Analysis device

Abstract

The purpose of the present invention is to provide an analysis device with which it is possible to carry out analysis which is simple and has a stable measured electric potential.　Provided is an analysis device having a flow path region surrounded by flow path walls provided inside of a porous substrate, said analysis device being characterized in that: the flow path region has a first flow path chamber, a second flow path chamber, and a flow path connecting the first flow path chamber and the second flow path chamber; a reference electrode is disposed in the first flow path chamber; a working electrode is disposed in the second flow path chamber; when using the advancing direction of a sample in the flow path region as a reference, a component A is disposed upstream relative to the first flow path chamber or upstream relative to a position which, when an electrolyte is disposed on the surface of the reference electrode, has the electrolyte disposed thereto; and the component A has the effect of changing the three-dimensional structure of a protein in the sample.

Description

analytical devices

[0001] The present invention relates to an analytical device having a flow path region formed inside a porous substrate, and an electrolyte concentration measurement system and electrolyte concentration measurement method that use the analytical device.

[0002] In recent years, devices that utilize microchannels to perform efficient (trace-volume, rapid, and simple) chemical analysis within a single chip have been attracting attention in a wide range of fields, including biochemical research, medicine, drug discovery, healthcare, the environment, and food.

[0003] Among analytical devices, those based on paper are called "μPADs" (microfluidic paper-based analytical devices), and they allow chemical analysis by using the capillary action of the paper to allow specimens or test solutions to penetrate. The advantages of these devices include their small size, low cost, portability, high disposability (simply burn them), and the lack of the need for large-scale equipment. It is said that the practical application of these new analytical devices will enable anyone to easily and inexpensively perform point-of-care (POC) testing and diagnosis.

[0004] In addition, due to the advantages of μPADs mentioned above, it is expected that they will be used in developing countries and depopulated areas that lack adequate medical facilities, at disaster sites where rapid emergency response is required, at airports where the spread of infectious diseases must be stopped at the border, etc. They are also attracting attention as healthcare devices for managing and monitoring daily health conditions, and as various pathological diagnostic devices in regular medical settings, and their applications are diverse.

[0005] One of the biochemical tests used in the pathological diagnosis is an electrolyte test. Electrolytes (such as sodium ions, potassium ions, and chloride ions) are essential for maintaining a constant body fluid level and pH, as well as for normal nerve transmission and muscle function, and are essential for maintaining life. An electrolyte test measures the concentration of electrolyte ions in the blood or urine to confirm the balance of ion concentrations in the body.

[0006] Generally, if there is a change in electrolyte concentration in the body, there is a high possibility that abnormalities in kidney function or hormone activity are occurring. For this reason, electrolyte testing is an essential test for disease screening. It is also a very important test for checking the physiological function (life support) of patients at disaster sites, etc. Research is being conducted at various universities and companies with the aim of using μPADs to measure electrolytes.

[0007] Non-Patent Document 1 proposes an analytical device for measuring the concentrations of Na ions and K ions. This analytical device has a dispensing section for dispensing a sample. The dispensed sample permeates from the dispensing section into the working electrode and reference electrode regions, electrically connecting the two electrodes and measuring the potential difference. In this analytical device, KCl ion crystals are deposited on the reference electrode to obtain a stable potential at the reference electrode. During measurement, KCl dissolves in the sample, maintaining a high concentration of Cl ions in the reference electrode region and obtaining a stable reference electrode potential. Patent Document 1 also discloses a multi-ion sensor plate, which is not a μPAD (i.e., the substrate used is not a porous substrate), in which a stable potential is obtained by contacting the analyte solution supplied to the working electrode region with the reference solution supplied to the reference electrode region at a liquid junction.

[0008] Japanese Unexamined Patent Publication No. 11-194109

[0009] Nipapan Ruecha, Orawon Chailapakul, Koji Suzuki and Daniel Citterio, “Fully Inkjet-Printed Paper-Based Potentiometric Ion-Sensing Devices”, Analytical chemistry, August 29, 2017 Published, 89, pp.10608-10616

[0010] However, in the configuration described in Non-Patent Document 1, when measuring samples containing protein, such as serum or plasma, the hydration water of the hydrated protein is easily taken away by the electrolyte as the sample permeates the electrolyte layer (KCl layer), which can easily cause salting out or aggregation of the protein. As a result, the salting out or aggregation of the protein can inhibit the permeation of the sample in the flow channel, resulting in unstable measurement potential and prolonged measurement times.

[0011] Furthermore, Patent Document 1 discloses two configurations: one in which a surfactant is added to the sample to maintain the dispersibility of hydrophobic substances (hematocrit or similar substances) contained in the sample liquid and prevent their precipitation (precipitation), and the other in which a surfactant coating is provided in the flow path leading to the sample liquid side electrode. However, in the configuration of Patent Document 1, in order to incorporate the surfactant into the sample liquid in a sufficiently mixed state in advance, a sample volume of several mL is required to stir the mixed liquid, which may place a heavy burden on the patient. Furthermore, when a surfactant coating is provided in the flow path, the coating on the flow path wall is a constraint, preventing uniform contact with the sample flowing through the flow path, which may result in insufficient mixing with the sample liquid.

[0012] The present invention aims to provide an analytical device in which a layer that suppresses salting out and aggregation of proteins is positioned at an appropriate location, which exhibits an appropriate reference electrode potential, and which enables simple and stable analysis.

[0013] The present invention relates to an analytical device having a flow path region surrounded by a flow path wall provided inside a porous substrate, the flow path region having a first flow path chamber, a second flow path chamber, and a flow path connecting the first flow path chamber and the second flow path chamber, a reference electrode being arranged in the first flow path chamber, and a working electrode being arranged in the second flow path chamber, and based on the direction of travel of a sample in the flow path region, an electrolyte being arranged upstream of the first flow path chamber or on the surface of the reference electrode, and component A being arranged upstream of the position where the electrolyte is arranged, and the component A is a component that has an effect of changing the three-dimensional structure of a protein in the sample.

[0014] The present invention provides an analytical device that can perform simple analysis with stable measured potential. By disposing component A in the porous flow path, the sample and component A can be effectively mixed, which prevents salting out of the electrolyte disposed near the reference electrode, thereby enabling testing with a small amount of sample.

[0015] 2B is a diagram illustrating the configuration of a reference electrode. FIG. 1A is a cross-sectional view of the reference electrode along the dashed line in FIG. 1A. FIG. 2B is a cross-sectional view of the configuration of a conventional example along the dashed line in FIG. 2B. FIG. 2B is a diagram illustrating the configuration of a conventional example. FIG. 3 is a diagram illustrating the change in potential over time when a serum sample is measured with the configuration of the conventional example. FIG. 4 is a diagram illustrating the change in potential over time when an ultrafiltered serum sample is measured with the configuration of the conventional example. FIG. 4 is a diagram illustrating the configuration of an analytical device of Example 1. FIG. 5 is a diagram illustrating the configuration of a flow path pattern. FIG. 6 is a diagram illustrating the change in potential over time when a serum sample is measured with only the reference electrode of Example 1. FIG. 7 is a diagram illustrating the change in potential over time when a serum sample is measured with only the reference electrode of Example 1. FIG. 8 is a diagram illustrating the change in potential over time when a serum sample is measured with a configuration in which the reference electrode and working electrode are combined of Example 1. FIG. 9 is a diagram illustrating the change in potential over time when a serum sample is measured with the configuration of a comparative example of Example 1. FIG. 10 is a diagram illustrating the change in potential over time when a serum sample is measured with only the reference electrode of Example 2. FIG. 11 is a diagram illustrating the change in potential over time when a serum sample is measured with only the reference electrode of Example 2.

[0016] The function of the present invention will be explained in detail. The role of the reference electrode in measuring the electrolyte concentration of the present invention is to provide a reference potential for the potential generated at the working electrode. Therefore, if the potential of the reference electrode is not stable, the potential difference between the reference electrode and the working electrode will be inaccurate.

[0017] Generally, an Ag / AgCl electrode is used as the base electrode of a reference electrode. In an Ag / AgCl electrode, the following equilibrium reaction occurs at the interface with the sample, and Cl － The potential is determined by the concentration of Therefore, Cl － By keeping the concentration constant, the potential can be stabilized.

[0018] Therefore, in saturated sodium chloride (NaCl) or potassium chloride (KCl) solutions, Cl － Since the concentration is constant, if saturated NaCl or KCl solution is reacted with an Ag / AgCl electrode, the same interfacial potential will always be obtained. When the sample is a NaCl solution, once the concentration reaches the saturated concentration of 5.2 mol / L to 5.4 mol / L, it will no longer dissolve, so Cl － The concentration becomes constant, the above-mentioned equilibrium state is reached, and the potential does not change any more.

[0019] Based on this principle, saturated KCl or NaCl is used as the internal solution in internal liquid type reference electrodes, while in solid electrode type reference electrodes, this saturated solution is directly dispensed onto the Ag / AgCl electrode as the reference solution to ensure a stable potential.

[0020] As an example of a solid electrode type reference electrode, Non-Patent Document 1 discloses a configuration in which an electrolyte layer 4 (KCl layer) is laminated on a supporting electrolyte film 7 (provided to further stabilize the contact potential at the interface between the analyte and the Ag / AgCl electrode) provided on a reference electrode 3 (Ag / AgCl electrode), as shown in Figures 2A and 2B. In this configuration, the analyte itself dissolves the electrolyte layer 4 during penetration, resulting in Cl. － The concentration is kept constant. － By supplying the specimen with a constant concentration to the supporting electrolyte membrane 7 and the Ag / AgCl electrode, it becomes possible to perform measurements without using a reference solution.

[0021] Patent Document 1, which also has a solid-electrode-type reference electrode, employs a method of dispensing a reference solution separately from the sample for measurement. While this method allows for a stable measurement potential, the use of a reference solution can lead to issues with measurement simplicity and cost. Patent Document 1 also discloses two configurations to prevent protein aggregation, as described below: one in which a surfactant is incorporated into the sample, and another in which a surfactant coating is provided in the flow path leading to the sample liquid electrode. However, with the configuration of Patent Document 1, in order to thoroughly mix the surfactant into the sample liquid in advance, a sample volume of several mL is required to stir the mixture, which can be a significant burden on the patient. Furthermore, providing a surfactant coating in the flow path imposes limitations on the coating on the flow path walls, preventing uniform contact with the sample flowing through the flow path and potentially resulting in insufficient mixing with the sample liquid.

[0022] Configurations such as that described in Non-Patent Document 1, which have an electrolyte layer, have the following problem. When measuring samples containing protein, such as serum or plasma, as the sample permeates the electrolyte layer (KCl layer), the hydration water of the hydrated protein is easily taken away by the electrolyte, which can easily cause salting out or aggregation of the protein. As a result of the salting out or aggregation of the protein, the permeation of the sample within the flow path is hindered, resulting in an unstable measurement potential and a long measurement time.

[0023] This section describes the results of measuring a serum sample using a conventional configuration with an electrolyte layer on the reference electrode side, as shown in Figure 2B. Two devices with the same configuration were prepared, and Figure 3 shows the measurement results of the reference electrode potential difference when serum was used as the sample. In this experiment, to focus solely on the performance of the reference electrode 3, a commercially available reference electrode (RE-1BP, BAS Corporation) was placed on the side of the analytical device where the working electrode 6 would normally be formed, instead of the working electrode 6. The commercially available electrode and reference electrode 3 were connected via the sample, and the potential difference between the two electrodes was measured. The measurement results show that the measured potential fluctuated significantly for approximately 100 seconds after dispensing. After that, the measured potential fluctuations decreased over time, but a stable potential could not be maintained, and a difference of approximately Δ0.9 mV was confirmed after 200 seconds. Furthermore, with this configuration, sample penetration within the electrolyte layer was unstable and slow, and clumps of material were observed in the flow path.

[0024] For comparison, a serum sample was ultrafiltered using a commercially available filter (Amicon Ultra-0.5 Device (NMWL: 30K), Merck) with the same configuration as above to remove proteins and other molecules, and the results are shown in Figure 4. It was confirmed that this sample had smooth permeation within the electrolyte layer and a stable potential.

[0025] From the above experiments, it was confirmed that in order to quickly stabilize the potential of the reference electrode, it is necessary for the sample not to aggregate in the flow path. The analytical device of the present invention does not slow down the permeation of the sample, and the Cl of the sample is also effective in measuring samples containing protein, such as serum and plasma. － The reference electrode can be stably contacted with the electrolyte layer when the concentration is saturated. That is, the analytical device of the present invention has a configuration in which, when the direction of movement of the sample in the flow path region is used as a reference, a layer that suppresses salting out and aggregation of proteins is disposed upstream of the position where the electrolyte layer is disposed.

[0026] Furthermore, the analytical device of the present invention may have a configuration in which, in addition to the first flow channel chamber and the second flow channel chamber, a plurality of third flow channel chambers each having a working electrode disposed therein are connected by a flow channel. The number of flow channel chambers disposed on the analytical device is not particularly limited.

[0027] Hereinafter, exemplary embodiments of the present invention will be described with reference to the drawings. Note that the following embodiments are merely examples, and the present invention is not limited to the contents of the embodiments. Furthermore, in the following drawings, components that are not necessary for explaining the embodiments are omitted from the drawings.

[0028] Example 1 Example 1 will be described with reference to FIGS. 5 to 8. FIG. 5 shows the analytical device of Example 1. The analytical device has a flow path region surrounded by a flow path wall 2 provided inside a porous substrate. The flow path region includes a first flow path chamber 9, a second flow path chamber 10, and a flow path 1 connecting the first flow path chamber 9 and the second flow path chamber 10. The flow path region may or may not include a third flow path chamber 11 and a fourth flow path chamber 12. A reference electrode 3 is disposed in the first flow path chamber 9. An electrolyte layer 4 is disposed upstream of the reference electrode 3, relative to the direction of sample flow (i.e., the upstream side is the dispensing section). By disposing the electrolyte layer 4 upstream of the reference electrode 3, the electrolyte concentration in the sample reaches the reference electrode 3 stably in a saturated state. A component A layer 5, which acts to change the three-dimensional structure of proteins in the sample, is disposed upstream of the electrolyte layer 4. A working electrode 6 is disposed in the second flow channel chamber 10, and the working electrode 6 is composed of a base electrode 6b of the working electrode and an ion selective membrane 6a provided so as to cover it.

[0029] The flow path was formed by the method described in (JP 2021-37612 A). Specifically, a desired flow path pattern was formed on filter paper in an unfixed state by electrophotography using flow path forming particles (toner) characterized by melting properties, and then the flow path pattern was penetrated into the paper using an oven or heater to form the flow path pattern.

[0030] The formed flow path pattern is as shown in Figure 6, where reference numeral 1 denotes the flow path and reference numeral 2 denotes the flow path wall formed by the penetration of flow path forming particles. Next, a reference electrode 3 and a working electrode 6 are formed on the flow path pattern by screen printing, an inkjet device (IJ), a dispenser, or the like. Since this patent is an invention related to the reference electrode, the configuration will be explained mainly with reference electrodes.

[0031] As shown in FIG. 1A, an Ag / AgCl electrode was printed by screen printing on the flow path pattern as a reference electrode, and further, as shown in FIGS. 1A and 1B, an electrolyte (NaCl or KCl) was printed by an ink jet or dispenser at a position on the flow path upstream of the Ag / AgCl electrode in the direction of sample flow. By disposing the electrolyte layer (NaCl or KCl) at such a position, the sample that is flowing through the electrolyte layer will always pass through. At this time, the electrolyte is dissolved, and the concentration of the electrolyte in the sample (Cl － The concentration of the sample reaches saturation, and in this state the sample reaches the reference electrode (Ag / AgCl electrode). As a result, the potential of the reference electrode (Ag / AgCl electrode) is stabilized.

[0032] The electrolyte is preferably a chloride, and particularly preferably sodium chloride (NaCl) or potassium chloride (KCl), which are easy to handle. The following description will be given using NaCl, which has a more stable temperature dependence of solubility.

[0033] The electrolyte (NaCl) was added to the flow path before the reference electrode in a concentration of 3.0 × 10 per 1 L of sample volume supplied to the reference electrode 3. ２ g / L or more, preferably 3.4 × 10 ２ The saturated concentration of the electrolyte is also dependent on the amount of sample, so the amount of NaCl required for saturation is divided by the amount of sample supplied to the reference electrode 3. The saturated concentration is 3.0 x 10 per 1 L of sample supplied to the reference electrode 3. ２ If NaCl of 3.4 × 10 g / L or more is placed, the concentration of the sample that has passed through the NaCl layer will be 4.6 mol / L or more, which is close to saturation, and stable measurement will be possible. ２In this case, the concentration of the analyte that passes through the NaCl layer is 5.2 mol / L or more, and the Cl － The concentration of the ions is sufficient to maintain a saturated state.

[0034] The appropriate amount of sample to be supplied to the dispensing unit 8 depends on the size and performance of each device, and is generally about 10 μL to 50 μL. For example, in a small device, the reference electrode is 3 mm×3 mm in size, the working electrode 6 is 3 mm×3 mm in size, and the paper is 200 μm thick, the volume of the entire flow path, including the volume of the flow path between them, is 3.6×10 －９ m ３ (3.6 μL) (1.8 μL each for the reference electrode and working electrode). Therefore, approximately 10 μL of sample is sufficient to supply the sample to the reference electrode and working electrode.

[0035] The larger the device size, the larger the required sample volume, but since the sample is based on human blood or urine, a smaller volume is preferable, generally 50 μL or less. Although the present invention does not depend on the size of the device or the required sample volume, for the reasons mentioned above, the present invention will be described using a sample volume range of approximately 10 μL to 50 μL.

[0036] In the analytical device of Example 1, 10 μL of sample is dispensed into dispenser 8 located in the center of the flow path region, about half of which is supplied to the reference electrode side, and the other half is supplied to the working electrode side, so the amount of sample supplied to the reference electrode is about 5 μL. In this example, an electrolyte with a saturated concentration is placed upstream of the first flow path chamber in FIG. 1A.

[0037] For example, if the amount of sample supplied to the reference electrode is 5 μL, placing 1.5 mg of NaCl will result in a NaCl concentration of 4.6 mol / L or more. Therefore, if NaCl is placed in a 3 mm × 3 mm area with a thickness of 200 μm, 8.3 × 10 per unit volume of the NaCl placement area will be ５ g / m ３ If more than 9.4 × 10 NaCl is placed, the NaCl concentration of the sample that has passed through the NaCl layer will be 4.6 mol / L or more, which is close to saturation. ５ g / m３ With this amount of NaCl, the concentration of the analyte that passes through the NaCl layer is 5.2 mol / L, which is sufficient to bring the Ag / AgCl electrode into equilibrium.

[0038] When the sample reaches the electrolyte (NaCl) layer, it dissolves the electrolyte (NaCl) and moves toward the reference electrode (Ag / AgCl electrode). － The concentration of is always kept saturated or close to saturated, and this saturated sample is continuously supplied to the reference electrode (Ag / AgCl electrode), so a stable potential is obtained.

[0039] In this configuration, the reference electrode and the electrolyte layer are not in contact with each other, but the electrolyte layer may be in contact with the reference electrode or may overlap the reference electrode. The important point is that the analyte reaches the reference electrode after the electrolyte concentration in the analyte becomes saturated.

[0040] A surfactant (Tween 20, chemical formula: C) is added as a component A layer 5 at a position upstream of, and preferably adjacent to, the electrolyte layer 4. ５８ H １１４ O ２６An aqueous solution containing HCl was printed using an inkjet printer or dispenser. These techniques allow for uniform distribution of the surfactant in the desired flow channel region on the porous substrate. Additionally, the large surface area of ​​the porous structure can be utilized, increasing the contact area between the sample and surfactant (compared to a non-porous substrate). Based on the BET specific surface area of ​​the paper, the surface area can be increased by approximately 1,000 times compared to the configuration described in Patent Document 1, which favors mixing of the surfactant and sample. The sample permeates the flow channel via capillary action and is stirred between the fibers, allowing even small amounts of sample (on the order of μL) to be sufficiently mixed with the surfactant. Any porous substrate (e.g., filter paper, glass filter paper) can be used for this purpose as long as it has a porous structure. The important thing is to uniformly distribute a predetermined amount of surfactant in the desired flow channel region. Furthermore, considering the cross-section of the porous flow channel from the perspective of sample usage, for example, if a substrate with a porosity of 50% is used, the flow channel can be filled with half the amount of sample compared to a hollow flow channel with the same cross-sectional area (such as the flow channel described in Patent Document 1). As described above, the principle of reference electrode potential generation is based on Cl. － Because the effect depends on the concentration of the surfactant, as long as there is a liquid junction between the working electrode and the sample, half the sample volume can achieve the same effect. Therefore, with the above configuration, even with small amounts of sample (on the order of μL), mixing with a surfactant to form micelles (changing the protein's three-dimensional structure) can suppress aggregation during penetration into the electrolyte layer, and a stable measurement potential can be expected. Surfactants can be cationic, anionic, amphoteric, nonionic, etc., but for electrolyte measurement chips, nonionic surfactants are preferred because they do not inhibit the potential development on the Ag / AgCl electrode or ion-selective membrane. While a surfactant was used in this example, any substance that can change the three-dimensional structure of the protein can be used, including salts (e.g., guanidine hydrochloride as a chaotropic salt), acids (e.g., citric acid), organic solvents (e.g., ethanol), and water-soluble polymers (e.g., polyethylene glycol).

[0041] As with the formation of the electrolyte layer, the formation of the surfactant layer also depends on the amount of sample supplied to the reference electrode. The following experiment was conducted to confirm the appropriate amount of surfactant that can suppress protein aggregation while saturating the NaCl concentration in the electrolyte layer when the amount of sample supplied to the reference electrode is 5 μL.

[0042] Three types of analytical devices were prepared, each containing 0.012 mg, 0.058 mg, and 0.115 g of the nonionic surfactant Tween 20, and the potential of the reference electrode was measured. In this experiment, to focus only on the performance of the reference electrode, a commercially available reference electrode was placed on the side of the analytical device where the working electrode would normally be formed, and the commercially available electrode and reference electrode were connected with a sample, and the potential difference between the two electrodes was measured.

[0043] The results for 0.012 mg are shown in Figure 7, the results for 0.058 mg in Figure 8, and the results for 0.115 mg in Figure 9. It can be seen that the potential difference after 200 seconds for the conventional configuration was Δ0.9 mV, but this was improved for the amounts of 0.012 mg and 0.058 mg. It can also be seen that the potential was unstable for 0.115 mg. This is because the application of a large amount of surfactant caused the sample to permeate too quickly, resulting in Cl formation in the electrolyte layer. － This is thought to be because the concentration of Tween 20 reached the Ag / AgCl electrode without reaching the saturated concentration.

[0044] Furthermore, as shown in Figure 5, a working electrode 6, which is an ion-selective electrode, was placed on one side, and with both electrodes in place, a sample was dispensed and the potential difference with the reference electrode was measured. The results are shown in Figure 10. Furthermore, as a comparative example, the results for a configuration without a surfactant layer are shown in Figure 11. It was confirmed that, in the configuration of Example 1 in Figure 10, the measured potential stabilized from about 60 seconds after dispensing, but in the configuration of the comparative example in Figure 11, the potential was unstable.

[0045] The working electrode (ion selective electrode) used was a solid contact type ion selective electrode in which an ion selective membrane 6a having selectivity for the target ion was laminated on a base electrode 6b. ＋ was used as an ion selective electrode for the target ion.

[0046] For the base electrode of the working electrode, efforts using Ag / AgCl, carbon, and PEDOT (poly(3,4-ethylenedioxyphene)) / PSS (poly(4-styrenesulfonate)) have been proposed, and these can be used without any particular limitations in the present invention. It is sufficient to select a base electrode that matches the required characteristics of the device, such as cost and performance, and in this example, an Ag / AgCl electrode was used as the base electrode.

[0047] The ion-selective membrane may be any commonly used membrane, as long as it is sensitive to the target ion and sufficiently selective to interfering ions. Examples of materials used for the ion-selective membrane include ionophores such as valinomycin, anion decontaminants such as potassium tetraphenylborate (KTPB), plasticizers such as NPOE (o-nitrophenyl octyl ether) and DOS (di(2-ethylhexyl) sebacate), and polymers such as PVC (polyvinyl chloride) and copolymers of polyvinyl chloride and polyvinyl acetate.

[0048] The respective components are mixed in appropriate amounts, dissolved or dispersed in a solvent such as tetrahydrofuran (THF) or cyclohexanone, and the resulting solution is applied by inkjet printing to a base electrode (Ag / AgCl electrode) on which an intermediate layer such as NaCl is laminated. The application method does not necessarily have to be inkjet printing, and the ion-selective membrane can be laminated on the base electrode after adjusting the viscosity of the solution to suit each printing method, such as dispenser printing or screen printing.

[0049] The present invention is not limited to analytical devices using a single Ag / AgCl electrode as the reference electrode. As described in Non-Patent Document 1, the reference electrode may be configured with an Ag / AgCl base electrode, with a supporting electrolyte layer provided on its surface to stabilize the interfacial potential with the analyte and reduce the influence of interfering ions. For example, as described in Non-Patent Document 1, a supporting electrolyte layer can be formed by mixing appropriate amounts of TBA-TBB (tetrabutylammonium tetrabutylborate) or TDMACl (tridodecylmethylammonium chloride), a plasticizer, and PVC, and then mixing the mixture with a solvent such as THF or cyclohexanone, followed by application and drying. Furthermore, in the present invention, laminate layers may be provided on the front and back surfaces of the reference electrode to prevent leakage or contamination of the electrolyte layer and component A layer during handling of the analytical device. As described above, according to the present invention, by disposing component A in the porous flow path, the analyte and component A can be effectively mixed, thereby suppressing salting out of the electrolyte disposed near the reference electrode and enabling testing with a small amount of analyte.

[0050] Example 2 In this example, the component A layer was treated with L-(+)-arginine hydrochloride (chemical formula: C ６ H １４ N ４ O ２L-(+)-arginine hydrochloride (HCl) was used. Because L-(+)-arginine hydrochloride is not a surfactant, it does not form micelles with proteins in the sample. However, it has the effect of solubilizing proteins by hydrophobic interactions with specific hydrophobic residues (e.g., aromatic amino acid residues) in the sample. This prevents protein aggregation as the sample penetrates the electrolyte layer. Additionally, when a surfactant is used in the component A layer, depending on the combination of surfactant and ion-selective membrane material, the surfactant may disrupt the membrane structure of the ion-selective membrane and affect the measured potential. Therefore, it was necessary to pay attention to factors that could cause the surfactant to diffuse and come into contact with the ion-selective membrane (e.g., the distance between the reference electrode and the working electrode, the measurement time, etc.). On the other hand, L-(+)-arginine hydrochloride solubilizes proteins by hydrophobic interactions with specific hydrophobic residues (e.g., aromatic amino acid residues), so it has little effect on the ion-selective membrane and allows for greater flexibility in the selection of membrane materials. Two analytical devices containing 0.13 mg and 0.65 mg of L-(+)-arginine hydrochloride were prepared, and the potential of the reference electrode was measured. In this experiment, in order to focus only on the performance of the reference electrode, a commercially available reference electrode was placed instead of the working electrode on the side of the analytical device where the working electrode would normally be formed, and the commercially available electrode and reference electrode were connected with a sample, and the potential difference between the two electrodes was measured.

[0051] The results for 0.13 mg are shown in Figure 12, and the results for 0.65 mg are shown in Figure 13. It can be seen that the time stability of the potential was improved with an amount of 0.13 mg, compared to a potential difference of Δ0.9 mV after 200 seconds with the conventional configuration. For 0.65 mg, the potential difference was slightly larger at Δ1.2 mV. Therefore, for more accurate measurements, it is preferable that the amount of L-(+)-arginine hydrochloride be approximately 0.13 mg. Therefore, this experiment confirmed that optimizing the amount of L-(+)-arginine hydrochloride placed as the component A layer improves the time stability of the potential and enables measurements to be made in a short time.

[0052] The present invention is not limited to the above-described embodiments, and various modifications and variations can be made without departing from the spirit and scope of the present invention. Therefore, the following claims are appended to apprise the public of the scope of the present invention.

[0053] This application claims priority based on Japanese Patent Application No. 2023-097172 filed on June 13, 2023, and Japanese Patent Application No. 2024-067014 filed on April 17, 2024, the entire contents of which are incorporated herein by reference.

[0054] DESCRIPTION OF SYMBOLS 1: Flow path 2: Flow path wall 3: Reference electrode 4: Electrolyte layer 5: Component A layer 6: Working electrode 6a: Ion selective membrane 6b: Base electrode of working electrode 7: Supporting electrolyte membrane 8: Dispensing section 9: First flow path chamber 10: Second flow path chamber 11: Third flow path chamber 12: Fourth flow path chamber

Claims

1. An analytical device having a flow path region surrounded by flow path walls provided inside a porous substrate, the flow path region having a first flow path chamber, a second flow path chamber, and a flow path connecting the first flow path chamber and the second flow path chamber, a reference electrode being arranged in the first flow path chamber, and a working electrode being arranged in the second flow path chamber, an electrolyte being arranged upstream of the first flow path chamber or on the surface of the reference electrode when the direction of sample travel in the flow path region is used as a reference, and component A being arranged upstream of the position where the electrolyte is arranged, the analytical device characterized in that component A is a component that has the effect of changing the three-dimensional structure of a protein in the sample.

2. The analytical device according to claim 1, wherein said component A and said electrolyte have an area in which they are adjacent or mixed.

3. The analytical device according to claim 1, wherein said component A is a component having the ability to form micelles together with proteins.

4. The analytical device according to claim 3, wherein said component A is a surfactant.

5. The analytical device according to claim 4, wherein said component A is a non-ionic surfactant.

6. The analytical device according to claim 5, wherein said component A is Tween 20.

7. The analytical device according to claim 1, wherein said component A is a water-soluble polymer.

8. The analytical device according to claim 1, wherein said component A is a salt.

9. The analytical device according to claim 8, wherein said component A is a chaotropic salt.

10. The analytical device according to claim 8, wherein said component A is L-(+)-arginine hydrochloride.

11. The analytical device according to claim 1, wherein said component A is an acid.

12. The analytical device according to claim 1, wherein said component A is an organic solvent.

13. The analytical device according to any one of claims 1 to 12, wherein the base electrode of the reference electrode is an Ag / AgCl electrode.

14. The analytical device according to any one of claims 1 to 13, wherein the reference electrode has a laminate layer on a surface of the reference electrode.

15. The analytical device according to any one of claims 1 to 14, wherein the reference electrode is a reference electrode in which a supporting electrolyte membrane is laminated on a base electrode.

16. The analytical device according to any one of claims 1 to 15, wherein the working electrode is an ion selective electrode having an ion selective membrane laminated on a base electrode of the working electrode.

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