Microanalysis chip

Abstract

To provide a micro analysis chip that performs electric potential difference ion concentration measurement, and that is excellent in measurement sensitivity and time stability of electric potential.SOLUTION: A micro analysis chip has a first porous base material and a second porous base material laminated thereon. Inside the first porous base material, a channel wall forms a first channel chamber, a second channel chamber, and a first channel connecting the first channel chamber and the second channel chamber to each other. A reference electrode is arranged in the first channel chamber. A working electrode is arranged in the second channel chamber. The second porous base material is provided on a surface of the first porous base material to overlap at least part of an area where the working electrode is formed.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a microanalysis chip in which a microchannel is formed inside a porous substrate. [Background technology]

[0002] In recent years, microanalysis chips, which utilize micro-sized fine channels to efficiently perform biochemical 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. In the early 1990s, photolithography and molds were used to form micrometer-sized fine channels on glass or silicon, and microanalysis chips were developed that could perform sample pretreatment, stirring, mixing, reaction, and detection on a single chip. As a result, miniaturization of testing systems, rapid analysis, and reductions in specimen and waste liquid volumes were realized.

[0003] Electrochemical analysis, which measures the potential between electrodes immersed in the sample to be analyzed, is widely used in fields such as medicine and the environment. Conventional electrochemical analysis is performed by technicians using sophisticated equipment, which limits the fields and resources available for measurement. However, there is a need for inexpensive, easy-to-use, and disposable microanalytical chips for electrochemical analysis, such as those for use in developing countries with insufficient medical facilities, remote areas, disaster sites, and airports where the spread of infectious diseases must be prevented at the border.

[0004] Non-Patent Document 1 proposes a filter paper-based device for measuring the concentrations of Na and K ions. This device is fabricated on a single piece of filter paper and has a dispensing section for dispensing the sample. The dispensed sample penetrates from the dispensing section into the working electrode and reference electrode regions, electrically connecting the two electrodes and enabling potential difference measurement. To achieve a stable potential at the reference electrode, the device also deposits KCl ion crystals on the reference electrode. During measurement, the KCl ion crystals dissolve into the sample, maintaining a high concentration of Cl ions in the reference electrode region and achieving a stable reference electrode potential. Furthermore, a K ion-selective membrane formed over the working electrode selects only the target ion, allowing measurement to be performed without being affected by other ions.

[0005] Patent Document 1 proposes a microanalysis chip for electrochemical analysis. This device has a two-layer structure, with a dispensing section for dispensing a sample in the upper layer. The dispensed sample essentially permeates the thickness of the porous hydrophilic layer and penetrates into the upper surface of the electrode assembly on the lower substrate. Then, multiple electrodes are electrically connected, enabling electrochemical measurements. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] US Patent Application Publication No. 2012 / 0181184 [Non-patent literature]

[0007] [Non-Patent Document 1] Nipapan Ruecha, Orawon Chailapakul, Koji Suzuki and Daniel Chitterio “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]

[0008] However, the above-described configuration may have undesirable effects on the accuracy of analyte concentration measurement. Specifically, it may have undesirable effects on the measurement sensitivity of electrolyte concentration, ion selectivity for specific ions, potential stability during measurement, etc. One possible cause of this is insufficient contact area between the analyte and the ion-selective membrane or unstable contact.

[0009] In the configuration disclosed in Non-Patent Document 1, the conditions for forming the ion-selective membrane had to be precisely controlled to obtain good measurement accuracy. Specifically, it was necessary to make it easier for the ion-selective membrane to penetrate into the voids of the porous substrate so that the contact area between the porous substrate and the ion-selective membrane within the porous substrate would be large. However, Non-Patent Document 1 did not disclose details of a method for controlling penetration. Furthermore, increasing the area of ​​the ion-selective membrane in order to increase the area of ​​the interface between the ion-selective membrane and the porous substrate would result in an increase in the size of the flow path device.

[0010] In the configuration disclosed in Patent Document 1, the sample is supplied by contacting the porous hydrophilic layer with the upper surface of the electrode assembly. However, because the substrate supporting the electrode assembly does not have a flow path through which the sample can penetrate, the sample flow rate due to capillary action is low, which can result in a long time from dispensing to the completion of measurement. Furthermore, sample contact with the side of the electrode assembly, which does not have a flow path, can be unstable, which can have an undesirable effect on the measured potential. In view of the above problems, the present disclosure aims to provide a microanalysis chip that can quickly achieve good electrolyte concentration measurement sensitivity, ion selectivity, and potential stability. [Means for solving the problem]

[0011] In order to achieve the above object, according to one aspect of the present disclosure, A microanalysis chip configured by laminating a first porous substrate and a second porous substrate, a first flow path chamber, a second flow path chamber, and a first flow path connecting the first flow path chamber and the second flow path chamber are formed by flow path walls inside the first porous substrate; a reference electrode is disposed in the first flow chamber; A working electrode is disposed in the second flow chamber, The second porous substrate is provided on the surface of the first porous substrate so as to overlap at least a portion of the region where the working electrode is formed, thereby providing a microanalysis chip. [Effects of the Invention]

[0012] According to one aspect of the present disclosure, it is possible to provide a microanalysis chip that is excellent in measurement sensitivity of electrolyte concentration, ion selectivity, and time stability of potential. [Brief explanation of the drawings]

[0013] [Figure 1] 1 is a simplified cross-sectional view showing the configuration of a micro-analysis chip P1 according to Example 1. FIG. [Figure 2] FIG. 1 is a diagram showing a simplified relative positional relationship and size relationship between a flow channel chamber 1, a flow channel chamber 2, a flow channel 3, a flow channel wall 5, a dispensing section 6, a reference electrode 7, a working electrode 8, an ion selective membrane 9, and an ion crystal 10 of a micro-analysis chip P1 according to Example 1. [Figure 3] 1 is a simplified cross-sectional view showing the configuration of a micro-analysis chip P2 according to Comparative Example 1. FIG. [Figure 4] 1 shows an example of measurement results in Example 1 and Comparative Example 1. [Figure 5]1 shows an example of measurement results in Example 1 and Comparative Example 1. [Figure 6] FIG. 2 is a simplified cross-sectional view showing the filtering effect of Example 1. [Figure 7] FIG. 1 is a simplified cross-sectional view showing the filtering effect of Comparative Example 1. [Figure 8] 10 is a simplified cross-sectional view showing the configuration of a micro-analysis chip P3 according to Comparative Example 2. FIG. [Figure 9] FIG. 1 is an image diagram showing the time course of behavior of a sample after dispensing in Example 1 and Comparative Example 1. [Figure 10] 10 is a simplified cross-sectional view showing the configuration of a micro-analysis chip P2 according to a first modified example of the first embodiment. FIG. DETAILED DESCRIPTION OF THE INVENTION

[0014] In this specification, unless otherwise specified, the expressions "XX or more and YY or less" and "XX to YY" representing a numerical range mean a numerical range including the lower and upper limits, which are the endpoints. Furthermore, when a numerical range is described in stages, any combination of the upper and lower limits of each numerical range is disclosed. The microanalysis chip according to the present invention for solving the above-mentioned problems will be described based on the following embodiments. Note that the embodiments shown below are merely examples and are not intended to limit the technical scope of the present invention. FIG. 1 is a simplified cross-sectional view of the micro-analysis chip P1 taken along the line AA in FIG. Figure 2 shows a simplified diagram of the relative positions and size relationships of the flow channel chamber 1, flow channel chamber 2, flow channel 3, flow channel wall 5, dispensing section 6, reference electrode 7, working electrode 8, ion selective membrane 9, and ion crystal 10 of the microanalysis chip P1 of Example 1.

[0015] The micro-analysis chip P1 is composed of a porous substrate S1 and a porous substrate S2. The porous substrate S1 and the porous substrate S2 are laminated in the thickness direction. The porous substrate S1 has a first flow path region surrounded by a flow path wall 5. The flow path pattern in the porous substrate S1 is composed of a flow path chamber 1 (first flow path chamber), a flow path chamber 2 (second flow path chamber), and a flow path 3. The flow path 3 (first flow path) connects the flow path chamber 1 (first flow path chamber) and the flow path chamber 2 (second flow path chamber). A reference electrode 7 is disposed in the flow channel chamber 1, and ion crystals 10 having analyte solubility are disposed on the surface of the reference electrode 7. In the flow channel chamber 2, a working electrode 8 is disposed. The porous substrate S2 has a dispensing section 6 for dispensing a sample, and a flow path 4 (second flow path) that connects the dispensing section 6 with the region where the working electrode 8 is formed. The flow path 4 is provided on the surface of the porous substrate S1 so as to overlap at least a part of the region where the working electrode 8 is formed.

[0016] It is preferable that the upper surface of the porous substrate S2, at least the position connecting the dispensing unit 6 and the working electrode 8, be covered with a restricting member 11 that is impermeable to the sample. This is because, for example, this may be advantageous for filtering out interfering particles, as described below, or the influence of the outside air may be reduced by reducing the area of ​​the sample exposed to the outside air, making it easier to stabilize the measured potential. The lower surface of the porous substrate S1 and the upper surface of the porous substrate S2 are preferably covered with a restricting member 11, respectively.

[0017] <Porous base material> In the following examples, a paper substrate was used as the porous substrate, but the porous substrate is not limited to paper. The porous substrate may be any material that generates capillary action in a liquid, and may have a porous structure such as open cells or nanofibers inside, or a mesh structure. Resin, glass, an inorganic substrate, fabric, metal paper, etc. may also be used. For example, porous substrates with a large thickness or a large porosity are suitable for rapid measurement because they have a large sample flow rate due to capillary action. In the following examples, the porous substrate S1 and the porous substrate S2 each consist of one sheet of the same paper substrate, but this combination is not limiting. A flow path region equivalent to that of this example may be formed by folding back a single porous substrate S1. Also, different materials may be used for the porous substrate S1 and the porous substrate S2, and depending on the material combination, it may be possible to provide the device with effects such as sample flow control and sample filtration.

[0018] <Channel wall> In the following examples, a flow path pattern (flow path wall) was formed by disposing a hydrophobic resin and then thermally fixing it, but the method for forming the flow path pattern is not limited to this. It is sufficient to form a flow path pattern by cutting a paper porous substrate to leave only the flow path shape, or the flow path wall may be formed by a wax printer.

[0019] <Regulating member> The restricting member may be any member that is impermeable to the sample. Specifically, a laminate film made of PET (polyethylene terephthalate) can be mentioned.

[0020] A reference electrode 7 is placed in the flow channel chamber 1 (first flow channel chamber). The top and side surfaces of the reference electrode 7 are covered with ion crystals 10. The reference electrode 7 has a lead wire that extends continuously from inside the flow channel chamber 1 onto the flow channel wall 5 as a contact point during measurement. A working electrode 8 is disposed in the flow channel chamber 2 (second flow channel chamber). The top and side surfaces of the working electrode 8 are covered with an ion-selective membrane 9 containing a component having ion selectivity. The working electrode 8 has a lead wire that continuously extends from inside the flow channel chamber 2 onto the flow channel wall 5. In the following examples, one flow channel chamber (first flow channel chamber) for the reference electrode 7 and one flow channel chamber (second flow channel chamber) for the working electrode 8 are used, but the number of flow channel chambers is not limited to this. If there are multiple ions whose concentrations are to be measured, the number of working electrodes and flow channel chambers may be increased by the number of ion species.

[0021] The porous substrate S2 has a dispensing section 6 and a flow path 4. The dispensing section 6 is arranged so as to overlap the flow path 3 of the porous substrate S1. The flow path 4 is arranged so as to overlap the flow path 3 and the ion selective membrane 9. The flow path 4 of the porous substrate S2 does not necessarily need to cover the entire upper surface of the working electrode 8; for example, in the case of a working electrode that has good wettability with the analyte, the same effect as in this embodiment can be obtained by covering only a part of the upper surface of the working electrode.

[0022] [Example 1] <Flow path configuration> In Example 1, porous substrates S1 and S2 made of paper with a thickness L1 of 0.1 mm and a porosity of 50% were used. The porosity (%) was calculated using the following formula. Porosity (%) = (true density - apparent density) / true density x 100 Also, the apparent density (g / cm 3 ) was calculated using the following formula: Apparent density (g / cm 3 )=Basic weight (g / m 2 ) / thickness (mm) x 1000

[0023] After placing a hydrophobic resin on the surface of the porous substrate S1, the resin was thermally fixed to form a flow path pattern as a flow path wall 5 that is impermeable to samples. The porous substrate S2 was placed on top of the flow path 3 and the ion selective membrane 9. The porous substrates S1 and S2 were surrounded by a restricting member that was impermeable to samples, and in this example, a laminate film made of PET (polyethylene terephthalate) was used as the restricting member. The sizes of the flow channel chambers 1 and 2 and the dispensing part 6 were as follows. Flow channel chamber 1: L11 = 6 mm, L12 = 6 mm Flow channel chamber 2: L21 = 6 mm, L22 = 6 mm Dispensing part 6: 3mm diameter circle Shortest distance L41 from the center of the dispensing part 6 to the ion crystal 10: 7 mm Shortest distance L42 from the center of the dispensing part 6 to the ion selective membrane 9: 6.5 mm Width of flow path 3 L31: 3 mm, width of flow path 4 L43: 3 mm

[0024] <Specimen> The sample used was an aqueous solution containing NaCl, the ion to be measured, and KCl, a coexisting ion.

[0025] <Electrode formulation> The formulation of the electrode according to Example 1 will be described. A reference electrode 7 using Ag / AgCl was provided in the flow channel chamber 1. 3.5 mg of KCl ion crystals were placed on the reference electrode 7 as ion crystals 10. The size of the reference electrode 7 was as follows. Reference electrode 7: L71=4mm, L72=4mm

[0026] On the other hand, a working electrode 8 made primarily of carbon is provided in the flow channel chamber 2. Note that instead of the carbon electrode, a working electrode 8 made of a conductive polymer such as PEDOT:PSS (a dispersion of polyethylenedioxythiophene and polystyrene sulfonic acid) may be used. Also, a material such as Ag / AgCl, which has traditionally been used as the base of a reference electrode, may be used. The working electrode 8 had the following dimensions: Working electrode 8: L81=5.5mm, L82=5.5mm

[0027] A Na ion selective membrane 9 was formed so as to cover the working electrode 8. The ion selective membrane 9 was made of the following material. Ion-selective material Bis(12-crown-4) 3.0 wt% Potassium tetrakis (4-chlorophenyl) borate 0.5wt% as an anion scavenger o-nitrophenyl octyl ether 64.0wt% Polyvinyl chloride 32.5wt% In this example, the reference electrode 7, the working electrode 8, and the ion selective membrane 9 are formed in the shapes, sizes, and materials described above, but are not limited to these.

[0028] <Ionic Crystal> An ionic crystal 10 was formed so as to cover the reference electrode 7 . In this embodiment, KCl ion crystals were used as the ion crystals 10. However, the material of the ion crystals 10 is not limited to KCl ion crystals as long as it contains Cl ions. The mass of the ion crystals 10 to be placed is not limited to this, but is within the range of mass that results in a saturated solution when the KCl ion crystals are dissolved in pure water with a volume equivalent to that of the solution in the flow channel chamber 1. Furthermore, although the ion selective membrane 9 is provided in this embodiment, the ion selective membrane 9 is not necessarily required when measuring the total amount of ions in a sample.

[0029] <Sample penetration> The penetration of the specimen will now be described. After the sample is dispensed into the dispensing section 6 on the porous substrate S2, it simultaneously penetrates the paper porous substrates S1 and S2 in both the flow path chamber 1 direction and the flow path chamber 2 direction by capillary action according to the flow path pattern. While the dispensed sample permeates into flow channel chamber 1, ions contained in the sample are selected by ion selective membrane 9 in flow channel chamber 2, and the measurement potential of working electrode 8 required for measuring the electrolyte concentration is stabilized. In this embodiment, the position of the dispensing unit 6 is set to the midpoint where the distance from the flow channel chamber 1 and the distance from the flow channel chamber 2 are approximately equal, but the position of the dispensing unit 6 is not limited to this. Depending on the position of the dispensing unit relative to the ion selective membrane and the material of the porous substrate, it may be possible to provide the device with a filtering function for specimens that contain fine particles of about several μm in size, thereby improving sensitivity, selectivity, etc.

[0030] <Measurement of sample concentration> The measurement of analyte concentration will now be described. When the sample penetrates and reaches the ion crystals 10 covering the reference electrode 7, the KCl ion crystals dissolve in the sample, and the Cl ion concentration in the solution in the flow channel chamber 1 becomes saturated. If the measured potential of the working electrode 8 is stable at this time, the sample concentration can be measured, and the measurement of the sample concentration ends after a predetermined measurement time has elapsed. If the dispensed volume of the sample is equal to or greater than the volume that can reach flow channel chambers 1 and 2 via the flow channel 3 of the porous substrate S2, a sufficient amount of sample can be brought into contact with the ion selective membrane 9, and the sample concentration can be measured with good accuracy between the working electrode 8 and the reference electrode 7.

[0031] [Comparative Example 1] In order to explain the effects of Example 1 in more detail, Comparative Example 1 will be described with reference to FIG. <Flow path configuration> The micro-analysis chip P2 has a porous substrate S1 but does not have a porous substrate S2. In the micro-analysis chip P2, the shapes of the porous substrate S1 and the flow path wall were the same as in Example 1. The configurations (shapes and sizes) of the dispensing section 6, reference electrode 7, working electrode 8, ion selective membrane 9, ion crystal 10, etc. were also the same as in Example 1. However, the dispensing section 6 was formed in the porous substrate S1. As described above, the porous substrate S2 was not used.

[0032] <Sample penetration> The dispensed sample began to permeate simultaneously from dispenser 6 in both the flow channel chamber 1 side direction and the flow channel chamber 2 side direction, and reached flow channel chamber 1 and flow channel chamber 2 simultaneously. The specimen that reached the flow channel chamber 1 dissolved the ion crystals 10 covering the reference electrode 7 in the same manner as in Example 1, forming a saturated Cl ion solution. On the other hand, the sample that reached the flow channel chamber 2 came into contact with the ion selective membrane 9, but the main contact was at the boundary between the porous substrate S1 and the ion selective membrane 9 in the cross section of the porous substrate S1, which in Comparative Example 1 corresponds to the side surface of the ion selective membrane 9. The sample did not come into contact with the upper surface of the ion selective membrane 9. For this reason, depending on the state of penetration of the ion selective membrane 9 relative to the porosity of the porous substrate S1 and the shape in which the ion selective membrane 9 was arranged within the porous substrate S1, sufficient measurement sensitivity and ion selectivity were sometimes not obtained.

[0033] [Effects of Example 1] [Advantages of Example 1 over Comparative Example 1] Three advantages of Example 1 over Comparative Example 1 will be described.

[0034] <Measurement of sample concentration> The electrolyte concentration was measured by the mixed solution method specified in JIS K0122, General Rules for Ion Electrode Measurement Methods. The solution containing the ions to be measured was NaCl, and the solution containing the coexisting ions was KCl. The KCl concentration was fixed at 10 mmol / L, and the NaCl concentration was varied in five steps: 100 μmol / L, 1 mmol / L, 10 mmol / L, 100 mmol / L, and 800 mmol / L. The sample volume was 30 μL to 50 μL. For this measurement, a commercially available silver-silver chloride electrode (RE-1BP, BAS Corporation) was used as the reference electrode to compare the effect of the porous substrate S2 on the working electrode 8.

[0035] The measurement results are shown in Figure 4. Figure 4 shows the logarithmic Na ion concentration (logC Na +) and the measured potential (potential after stabilization). Sensitivity represents the slope of the region where the slope of the measured potential versus logarithmic Na ion concentration is non-negative and constant. The ion selectivity coefficient indicates how many orders of magnitude smaller the target ion can be detected than the coexisting K ion; the larger the negative value, the better. As shown in Figure 4(a), the non-negative linear rise point in Example 1 was near a logarithmic Na ion concentration of -3 (mol / L). In this example, the logarithmic concentration of the coexisting ions was -2, which indicates that the target Na ions can be measured at concentrations that are one order of magnitude lower. On the other hand, as shown in FIG. 4(b), in Comparative Example 1, it was difficult to determine the region where the measurement potential rose, and the determination of the ion selectivity coefficient and sensitivity was unstable.

[0036] Furthermore, as shown in Figure 5, differences in behavior can be seen in the time stability of the measured potential between Example 1 and Comparative Example 1. Figure 5(a) shows the time transition of the stable potential in Example 1, while Figure 5(b) shows both sudden changes in the potential and continuous gradual changes. From this, it was shown that Example 1 can quickly and stably supply the sample required for measurement to the ion-selective membrane, and has excellent measurement sensitivity, ion selectivity, and time stability of potential, making it effective for measuring electrolyte concentrations.

[0037] <Filtration of interfering particles> The filtering of interfering particles will now be described. In the case of a sample containing particles that may interfere with electrolyte concentration measurement, it is generally desirable for stable potential measurement that the interfering particles do not come into contact with the ion selective membrane. 6 and 7 show schematic cross-sectional views of the micro-analysis chip P1 used in Example 1 and the micro-analysis chip P2 used in Comparative Example 1, respectively, when 30 μL to 50 μL of a specimen containing particles 61 composed mainly of styrene acrylic and having a number-average particle size of approximately 8 μm was dispensed. After dispensing, the interfering particles (particles 61) were observed to remain near the interface between the porous substrate having the dispensing portion and the regulating member in both Example 1 and Comparative Example 1.

[0038] As shown in Figure 6, in the micro-analysis chip P1 of Example 1, interfering particles remain at the interface between the porous substrate S2 and the regulating member 11, making it difficult for the interfering particles to diffuse into the interior of the porous substrate S1, making this a suitable configuration for preventing the interfering particles from coming into contact with the ion selective membrane 9. On the other hand, as shown in Figure 7, in the microanalysis chip P2 of Comparative Example 1, depending on the distance between the dispensing section 6 and the ion selective membrane 9, interfering particles may penetrate and reach the surface of the ion selective membrane 9, affecting the electrochemical measurement. Therefore, when measuring a sample that contains particles that may interfere with electrochemical measurement, by using the configuration of Example 1, it is possible to filter out the interfering particles without performing pre-measurement on the sample, making it suitable for measuring electrolyte concentrations.

[0039] Comparative Example 2 8 is a simplified cross-sectional view showing the configuration of a micro-analysis chip P3 according to Comparative Example 2. The configuration of the micro-analysis chip P3 shown in FIG. 8 is modeled after the configuration disclosed in Patent Document 1. <Flow path configuration> The micro-analysis chip P3 has a porous substrate S1 and a porous substrate S2. The porous substrate S1 has a configuration in which the area other than the area where the reference electrode 7 and the working electrode 8 are formed is filled with a channel wall 5 that is impermeable to the sample. The porous substrate S2 has a dispensing section 6 and a flow path 4. The dispensing section 6 is disposed approximately directly above the midpoint between the reference electrode 7 and the working electrode 8 of the porous substrate S1. The flow path 4 is disposed so as to overlap the end of the ionic crystal 10 on the dispensing section 6 side and the ion selective membrane 9 of the working electrode 8.

[0040] <Reduction of measurement time> The reduction of measurement time will be explained. FIG. 9 shows the behavior of the specimen over time after dispensing in Example 1, Comparative Example 1, and Comparative Example 2. ΔT1 represents the time it takes for the sample to fill the flow path area after dispensing before reaching the working electrode. ΔT2 represents the time required for the sample to reach the working electrode, cover the surface of the working electrode, and begin measurement. ΔT3 represents the time from the start of electrochemical measurement until the potential stabilizes and the measurement is completed. Table 1 shows the results of the behavior of the specimen over time in Example 1, Comparative Example 1, and Comparative Example 2. The results were obtained by averaging the values ​​measured during the five concentration levels mentioned above (the KCl concentration was fixed at 10 mmol / L, and the NaCl concentration was changed to five levels: 100 μmol / L, 1 mmol / L, 10 mmol / L, 100 mmol / L, and 800 mmol / L). In Example 1, since a flow path is formed in the porous substrate S2, the flow path volume can be increased, and the flow rate of the specimen permeating into the flow path can be increased compared to Comparative Examples 1 and 2. As a result, Example 1 achieved time reductions for all of ΔT1, ΔT2, and ΔT3 compared to Comparative Examples 1 and 2, and it was shown that the total measurement time T (=ΔT1+ΔT2+ΔT3) could be reduced.

[0041] [Table 1]

[0042] Table 2 summarizes the advantages of Example 1 over Comparative Examples 1 and 2. In Example 1, good sensitivity and ion selectivity were obtained from the stable measured potential over time. The sensitivity and ion selectivity were calculated in accordance with the aforementioned JIS K0122 general rules for ion electrode measurement methods. On the other hand, in Comparative Example 1, the ion selectivity was not constant in each measurement in which the ion concentration was changed (the KCl concentration was fixed at 10 mmol / L, and the NaCl concentration was changed to five levels: 100 μmol / L, 1 mmol / L, 10 mmol / L, 100 mmol / L, and 800 mmol / L), and it was shown that the concentration measurement may become unstable. In Comparative Example 2, the change in potential over time was more unstable than in Example 1, and the total measurement time was longer. Furthermore, the configuration of Example 1 makes it possible to increase the area of ​​the ion selective membrane 9 that comes into contact with the sample, while rapidly supplying a sufficient amount of highly pure sample that reacts with the ion selective membrane 9 (FIG. 6). Furthermore, it has been shown to be effective in measuring electrolyte concentrations accurately in a short time, with excellent measurement sensitivity and ion selectivity (FIGS. 4(a)(b)) and time stability of the measured potential (FIGS. 5(a)(b)).

[0043] [Table 2]

[0044] [Modification 1 of Example 1] The microanalysis chip P4 in the first modification of the first embodiment will be described. 10 is a cross-sectional view showing a schematic configuration of a micro-analysis chip P4. In this modification, the dispensing part 6 is disposed on the upper surface (substantially directly above) of the working electrode 8. This allows the distance between the reference electrode 7 and the working electrode 8 to be shortened while maintaining good measurement performance such as sensitivity, ion selectivity, and time stability, and makes it possible to miniaturize the device. [Explanation of symbols]

[0045] 1. First flow chamber including reference electrode 7 2. A second flow chamber including a working electrode 8 3: First flow channel connecting the flow channel chamber 1 and the flow channel chamber 2 4. A second flow path connecting the dispensing unit 6 and the working electrode 8 5. Channel wall 6. Dispensing section 7...Reference electrode 8...Working electrode 9. Ion-selective membrane Ionic crystals containing 10···Cl ions 11. Regulating member 12. Particles contained in the specimen S1: A porous substrate (first porous substrate) including a working electrode 8 S2: A porous substrate (second porous substrate) arranged on the upper surface of the flow channel 3 and the ion selective membrane 9 L1: Thickness of porous substrate

Claims

1. A microanalysis chip configured by laminating a first porous substrate and a second porous substrate, a first flow path chamber, a second flow path chamber, and a first flow path connecting the first flow path chamber and the second flow path chamber are formed by flow path walls inside the first porous substrate; a reference electrode is disposed in the first flow chamber; A working electrode is disposed in the second flow chamber, A microanalysis chip characterized in that the second porous substrate is provided so as to overlap at least a portion of the region on the surface of the first porous substrate where the working electrode is formed.

2. 2. The microanalysis chip according to claim 1, wherein the working electrode is covered with an ion-selective membrane containing a component having ion selectivity.

3. The micro-analysis chip according to claim 1 , wherein the second porous substrate has a dispensing portion for dispensing a sample.

4. a restricting member that is impermeable to samples is disposed on an upper surface of the second porous substrate; 4. The micro-analysis chip according to claim 3, wherein the restricting member has an opening in the dispensing section, and the second porous substrate is exposed.

5. 2. The microanalysis chip according to claim 1, wherein analyte-soluble ionic crystals are disposed on the surface of the reference electrode.

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