showing the absorbance peak or a wavelength near the peak (a wavelength with a difference of 20 nm or less from the peak wavelength) should be set as the measurement wavelength. Such measurement wavelengths can be, for example, around 400-440 nm or around 540-650 nm.

[0020] In this embodiment, "water-soluble formazan" means that the resulting formazan has sufficiently high solubility in water. Not only when the resulting formazan aggregates to form a visible precipitate, but even when the aggregate is difficult to see, if the solubility in water is insufficient, the measured value when performing optical measurements may decrease over time, resulting in insufficient measurement accuracy. Therefore, the tetrazolium salt that produces water-soluble formazan should specifically satisfy the following conditions.

[0021] Specifically, 195 μL of a tetrazolium salt solution containing a tetrazolium salt with a final concentration of 0.01–0.1% in the reaction mixture, 0.5 M potassium sodium tartrate, and 0.05 M sodium hydroxide is mixed with 5 μL of glucose solution at concentrations of 0 mM or 10 mM. The reaction mixture is heated at 100°C for 5 minutes or at 25°C for 60 minutes, then cooled. Immediately after cooling (at 0 minutes), the absorbance is measured at the measurement wavelength corresponding to the tetrazolium salt. Ten minutes after the start of cooling, the absorbance is measured again at the same measurement wavelength. For the measurement with a glucose concentration of 10 mM, the measurement at 10 minutes is subtracted from the measurement at 0 minutes, and the difference is calculated. At this time, for the measurement with a glucose concentration of 10 mM, the measurement with a glucose concentration of 0 mM, which has the same elapsed time, is subtracted as background beforehand. If the difference value described above is less than 10% of the value measured at 0 minutes with a glucose concentration of 10 mM, the resulting formazan can be evaluated as a "water-soluble formazan" with sufficiently high solubility in water. The "measurement wavelength corresponding to the tetrazolium salt" described above can be determined by using the glucose solution as a reducing sugar-containing sample, performing the reducing sugar quantification method shown in Figure 1, and measuring the absorbance while varying the wavelength. The measurement wavelength should be the wavelength that shows the absorbance peak or a wavelength near the peak wavelength (a wavelength with a difference of 20 nm or less from the peak wavelength). Such measurement wavelengths can be, for example, around 400-440 nm or around 540-650 nm. Although the properties of tetrazolium salts, such as solubility in water, change depending on the structure of the side chain, any tetrazolium salt that satisfies the above conditions can be used well as a "water-soluble formazan" in the reducing sugar quantification method of this embodiment.

[0022] According to the method for quantifying reducing sugars of this embodiment, configured as described above, a tetrazolium salt that produces water-soluble formazan when reduced is used to quantify the reducing sugar. This improves the stability of the measurement values ​​when optically measuring the resulting formazan, enabling more sensitive measurements. ＋Because this reaction utilizes the reducing properties of the aldehyde group of a reducing sugar to directly reduce a tetrazolium salt without the need for coenzymes such as NADH or electron carriers (electron mediators), the process for composing and quantifying the reaction solution can be simplified.

[0023] Furthermore, it becomes possible to set the reaction temperature for reducing tetrazolium salts with reducing sugars to a temperature lower than 100°C, simplifying the configuration of the measurement apparatus and improving the safety of the measurement operation. In particular, when using the "low-temperature reactive tetrazolium salt" described above, the reaction temperature for reducing the tetrazolium salt with reducing sugars can be set to a relatively low temperature, such as room temperature, thus reducing or eliminating the need for special heating means for the reduction of tetrazolium salts. In addition, it becomes possible to broaden the measurement range for the concentration in which reducing sugars can be quantified and to improve the measurement sensitivity of reducing sugars.

[0024] Another embodiment of the present disclosure is a "reducing sugar content measurement kit for measuring the amount of reducing sugar in a reducing sugar-containing sample containing reducing sugars." Specifically, this kit includes a "tetrazolium salt that is reduced to produce water-soluble formazan" and a "strong alkaline solution for preparing a reaction solution with strongly alkaline conditions, which is used as a reaction solution for reacting the reducing sugar-containing sample with the tetrazolium salt." Using such a reducing sugar content measurement kit, the method for quantifying reducing sugars in the embodiments described above can be easily carried out. In the reducing sugar content measurement kit, the tetrazolium salt and the strong alkaline solution may be provided separately or as a mixed solution.

[0025] <Confirmation of the properties of tetrazolium blue> Non-patent document 3 reports that tetrazolium blue TB (3,3'-[3,3'-Dimethoxy-(1,1'-biphenyl)-4,4'-diyl]bis(2,5-diphenyl-2H-tetrazolium) can be used to quantify reducing sugars by reducing them to produce blue formazan. The properties of tetrazolium chloride were confirmed. Following the method described in Non-Patent Document 3, the concentration of glucose, a reducing sugar, was varied to directly reduce TB with glucose, and the absorbance after the reaction was measured to derive a regression line. Specifically, a solution containing TB (product number: 05107-41, manufactured by Nacalai Tesque Co., Ltd.) with a final concentration of 0.1% in the reaction solution, 0.5 M sodium potassium tartrate, and 0.05 M sodium hydroxide was prepared as a tetrazolium salt solution. 5 μL of glucose solution (0-20 mM) was added to 195 μL of the obtained tetrazolium salt solution to prepare the reaction solution, which was heated at 100°C for 5 minutes and then cooled on ice. Immediately after the start of cooling (stopping the reaction), 150 μL of the reaction solution was transferred to an assay plate (Micro well plate GDMP-96F, AS ONE Corporation), and the absorbance at a measurement wavelength of 650 nm was measured using a plate reader (multi-detection mode microplate reader Spark, manufactured by TECAN).

[0026] Figure 2 is an explanatory diagram showing the relationship between glucose concentration and absorbance as measurement results. Figure 3 is an explanatory diagram showing the results of deriving a regression line for the glucose concentration measurements in the range of 0 to 2 mM shown in Figure 2. In Figures 2 and 3, the horizontal axis shows the glucose concentration in the added glucose solution (hereinafter also simply referred to as "glucose concentration"), and the vertical axis shows the measured absorbance at a measurement wavelength of 650 nm. As shown in Figure 3, in the glucose concentration range of 2 mM or less, a regression line (y = 0.1656x + 0.0051) is obtained, and the coefficient of determination R ２The value was 0.99. Thus, if the glucose-equivalent concentration of the reducing sugar to be quantified is within the concentration range corresponding to the case where the added glucose concentration is 0 to 2 mM as described above, it is considered possible to use the above regression line as a calibration curve for quantifying reducing sugars in glucose equivalent.

[0027] Furthermore, regarding TB, the stability of the measured values ​​of formazan produced by reduction was investigated by examining the time change of the measured value of the reducing sugar content of glucose (absorbance at 650 nm) as measured above. Specifically, as previously described, a reaction solution was prepared by adding 5 μL of glucose solution (0-20 mM) to 195 μL of TB solution, heating it at 100°C for 5 minutes, and then cooling it with ice. After that, the measured value immediately after the start of cooling (0 minutes) and the measured value 15 minutes after the start of cooling were compared.

[0028] Figure 4 is an explanatory diagram showing a comparison of measurements taken immediately after the start of cooling and measurements taken 10 minutes after the start of cooling. As shown in Figure 4, the measurement taken 10 minutes after the start of cooling was smaller than the measurement taken immediately after the start of cooling, confirming that the measurement value decreased over time. At this time, the degree of decrease in absorbance over time increased with increasing glucose concentration. For the measurement taken with a glucose concentration of 10 mM, as described above, the difference was calculated by subtracting the measurement taken 10 minutes after the start of cooling from the measurement taken 0 minutes after subtracting the background, and the difference value was found to be more than 10% of the measurement taken 0 minutes after the start of cooling, confirming that the stability of the measurement value after the reaction was insufficient. In addition, in the well of the reaction solution to which a 10 mM glucose solution was added, small granular precipitate was visually observed 10 minutes after the start of cooling. Therefore, TB considers that the absorbance decreased due to the aggregation of formazan because the water solubility of the generated formazan was insufficient.

[0029] <Direct reduction of tetrazolium salts using reducing sugars> [Comparison of various tetrazolium salts]Direct reduction of various tetrazolium salts was performed using reducing sugars. Here, four types of tetrazolium salts were used: WST-1 (2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt, product number: 342-06451, manufactured by Fujifilm Wako Pure Chemical Corporation), MMT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, product number: 345-01821, manufactured by Fujifilm Wako Pure Chemical Corporation), NTB (3,3'-[3,3'-Dimethoxy-(1,1'-biphenyl)-4,4'-diyl]-bis[2-(4-nitrophenyl)-5-phenyl2H-tetrazolium chloride], product number: 24720-01, manufactured by Nacalai Tesque, Inc.), and INT (2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride, product number: I10406, manufactured by Sigma-Aldrich). These tetrazolium salts are generally used as chromogenic agents during enzyme activity measurements and are used in reaction systems via coenzymes such as NAD ＋ and coenzymes and electron carriers (electron mediators) such as NADH.

[0030] When performing the reduction reaction, for each tetrazolium, a solution containing a tetrazolium salt with a final concentration of 0.1% in the reaction solution, 0.5 mM sodium potassium tartrate, and 0.05 M sodium hydroxide was prepared as the tetrazolium salt solution. To 195 μL of each obtained tetrazolium salt solution, 5 μL of a glucose solution (0 - 20 mM) was added, heated at 100°C for 5 minutes, and then cooled. Immediately after the start of cooling (reaction stop), 150 μL of the reaction solution was transferred to an assay plate (Micro well plate GDMP-96F, AS ONE Corporation), and the absorbance was measured using a plate reader (Multi-detection mode microplate reader Spark, manufactured by TECAN). A regression line was derived from the measured values within the glucose concentration range of 0 - 20 mM.

[0031] Figure 5 is an explanatory diagram showing the measurement results for WST-1, Figure 6 is an explanatory diagram showing the measurement results for MMT, Figure 7 is an explanatory diagram showing the measurement results for NTB, and Figure 8 is an explanatory diagram showing the measurement results for INT. Also, Figure 9 is an explanatory diagram showing an image of the assay plate within 10 minutes after the absorbance measurement and after the reaction stop. In Figures 5 to 8, the horizontal axis shows the glucose concentration in the glucose solution added to the tetrazolium salt solution (hereinafter also simply referred to as "glucose concentration"), and the vertical axis shows the measured value of the absorbance at a measurement wavelength of 650 nm.

[0032] As shown in Figure 5, in the case of WST-1, the absorbance increased with the increase in glucose concentration, and it was confirmed that WST-1 was directly reduced by glucose, which is a reducing sugar. Also, as shown in Figure 9, no aggregation of the formed formazan was observed. When using WST-1, the absorbance (background) in the case without glucose addition (glucose concentration = 0 mM) was relatively high, and when the added glucose concentration was 10 mM or more, the measured values exceeded the detection limit. In contrast, for MMT, there was no change in absorbance, and no formation of formazan was observed. For NTB, the absorbance increased with the increase in glucose concentration, and linearity was observed as the relationship between glucose concentration and absorbance up to a glucose concentration of 20 mM (the determination coefficient R of the derived regression line ２(=0.9978), it was confirmed that NTB was directly reduced by glucose, a reducing sugar. However, the absorbance was relatively high when no glucose was added (glucose concentration = 0 mM), and as shown in Figure 9, aggregation of the generated formazan was observed when the concentration of the added glucose solution was 1 mM or higher. In INT, the absorbance increased with the addition of glucose, suggesting that formazan was generated, but no linearity was observed in the relationship between glucose concentration and absorbance. Furthermore, as shown in Figure 9, aggregation of the generated formazan was also observed.

[0033] [Measurement Wavelength for WST-1] As shown in Figure 5, for WST-1, in which direct reduction by reducing sugars is considered to have proceeded well, the relationship between measurement wavelength and absorbance was further investigated. Here, absorbance was measured using wavelengths in the range of 320 nm to 760 nm for samples using WST-1 as the tetrazolium salt shown in Figure 5, with glucose concentrations of 0 mM, 2 mM, 4 mM, and 10 mM.

[0034] Figure 10 is an explanatory diagram showing the results of measuring absorbance in the measurement wavelength range of 320 nm to 760 nm for samples with glucose solutions added to tetrazolium salt solution at concentrations of 0 mM, 4 mM, and 10 mM. Figure 11 is an explanatory diagram showing the results of measuring absorbance in the measurement wavelength range of 460 nm to 700 nm for samples with glucose concentrations of 0 mM and 2 mM. As shown in Figures 10 and 11, in the sample with a glucose concentration of 0 mM, almost no increase in absorbance was observed even when the measurement wavelength was changed. In contrast, in samples with a glucose concentration of 2 mM or higher, an increase in absorbance was observed with changes in measurement wavelength, and absorbance peaks were observed at wavelengths of 420 nm and 590 nm. It should be noted that in the samples with glucose concentrations of 4 mM and 10 mM, the measured absorbance is considered to have reached the detection limit when the measurement wavelength is near 590 nm. Based on these results, when using WST-1 as the tetrazolium salt and performing direct reduction with reducing sugars, the measurement wavelengths for quantifying the resulting water-soluble formazan are considered to be preferably around 420 nm and around 590 nm.

[0035] [Concentration of WST-1 during reaction] For WST-1, the final concentration in the reaction solution was varied within the range from 0.1% to 0.0025%, and the reaction was carried out under the same conditions as the measurement results shown in FIG. 5 (reaction conditions: 100° C. for 5 minutes) except for the concentration of the tetrazolium salt. When 0 to 20 mM of glucose was added to the tetrazolium salt solution, the amount of formazan produced was measured. The absorbance in the case without glucose addition (glucose concentration 0 mM) was low, and from the measured values, the determination coefficient R of the regression straight line derived ２ was closer to 1 (excellent linearity). From this viewpoint, the tetrazolium salt concentration was most excellent when the final concentration of WST-1 in the reaction solution was 0.05% (data not shown).

[0036] FIG. 12 is an explanatory diagram showing the measurement results of the absorbance at a measurement wavelength of 420 nm when the final concentration of WST-1 in the reaction solution is 0.05%. FIG. 12(A) shows the result of plotting the measured values in the same manner as in FIG. 5, and FIG. 12(B) shows the regression straight line derived from the measured values in the range of glucose concentration 0 mM to 10 mM. In FIG. 12(B), the value obtained by subtracting the measured value of glucose concentration 0 mM shown in FIG. 12(A) from the measured values at each glucose concentration shown in FIG. 12(A) as the background is used. As shown in FIGS. 12(A) and 12(B), the absorbance increases as the glucose concentration increases. As shown in FIG. 12(B), the measured values in the range of glucose concentration 0 mM to 10 mM show high linearity, and the determination coefficient R of the regression straight line (y = 0.2008x - 0.0005) derived from the measured values ２ was 0.9997. Thus, by setting the final concentration of WST-1 in the reaction solution to 0.05% for example, when the glucose-equivalent concentration of the reducing sugar in the reducing sugar-containing sample to be quantified corresponds to the concentration range of the above-mentioned glucose concentration 0 to 10 mM, it was confirmed that good quantification becomes possible. Also, when the final concentration of WST-1 was 0.05%, the absorbance in the case without glucose addition was a relatively low value of 0.3 or less.

[0037] [Stability of measurement values ​​when using WST-1] Using WST-1 as the tetrazolium salt, the reaction was carried out under the same conditions as the measurement results shown in Figure 5 (reaction conditions: 100°C for 5 minutes), except that the final concentration of WST-1 in the reaction solution was 0.05%. The change in absorbance was compared between immediately after the reaction stopped (0 min) and 10 minutes after the reaction stopped (10 min).

[0038] Figure 13 is an explanatory diagram showing the relationship between glucose concentration and absorbance, with the measured value immediately after reaction cessation (0 min) and the measured value 10 minutes after reaction cessation (10 min) superimposed. As shown in Figure 13, the absorbance did not decrease even after 10 minutes had elapsed since reaction cessation, confirming that stable measurement was possible. Specifically, in the sample shown in Figure 13, the following holds true. That is, for the measured value of the added glucose solution concentration of 10 mM, the measured value at 10 minutes was subtracted from the measured value at 0 minutes, and the difference was calculated. At this time, for the measured value of glucose concentration of 10 mM mentioned above, the measured value of glucose concentration of 0 mM, which had the same elapsed time, was subtracted as background beforehand. Then, the value of the difference mentioned above was compared with the value at 0 minutes when the glucose concentration was 10 mM. As a result, the value of the difference mentioned above was less than 10% of the value at 0 minutes mentioned above.

[0039] [Reaction Temperature of WST-1] Using WST-1 as the tetrazolium salt, the reaction temperature for direct reduction with reducing sugars was changed from 100°C, and the measurement results of the amount of formazan produced were compared. Here, a solution containing WST-1 with a final concentration of 0.05% in the reaction mixture, 0.5 M potassium sodium tartrate, and 0.05 M sodium hydroxide was prepared as the tetrazolium salt solution. 5 μL of glucose solution (0-20 mM) was added to 195 μL of each obtained tetrazolium salt solution, and the mixture was heated at 60°C for 5 minutes and then cooled. Immediately after the start of cooling (stopping the reaction), 150 μL of the reaction mixture was transferred to an assay plate (Micro well plate GDMP-96F, AS ONE Corporation), and the absorbance at a measurement wavelength of 420 nm was measured using a plate reader (Multi-detection mode microplate reader Spark, TECAN). Then, a regression line was derived from the measured values ​​in the glucose concentration range of 0-20 mM. Furthermore, the absorbance at the measurement wavelength of 420 nm was measured in the same manner 10 minutes (10 min) after the reaction stopped.

[0040] Furthermore, the reaction conditions were changed from heating at 60°C for 5 minutes as described above to leaving the mixture at room temperature (25°C) for 30 minutes, or leaving it at room temperature (25°C) for 90 minutes, and the same measurements were performed.

[0041] Figure 14 is an explanatory diagram showing the absorbance measurement results at a measurement wavelength of 420 nm when the reaction conditions were heating at 60°C for 5 minutes. Figure 14(A) shows the results of plotting the measured values ​​in the same manner as in Figure 5, and Figure 14(B) shows the regression line derived from the measured values ​​in the range of glucose concentrations from 0 mM to 20 mM. Note that in Figure 14(B), the measured values ​​for each glucose concentration shown in Figure 14(A) were subtracted from the measured values ​​for the glucose concentration of 0 mM shown in Figure 14(A) as background. As shown in Figure 14, when the reaction conditions were heating at 60°C for 5 minutes, formazan formation was observed, similar to the case of heating at 100°C for 5 minutes shown in Figure 12. As shown in Figure 12, when the reaction conditions were heating at 100°C for 5 minutes, linearity was observed in the relationship between glucose concentration and absorbance in the range of added glucose concentrations up to 10 mM (coefficient of determination R of the derived regression line). ２= 0.9997). In contrast, as shown in Figure 14, when the reaction conditions were 60°C for 5 minutes, linearity was observed in the relationship between glucose concentration and absorbance in the range up to 20 mM of added glucose concentration. That is, the coefficient of determination R of the regression line (y = 0.116x + 0.0127) shown in Figure 14(B) ２ The value was 0.9989.

[0042] Figure 15 is an explanatory diagram showing the relationship between glucose concentration and absorbance in a sample heated at 60°C for 5 minutes under the reaction conditions shown in Figure 14, with the measured values ​​immediately after reaction cessation (0 min) and 10 minutes after reaction cessation (10 min) superimposed. As shown in Figure 15, the absorbance did not decrease even after 10 minutes had elapsed since reaction cessation, confirming that stable measurement was possible.

[0043] Figure 16 is an explanatory diagram showing the absorbance measurement results at a measurement wavelength of 420 nm when the reaction conditions were left at room temperature (25°C) for 30 minutes (30 min) and 90 minutes (90 min). Figure 17 is an explanatory diagram showing the regression line derived from the measured values. Figure 17(A) shows the results after reacting at room temperature (25°C) for 30 minutes, and Figure 17(B) shows the results after reacting at room temperature (25°C) for 90 minutes. Note that in Figures 17(A) and 17(B), the measured values ​​at each glucose concentration shown in Figure 16 were subtracted from the measured values ​​at 0 mM glucose concentration shown in Figure 16 as background. As shown in Figures 16, 17(A), and 17(B), even when the reaction temperature was room temperature (25°C), the measured absorbance increased in proportion to the amount of reducing sugar added, and the formation of formazan was observed. Furthermore, in both cases where the reaction conditions were 30 minutes at room temperature (25°C) and 90 minutes at room temperature (25°C), linearity was observed in the relationship between glucose concentration and absorbance up to a glucose concentration of 20 mM. That is, as shown in Figure 17(A), in the case of 30 minutes at room temperature, the coefficient of determination R of the derived regression line (y = 0.007x - 0.0037) ２ The coefficient of determination R for the derived regression line (y = 0.0284x - 0.0095) is 0.9980, and as shown in Figure 17(B), in the case of 90 minutes at room temperature,２ The value was 0.9965. Thus, when using WST-1 as the tetrazolium salt, it was confirmed that even when the reaction temperature is changed from 100°C to 60°C or room temperature (25°C), it is possible to quantify the reducing sugar by direct reduction with the reducing sugar.

[0044] For example, comparing the results when the reaction temperature is 60°C with the results when the reaction temperature is 100°C shown in Figure 12, it can be seen that even when the reaction temperature is lowered to 60°C, the measured values ​​remain at approximately 60% of the value when the reaction temperature is 100°C. In contrast, Non-Patent Document 3, which discloses the use of TB as the tetrazolium salt, states that the measured values ​​when the reaction conditions are heated at 60°C for 3 minutes decrease to about 5% of those when heated at 100°C for 3 minutes. Furthermore, as shown in Figure 3, when TB is used as the tetrazolium salt, the range in which sufficient linearity is observed in the relationship between glucose concentration and absorbance is limited to when the glucose concentration is 2 mM or less. Thus, it has been confirmed that when WST-1 is used as the tetrazolium salt, it is possible to set a lower reaction temperature for direct reduction by reducing sugars, and the range of detectable reducing sugar concentrations can be widened.

[0045] Furthermore, using WST-1 as the tetrazolium salt, we varied the reaction conditions for direct reduction with reducing sugars and compared the measurement results of the amount of formazan produced. Here, the direct reduction reaction with reducing sugars was carried out under the same conditions as those shown in Figure 14, except for the reaction conditions (reaction temperature and reaction time). Seven different reaction conditions were adopted: 4°C for 16 hours, 25°C for 60 minutes, 30°C for 60 minutes, 40°C for 15 minutes, 50°C for 10 minutes, 60°C for 10 minutes, and 100°C for 5 minutes. Two wavelengths were used for measurement: 420 nm and 600 nm.

[0046] Figure 18 is an explanatory diagram showing the absorbance measurement results at a measurement wavelength of 420 nm as a result of each reaction condition, and Figure 19 is an explanatory diagram showing the absorbance measurement results at a measurement wavelength of 600 nm. As shown in Figure 18, in the case of a measurement wavelength of 420 nm, linearity was observed in the relationship between glucose concentration and absorbance over a reaction temperature range of 4°C to 100°C and over a glucose concentration range of 0 mM to 10 mM. Furthermore, in the reaction temperature range of 4°C to 50°C, sufficient linearity was observed in the relationship between glucose concentration and absorbance over a glucose concentration range of 0 mM to 20 mM.

[0047] Furthermore, as shown in Figure 19, when the measurement wavelength was 600 nm, the measured values ​​were relatively larger compared to when the measurement wavelength was 420 nm, and relatively large values ​​were obtained even when the measurement temperature was between 4°C and 30°C. In addition, when the measurement wavelength was 600 nm, sufficient linearity was observed in the relationship between glucose concentration and absorbance in the temperature range of 4°C to 30°C, with the added glucose concentration in the range of 0 mM to 20 mM.

[0048] Therefore, when setting the reaction temperature to 4°C to 30°C, it is desirable to use a wavelength of 600 nm or near 600 nm as the measurement wavelength. Furthermore, when using a reaction temperature of 40°C or higher, it is desirable to use a wavelength of 420 nm or near 420 nm as the measurement wavelength, from the viewpoint of making it easier to keep the detected value within a general measurement range. In conclusion, it has been confirmed that by changing the tetrazolium salt concentration in the reaction solution, as well as appropriately adjusting the measurement wavelength and reaction conditions (reaction temperature and reaction time), it is possible to detect reducing sugars over a wider concentration range with greater accuracy.

[0049] [Reaction temperatures of various tetrazolium salts] Furthermore, various tetrazolium salts were subjected to direct reduction using reducing sugars. In this study, in addition to WST-1 as previously described, WST-8 (1-(2-Methoxy-4-nitrophenyl)-3-(2,4-disulfophenyl)-5-(4-nitrophenyl) formazan, disodium salt hydrate, product number: S7764, manufactured by Selleck Chemicals), MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, product number: G1112, manufactured by Promega), and XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium, inner salt, monosodium salt. Hydrate, product number: 14919, manufactured by Funakoshi Co., Ltd.) were used as tetrazolium salts. These tetrazolium salts are generally used as chromogenic agents to measure enzyme activity, such as in the measurement of viable cell counts, and NAD ＋ It is used in reaction systems that involve coenzymes such as NADH and electron carriers (electron mediators). Below, in addition to the above-mentioned WST-1, WST-8, MTS, and XTT, TB was also used as a tetrazolium salt, and the results of direct reduction by reducing sugars at reaction temperatures of 25°C and 4°C are shown.

[0050] The direct reduction of each tetrazolium salt with reducing sugars was carried out as follows. First, for each tetrazolium salt, a solution containing the tetrazolium salt, 0.5 M potassium sodium tartrate, and 0.05 M sodium hydroxide was prepared as a tetrazolium salt solution. Each tetrazolium salt solution was prepared so that the final concentration of the tetrazolium salt in the reaction mixture was 0.05% for WST-1, 0.05% for WST-8, 0.025% for MTS, 0.05% for XXT, and 0.1% for TB. Then, 5 μL of glucose solution (0-20 mM) was added to 195 μL of the obtained tetrazolium salt solution, and the mixture was left at room temperature (25°C) for 60 minutes without heating, or protected from light at 4°C for 16 hours, and then cooled on ice. Immediately after cooling began (reaction stopped), 150 μL of the reaction solution was transferred to an assay plate (Micro well plate GDMP-96F, AS ONE Corporation), and the absorbance at a measurement wavelength of 600 nm was measured using a plate reader (Multi-detection mode microplate reader Spark, TECAN). A regression line was derived from the measured values ​​in the glucose concentration range of 0 to 20 mM.

[0051] Figures 20 to 24 are explanatory diagrams showing the regression lines derived from the measured values. Figure 20 shows the results of WST-1, Figure 21 shows the results of WST-8, Figure 22 shows the results of MTS, Figure 23 shows the results of XTT, and Figure 24 shows the results of TB. Furthermore, Figures 20(A), 21(A), 22(A), 23(A), and 24(A) show the results when the reaction conditions are 25°C for 60 minutes, while Figures 20(B), 21(B), 22(B), 23(B), and 24(B) show the results when the reaction conditions are 4°C for 16 hours.

[0052] As shown in Figures 20(A), 21(A), 22(A), and 23(A), when the reaction conditions are 25°C for 60 minutes, and WST-1, WST-8, MTS, or XTT are used as the tetrazolium salt, the absorbance increases with increasing glucose concentration, and the measured values ​​in the glucose concentration range of 0 mM to 20 mM show high linearity (coefficient of determination R of the regression line). ２The coefficient of determination R of the regression line (y = 0.0858x - 0.0163) was 0.99 or higher in WST-1. ２ The coefficient of determination R in WST-8 is 0.9994, and the coefficient of determination of the regression line (y = 0.0829x - 0.0094) is R. ２ The coefficient of determination R in MTS is 0.9995, and the coefficient of determination of the regression line (y = 0.0385x - 0.0126) is R. ２ The coefficient of determination R for the regression line (y = 0.0512x + 0.0008) in XTT is 0.9992. ２ The value was 0.9987. In contrast, no increase in absorbance was observed with increasing glucose concentration in TB, suggesting that formazan was not produced.

[0053] Furthermore, for each sample shown in Figures 20(A), 21(A), 22(A), and 23(A), in addition to measurements taken immediately after reaction cessation (0 minutes), absorbance was also measured 10 minutes after reaction cessation (data not shown). As a result, it was confirmed that the absorbance did not decrease even after 10 minutes had elapsed since reaction cessation, demonstrating stable measurement capabilities. Specifically, for the samples shown in Figures 20(A), 21(A), 22(A), and 23(A), the following was observed: For the measurement value with an added glucose solution concentration of 10 mM, the measurement value at 10 minutes was subtracted from the measurement value at 0 minutes to find the difference. At this time, for the measurement value with a glucose concentration of 10 mM, a measurement value with a glucose concentration of 0 mM, which had the same elapsed time, was subtracted as background data. The difference value was then compared with the value at 0 minutes for the glucose concentration of 10 mM. As a result, the difference value was less than 10% of the value at 0 minutes.

[0054] As shown in Figures 20(B), 21(B), 22(B), and 23(B), when the reaction conditions are 4°C for 16 hours, and WST-1, WST-8, MTS, or XTT are used as the tetrazolium salt, the absorbance increases with increasing glucose concentration, and the measured values ​​in the glucose concentration range of 0 mM to 20 mM show high linearity (coefficient of determination R of the regression line). ２The coefficient of determination R of the regression line (y = 0.0582x + 0.0073) was 0.99 or higher in WST-1. ２ The coefficient of determination R in WST-8 is 0.9984, and the coefficient of determination of the regression line (y = 0.0598x - 0.0101) is R. ２ The coefficient of determination R in MTS is 0.9993, and the coefficient of determination of the regression line (y = 0.0218x - 0.0003) is R. ２ The coefficient of determination R in XTT is 0.9974, and the coefficient of determination of the regression line (y = 0.0128x - 0.0036) is R. ２ The value was 0.9955. In contrast, no increase in absorbance was observed with increasing glucose concentration in TB, suggesting that formazan was not produced.

[0055] As described above, when using WST-1, WST-8, MTS, or XTT as tetrazolium salts to perform direct reduction with reducing sugars, it was confirmed that a regression line with excellent linearity could be obtained not only at high reaction temperatures such as 100°C, but also at lower temperature conditions such as 25°C (room temperature) or 4°C. Therefore, when using these tetrazolium salts, it is considered possible to arbitrarily set the reaction temperature over a wide temperature range from 4°C to 100°C and quantify reducing sugars by direct reduction with reducing sugars.

[0056] For each of the tetrazolium salts described above (WST-1, WST-8, MTS, XTT, and TB), direct reduction with reducing sugars was performed by varying the reaction temperature and reaction time. The direct reduction of each tetrazolium salt with reducing sugars was carried out under the same conditions as the previously described reactions, except for the reaction temperature and reaction time, as shown in Figures 20 to 24. In addition to "4°C, 16 hours" and "25°C, 60 minutes," the reaction temperatures and times used were "30°C, 60 minutes," "50°C, 10 minutes," "60°C, 10 minutes," and "100°C, 5 minutes." The absorbance at a measurement wavelength of 600 nm was measured immediately after reaction cessation (0 minutes) and 10 minutes after reaction cessation.

[0057] Figure 25 is an explanatory diagram showing the results of reactions carried out with the five types of tetrazolium salts described above using the combinations of reaction temperature and reaction time described above. Specifically, it is an explanatory diagram showing the results of investigating whether or not direct reduction by reducing sugars occurred, and, if so, whether or not "water-soluble formazan" was produced. In particular, for samples in which the absorbance hardly increased even when the added glucose concentration was increased, it was determined that direct reduction by reducing sugars did not occur for the tetrazolium salt, and this was indicated as "unreacted" in Figure 25. Also, for samples in which the absorbance could not be measured due to a dark discoloration after the reaction, this was indicated as "unmeasurable" in Figure 25.

[0058] Furthermore, when direct reduction by reducing sugars occurred, the formation of "water-soluble formazan" was determined as follows: For the measurement value of the added glucose solution concentration at 10 mM, the measurement value at 10 minutes was subtracted from the measurement value at 0 minutes, and the difference was calculated. At this time, for the measurement value of 10 mM glucose, a measurement value of 0 mM glucose concentration with the same elapsed time was subtracted as background beforehand. The difference value was then compared with the value at 0 minutes for the glucose concentration of 10 mM. As a result, if the difference value was 10% or less of the value at 0 minutes, it was evaluated that "water-soluble formazan" had been formed, and this was indicated by "○" in Figure 25.

[0059] Figure 26 is an explanatory diagram showing images of assay plates after absorbance measurement for samples reacted with reducing sugars using WST-8 and MTS, two of the five tetrazolium salts described above, under reaction conditions of "25°C, 60 minutes," "60°C, 10 minutes," and "100°C, 5 minutes." As shown in Figure 26, when WST-8 was used as the tetrazolium salt, aggregation was observed under reaction temperature conditions of 100°C. Furthermore, when MTS was used as the tetrazolium salt, the sample showed a dark discoloration under reaction temperature conditions of 50°C to 100°C.

[0060] As shown in Figure 25, when WST-1 or XXT was used as the tetrazolium salt, water-soluble formazan was produced under any reaction temperature range of 4°C to 100°C. Therefore, it was confirmed that when WST-1 or XXT is used as the tetrazolium salt, the reducing sugar can be quantified by reacting it in the presence of the reducing sugar in the temperature range of 4°C to 100°C.

[0061] As shown in Figure 25, when using any of WST-1, XXT, or WST-8 as the tetrazolium salt, water-soluble formazan was produced under any reaction temperature range of 4°C to 60°C. Therefore, it was confirmed that when using any of WST-1, XXT, or WST-8 as the tetrazolium salt, the reducing sugar can be quantified by reacting in the presence of the original sugar at a temperature range of 4°C to 60°C.

[0062] As shown in Figure 25, when using any of WST-1, XXT, WST-8, and MTS as the tetrazolium salt, water-soluble formazan was produced under any reaction temperature range of 4°C to 30°C. Therefore, it was confirmed that when using any of WST-1, XXT, WST-8, and MTS as the tetrazolium salt, the reducing sugar can be quantified by reacting in the presence of the original sugar at a temperature range of 4°C to 30°C.

[0063] However, as shown in Figures 20 to 24 described above, WST-1 and WST-8, in particular, showed higher absorbances compared to other tetrazolium salts as a result of direct reduction by reducing sugars. Therefore, for WST-1 and WST-8, the regression lines obtained by measuring the absorbance after the reaction while changing the added glucose concentration are considered desirable as particularly suitable calibration curves for the quantification of reducing sugars.

[0064] [Reaction pH during direct reduction] The pH-related composition of the reaction solution for direct reduction by reducing sugars was changed, and the measurement results of the amount of formazan produced were compared. In each of the samples shown in Figures 2 to 26 described above, the tetrazolium salt solution prepared for direct reduction by reducing sugars contained 0.5 M sodium potassium tartrate and 0.05 M sodium hydroxide along with the tetrazolium salt. The reaction solutions prepared by adding glucose solution to the tetrazolium salt solution were all strongly alkaline with a pH of 12 or higher. Therefore, WST-1 was used as the tetrazolium salt, and the pH of the reaction solution was changed by changing the composition of the tetrazolium salt solution to investigate the extent to which direct reduction by reducing sugars proceeded.

[0065] For "Condition A," the tetrazolium salt solution used was the same solution as the reaction results shown in Figures 2 to 26 described above, namely an aqueous solution composed of tetrazolium salt, sodium potassium tartrate, and sodium hydroxide, with a concentration of 0.5 M sodium potassium tartrate and a concentration of 0.05 M sodium hydroxide (0.05 M NaOH + 0.5 M PST). For "Condition B," the tetrazolium salt solution used was an aqueous solution composed only of tetrazolium salt and sodium hydroxide, with a concentration of 0.05 M sodium hydroxide (0.05 M NaOH). For "Condition C," the tetrazolium salt solution used was an aqueous solution composed only of tetrazolium salt and sodium potassium tartrate, with a concentration of 0.5 M sodium potassium tartrate (0.5 M PST). Furthermore, for "Condition D," a Tris buffer with a pH of 9.0 containing tetrazolium salt, further containing 0.5 M sodium potassium tartrate (Tris pH9 + 0.5 M PST), was used as the tetrazolium salt solution. The tetrazolium salt concentration in the tetrazolium salt solution for each condition was adjusted so that the final concentration in the reaction solution was 0.05%.

[0066] To 195 μL of tetrazolium salt solution under each condition, 5 μL of glucose solution (0–20 mM) was added, and the mixture was heated at 30°C for 60 minutes and then cooled. Immediately after the start of cooling (stopping the reaction), 150 μL of the reaction mixture was transferred to an assay plate (Micro well plate GDMP-96F, AS ONE Corporation), and the absorbance at a measurement wavelength of 600 nm was measured using a plate reader (Multi-detection mode microplate reader Spark, TECAN).

[0067] Figure 27 is an explanatory diagram showing the results of direct reduction by reducing sugars using different tetrazolium salt solutions, as indicated by conditions A to D. As shown in Figure 27, the measured value was highest when using the tetrazolium salt solution under "Condition A," followed by the tetrazolium salt solution under "Condition B." In contrast, when using the tetrazolium salt solutions under "Condition C" and "Condition D," no increase in absorbance was observed with increasing glucose concentration, suggesting that formazan was not produced. From the above, it was confirmed that the quantitative determination of reducing sugars by directly reducing tetrazolium salts with reducing sugars requires the reaction to be carried out under strongly alkaline conditions, that potassium sodium tartrate is not essential in the reaction solution, and that adding potassium sodium tartrate to the reaction solution increases the measured absorbance and improves the detection sensitivity.

[0068] In addition to WST-1 (final concentration in reaction solution 0.05%), WST-8 (final concentration in reaction solution 0.05%), MTS (final concentration in reaction solution 0.025%), XTT (final concentration in reaction solution 0.05%), and TB (final concentration in reaction solution 0.1%) were used as tetrazolium salt solutions. Tris buffer at pH 9.0 containing the tetrazolium salts was used as the tetrazolium salt solution, and the treatment for direct reduction by reducing sugars was performed for comparison. Specifically, 5 μL of glucose solution (0-20 mM) was added to 195 μL of each of the above-mentioned tetrazolium salt solutions, and the mixture was heated at 30°C for 60 minutes, or heated at 60°C for 10 minutes and then cooled. Immediately after cooling began (reaction stopped), 150 μL of the reaction solution was transferred to an assay plate (Micro well plate GDMP-96F, AS ONE Corporation), and the absorbance at a measurement wavelength of 600 nm was measured using a plate reader (Multi-detection mode microplate reader Spark, TECAN). In all cases, regardless of the tetrazolium salt used or the reaction conditions, no increase in absorbance with increasing glucose concentration was observed, and formazan was not produced (data not shown). From the above, it is considered that the reaction to produce water-soluble formazan by the direct reduction of tetrazolium salts with reducing sugars needs to be carried out under strongly alkaline conditions.

[0069] This disclosure is not limited to the embodiments described above, and can be implemented in various configurations without departing from its spirit. For example, the technical features in the embodiments corresponding to the technical features in each form described in the summary of the invention can be replaced or combined as appropriate in order to solve some or all of the above-mentioned problems, or to achieve some or all of the above-mentioned effects. Furthermore, if a technical feature is not described as essential in this specification, it can be deleted as appropriate.

Claims

1. A method for quantifying reducing sugars, comprising: adding a tetrazolium salt that generates water-soluble formazan by reduction to a reducing sugar-containing sample containing reducing sugars; directly reducing the tetrazolium salt to generate water-soluble formazan under strongly alkaline conditions due to the reducing properties of the reducing sugars in the reducing sugar-containing sample; and optically quantifying the resulting water-soluble formazan to measure the amount of reducing sugars in the sample.

2. A method for quantifying reducing sugars according to claim 1, wherein the tetrazolium salt is any of the following: WST-1 (2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt), WST-8 (1-(2-Methoxy-4-nitrophenyl)-3-(2,4-disulfophenyl)-5-(4-nitrophenyl) formazan, disodium salt hydrate), MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt), and XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium, inner salt, monosodium salt hydrate).

3. A method for quantifying reducing sugars according to claim 2, wherein the tetrazolium salt is WST-1 (2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) or WST-8 (1-(2-Methoxy-4-nitrophenyl)-3-(2,4-disulfophenyl)-5-(4-nitrophenyl) formazan, disodium salt hydrate).

4. A method for quantifying reducing sugars according to claim 1 or 2, wherein the tetrazolium salt is WST-1 (2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) or XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium, inner salt, monosodium salt, hydrate), and the step of directly reducing the tetrazolium salt by the reducing properties of the reducing sugar to produce a water-soluble formazan is performed in a temperature range of 4°C to 100°C.

5. A method for quantifying reducing sugars according to claim 1 or 2, wherein the tetrazolium salt is any of WST-1 (2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt), WST-8 (1-(2-Methoxy-4-nitrophenyl)-3-(2,4-disulfophenyl)-5-(4-nitrophenyl) formazan, disodium salt hydrate), and XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium, inner salt, monosodium salt hydrate), and the step of directly reducing the tetrazolium salt by the reducing properties of the reducing sugar to produce a water-soluble formazan is performed in a temperature range of 4°C to 60°C.

6. A method for quantifying reducing sugars according to claim 2, wherein the step of directly reducing the tetrazolium salt by the reducing properties of the reducing sugar to produce a water-soluble formazan is performed in a temperature range of 4°C to room temperature.

7. A method for quantifying reducing sugars according to any one of claims 1 to 6, wherein the step of optically quantifying the water-soluble formazan is a method for quantifying reducing sugars in which at least one of the wavelengths of 400 to 440 nm and 540 to 650 nm is used as the measurement wavelength.

8. A method for quantifying a reducing sugar according to any one of claims 1 to 7, wherein the step of directly reducing the tetrazolium salt by the reducing properties of the reducing sugar to produce a water-soluble formazan is performed in a temperature range of 4°C to 30°C, and the step of optically quantifying the water-soluble formazan is performed with a measurement wavelength of 540 to 650 nm.

9. A reducing sugar content measurement kit for measuring the amount of reducing sugar in a reducing sugar-containing sample, comprising: a tetrazolium salt that is reduced to produce a water-soluble formazan; and a strongly alkaline solution for preparing a reaction solution under strongly alkaline conditions, which is used as a reaction solution for reacting the reducing sugar-containing sample with the tetrazolium salt.

Images