Sulfur component detection method

Abstract

To provide a sulfur component detection method capable of easily detecting a sulfur component of a sulfur compound, etc., in an analyte in a short time by the use of an electrode sensor.SOLUTION: A sulfur component detection method detects a sulfur component in an analyte 2 by the use of an electrode sensor 1 formed of an anode electrode body 10 and a cathode electrode body 12 in one body with an insulator 11 interposed, and comprises: measuring a galvanic current flowing between both the electrode bodies through electrochemical reaction between a sulfur component in the analyte 2 and the anode electrode body 10 with the analyte 2 in contact with both the electrode bodies; and detecting the sulfur component in the analyte 2 based upon the measured current value.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a sulfur component detection method for detecting trace amounts of sulfur compounds in a specimen using an electrode sensor. [Background technology]

[0002] Sulfur (symbol: S) is known as an essential element for life activities, and various sulfur compounds in living tissues play important biological functions. It is also known that sulfur is involved in a wide range of redox reactions in living tissues, and attempts have been made to analyze redox reactions in living tissues by detecting sulfur components.

[0003] For example, Patent Document 1 describes the use of an X-ray fluorescence analyzer to measure the content ratio of sulfur derived from methinene contained in a biological fluid derived from a subject that can be collected by the subject. Patent Document 2 describes the use of a solution obtained by adding a treatment liquid to a breath condensate collected from the breath to quantitatively analyze the sulfur metabolites using a mass spectrometer in order to diagnose an infectious disease based on the sulfur metabolites in the breath. Patent Document 3 describes the use of a sulfur compound sensor having a detection layer with exposed silver particles, in which a liquid or paste-like specimen is directly brought into contact with the detection layer, and a transmission or reflection spectrum is measured using a spectrometer to detect sulfur compounds.

[0004] The present inventors have proposed a microorganism detection device that detects a current change caused by the metabolites of microorganisms in a culture medium by measuring the galvanic current flowing between an electrode sensor in which an anode electrode body and a cathode electrode body are integrally formed via an insulator, while the electrode sensor is in contact with the culture medium, as shown in Patent Document 4. By using such an electrode sensor, it has become possible to detect microorganisms easily and accurately by detecting minute current changes caused by metabolites produced by microorganisms in the culture medium. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 6734489 [Patent Document 2] Patent Publication No. 2022-178150 [Patent Document 3] Retable 2019 / 167567 [Patent Document 4] Patent No. 6804063 Summary of the Invention [Problem to be solved by the invention]

[0006] In Patent Documents 1 to 3, an analytical instrument such as an X-ray fluorescence analyzer is required to detect sulfur compounds, and as described in Patent Document 2, pretreatment such as adding a treatment liquid is required, which poses the problem that it is difficult to perform detection treatment simply and quickly.

[0007] In particular, when analyzing redox reactions in biological tissues, it is desirable to perform the analysis without affecting the biological reactions as much as possible. However, conventional detection methods require steps such as sample extraction and pretreatment, which have the drawback that these steps are likely to affect the biological reactions.

[0008] Therefore, an object of the present invention is to provide a method for detecting sulfur components, which can easily and quickly detect sulfur components such as sulfur compounds in a sample using an electrode sensor. [Means for solving the problem]

[0009] The sulfur component detection method according to the present invention is a method for detecting sulfur components in a specimen using an electrode sensor in which an anode electrode body and a cathode electrode body are integrally formed via an insulator, and in a state in which the specimen is in contact with both electrode bodies, a galvanic current flowing between the two electrode bodies due to an electrochemical reaction occurring between the sulfur components in the specimen and the anode electrode body is measured, and the sulfur components in the specimen are detected based on the measured current value. Furthermore, the specimen contains sulfur compounds derived from biological tissue. Furthermore, the specimen contains sulfur compounds derived from metabolites of living organisms. Furthermore, at least the exposed surface of the anode electrode body that comes into contact with the specimen is coated with silver, and sulfur compounds are generated on the exposed surface due to an electrochemical reaction occurring between the silver and the sulfur components. Furthermore, at least the exposed surface of the anode electrode body is coated with a layer of silver having a thickness of 10 μm or more. Effect of the Invention

[0010] According to the present invention, since an electrochemical reaction occurring between a sulfur component in a sample and the anode electrode assembly is reflected in a galvanic current flowing between the anode electrode assembly and the cathode electrode assembly, it is possible to detect a trace amount of sulfur components in a sample based on the measured galvanic current. Furthermore, since detection can be performed simply by bringing the electrode sensor into contact with the sample, and no pretreatment or other steps are required, it is possible to detect sulfur components in a sample simply and in a short time. [Brief description of the drawings]

[0011] [Figure 1] 1 is a configuration example of a sulfur component detection device for carrying out a sulfur component detection method according to the present invention. [Diagram 2] 3A and 3B are a plan view and a partially enlarged cross-sectional view of an electrode sensor. [Diagram 3] 1 is a graph showing changes in current value and hydrogen sulfide H2S concentration over time. [Figure 4] 1 is a graph showing the correlation between measured current values ​​and concentrations. [Diagram 5]1 is a graph showing the time course of OD values ​​corresponding to the number of bacteria and measured current values. [Figure 6] 13 is a graph showing the measurement results of a detection test using an electrode sensor for the first time, after a first regeneration process, and after a second regeneration process in the case of regeneration process by energization. [Figure 7] 1 is a graph showing the measurement results of a detection test using an electrode sensor after the initial regeneration treatment, the first regeneration treatment, and the second regeneration treatment in the case of regeneration treatment with thiourea. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Hereinafter, embodiments of the present invention will be described with reference to the drawings. Note that the embodiments described below are preferred examples for carrying out the present invention, and therefore various technical limitations are imposed thereon, but the present invention is not limited to these embodiments unless otherwise specified in the following description to the effect that the present invention is limited thereto.

[0013] 1 shows an example of the configuration of a sulfur component detection device for carrying out a sulfur component detection method according to the present invention. The sulfur component detection device includes an electrode sensor 1 in which an anode electrode body and a cathode electrode body are integrally formed via an insulator, a liquid or paste-like specimen 2 containing a sulfur component, and a detection unit 3 that detects a current change due to the sulfur component in the specimen 2 by measuring a galvanic current flowing between at least both electrodes of the electrode sensor 1 while the electrodes are in contact with the specimen 2. In this example, the electrode sensor 1 is set in a storage container 4, and the specimen 2 is dropped on the upper surface of the electrode sensor 1 and set in a state where it is attached to both electrodes, thereby detecting the sulfur component in the specimen 2.

[0014] 2A and 2B are a plan view and a partially enlarged cross-sectional view, respectively, of the electrode sensor 1. FIG. 2B shows a cross section taken along the left-right direction in a region enclosed by a circle.

[0015] In the electrode sensor 1, an insulator 11 is laminated in layers on the upper surface of a rectangular anode electrode body 10, and a cathode electrode body 12 is laminated in layers on the upper surface of the insulator 11. Narrow cutouts are formed in parallel in a comb shape in the central parts of the insulator 11 and the cathode electrode body 12, exposing the surface of the anode electrode body 10. Then, by bringing a specimen 2 into contact with both electrode bodies of the electrode sensor 1 and measuring the galvanic current flowing between the anode electrode body 10 and the cathode electrode body 12 exposed in the cutouts, a change in current due to the sulfur component in the specimen 2 can be detected.

[0016] A galvanic current is generated between the two electrodes by the electrode surface of the anode electrode body 10 exposed in the cutout becoming a site where an oxidation reaction occurs to generate electrons, and by the electrode surface of the comb-shaped electrode portion of the cathode electrode body 12 becoming a site where a reduction reaction occurs to lose electrons. Therefore, the electrode surface of the cathode electrode body 12 is set to a combination of dissimilar metals for both electrodes so that it has a more noble potential than the electrode surface of the anode electrode body 10 under environmental conditions in which an electrochemical reaction occurs.

[0017] In the electrode sensor 1, the two electrode bodies are integrally formed in the comb-shaped electrode portion so as to be arranged opposite to each other with an insulator interposed therebetween, and a galvanic current is generated by causing an electrochemical reaction at the part in contact with the two electrode bodies with a specimen interposed between them. The output of the generated galvanic current is proportional to the length of the boundary of the contact area with the specimen at the boundary part of the two electrode bodies arranged opposite to each other with the insulator interposed therebetween, and it is known that the detection sensitivity increases as the distance between the boundary parts becomes narrower.

[0018] For this reason, in the above-mentioned example, the electrode sensor is arranged such that the anode electrode body and the cathode electrode body are stacked with an insulator interposed therebetween, but is not limited to this configuration. For example, the electrode sensor may be arranged such that both electrode bodies are arranged in parallel on a substrate, the boundary portion where the electrodes are arranged facing each other is formed in a comb-like shape, and the formed narrow electrode portions are arranged in parallel alternately.

[0019] It is also possible to form such a comb-tooth electrode structure so as to surround a stick-shaped substrate, or to form a comb-tooth electrode structure on the inside of a cylindrical substrate to create a compact electrode sensor, and the compact electrode sensor may be inserted into a specimen to detect sulfur components.

[0020] The insulator 11 is made of a non-conductive insulating material that electrically insulates the anode electrode body 10 and the cathode electrode body 12. There are no particular limitations on the insulating material, and examples include resin materials and ceramic materials. As shown in the above example, when forming a laminated structure, it can be formed by a printing method using a paste made of an insulating material, in the same way as the cathode electrode body described later.

[0021] A metal thin film is preferably formed on the electrode surfaces of the anode electrode body 10 and the cathode electrode body 12. The metal thin film is preferably a single composition of gold, platinum, silver, titanium, nickel, chromium, or carbon, or a composition containing one or more of these. In addition, it is preferable that the surfaces of the anode electrode body and the cathode electrode body have a low resistance value, specifically, it is preferable to set the resistance value to 0.1 Ω or less. The surface resistance value can be measured by connecting measurement terminals to both ends of the center line of the electrode body.

[0022] When silver is used as the metal of the anode electrode body 10, the metal used in the cathode electrode body 12 may be a metal having a composition that has a more noble potential than silver. When silver is used in the anode electrode body 10, the thickness of the metal thin film is preferably 10 μm or more. By making the thickness 10 μm or more, the electrical resistance value decreases and the current output improves. When the thickness of the silver metal thin film is thinner than 10 μm, problems such as peeling of the thin film are likely to occur when used for a long period of time.

[0023] The anode electrode body 10 can be a silver plate, but can also be configured by forming a metal film made of silver on the substrate surface by wet film formation, dry film formation, or sintering a conductive paste. Examples of wet film formation include electroplating, electroless plating, and composite plating, and examples of dry film formation include vapor deposition and sputtering.

[0024] The cathode electrode body 12 has a conductive material including a metal material at least on the electrode surface, and when a plate-shaped body is used for the anode electrode body 10 serving as the substrate as shown in Fig. 2, the cathode electrode body 12 is preferably formed by a printing method using a conductive paste. By forming the cathode electrode body by such a printing method, the layer thickness is formed to be thick at 5 to 100 µm, the electrical resistance value is reduced, and it becomes possible to accurately and stably measure the current generated between the two electrode bodies.

[0025] In addition, when both electrodes are formed on an insulating substrate, they can be formed by a printing method using a conductive paste. Examples of printing methods using a conductive paste include letterpress printing, lithographic printing, intaglio printing, stencil printing, electrostatic printing, inkjet printing, and laser printing. The electrode sensor shown in FIG. 2 can be created by screen printing, which is a stencil printing method, but other printing methods may also be used, and are not particularly limited.

[0026] In the electrode sensor 1, a galvanic current flows due to an electrochemical reaction that occurs between the anode electrode body 10 and the cathode electrode body 12 due to a trace amount of sulfur contained in the specimen 2 that contacts both electrodes. For example, in the case of hydrogen sulfide, a sulfur compound contained in the metabolism of microorganisms, it is believed that the following reaction occurs, causing a change in the current flowing between the two electrodes. <Sample> H2S → HS - +H + <Cathode electrode body> O2+2H2O+4e - →4OH - <Anode electrode body> 2Ag+H2S →Ag2S+2H + +2e - 2Ag+HS - →Ag2S+H + +2e - 2Ag+S 2- →Ag2S+2e -

[0027] In the above reaction, silver ions (Ag) are produced in the anode electrode as the oxidation reaction progresses. + occurs at the interface with the sample, causing a current to flow, but silver ions (Ag + are the sulfur components in the sample, namely hydrogen sulfide (H2S) and hydrogen sulfide ion (HS - and sulfide ion S 2- Immediately after generation, it combines with silver sulfide Ag2S to form the silver sulfide Ag2S. The silver sulfide formed, Ag2S, is insoluble in water and so deposits on the surface of the anode electrode body.

[0028] The deposition of silver sulfide (Ag2S) causes the formation of silver ions (Ag) at the interface between the anode electrode and the specimen. + disappears, but the oxidation reaction progresses and silver ions Ag + Therefore, silver ions (Ag) are generated between the anode electrode and the sulfur component in the sample. + A series of electrochemical reactions occurs, consisting of the supply of hydrogen sulfide and the precipitation of silver sulfide (Ag2S). Therefore, the change in the concentration of hydrogen sulfide in the sample causes the silver ion (Ag + It is thought that the amount of generated current changes, resulting in a change in the current.

[0029] Furthermore, when measuring metabolites produced by microorganisms, no pretreatment using reagents or the like is required while maintaining the environmental conditions, so it is possible to measure in a natural state without damaging the microorganisms, and the state of the microorganisms can be grasped in real time. Similarly, in the case of biological tissues other than microorganisms, when detecting sulfur compounds in biological reactions or products produced by biological reactions, it is possible to easily detect them without changing the environmental conditions or the like.

[0030] When the surface of the anode electrode body on which silver sulfide (Ag2S) is deposited is covered with silver sulfide (Ag2S), silver ions (Ag + Therefore, it is preferable to perform a regeneration process for the electrode sensor to remove silver sulfide Ag2S as needed.

[0031] As a method for regenerating the electrode sensor, it is preferable to remove silver sulfide Ag2S, which is a sulfur compound formed on the exposed surface of the anode electrode body, by a reduction reaction. Specifically, the silver sulfide Ag2S formed on the exposed surface can be removed by a reduction reaction similar to that of a cathodic reduction method that occurs when an electric current is applied to the anode electrode body in an electrolyte. The electrolyte may be any liquid that can be electrically conducted, and is not particularly limited, but may be, for example, an aqueous potassium chloride solution. As a regeneration device for applying electric current, a known electrochemical reaction measurement device such as a galvanostat may be used, and electric current may be applied to the anode electrode body as a working electrode.

[0032] In addition, it is also possible to remove silver sulfide Ag2S formed on the exposed surface by a reduction reaction caused by contacting at least the exposed surface of the anode electrode body with a sulfuric acid solution containing thiourea. The sulfuric acid solution containing thiourea may be set to a thiourea concentration of 0.1 to 0.5 M (molar), and silver sulfide Ag2S can be easily removed by dropping the solution onto the exposed surface and leaving it for a predetermined period of time.

[0033] By removing silver sulfide (Ag2S) from the exposed surface of the anode electrode, silver ions (Ag) are generated by oxidation at the interface with the specimen. + is generated, making it possible to detect sulfur components.

[0034] The detection unit 3 constantly measures the galvanic current occurring between the anode electrode assembly 10 and the cathode electrode assembly 12 using a non-resistance ammeter connected between the two electrodes. The measurable current range is preferably set to 0.1 nA to 1000 nA.

[0035] The detection unit 3 can be made compact and portable by supplying power from a battery such as a cell or from an AC adapter from a commercial power source. The measurement unit 12 can also be provided with a function for measuring in multiple channels, and current changes of multiple specimens can be measured in parallel.

[0036] In order to measure minute changes in the current, it is preferable to measure the current at short time intervals, specifically, the time interval is set to 0.1 to 60 seconds. The measured current value is stored, and the presence or absence and transition of sulfur components in the sample can be detected and analyzed based on the change in the current value.

[0037] The substance to be detected is not particularly limited as long as it contains a sulfur component. For example, inorganic compounds include hydrogen sulfide, sodium sulfide, sulfurous acid, sulfur monoxide, sulfur dioxide, sulfur trioxide, disulfur monoxide, and dithionous acid. Organic compounds include cysteine, propanethiol, ethanedithiol, thiophenol, glutathione, and 4-mercaptobenzoic acid. Of the organic compounds, organic compounds having a thiol group are preferred.

[0038] The specimen to be detected is preferably in a paste or liquid form, and may be prepared by dissolving the substance to be detected in a solvent, etc. Examples of specimens include body fluids such as blood, urine, and sweat, biological tissues such as brain and liver, and foods such as milk and meat.

[0039] When a metabolite of a microorganism is used as a specimen, a culture medium in which the microorganism is cultured can be used as the specimen. The culture medium is not particularly limited as long as it can culture a wide range of microorganisms, and known culture media such as liquid culture medium, solid culture medium, gel culture medium, and membrane culture medium can be used. The microorganism may be any type of living microorganism that produces a metabolite, and examples of the microorganism include bacteria, actinomycetes, and fungi. For example, the causative bacteria of food poisoning or infectious diseases can also be determined.

[0040] By removing the microorganisms from the environment in which they live and adding them to a culture medium to prepare a sample, sulfur compounds contained in the metabolites of the microorganisms can be easily detected. The sample can be used as is, and the detection process can be performed without stressing the microorganisms. In addition, the microorganisms may be concentrated as necessary, and the detection process can be performed with simple operations. It is preferable to select a combination of the medium and the metal material used for the electrode body so that the current flowing between the two electrode bodies is as stable and low as possible when the two electrode bodies are in contact with a culture medium containing no microorganisms. EXAMPLES

[0041] The present invention will now be described in detail with reference to examples, but the present invention is not limited to these examples.

[0042] <About electrode sensors> The electrode sensor shown in Figure 2 was prepared. The electrode sensor was formed by laminating a cathode electrode body made of a conductive layer containing carbon on the surface of a rectangular anode electrode body on which a silver metal thin film was formed, via an insulator made of a resin layer. The cathode electrode body was formed into a circular shape with a radius of 35 mm, and in the center, narrow notches with a width of 1 mm were arranged in a comb shape at intervals of 1 mm. In the narrow notches, no resin layer was formed, resulting in an electrode structure in which the silver metal thin film of the anode electrode body was exposed.

[0043] A commercially available iron substrate (width 64 mm × length 64 mm × thickness 0.8 mm) was used as the anode electrode body, and a silver metal thin film was formed on one exposed surface of the substrate by wet film formation. In the wet film formation process, the substrate was immersed in a commercially available plating solution containing silver cyanide and potassium cyanide, and then a current of 120 C was applied to the substrate at 20°C. The thickness of the silver metal thin film formed on the surface of the substrate was 12 μm.

[0044] Next, a resin layer was formed as an insulator on the surface of the metal thin film formed on the substrate. The resin layer was formed by screen printing a resin paste (manufactured by Henkel Ablestick Japan Co., Ltd.) made of an epoxy resin material with comb-shaped notches, and then heating and curing it at 150°C for 1 hour in a nitrogen atmosphere. The thickness of the resin layer was 20 μm.

[0045] Next, a conductive layer that would become a cathode electrode body was formed so as to be laminated on the resin layer. The conductive layer was formed by screen printing a conductive paste (manufactured by Tamura Corporation) as a conductive material containing a carbon material, forming comb-shaped notches so as to overlap the resin layer, and then heating and curing it at 130°C for 1 hour in a nitrogen atmosphere. The thickness of the conductive layer was 20 μm.

[0046] <About the detector> The detection device used was assembled as shown in FIG. 1. In a sterile chamber, an electrode sensor was placed on the upper surface of a resin bottom plate with the cathode electrode body facing up, and a glass cylinder (inner diameter 42 mm, height 32 mm) was placed on the cathode electrode body via a silicon packing material. A resin cover plate was placed to cover the upper opening of the cylinder, and the four corners of the bottom plate and cover plate were fixed with fasteners consisting of bolts and nuts so that each part was in close contact with each other, and the storage container was assembled. An opening was formed in the center of the cover plate for introducing the sample. Each part of the assembled storage container was autoclaved at 121°C for 15 minutes and placed in a thermostatic bath at 37°C.

[0047] One end of a conductive wire was connected to the exposed portion of the anode electrode body and the cathode electrode body of the electrode sensor outside the container, and the other end was connected to a resistanceless ammeter (manufactured by Syrinx Co., Ltd.) to measure the current between the two electrodes. Detection processing was performed using an information processing device that stores the measurement results and analyzes the transition of the current value.

[0048] <About the culture medium used to prepare specimens> LB culture medium was used as a medium for culturing microorganisms. LB culture medium was prepared by adding tryptone (10.0 g; BD Biomedicals), yeast extract (5.0 g; BD Biosciences), and NaCl (5.0 g; Wako) to 1 liter of pure water and stirring, and then sterilizing with high-pressure steam at 121°C for 15 minutes using an autoclave.

[0049] [Example 1] Detection tests were conducted using test solutions containing various sulfur compounds as samples.

[0050] <About the test solution> As sulfur compounds, sodium sulfide (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.), cysteine ​​(manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.), cystine (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.), methionine (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.), propanethiol (manufactured by Wako Pure Chemical Industries, Ltd.), and ethanedithiol (manufactured by Wako Pure Chemical Industries, Ltd.) were added to the LB culture solution to a predetermined concentration to prepare a test solution. The added concentrations were set to 0.312 mM (millimolar) for sodium sulfide, 0.825 mM for cysteine, 4.16 mM for cystine, 6.70 mM for methionine, 13.1 mM for propanethiol, and 10.6 mM for ethanedithiol. In addition, a test solution was prepared by dissolving sodium sulfide in degassed pure water. The added concentration was set to 0.312 mM. For comparison, a test solution containing only LB culture solution without the addition of sulfur compounds was also prepared.

[0051] <About detection tests> After dropping 10 mL of each test solution onto the top surface of the cathode electrode of the electrode sensor through the opening of the container of the detection device, the current flowing between the anode electrode and the cathode electrode was measured. During the measurement, the opening of the container was set in an open state.

[0052] The stable current values ​​100 minutes after dropping the test solution were 180nA (nanoamperes) for sodium sulfide, 215nA for cysteine, below the detection limit (10nA) for cystine, below the detection limit for methionine, 104nA for propanethiol, and 272nA for ethanedithiol. In addition, the sodium sulfide test solution using pure water had a current of 620nA, while the test solution containing only LB culture medium had a current below the detection limit.

[0053] <About the measurement results> The measurement results showed that different current values ​​were measured depending on the type of sulfur compound. The current value per measured concentration was highest for sodium sulfide, followed by cysteine, ethanediol, and propanethiol, in that order. No current was detected for cystine or methionine.

[0054] In the case of sodium sulfide, hydrogen sulfide ions (HS) are present in the test solution. - or sulfur ion S 2- It is believed that a sulfur compound having a thiol group is produced. In addition, since a current value is measured for a sulfur compound having a thiol group, it is believed that a sulfur compound having a thiol group is detected.

[0055] In the test solution for sodium sulfide, the current value was higher when it was added to pure water than when it was added to LB culture solution. This is because hydrogen sulfide ions HS, a sulfur component in the test solution, - or sulfur ion S 2- It is presumed that the concentration of sulfur components in the LB culture medium is lower than that in pure water because it is adsorbed to proteins and other substances contained in the LB culture medium.

[0056] [Example 2] A detection test was carried out using a bacterial liquid containing live bacteria as a sample.

[0057] <Preparation of bacterial solution> Commercially available Escherichia coli (E. coli DH5α) was used for the bacterial suspension. E. coli was cultured on a plate medium, and independent colonies were picked up and cultured in LB culture medium, followed by centrifugation. The resulting precipitated bacterial cells were washed with LB culture medium to remove any metabolites produced, and then resuspended in LB culture medium. The bacterial count in the bacterial suspension was measured using a spectrophotometer (Hitachi High-Tech Corporation) to measure the optical density (OD) at 600 nm; an OD of 0.1 was considered to be a count of 10 7 The standard is equivalent to cfu / ml, and the number of bacteria is 10 6 The cells were diluted with LB culture medium to give cfu / ml.

[0058] <About detection tests> After dropping 10 ml of the bacteria liquid onto the upper surface of the cathode electrode of the electrode sensor through the opening of the container of the detection device, the current flowing between the anode electrode and the cathode electrode was continuously measured at 0.5 second intervals. During the measurement, the opening of the container was set in an open state.

[0059] <Quantitative analysis of hydrogen sulfide (H2S) concentration in the bacterial solution>\ In parallel with the measurement of the current value, the hydrogen sulfide (H2S) concentration in the bacterial solution was quantitatively analyzed. 0.5 ml samples were taken from the bacterial solution during measurement at appropriate times, and quantitative analysis of the samples was performed using an ion chromatograph (ICS-3000 electrochemical detector manufactured by Thermo Fisher Scientific Co., Ltd.) using the calibration curve method.

[0060] <About the measurement results> Figure 3 shows the time course of the current and hydrogen sulfide H2S concentration, with the horizontal axis representing time (minutes), the left vertical axis representing current (nA), and the right vertical axis representing concentration (μM (micromolar)). Figure 4 is a graph showing the correlation between the measured current and concentration, with the horizontal axis representing concentration (μM) and the vertical axis representing current (nA).

[0061] It is known that E. coli releases minute amounts of hydrogen sulfide (H2S) during proliferation from the metabolic decomposition of cysteine, etc. As shown in Figure 3, the quantitative analysis confirmed that a small amount of hydrogen sulfide (H2S) was released into the LB culture medium as E. coli proliferated, and an increase in the current value was confirmed accordingly.

[0062] In Figure 4, a high positive correlation (correlation coefficient R2 = 0.993) was confirmed between the hydrogen sulfide (H2S) concentration in the LB culture medium and the current value of the electrode sensor, and a linear relationship equation (y = 530x - 1.70) was obtained that quantitatively shows the correlation between the two.

[0063] It was also shown that the electrode sensor can detect trace concentrations of hydrogen sulfide H2S of 0.022 μM to 0.70 μM. Based on the obtained linear relationship equation, it is possible to detect trace amounts of hydrogen sulfide H2S in a sample from the current value of the electrode sensor, and the detection limit based on the linear relationship equation is a trace concentration of 0.003 μM.

[0064] The above detection test results confirmed that even in a solution containing many impurities such as proteins and amino acids, such as LB culture medium, trace amounts of hydrogen sulfide (H2S) present in the solution can be directly detected without pretreatment such as pH adjustment or extraction.

[0065] [Example 3] A detection test was carried out by changing the thickness of the metal thin film of the anode electrode body of the electrode sensor.

[0066] <About detection tests> Four types of electrode sensors, in which the thickness of the silver metal thin film formed on the surface of the substrate as the anode electrode body was 5, 8, 10, and 15 μm, were manufactured in the same manner as in Example 1. For comparison, an electrode sensor manufactured in the same manner as in Example 1 was prepared, using a silver plate as the substrate and no metal thin film.

[0067] Using the same medium and microorganisms as in Example 2, 8A sample containing cfu / milliliter was prepared, and the current value was measured by the same detection test as in Example 2.

[0068] <About the measurement results> From the obtained measurement results, looking at the relationship between the silver film thickness and the maximum current value, when the film thickness was 5 μm, the maximum current value was 350 nA, and when it was 8 μm, the maximum current value was 400 nA, whereas when the film thickness was 10 μm, it was 610 nA, when the film thickness was 12 μm in Example 2, it was 650 nA, when the film thickness was 15 μm, and it was 650 nA when a silver plate was used.

[0069] From these measurement results, it was confirmed that by forming the silver film thickness of the anode electrode body to 10 μm or more, the maximum current value can be increased and stable measurement results can be obtained.

[0070] [Example 4] A confirmation test was conducted to examine the effect of the detection test on live bacteria in the specimen.

[0071] <About confirmation tests> Using the electrode sensor manufactured in the same manner as in Example 1, a detection test was carried out on a specimen containing live bacteria in the same manner as in Example 2. The number of live bacteria was 10 5 A bacterial solution adjusted to cfu / milliliter was used.

[0072] Three milliliters of the bacterial liquid on the electrode sensor in the storage container were sampled every 30 minutes, and the OD value of the sampled bacterial liquid at 600 nm was measured using a spectrophotometer in the same manner as in Example 2. Meanwhile, a bacterial liquid similar to the specimen used in the detection test was left to stand in a commercially available plastic container at 37°C, and the OD value of the bacterial liquid at 600 nm was measured every 30 minutes in the same manner.

[0073] <About the measurement results> The measurement results are shown in Figure 5. In Figure 5, the horizontal axis represents time (minutes), the left vertical axis represents OD value, and the right vertical axis represents current value (n). Comparing the time transition of the OD value corresponding to the number of bacteria and the measured current value, it was confirmed that the proliferation of bacteria did not coincide with the increase in current value, and that the current value began to increase about 100 minutes after the bacteria began to proliferate, and that the bacteria continued to proliferate while the current value was increasing.

[0074] In addition, the changes in OD value of the bacteria liquid on the electrode sensor and the bacteria liquid in the plastic container were consistent, and it is considered that the growth rates of both are the same. Specifically, the doubling time of E. coli was calculated from the slope of the time change in OD value during the logarithmic growth period of 60 to 210 minutes, and the doubling time was 27 minutes for both. In other words, it was confirmed that the growth of live bacteria was not hindered at all when in contact with the electrode sensor.

[0075] [Example 5] A regeneration test was carried out on the electrode sensor used in the detection test.

[0076] <About regeneration testing> The same detection test as in Example 2 was performed with the number of bacteria being 10 7 The test was performed using a bacterial solution containing cfu / ml of bacteria. After the detection test, the electrode sensor had turned brown due to the formation of silver sulfide on the exposed silver-plated surface of the anode electrode body.

[0077] A regeneration test was conducted using a used electrode sensor as a test specimen, using a potentiogalvanostat (SI1287, manufactured by Toyo Corporation) with a reduction reaction similar to that of the cathodic reduction method. The test specimen was immersed in a 0.1N potassium chloride (KCl) electrolyte, and a current of -125μA was applied for 1000 seconds using a three-electrode method in which the anode electrode of the test specimen was used as the working electrode, SCE as the reference electrode, and platinum as the counter electrode.

[0078] In addition, a test solution was prepared by dissolving thiourea at a concentration of 0.5 molar in a 0.01N aqueous sulfuric acid solution, and a regeneration test was conducted by a reduction reaction of thiourea using the test solution. A used electrode sensor was used as a test specimen, and 6 milliliters of the test solution was dropped onto the discolored surface of the specimen and left at room temperature for 1 minute.

[0079] After the regeneration test, the electrode sensor was washed with running water and dried indoors. Next, the electrode sensor that had been subjected to the first regeneration treatment was used to perform a detection test similar to the above-mentioned detection test. Then, the electrode sensor used in the second detection test was subjected to a second regeneration treatment, and the second detection test was performed similarly to the above-mentioned detection test.

[0080] <About the measurement results> Figure 6 shows the measurement results of the detection test using the electrode sensor for the first time, after the first regeneration process, and after the second regeneration process in the case of regeneration process by current flow. In Figure 6, the horizontal axis is time (minutes) and the vertical axis is current value (μA), showing the change in current value over time. The maximum current value was 284nA for the first time, 273nA for the first regeneration process, and 242nA for the second regeneration process.

[0081] In addition, Figure 7 shows the measurement results of the detection test using the electrode sensor after the first regeneration treatment, the first regeneration treatment, and the second regeneration treatment in the case of regeneration treatment with thiourea. Figure 7 shows the time transition of the current value, as in Figure 6. The maximum current value was 305 nA in the first regeneration treatment, 284 nA in the first regeneration treatment, and 251 nA in the second regeneration treatment.

[0082] It was visually confirmed that the discolored surface of the anode electrode of the electrode sensor changed from brown to white after the regeneration treatment. It is believed that the silver sulfide precipitated on the surface was removed by the regeneration treatment using a reduction reaction. Regardless of the reduction reaction used for the regeneration treatment, the electrode sensor after the first regeneration treatment gave detection results equivalent to those of the initial electrode sensor. Therefore, it was confirmed that detection tests can be performed using electrode sensors that have been regenerated for the first time.

[0083] In the case of the electrode sensor that had been regenerated a second time, the current value increased in the same way as the first time, but the maximum current value decreased by about 15%. However, it was confirmed that it was possible to detect the presence or absence of sulfur components. [Explanation of symbols]

[0084] REFERENCE SIGNS LIST 1 electrode sensor, 2 specimen, 3 detection unit, 4 storage container, 10 anode electrode body, 11 insulator, 12 cathode electrode body

Claims

1. A sulfur component detection method for detecting sulfur components in a specimen using an electrode sensor in which an anode electrode body and a cathode electrode body are integrally formed via an insulator, the method measuring a galvanic current flowing between the two electrode bodies due to an electrochemical reaction that occurs between the sulfur components in the specimen and the anode electrode body while the specimen is in contact with the two electrode bodies, and detecting the sulfur components in the specimen based on the measured current value.

2. The method for detecting a sulfur component according to claim 1 , wherein the specimen contains a sulfur compound derived from biological tissue.

3. 3. The method for detecting a sulfur component according to claim 1, wherein the specimen contains a sulfur compound derived from a metabolic product of an organism.

4. 3. The method for detecting a sulfur component according to claim 1, wherein at least the exposed surface of the anode electrode body that comes into contact with the sample is coated with silver, and a sulfur compound is produced on the exposed surface in association with an electrochemical reaction that occurs between the silver and the sulfur component.

5. The method for detecting a sulfur component according to claim 4, wherein at least the exposed surface of the anode electrode body is covered with a layer of silver having a thickness of 10 μm or more.

6. 5. A method for regenerating the electrode sensor used in the method for detecting sulfur components according to claim 4, comprising removing sulfur compounds formed on the exposed surface of the anode electrode body by a reduction reaction.

7. 7. The method for regenerating an electrode sensor according to claim 6, further comprising removing sulfur compounds formed on the exposed surface by a reduction reaction caused by passing a current through the anode electrode body in an electrolyte.

8. The method for regenerating an electrode sensor according to claim 6, further comprising the step of contacting at least the exposed surface of the anode electrode body with a sulfuric acid solution containing thiourea to cause a reduction reaction, thereby removing sulfur compounds formed on the exposed surface.

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