JP4798502B2 - BOD measurement method - Google Patents

Description

  The present invention relates to a method for measuring BOD. More specifically, the present invention relates to a method for measuring BOD using eukaryotic microorganisms.

  BOD (Biochemical Oxygen Demand) is an important item for water quality management of rivers and industrial wastewater, and is an international indicator of organic water pollution. Water pollution caused by organic compounds is reduced and eliminated by the oxidation reaction by aerobic microorganisms, and dissolved oxygen is consumed corresponding to the concentration of the organic substance. Therefore, by measuring the amount of oxygen consumed, water quality The pollution becomes clear. Accordingly, BOD is an indirect representation of organic compound concentration by oxygen content.

Various methods have been proposed as BOD measurement methods. For example, in Patent Document 1, yeasts belonging to the genus Trichosporon are fixed on the surface of a dissolved oxygen electrode, and the concentration of dissolved oxygen consumed by the yeast assimilating organic pollutants contained in rivers and wastewater is determined as an oxygen electrode. The method of measuring by is proposed. According to this method, although the total amount of various organic substances contained in rivers and wastewater can be measured together as organic pollutants, the concentration of oxygen that is only slightly dissolved in the measurement solution is highly sensitive. Since it is necessary to measure, there is a problem that the measurement system will inevitably become large, and it is suitable for BOD measurement that requires not only indoors such as test rooms and factories but also on-site measurement. There was a case. In addition, due to the measurement system, there is a limit that it is difficult to measure an accurate value in a waste liquid with very low dissolved oxygen.
Japanese Patent Publication No.61-7258

In response to this problem, a BOD measurement method using a mediator that enables accurate measurement of BOD even when the amount of dissolved oxygen is low has been developed. Such a measurement method is an electrochemical (electrode) measurement type that uses the concentration of electrons whose dissolved concentration in liquid is higher than that of dissolved oxygen as an index, and enables accurate measurement of BOD and downsizing of the measuring device. It was groundbreaking.
Japanese Patent Laid-Open No. 07-167824

  However, since this measurement method detects the movement of electrons in the electron transport system due to the metabolism of organic substances by microorganisms, when using eukaryotic microorganisms in which electron transfer of the respiratory chain occurs in mitochondria, However, it is said that the measurement sensitivity is poor, and only a number of measurement methods using prokaryotic microorganisms have been proposed. However, prokaryotic microorganisms have problems in operability and storage stability in culturing and pretreatment, etc., and have become barriers when applied to various measurement methods and put to practical use. Mediator type using eukaryotic microorganisms Development of BOD measurement method is desired.

  An object of the present invention is to provide a method for measuring BOD with high sensitivity using a eukaryotic microorganism.

The purpose of such invention, the organic substance is metabolized by the yeast, by measuring the metabolic activity of the yeast, in the measuring method of BOD detecting or quantifying organic mass in a solution containing organic substances, yeast organic This is achieved by a BOD measurement method in which an oxidized fat-soluble mediator and an oxidized hydrophilic mediator are present when a substance is metabolized, and the resulting reduced hydrophilic mediator is detected or quantified by an electrode.

  According to the method of the present invention, BOD can be measured without using a prokaryotic microorganism having problems in operability and storage stability in culture, pretreatment, etc., and the measurement sensitivity has been proposed in the past. Compared with the BOD measurement method using a nuclear microorganism, even when a small amount of sample such as 1 to 350 μl is used, it has an excellent effect of being several times higher in sensitivity. Furthermore, when a fat-soluble mediator is used by being dissolved in dimethyl sulfoxide, it is possible to achieve an improvement in measurement sensitivity and a reduction in bag.

Examples of yeast include Saccharomyces cerevisiae , Trichosporon cutaneum , Trichosporon fermentans , Trichosporon brassicae , Candida genus, Aspergillus genus, etc., and preferably Saccharomyces cerevisiae , Trichosporon cutaneum, etc. well known as baker's yeast are used. Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) can use a commercial item as it is. Here, the microorganism to be used can be used in the state of viable bacteria, preferably in the logarithmic growth phase, or in a form that can be stored by some method, for example, in the state of dead bacteria, considering practicality. You can also. When using live bacteria, the concentration of the bacterial cells during the reaction is OD ₆₀₀ = 0.1 or more, preferably about 0.5 to 20, more preferably about 1.5 to 5.

  Oxidized fat-soluble mediators include menadione (2-methyl-1,4-naphthoquinone), benzoquinone, 1,2-naphthoquinone, ubiquinone, hydroquinone, 2,6-dichlorobenzoquinone, 2-methylbenzoquinone, 2,5- Quinones such as dihydroxybenzoquinone and 2-hydroxy-1,4-naphthoquinone, and benzoamines such as 2,3,5,6-tetramethyl-1,4-phenylenediamine and N, N-dimethyl-p-phenylenediamine Menadione, benzoquinone, 1,2-naphthoquinone, 2,3,5,6-tetramethyl-1,4-phenylenediamine and N, N-dimethyl-p-phenylenediamine are preferably used. Here, the fat-soluble mediator is preferably dissolved in dimethyl sulfoxide. When menadione is dissolved in DMSO and used, the concentration of menadione in the reaction solution is 20 to 100 μM, preferably 100 to 400 μM. When menadione is used at a concentration higher than this, it becomes difficult to reuse the electrode. On the other hand, when it is used at a concentration lower than this, the response to organic substances is reduced. DMSO is used so that it is 3% or less, preferably 1% or less in the reaction solution. When DMSO is added to the reaction solution at a rate higher than this, the sensor response value is lowered, which is not preferable.

  Oxidized hydrophilic mediators include riboflavin, L-ascorbic acid, flavin adenine dinucleotide, flavin mononucleotide, nicotine adenine dinucleotide, lumicochrome, glutathione, peroxidase, cytochrome C, ferredoxin and other biological redox substances or their derivatives , Hexacyanoiron (III) potassium, hexamethylenetetramine ruthenium, carboxymethylated ferrocene, DCIP, 1-M-PMS, 9-dimethylaminobenzo-α-phenazoxonium chloride, Fe-EDTA, Mn-EDTA, Zn- EDTA, mesosulfate, methyl viologen, durohydroquinone, ferrocenecarboxylic acid, phenazine methosulfate, etc. are used, preferably hexacyanoiron (III) potassium, hexamethylenetetramineruthenium, carboxymethylated ferro Emissions, DCIP, 1-M-PMS, 9- dimethylaminobenzoate -α- phenazopyridine sulfoxonium chloride is more preferably hexacyanoferrate (III) potassium, hexamethylenetetramine ruthenium, carboxymethylated ferrocene used.

  Oxidized fat-soluble mediator and oxidized hydrophilic mediator are used in such a combination that reduced lipid-soluble mediator generates reduced hydrophilic mediator from oxidized hydrophilic mediator. The concentration of is generally 40 nM or more, preferably 50 nM to 500 mM, more preferably about 100 nM to 50 mM. Thus, the combined use of an oxidized fat-soluble mediator and an oxidized hydrophilic mediator provides an excellent effect that BOD can be measured using a small amount of sample such as 1 to 350 μl.

In addition to the above yeast and mediator, it is preferable to use superoxide dismutase (EC 1.15.1.1) in the method of the present invention. Superoxide dismutase, also called superoxide dismutase, acts to catalyze the disproportionation reaction of superoxide anion radicals, so it generates oxygen and hydrogen peroxide from oxygen radicals. Can be up.

Measurements of BOD in the presence of fat-soluble mediators and oxidized hydrophilic mediator oxidized form, preferably further in the presence of superoxide dismutase, to metabolize organic substances in the solution by the yeast, the metabolic activity of the yeast This is done by measuring. Here, in order to keep the activity of the cells during metabolism of the organic substance in good condition, it is desirable that the reaction tank contains at least about 10 mM phosphate ions. This, 10 mM phosphate buffer (phosphate buffer; PB dissolves _{Na 2 HPO 4 · 12H 2 O} 3.58g or _{NaH 2 PO 4 · 2H 2 O} 1.56g of the pure water, respectively, each solution and 1l, Both were adjusted to pH 7.0 and autoclaved at 121 ° C for 15 minutes, or 1x phosphate buffer saline (PBS: NaCl 8g, Na ₂ HPO ₄ · 12H ₂ O 2.9 g, KCl 0.2g, KH ₂ PO ₄ 0.2g dissolved in pure water and adjusted to pH 7.0, then made up to 1L with pure water, autoclaved at 121 ° C for 15 minutes, preferably phosphate buffer Here, when phosphate ions of about 10 mM are contained, the effect of buffering the influence of the pH of the sample liquid is also exhibited.When the BOD sensor described later is used, this is reused. From the viewpoint, a surfactant such as Triton-X is preferably contained in the reaction solution in the reaction solution, preferably 0.00001 to 0.1%, more preferably 0. As a result, it is possible to solve the problem that the response value of the sensor greatly decreases as the number of measurements increases.

  Metabolic activity is achieved by reducing the hydrophilic mediator produced in the solution between the electrodes when a potential difference is applied between the working electrode and the counter electrode after the electrode having at least the working electrode and the counter electrode is immersed in the solution. It is calculated by measuring the current flowing through

In the measurement, yeast , oxidized fat-soluble mediator and oxidized hydrophilic mediator, preferably superoxide dismutase was previously present on the electrode, and a solution containing an organic substance was added thereto. A BOD sensor that can measure the amount of organic substances can also be used. As such a sensor, for example, a working electrode and a counter electrode are provided on a substrate, and if necessary, a reference electrode is provided, and yeast , oxidized fat-soluble mediator and oxidized hydrophilic mediator on the working electrode and / or in the vicinity thereof, preferably Furthermore, a sensor provided with superoxide dismutase can be mentioned. Examples of such sensors include platinum, gold, carbon, and the like by screen printing, vapor deposition, sputtering, etc. on an insulating substrate such as ceramics, glass, plastic, paper, biodegradable materials (for example, microbial production polyester). Etc., and electrodes in which microorganisms are fixed on the working electrode and / or in the vicinity thereof by a crosslinking method, a covalent bonding method, an ionic bonding method, or the like are used.

Such sensors, preferably via a spacer to the bottom insulating substrate, an upper insulating substrate is a substrate provided with an electrode comprising at least a working electrode and a counter electrode, located on the surface of the electrode bottom insulation substrate side arranged such that, more preferably mediator layer or microorganisms on the working electrode and / or its periphery - is used that arranged mediator mixture layer. Unlike a general sensor, such a sensor has electrodes so as to be in contact with the sample liquid surface or soaked in the sample liquid. Therefore, particulate matter such as gravel is present in the sample liquid. There is an effect that the influence on the measurement value due to the deposition on the electrode surface can be eliminated. Furthermore, by placing a stirring stone in the reaction tank of the sensor and providing a stirring system below the sensor, it is possible to continuously stir the reaction solution. Therefore, it is possible to improve the reaction efficiency and repeatability. In addition, accurate electrochemical measurement can be performed by stopping stirring during measurement.

  The current value is measured by reacting a solution containing an organic substance with a microorganism, etc., and then measuring the change in current value over time from 0 to 60 minutes, preferably from 0.5 to 30.5 minutes. Using electrochemical analyzers such as the product SHV-100 and BAS product CHI-1202, using electrochemical measurement methods such as chronoamperometry, the response of the generated reduced mediator to the potential applied between the electrodes It is done by confirming. The potential applied between the electrodes is not particularly limited, but 0.7 to 1.1 V, preferably 0.8 to 1.0 V is selected for the purpose of measuring the generated hydrogen peroxide together.

  The electrode terminal is connected to a measuring device and measures an electrical value generated between the electrodes. This measuring apparatus includes a measuring unit that measures an electrical value at the electrode and a display unit that displays the measured value. As a measuring method in this measuring unit, a chronoamperometry method or the like can be used as described above.

  The apparatus can also be equipped with a memory for storing measured values. Further, when managing the measured values remotely, wireless means for transmitting measurement data to the measurement unit, preferably wireless means such as a non-contact type IC card or short-range wireless communication (for example, Bluetooth; registered trademark) is used. It can also be installed.

  Next, the present invention will be described with reference to examples.

Example 1
Take a very small amount of freeze-dried baker's yeast cells (Nisshin Flour Milled Product) with a spatula and put them in a YPD liquid medium (yeast extract 10 g, polypeptone 20 g, glucose 20 g dissolved in pure water) in a 18 mm × 180 mm test tube. Inoculate 2 ml after autoclaving at 121 ° C for 15 minutes), cover with a silico stopper, and shake culture at 28 ° C, 180 rpm, aerobic conditions for 18 hours. Went. Next, YPD agar medium (2 g yeast extract, 4 g polypeptone, 4 g glucose, 4 g agar was dissolved in pure water to make a total volume of 200 ml and autoclaved at 121 ° C for 15 minutes) About 20ml each in a petri dish and dried in a thermostat (ARNSTEAD / THERMOLYNE LAB-LINE 120-5JPN) in a thermostat, and the yeast solution cultured from dry yeast is a mesh pattern with platinum ears And pre-cultured at 28 ° C. for 2 nights.

Inoculate 2 ml of YPD liquid medium with a platinum loop from a colony on a YPD agar medium, shake culture at 28 ° C., 180 rpm, aerobic conditions for 13-14 hours, and further in a 500 ml Sakaguchi flask, A preculture solution was inoculated into 50 ml of YPD liquid medium to a final concentration of 0.5%, capped with a silico stopper, and main culture was performed at 28 ° C., 120 rpm, aerobic conditions for 9 to 10 hours. The growth phase of the cells reached the logarithmic growth phase (OD ₆₀₀ = 0.9 or more).

After transferring the entire amount of the main culture to a centrifuge tube, the cells were collected by centrifugation at 4 ° C. and 3000 rpm for 3 minutes. After collection, the cells were washed 3 times with 0.9% physiological saline, then the pelleted yeast was suspended in 25 ml of 0.9% physiological saline, aerated in a 50 ml centrifuge tube for 2 hours, and washed again. Repeated times. After washing, add 0.9% physiological saline so that the final cell concentration is OD ₆₀₀ = 15-75.7, and disperse the yeast solution well by vortex mixer and pipetting to prepare a cell suspension (yeast solution) did.

In the sensor chip, 30 μl of OD ₆₀₀ = 15 prepared yeast solution, 158 mg of potassium ferricyanide (Wako Pure Chemical Industries, Ltd.) dissolved in 2 ml of pH 7.0 PBS buffer, 50 μl of 0.24 M potassium ferricyanide, LB medium (yeast extract) Dissolve 0.25g, polypeptone 0.5g, NaCl 0.25g in pure water, adjust to pH 7.0 with sodium hydroxide aqueous solution, make the whole volume 50ml with pure water, autoclave sterilization under conditions of 121 ° C for 15 minutes PBS buffer solution so that the total reaction volume is 300 μl in 1.5 μl of 20 mM menadione in which 60 μl and menadione (MP Biomedicals product) 3.5 mg are dissolved in pure water 40 μl and ethanol 960 μl mixed solution (96% ethanol). In addition, after thoroughly stirring using a pipetman, the electric potential was set to 0.9 V, and the current value measured after 0.5 minutes, 5.5 minutes, 15.5 minutes and 30.5 minutes was measured using Hokuto Denko SHV-100. It was conducted.

Here, a sensor chip having an outer size of 40 × 18 × 4.5 mm and a reaction vessel size of 10 × 12.5 × 4.5 mm (reaction vessel internal volume 562.5 mm ³ ) having the shape shown in FIG. 1 was used as the sensor. Specifically, a transparent polyethylene styrene phthalate having a thickness of 188 μm is punched out so as to become a main body of 35 mm × 18 mm and a support portion 11 of 16 mm × 6.0 mm as shown in FIG. Was made. On this bottom insulating substrate, a lower spacer 12 having the same outer shape as the bottom insulating substrate and having a space 13 of 10 mm × 12.5 mm with a thickness of 1 mm as shown in II) is an acrylic adhesive (Nitto Product No. 5000N). On the lower spacer, an adhesive layer having a thickness of 25 μm was provided, and the upper insulating substrate 14 was further bonded. This upper insulating substrate is made of the same material as the bottom insulating substrate, and as shown in III), with a 40 mm x 18 mm body, a 21 mm x 6.0 mm support 11 and a 3 mm gap. A space 13 of 10 mm × 12.5 mm having an 8 mm × 3 mm comb-shaped substrate portion is formed. On the surface of the bottom insulating substrate side, two comb-shaped substrate portions are arranged with 7.5 mm × 2 mm. A carbon ink having a thickness of 10 μm was formed by a screen printing method with two conductors 15 and two conductors 15 of 21 mm × 2.6 mm at intervals of 0.6 mm in the support portion. On the upper insulating substrate, three upper spacers having the same material and shape as the lower spacer were bonded together using an adhesive. The internal volume of the reaction space of the biosensor produced in this way was about 560 mm ^{3 as} described above.

Comparative Example 1
In Example 1, menadione was not used.

Comparative Example 2
In Example 1, no potassium ferricyanide was used.

  The results obtained in Example 1 and Comparative Examples 1 and 2 are shown in FIG. As shown in FIG. 1, when potassium ferricyanide is used alone as a mediator, the metabolic activity due to the metabolism of the organic substance contained in the LB medium cannot be confirmed as a change with time. When menadione is used together with potassium cyanide, it is shown that the response value increases as the reaction time elapses, and the metabolic activity due to metabolism of organic substances contained in LB medium is confirmed as a change over time It was possible to do.

Example 2
In the sensor chip, 50 μl of prepared yeast solution with OD ₆₀₀ = 15, 10 μl of 0.05% Triton-X 100, 83 μl of 0.24 M potassium ferricyanide, 100 μl of LB medium and 2.5 μl of 20 mM menadione dissolved in ethanol, total reaction volume of 500 μl PBS buffer solution was added and the current value was measured in the same manner as in Example 1 after 0.5 minutes, 5.5 minutes, 15.5 minutes and 30.5 minutes after thorough stirring using Pipetteman. As the sensor, the sensor used in Example 1 with the lower spacer changed to three and the upper spacer changed to one was used.

Example 3
In Example 2, the same amount of LB medium with 7/10 concentration using pure water was used.

Example 4
In Example 2, the same amount was used as the LB medium with 4/10 concentration using pure water.

Example 5
In Example 2, the same amount was used as the LB medium at 1/10 concentration using pure water.

Example 6
In Example 2, the same amount of LB medium with a 1/20 concentration using pure water was used.

Example 7
In Example 2, no LB medium was used.

Example 8
In Example 2, 5 μl of SOD solution in which 4500 U of Superoxide Dismutase Solution (Wako Pure Chemical Industries) was dissolved in 450 μl of PBS buffer was used.

  The results obtained in Examples 2-8 above are shown in FIG. As shown in FIG. 2, it was shown that the increase in the metabolic activity of the microorganism accompanying the increase in the amount of organic substance can be measured by the response value (output current value). Furthermore, it was shown that the response value can be greatly enhanced by using Superoxide Dismutase.

Example 9
The yeast was cultured and washed in the same manner as in Example 1, 0.9% physiological saline was added so that the final cell concentration would be OD ₆₀₀ = 45, and the yeast solution was well dispersed by vortex mixer and pipetting. A liquid was prepared. As the sensor, the sensor used in Example 2 was used after placing a minute stirrer (manufactured by ASONE, Cellstar CC-303) under the chip.

  In the sensor chip, 25 μl of prepared yeast solution, 41.5 μl of 0.48 M potassium ferricyanide prepared with 10-fold PBS buffer, 350 μl of measurement sample solution, 5 μl of 0.05% (w / v) Triton-X 100, 20 mM dissolved in ethanol Add 500 μl of a mixture of 5 μl of menadione and 48.5 μl of pure water, stir with a stirring stone for 15 minutes, and adjust the potential to 0.9 by chronoamperometry using an electrochemical measuring instrument (BAS, CHI-1202). The current value was measured with V set. The measurement was performed 3 times for each standard solution.

Actual river water was used as a measurement sample. The actual river water was taken from the Yudon River (Higashi Bridge, 9:40 on August 30, 2006, weather: cloudy, temperature: 29.5 ° C, humidity 81.1%) flowing in Hachioji City, Tokyo. Examination of the water sampling in the field, a water temperature 24.8 ° C., pH 7.2, dissolved oxygen _{8.8mg O 2 / l, COD Mn} 6mg O 2 / l, conductivity of 0.24ms / cm, was 0% salt concentration, the actual river Since the COD _Mn value of water was extremely low, a 9: 1 mixture of actual river water and GGA standard solution was used as the measurement sample solution. Three types of GGA standard solutions were used: 1,500 mg O ₂ / l GGA solution, 750 mg O ₂ / l GGA solution, and 0 mg O ₂ / l GGA solution (pure water). The measurement was performed three times for each sample solution.

With respect to the obtained current value, BOD was calculated based on a calibration curve prepared in advance using the sensor. Here, as a sample solution for preparing a calibration curve, 15.0 mg of glucose and 15.0 mg of glutamic acid were dissolved in 10.0 ml of pure water and sterilized by a membrane filter having a pore of 0.22 μm, and the BOD value is 2,200 mg O ₂ / l. A 220 mg O ₂ / l standard solution prepared by diluting a glucose / glutamic acid (GGA) standard solution 10-fold with pure water and a 0 mg O ₂ / l standard solution containing no GGA were used.

The calculation results are shown in FIG. FIG. 5 shows the average value of the sensor response values by associating the results of measurement using the BOD ₅ method (based on JIS K 0102) with the results of three measurements using the sensor of the present invention. The values of standard deviation are shown as error bars as plots. Although a clear sensor response was not obtained for the sample liquid obtained by adding pure water to actual river water, the correlation between the sensor method and the BOD ₅ method was found to be very good (y = 0.922x-6.71, r = 0.998). This result suggests the possibility of measuring actual fresh water using the sensor of the present invention.

Example 10
In Example 9, seawater was used as the actual sample solution, and artificial seawater (2.65% NaCl, 0.326% MgCl ₂ , 0.207% MgSO ₄ , 0.136%) was used instead of pure water as the sample solution for preparing the calibration curve and the measurement sample solution. CaSO ₄ , 0.0714% KCl) was used. The actual seawater was taken from Odaiba, Koto-ku, Tokyo (18:30 on August 31, 2006, weather: fine, temperature: 27.1 ° C, humidity 61.2%). Examination of the water sampling in the field, a water temperature 25.4 ° C., pH 8.0, dissolved oxygen _{7.58mg O 2 / l, COD Mn} 7.5mg O 2 / l, conductivity of 29.6mS / cm, was the salt concentration 3.28%.

The obtained result is shown in FIG. FIG. 6 shows the results of measuring the sample solution using the BOD ₅ method and the results of measuring it once using the sensor of the present invention. As in Example 9, it was shown that the correlation between the sensor method and the BOD ₅ method was obtained (y = 1.24x-31.9, r = 0.989). In the sensor method, the sensor response to the sample solution in which artificial seawater was added to actual seawater at a ratio of 9: 1 showed a higher response than the BOD ₅ method. On the other hand, the results of the BOD ₅ method obtained for the sample solution prepared to 150 mg O ₂ / l GGA in real seawater showed values lower than 150 mg O ₂ / l, but the sensor method and BOD It was found that the correlation of ₅ methods was obtained. Therefore, the possibility that real seawater could be measured using the sensor of the present invention as in Example 9 was suggested.

Example 11
In Example 9, dimethis sulfoxide (DMSO) was used as a solvent for dissolving menadione, and the current value was measured when pure water was used as a measurement sample. The obtained results are shown in Table 1 together with the case of using ethanol as the menadione solvent. Changing the solvent for dissolving menadione from ethanol to DMSO has been shown to provide a greater difference in sensor response to 0 ppm and 220 ppm, and the use of DMSO has a lower baseline and more accuracy than ethanol. It was shown that a good sensor response can be obtained.

Example 12
The yeast was cultured and washed in the same manner as in Example 1. 0.9% physiological saline was added so that the final cell concentration would be OD ₆₀₀ = 75.7, and the yeast solution was well dispersed by vortex mixer and pipetting. A liquid was prepared. As the sensor, the sensor used in Example 2 was used after placing a minute stirrer (manufactured by ASONE, Cellstar CC-303) under the chip.

In the sensor chip, 305 μl of pure water as mixed reagent solution, 5 μl of 0.05% (w / v) Triton-X 100, 50 μl of 100 mM MPB buffer at pH 7.0, 50 μl of 0.4 M potassium ferricyanide dissolved in pure water, dissolved in DMSO Mix 5 μl of 20 mM menadione and 35 μl of 2200 mg O ₂ / l GGA standard solution with stirring stone, add 50 μl of the above yeast solution into the mixed solution as the initiator of the reaction, and add the volume of the reaction solution in the chip. 500 μl. The reaction was carried out accurately for 10 minutes under stirring conditions, the potential was set to 0.9 V by chronoamperometry using an electrochemical measuring instrument (CHI-1202), and the current value was measured under the same stirring conditions as during the reaction. Was done. The measurement was performed three times for each standard solution.

Example 13
In Example 12, the same amount of 10 times PBS with pH 7.0 was used instead of PB.

The results obtained in Examples 12 to 13 are shown in Table 2. As shown in this table, PB with less salt stress gave a higher response to GGA than PBS.

Example 14
In Example 12, the amount of pure water was 40 μl, and 0.4 M potassium ferricyanide dissolved in 10 mM PB buffer at pH 7.0 as potassium ferricyanide had a concentration of 0, 5.5, 11, 22, 55, 110 or 220 mg as a GGA standard solution. The measurement was carried out using 350 μl of O ₂ / l and changing to OD ₆₀₀ = 71.8 for the yeast solution.

The results obtained in Example 14 are shown in FIG. As shown in this figure, it was shown that the increase in the metabolic activity of the microorganism accompanying the increase in the concentration of the GGA standard solution can be measured by the electrode response value. Here, the upper limit of detection obtained by measuring each standard solution three times is 220 mg O ₂ / l GGA, the lower limit is 5.5 mg O ₂ / l GGA, and the coefficient of variation (CV value) is 0.86% (0 mg O ₂ / l GGA), 0.48% (5.5mg O 2 / l GGA), 2.22% (11mg O 2 / l GGA), 0.52% (22mg O 2 / l GGA), 2.70% (55mg O 2 / l GGA), 2.00% (110 mg O ₂ / l GGA), 0.72% (220 mg O ₂ / l GGA), the average value was 1.36%, and the correlation coefficient was r = 0.95. From the above, it was shown that a response showing a positive correlation with GGA can be obtained in a very wide range.

It is a front view of the sensor chip used in the example. It is a figure which shows the structure of the sensor chip used in the Example. It is a graph which shows the responsiveness by the presence or absence of menadione. It is a graph which shows the responsiveness with respect to each LB culture medium concentration. Using the actual river water prepared by adding GGA standard solution is a diagram showing in correspondence the results of measurement by the sensor method and BOD ₅ method. It is a figure which shows corresponding the result measured by the sensor method and BOD ₅ method using the real seawater prepared by adding GGA standard solution prepared with artificial seawater. It is a graph which shows the responsiveness by a GGA standard solution.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Sensor chip 2 Reaction tank 3 Electrode 4 Terminal 10 Bottom part insulating substrate 11 Support part 12 Lower spacer 13 Space 14 Upper insulating substrate 15 Conductor

Claims (11)

The organic material was metabolized by yeast by measuring metabolic activity of the yeast, in the measuring method of BOD detecting or quantifying organic mass in a solution containing organic substances,
Measurement of BOD, characterized by the presence of oxidized fat-soluble mediator and oxidized hydrophilic mediator when yeast metabolizes organic substances, and detection or quantification of the reduced hydrophilic mediator produced by electrodes Law. The method for measuring BOD according to claim 1, wherein the metabolism of the yeast organic substance is carried out in the presence of 10 mM or more of phosphate ions.   The method for measuring BOD according to claim 1, further comprising superoxide dismutase in addition to the oxidized fat-soluble mediator and oxidized hydrophilic mediator. The method for measuring BOD according to claim 1, wherein the fat-soluble mediator is used by being dissolved in dimethyl sulfoxide. The method for measuring BOD according to claim 1, wherein the oxidized fat-soluble mediator is a quinone or a benzoamine. The method for measuring BOD according to claim 5, wherein the quinones are 2-methyl-1,4-naphthoquinone, benzoquinone or 1,2-naphthoquinone. 6. The method for measuring BOD according to claim 5, wherein the benzoamine is 2,3,5,6-tetramethyl-1,4-phenylenediamine or N, N-dimethyl-p-phenylenediamine. The method for measuring BOD according to claim 1, wherein the oxidized hydrophilic mediator is hexacyano iron (III) potassium, hexamethylenetetramine ruthenium or carboxymethylated ferrocene. On the substrate, yeast, oxidized fat-soluble mediator and oxidized hydrophilic mediator are present on the electrode, and the organic substance is detected or quantified by adding a solution containing the organic substance to this. A BOD sensor with at least an electrode consisting of a working electrode and a counter electrode. The BOD sensor according to claim 9, wherein superoxide dismutase is further present on the electrode. An upper insulating substrate, which is a substrate provided with at least an electrode composed of a working electrode and a counter electrode on a bottom insulating substrate via a spacer, and is arranged so that the electrode is positioned on the surface on the bottom insulating substrate side. Item 11. The BOD sensor according to Item 9 or 10.

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