Analysis device

Abstract

To provide an analysis device that is able to analyze a measurement potential easily and stably.SOLUTION: An analysis device having a flow channel region surrounded by a flow channel wall provided inside a porous substrate. The flow channel region has a first flow channel chamber, a second flow channel chamber, and a flow channel connecting the first flow channel chamber and the second flow channel chamber. A reference electrode is disposed in the first flow channel chamber, and an action electrode is disposed in the second flow channel. When a traveling direction of a specimen in the flow channel region is a reference, an electrolyte is disposed upstream of the first flow path chamber or on a surface of the reference electrode, and a component A is disposed upstream of a position where the electrolyte is disposed. The component A is a component having an action of changing a three-dimensional structure of a protein in the specimen.SELECTED DRAWING: Figure 1A

Description

[Technical field]

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

[0002] In recent years, devices that use microfluidics to perform efficient (trace-volume, rapid, 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, paper-based devices are called "μPADs" (microfluidic paper-based analytical devices), and they can perform chemical analysis by using the capillary action of the paper to permeate samples or test liquids. The advantages of these devices include their small size, low cost, ease of transport, high disposability (simply burn them and dispose), and no need for large-scale equipment. It is said that the practical application of these new analytical devices will enable anyone to easily and cheaply perform point-of-care (POC) testing and diagnosis.

[0004] In addition, due to the advantages of μPADs mentioned above, they are expected to be used in developing countries and depopulated areas where medical facilities are insufficient, 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 in the above pathological diagnosis is the electrolyte test. Electrolytes (Na ions, K ions, Cl ions, etc.) are essential for maintaining life by maintaining a constant amount of water and pH in the body, as well as for normal nerve transmission and muscle function. The electrolyte test measures the concentration of electrolyte ions in the blood and 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 there is an abnormality in kidney function or hormone activity. For this reason, electrolyte tests are essential for screening for illnesses. They are also extremely important tests for checking the physiological functions (life support) of patients at disaster sites, etc. Research aimed at using μPADs to measure electrolytes is being conducted at various universities and companies.

[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 specimen, and the dispensed specimen permeates from the dispensing section into the working electrode and the reference electrode, electrically connecting both electrodes to measure 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, and when KCl dissolves in the specimen during measurement, the Cl ions in the reference electrode region are maintained at a high concentration, and a stable reference electrode potential can be obtained. In addition, although not μPADs (the substrate used is not a porous substrate), Patent Document 1 discloses a multi-ion sensor plate in which a specimen liquid supplied to the working electrode section and a reference liquid supplied to the reference electrode section come into contact with each other at a liquid junction to obtain a stable potential. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Japanese Patent Application Publication No. 11-194109 [Non-patent literature]

[0009] [Non-Patent Document 1] 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 Summary of the Invention [Problem to be solved by the invention]

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

[0011] In addition, Patent Document 1 discloses two configurations: one in which a surfactant is contained in the sample to maintain the dispersibility of hydrophobic substances (hematocrit or similar substances) contained in the sample liquid and prevent its precipitation (precipitation), and the other in which a surfactant coating is provided on the flow path leading to the sample liquid side electrode. However, in the configuration of Patent Document 1, in order to contain the surfactant in a sufficiently mixed state in advance in the sample liquid, a sample volume of about several mL is required to stir the mixed liquid, which may be a heavy burden on the patient. In addition, when a surfactant coating is provided in the flow path, coating on the flow path wall is a constraint, and the surfactant cannot be uniformly contacted 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 position, which exhibits an appropriate reference electrode potential, and which enables simple and stable analysis. [Means for solving the problem]

[0013] The present invention provides an analytical device having a flow path region surrounded by a flow path wall provided inside a porous substrate, The flow channel region includes a first flow channel chamber, a second flow channel 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 chamber, A working electrode is disposed in the second flow chamber, When the traveling direction of the sample in the flow path region is used as a reference, An electrolyte is disposed upstream of the first flow chamber or on the surface of the reference electrode; Component A is disposed upstream of the location where the electrolyte is disposed; The present invention relates to an analytical device, wherein the component A is a component that has an effect of changing the three-dimensional structure of a protein in a sample. Effect of the Invention

[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 specimen and component A can be effectively mixed, which makes it possible to suppress salting out of the electrolyte disposed near the reference electrode, and thus makes it possible to perform testing with a small amount of specimen. [Brief description of the drawings]

[0015] [Figure 1A] FIG. 2 is a diagram showing the configuration of a reference electrode. [Figure 1B] FIG. 1B is a cross-sectional view of the reference electrode taken along the dashed line in FIG. 1A. [Figure 2A] FIG. 3 is a cross-sectional view of the conventional configuration taken along the dashed line in FIG. 2B. [Figure 2B] FIG. 1 is a diagram showing a configuration of a conventional example. [Diagram 3] FIG. 13 is a diagram showing the change in potential over time when a serum sample is measured using a conventional configuration. [Figure 4]FIG. 13 is a diagram showing the change in potential over time when an ultrafiltered serum sample is measured using a conventional configuration. [Diagram 5] FIG. 1 is a configuration diagram of an analytical device according to a first embodiment. [Figure 6] FIG. 4 is a configuration diagram of a flow path pattern. [Figure 7] 1 is a graph showing the change in potential over time when a serum sample is measured using only the reference electrode of Example 1. FIG. [Figure 8] 1 is a graph showing the change in potential over time when a serum sample is measured using only the reference electrode of Example 1. FIG. [Figure 9] 1 is a graph showing the change in potential over time when a serum sample is measured using only the reference electrode of Example 1. FIG. [Figure 10] FIG. 2 is a graph showing the change in potential over time when a serum sample is measured using a combination of the reference electrode and working electrode of Example 1. [Figure 11] FIG. 13 is a graph showing the change in potential over time when a serum sample is measured using a comparative example configuration of Example 1. [Figure 12] 13 is a graph showing the change in potential over time when a serum sample is measured using only the reference electrode of Example 2. FIG. [Figure 13] 13 is a graph showing the change in potential over time when a serum sample is measured using only the reference electrode of Example 2. FIG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The operation of the present invention will now be described in detail. The role of the reference electrode in the electrolyte concentration measurement 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 [ka] 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, the same interfacial potential will always be obtained by reacting a saturated NaCl or KCl solution with an Ag / AgCl electrode. If the sample is a NaCl solution, once the concentration reaches the saturation 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 solution type reference electrodes, while in solid electrode type reference electrodes, this saturated solution is directly dispensed as the standard solution into the Ag / AgCl electrode to ensure a stable potential.

[0020] As an example of a solid electrode type reference electrode, Non-Patent Document 1 shows 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 Figs. 2A and 2B. In this configuration, the analyte itself dissolves the electrolyte layer 4 during penetration, producing Cl. - Keep the concentration constant. And Cl - When the specimen has a constant concentration, it is supplied to the supporting electrolyte membrane 7 and the Ag / AgCl electrode, making it possible to perform measurement without using a reference solution.

[0021] In Patent Document 1, which also has a configuration with a solid electrode type reference electrode, a method is adopted in which a reference liquid is dispensed separately from the sample to perform the measurement. This method allows a stable measurement potential to be obtained, but the use of a reference liquid tends to make the measurement easier and more costly. In addition, Patent Document 1 discloses two configurations: a configuration in which a surfactant is contained in the sample to prevent protein aggregation, which will be described later, and a configuration in which a surfactant coating is provided on the flow path leading to the sample liquid side electrode. However, in the configuration of Patent Document 1, in order to contain the surfactant in a sufficiently mixed state in advance in the sample liquid, a sample volume of about several mL is required to stir the mixed liquid, which may be a heavy burden on the patient. In addition, when a surfactant coating is provided in the flow path, coating on the flow path wall is a constraint, and the surfactant cannot be uniformly contacted with the sample flowing through the flow path, which may result in insufficient mixing with the sample liquid.

[0022] The configuration having an electrolyte layer, such as that disclosed in Non-Patent Document 1, has the following problems. This is because, when measuring samples that contain proteins in their components, such as serum or plasma, the hydration water of hydrated proteins is easily taken away by the electrolyte when the sample permeates the electrolyte layer (KCl layer), which can easily cause salting out or aggregation of the proteins.As a result of salting out or aggregation of proteins, the permeation of the sample in the flow path is hindered, and the measurement potential becomes unstable and the measurement time becomes longer.

[0023] 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 are described below. Two devices with the same configuration were prepared, and the measurement results of the reference electrode potential difference in the device when serum was used as a sample are shown in Figure 3. In this experiment, in order to focus only on the performance of the reference electrode 3, a commercially available reference electrode (RE-1BP, BAS Co., Ltd.) was placed instead of the working electrode 6 on the side where the working electrode 6 is originally formed in the analytical device, and the commercially available electrode and the reference electrode 3 were connected with a sample, and the potential difference between the two electrodes was measured. From the measurement results, it can be seen that the measured potential fluctuates greatly for about 100 seconds after dispensing. After that, the measured potential fluctuates less over time, but it was not possible to maintain a stable potential, and it was confirmed that a difference of about Δ0.9 mV occurred after 200 seconds. In addition, in this configuration, the permeation of the sample in the electrolyte layer was unstable and slow, and something like a clump was confirmed on 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 in the electrolyte layer and the potential was stable.

[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 specimen not to aggregate within the flow channel. The analytical device of the present invention is capable of preventing the permeation of samples containing proteins, such as serum and plasma, without slowing down the permeation of the samples, and preventing the Cl of the samples. - It is possible for the concentration to be saturated and to come into stable contact with the reference electrode. In other words, the analytical device of the present invention has a configuration in which, based on the direction of sample progression in the flow path region, a layer that suppresses salting-out and aggregation of proteins is disposed upstream of the position at which the electrolyte layer is disposed.

[0026] The analytical device of the present invention may have a plurality of third flow chambers in which a working electrode is arranged, in addition to the first flow chamber and the second flow chamber, and the third flow chambers may be connected to each other through a flow channel. The number of flow chambers arranged on the analytical device is not particularly limited. EXAMPLES

[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. In addition, in each of the following drawings, components that are not necessary for the description of the embodiments are omitted from the drawings.

[0028] <Example 1> The first embodiment will be described with reference to FIGS. FIG. 5 shows the analytical device of Example 1. It has a flow region surrounded by a flow wall 2 provided inside the porous substrate. The flow region has a first flow chamber 9, a second flow chamber 10, and a flow channel 1 connecting the first flow chamber 9 and the second flow chamber 10, and a dispensing section 8 is present in the flow channel 1. The flow region may have a third flow chamber 11 and a fourth flow chamber 12 of the electrode, or may not have these. A reference electrode 3 is arranged in the first flow chamber 9, and an electrolyte layer 4 is arranged upstream of the reference electrode 3 based on the traveling direction of the sample (i.e., the dispensing section side is upstream). By arranging 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 having an effect of changing the three-dimensional structure of the protein in the sample is arranged upstream of the electrolyte layer 4. A working electrode 6 is arranged in the second flow 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) having melting properties, and then the flow path pattern was permeated into the inside of the paper by an oven or heater to form the flow path pattern.

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

[0031] As shown in Figure 1A, an Ag / AgCl electrode was printed as a reference electrode on the flow path pattern by screen printing, and further, as shown in Figures 1A and 1B, an electrolyte (NaCl or KCl) was printed by an inkjet or dispenser at a position on the flow path upstream of the Ag / AgCl electrode in the direction of sample flow. By locating an electrolyte layer (NaCl or KCl) at such a position, the sample that moves forward always passes through the electrolyte layer. At that time, the electrolyte is dissolved, and the concentration of the electrolyte in the sample (Cl - The concentration of the sample in the sample solution is saturated, 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 more 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) is placed in the flow path just before the reference electrode, and the concentration is 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 electrolyte was arranged in layers so that the NaCl concentration was 3.0×10 g / L or more. This is because the saturated concentration of the electrolyte also depends 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. 2If NaCl of 3.4 × 10 g / L or more is placed, the concentration of the sample that passes through the NaCl layer will be 4.6 mol / L or more, which is close to saturation, making stable measurement possible. 2 In 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 1000 mg / kg of water is sufficient to keep the water saturated.

[0034] The amount of sample to be supplied to the dispensing unit 8 varies depending 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, about 10 μL of sample is enough 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, the smaller the required volume is, 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 above reasons, the present invention will be described with the sample volume in the range of about 10 μL to 50 μL.

[0036] In the case of the analytical device of Example 1, 10 μL of the specimen is dispensed and supplied to the dispenser 8 located in the center of the flow path area, about half of which is supplied to the reference electrode side, and the remaining half is supplied to the working electrode side, so that the amount of specimen 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.

[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 x 3 mm area with a thickness of 200 μm, 8.3 × 10 5 g / m 3 If more than 9.4 × 10 NaCl is placed, the NaCl concentration of the sample that passes through the NaCl layer will be 4.6 mol / L or more, which is close to saturation. 5 g / m 3 With this amount of NaCl placed, 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). At this time, the Cl of the sample that passes through the electrolyte (NaCl) layer - The concentration of is always kept at or near saturation, and this saturated sample is continuously supplied to the reference electrode (Ag / AgCl electrode), resulting in a stable potential.

[0039] In this configuration, the reference electrode and the electrolyte layer are not in contact, 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 concentration of the electrolyte in the analyte becomes saturated.

[0040] A surfactant (Tween 20, chemical formula: C) is used as a component A layer 5 in a position upstream of, and preferably adjacent to, the electrolyte layer 4. 58 H 114 O 26) was printed by an inkjet or dispenser. These methods allow the surfactant to be uniformly arranged in the desired flow channel region on the porous substrate. In addition, the surface area of ​​the porous structure can be utilized, so the contact area between the sample and the surfactant can be increased (compared to the case of a non-porous substrate). Estimated from the BET specific surface area of ​​the paper, the surface area can be increased by about 1000 times or more compared to the configuration of Patent Document 1, which works favorably for mixing the surfactant and the sample. The sample is stirred between the fibers while penetrating this flow channel by capillary action, so that even a small amount of sample on the order of μL can be sufficiently mixed with the surfactant. The porous substrate used at this time can be anything as long as it has a porous structure (filter paper, glass filter paper, etc.). The important thing is to uniformly arrange a predetermined amount of surfactant in the desired flow channel region. In addition, when focusing on the cross section of the porous flow channel from the viewpoint of the amount of sample used, for example, when 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 section (a flow channel like that of Patent Document 1). As described above, the principle of the potential development of the reference electrode is that Cl - Since the effect depends on the concentration of the surfactant, the same effect can be obtained even with half the amount of sample as long as there is a liquid junction with the working electrode. Therefore, with the above arrangement, even with a small amount of sample on the order of μL, it is possible to suppress aggregation during penetration into the electrolyte layer by mixing with the surfactant to form micelles (changing the three-dimensional structure of the protein), and it is expected that a stable measurement potential can be obtained. There are various types of surfactants, such as cationic, anionic, amphoteric, and nonionic, but for the electrolyte measurement chip, nonionic ones that are less likely to inhibit the potential development on the Ag / AgCl electrode or ion-selective membrane are preferable. In addition, although a surfactant was used in this embodiment, any one that can change the three-dimensional structure of the protein can be used, and it can also be a salt (for example, guanidine hydrochloride as a chaotropic salt), an acid (for example, citric acid), an organic solvent (for example, ethanol), or a water-soluble polymer (for example, polyethylene glycol).

[0041] The formation of the surfactant layer also depends on the amount of sample supplied to the reference electrode, just like the formation of the electrolyte layer. When the amount of sample supplied to the reference electrode is 5 μL, 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.

[0042] Three types of analytical devices were prepared, with the amounts of nonionic surfactant Tween 20 being 0.012 mg, 0.058 mg, and 0.115 g, 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 the 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, those for 0.058 mg in Figure 8, and those 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, whereas the amounts of 0.012 mg and 0.058 mg showed an improvement. 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 Ag / AgCl electrode was reached without reaching the saturation concentration of Tween 20. From the above, it can be seen that it is better to place Tween 20 in the right amount.

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

[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 they 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 embodiment, an Ag / AgCl electrode was used as the base electrode.

[0047] The ion-selective membrane may be any commonly used membrane that is sensitive to the target ion and has sufficient selectivity to interfering ions. Materials used in 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) alone and copolymers of polyvinyl chloride and polyvinyl acetate.

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

[0049] The present invention is not limited to an analytical device using a single Ag / AgCl electrode as a reference electrode. As described in Non-Patent Document 1, Ag / AgCl may be used as the base electrode of the reference electrode, and a supporting electrolyte layer may be provided on the surface of the base electrode to stabilize the interfacial potential with the sample 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) with a plasticizer and PVC, mixing them with a solvent such as THF or cyclohexanone, and applying and drying the resulting solution. In addition, in the present invention, a laminate layer may be provided on the front and back surfaces of the reference electrode to prevent the electrolyte layer and component A layer from flowing out or becoming contaminated when handling the analytical device. As described above, according to the present invention, by disposing component A in the porous flow path, the specimen and component A can be effectively mixed, thereby suppressing salting out of the electrolyte disposed near the reference electrode, and thus enabling testing with a small amount of specimen.

[0050] <Example 2> In this example, the component A layer was treated with L-(+)-arginine hydrochloride (chemical formula: CH 14 N4O2·HCl) was used. L-(+)-Arginine hydrochloride is not a surfactant and does not form micelles with proteins in the sample, but it has the effect of solubilizing the proteins by hydrophobic interaction with specific hydrophobic residues (aromatic amino acid residues, etc.) in the sample. Therefore, it is possible to suppress protein aggregation when the sample permeates the electrolyte layer. In addition, when a surfactant is used in the component A layer, depending on the combination of the surfactant and the material of the ion-selective membrane, the surfactant may destroy the membrane structure of the ion-selective membrane and affect the measured potential. Therefore, it was necessary to pay attention to factors that cause the surfactant to diffuse and come into contact with the ion-selective membrane (distance between the reference electrode and the working electrode, measurement time, etc.). On the other hand, in the case of L-(+)-arginine hydrochloride, the effect is to solubilize by hydrophobic interaction with specific hydrophobic residues (aromatic amino acid residues, etc.), so the effect on the ion-selective membrane is small and it has the effect of increasing the freedom of choice of membrane material. Two types of analytical devices were prepared, with the amount of L-(+)-arginine hydrochloride being 0.13 mg and 0.65 mg, 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 where the working electrode would normally be formed on the analytical device, and the commercially available electrode and the 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 is improved with an amount of 0.13 mg, compared to the conventional configuration, where the potential difference after 200 seconds is Δ0.9 mV. For 0.65 mg, the potential difference is slightly large at Δ1.2 mV. Therefore, when performing measurements with higher accuracy, it is preferable that the amount of L-(+)-arginine hydrochloride is about 0.13 mg. Therefore, this experiment confirmed that the time stability of the potential is improved and measurements can be made in a short time by optimizing the amount of L-(+)-arginine hydrochloride placed as the component A layer.

[0052] The embodiments of the present invention include the following configurations. [Configuration 1] An analytical device having a flow path region surrounded by a flow path wall provided inside a porous substrate, 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 chamber, A working electrode is disposed in the second flow chamber, an electrolyte is disposed upstream of the first flow channel chamber or on a surface of the reference electrode when a direction of travel of the sample in the flow channel region is taken as a reference; Component A is disposed upstream of the location where the electrolyte is disposed; An analytical device, wherein the component A is a component that has an effect of changing the three-dimensional structure of a protein in a sample. [Configuration 2] 2. The analytical device of claim 1, wherein the component A and the electrolyte have regions where they are adjacent or mixed. [Configuration 3] 2. The analytical device according to claim 1, wherein the component A is a component having an action of forming micelles together with a protein. [Configuration 4] 4. The analytical device according to configuration 3, wherein component A is a surfactant. [Configuration 5] 5. The analytical device according to claim 4, wherein component A is a non-ionic surfactant. [Configuration 6] 6. The analytical device according to configuration 5, wherein component A is Tween 20. [Configuration 7] 2. The analytical device according to claim 1, wherein the component A is a water-soluble polymer. [Configuration 8] 2. The analytical device according to claim 1, wherein component A is a salt. [Configuration 9] 9. The analytical device according to claim 8, wherein component A is a chaotropic salt. [Configuration 10] 9. The analytical device of claim 8, wherein component A is L-(+)-arginine hydrochloride. [Configuration 11] 2. The analytical device according to claim 1, wherein component A is an acid. [Configuration 12] 2. The analytical device according to claim 1, wherein component A is an organic solvent. [Configuration 13] 13. The analytical device according to any one of configurations 1 to 12, wherein the base electrode of the reference electrode is an Ag / AgCl electrode. [Configuration 14] 14. The analytical device according to any one of configurations 1 to 13, wherein the reference electrode has a laminate layer on a surface of the reference electrode. [Configuration 15] 15. The analytical device according to any one of configurations 1 to 14, wherein the reference electrode is a reference electrode in which a supporting electrolyte membrane is laminated on a base electrode. [Configuration 16] 16. The analytical device according to any one of configurations 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. [Explanation of symbols]

[0053] 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 passage chamber 10...Second channel chamber 11...Third channel chamber 12...Fourth channel chamber

Claims

1. An analytical device having a flow path region surrounded by a flow path wall provided inside a porous substrate, 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 chamber, A working electrode is disposed in the second flow chamber, an electrolyte is disposed upstream of the first flow channel chamber or on a surface of the reference electrode when a direction of travel of the sample in the flow channel region is taken as a reference; Component A is disposed upstream of the location where the electrolyte is disposed; An analytical device, wherein the component A is a component that has an effect of changing the three-dimensional structure of a protein in a sample.

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

3. 2. The analytical device according to claim 1, wherein the component A is a component having an action of forming micelles with a protein.

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

5. The analytical device according to claim 4 , wherein the component A is a nonionic surfactant.

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

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

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

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

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

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

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

13. 2. The analytical device according to claim 1, wherein the base electrode of the reference electrode is an Ag / AgCl electrode.

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

15. 2. The analytical device according to claim 1, wherein the reference electrode is a reference electrode in which a supporting electrolyte membrane is laminated on a base electrode.

16. 2. The analytical device according to claim 1, 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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