Microanalysis chip

Abstract

To provide a micro analysis chip which enables stable analysis.SOLUTION: A micro analysis chip having a flow channel area surrounded by a flow channel wall provided inside a porous base material is provided, the flow channel area comprising 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, where the first flow channel chamber has a reference electrode provided therein and the second flow channel chamber has a working electrode provided therein. An electrolyte is provided upstream of the reference electrode in a travel direction of a sample in the flow channel area.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, an electrolyte concentration measurement system using the microanalysis chip, and an electrolyte concentration measurement method.

Background Art

[0002] In recent years, the development of microchannel devices that can efficiently (in trace amounts, rapidly, and simply) perform biochemical analysis within a single chip using micro-sized microchannels has attracted attention in a wide range of fields such as biochemical research, medicine, drug discovery, healthcare, the environment, and food.

[0003] Among them, paper-based paper microanalysis chips can drive samples and test solutions without using electricity by using an inexpensive material such as paper as a substrate and utilizing the capillary action of the paper itself. Therefore, they are small, low-cost, easy to carry, highly disposable (can be discarded simply by burning), and do not require large-scale equipment, making it possible for anyone to easily perform POC (point of care) testing and diagnosis at low cost. Thus, it is expected worldwide as a testing device for medical activities in developing countries, remote areas, and disaster sites where medical facilities are not well-developed, as well as at airports where the spread of infectious diseases must be stopped at the border. It is also attracting attention as a healthcare device for managing and monitoring one's own health status and as various pathological diagnosis devices in ordinary medical settings.

[0004] One of the biochemical tests in the above pathological diagnosis is electrolyte measurement. Electrolyte measurement is a test for measuring ion concentrations (such as Na + , K + , Cl - etc.) in blood or urine. Electrolytes are essential for maintaining the body's ion concentration balance, which is necessary for maintaining the body's water volume and pH, and for normal functioning of nerves, the heart, and muscles.

[0005] In addition, when there are changes in electrolyte concentration, it is an essential test for disease screening, such as the high possibility of abnormalities in kidney function and hormonal functions. Furthermore, it is also a very important test for confirming the physiological functions (vital signs) of patients at disaster sites and the like.

[0006] Research on performing this electrolyte measurement with μPADs is being carried out at various universities and companies.

[0007] In Non-Patent Document 1, a filter paper-based measurement device for the concentrations of Na ions and K ions has been proposed. This device has a dispensing section for dispensing the sample. When the dispensed sample penetrates from the dispensing section into the regions of the working electrode and the reference electrode respectively, the two electrodes are electrically connected, enabling potential difference measurement. Also, in this device, in order to obtain a stable potential at the reference electrode, a KCl ion crystal is deposited on the reference electrode. During measurement, KCl dissolves into the sample, thereby maintaining a high concentration of Cl ions in the reference electrode region and obtaining a stable potential of the reference electrode. Furthermore, only the ions to be measured are selected by an ion-selective membrane formed to cover the working electrode, so that measurement can be performed without being affected by other ions.

[0008] Also, Patent Document 1 discloses a method of obtaining a stable potential at the reference electrode by dispensing the sample into the working electrode section and simultaneously dispensing a saturated solution of KCl or NaCl as a standard solution into the reference electrode section.

Prior Art Documents

Patent Documents

[0009]

Patent Document 1

Non-Patent Documents

[0010]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0011] However, the arrangement of the electrolyte layer (KCl layer) is extremely difficult. Simply laminating and arranging the KCl layer on the supporting electrolyte or AgCl does not result in a stable potential on the reference electrode side. Therefore, even for specimens of the same concentration, the variation between devices becomes large, making it difficult to obtain stable measurement results. This problem was discovered through the research of the inventors of the present application.

[0012] An object of the present invention is to provide a microanalysis chip in which the electrolyte layer is arranged at an appropriate position, shows the potential of an appropriate reference electrode, and enables stable analysis.

Means for Solving the Problems

[0013] The present invention relates to a microanalysis chip having a flow path region surrounded by flow path walls provided inside a porous substrate, wherein 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 arranged in the first flow path chamber, a working electrode is arranged in the second flow path chamber, and an electrolyte is arranged upstream of the reference electrode when based on the advancing direction of the specimen in the flow path region. The present invention relates to a microanalysis chip characterized by this.

Effects of the Invention

[0014] According to the present invention, a microanalysis chip capable of performing stable analysis can be provided.

Brief Description of the Drawings

[0015]

Figure 1

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Figure 12

Modes for Carrying Out the Invention

[0016] The operation of the present invention will be described in detail.

[0017] The role of the reference electrode is to serve as a reference potential with respect to the potential generated at the working electrode. Therefore, regardless of the concentration of the analyte, the reference electrode must always exhibit the same potential. If the potential of the reference electrode is unstable, the potential difference between the reference electrode and the working electrode will become inaccurate.

[0018] Generally, Ag / AgCl is often used as the base electrode of the reference electrode. At the interface with the analyte, the following equilibrium reaction occurs in Ag / AgCl, and the potential is determined by the concentration of Cl - .

[0019]

Equation

[0020] Therefore, by keeping the Cl - concentration constant, the potential can be stabilized.

[0021] In a saturated sodium chloride (NaCl) solution or potassium chloride (KCl) solution, since the concentration of Cl - is constant, if a saturated NaCl solution or KCl solution is reacted with AgCl, the same interfacial potential can always be obtained.

[0022] Figure 2(b) shows the measurement results of the potential when directly dispensing an NaCl solution onto the Ag / AgCl electrode 3 while changing the concentration of NaCl (Cl - concentration) using the flow channel of the microanalysis chip. During the measurement, as shown in Figure 2(a), a commercially available electrode 5 was placed on one side, the space between the Ag / AgCl electrode 3 and the commercially available electrode 5 was connected with an NaCl solution, the potential between the electrodes at that time was measured, and the change in potential due to the change in the concentration of NaCl was plotted.

[0023] As the concentration of NaCl, that is, the concentration of Cl - increases, the potential of the Ag / AgCl electrode decreases. When the concentration of NaCl reaches a concentration close to saturation (about 5.2 mol / L at a temperature of 25°C), since NaCl cannot dissolve any more, Cl -The concentration becomes constant, the above equilibrium reaction stops, and the potential no longer changes further. Since it is considered that the measurement is often carried out at room temperature (about 25°C), in the following description, it is assumed to be used at room temperature (about 25°C), and the design is based on the saturation concentration at 25°C. The amount of electrolyte arranged in the examples described later is also the amount assumed at 25°C. However, when use in different environments is assumed, the design should be according to the usage environment and is not limited to the configuration (electrolyte amount) assumed at 25°C.

[0024] Based on the above principle, usually, in an internal liquid type reference electrode, saturated KCl or NaCl is used as the internal liquid. In a solid electrode type, this saturated solution is directly dispensed onto the Ag / AgCl electrode as the standard solution, and a method is taken to ensure the potential of a stable reference electrode.

[0025] However, in that case, it is necessary to prepare a reference solution separately from the sample, and in a microanalysis chip, which is a simple and low-cost inspection device, the labor and cost are issues.

[0026] To solve that problem, in Non-Patent Document 1, as shown in FIGS. 7(a) and 7(b), an electrolyte layer 3a is laminated on a supporting electrolyte membrane 9 provided on a reference electrode (Ag / AgCl electrode) 3 (provided to make the contact potential at the interface between the sample and the Ag / AgCl electrode more stable). The sample dissolves this electrolyte layer, and a sample saturated in concentration is supplied to the supporting electrolyte membrane and the Ag / AgCl electrode to stabilize the interfacial potential.

[0027] However, it has been difficult to obtain a stable potential even when the electrolyte is laminated on the Ag / AgCl electrode or the supporting electrolyte membrane by various printing methods such as inkjet and dispenser, and at various concentrations and pitch intervals.

[0028] In this configuration, a test was conducted as follows to confirm that it is difficult to obtain a stable potential.

[0029] A sheet 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 was prepared. In the first flow path chamber of this sheet, a reference electrode (Ag / AgCl electrode) 3 was arranged, a supporting electrolyte membrane 9 was laminated on the electrode, and further, an electrolyte layer (KCl layer) 3a was laminated on the supporting electrolyte membrane 9. Also, a commercially available electrode 5 was installed in the second flow path chamber to fabricate a microanalysis chip. In this microanalysis chip, a KCl solution with a varying concentration was dropped as a sample into the flow path portion connecting the Ag / AgCl electrode and the commercially available electrode, and the potential difference between the Ag / AgCl electrode and the reference electrode was measured. The KCl solution was varied in the range of 10 -4 ~800 mmol / L. The results of the test are shown in Fig. 8(c). Originally, even if the concentration of the sample is changed, the potential of the Ag / AgCl electrode should be constant, but the variation in potential became nearly 15 mV.

[0030] The reason for the variation in potential is considered as follows.

[0031] The sample that first reaches the electrolyte layer dissolves the electrolyte and contacts the Ag / AgCl electrode with the Cl - concentration saturated, but is washed away by the sample flowing in later (Figs. 8(a) and (b)). Then, the sample flowing in later contacts the Ag / AgCl electrode with the Cl - concentration not reaching saturation because the electrolyte has already been dissolved and does not exist (or has been washed away). Therefore, both the sample with the Cl - concentration saturated and the sample not saturated contact the Ag / AgCl electrode simultaneously, and it is considered that the potential becomes unstable.

[0032] Also, as shown in Figs. 9(a) and (b), even when the filter paper 10 impregnated with the electrolyte was placed on the reference electrode (Ag / AgCl electrode) 3 or the supporting electrolyte membrane 9, the results were the same. Fig. 9(b) is a cross-section of the dotted line portion in Fig. 9(a).

[0033] That is, Cl -It was confirmed that the measured interfacial potential was unstable unless the sample with a saturated concentration was stably supplied to the Ag / AgCl electrode.

[0034] The microanalysis chip of the present invention is characterized in that, when based on the traveling direction of the sample in the flow path region, an electrolyte is arranged upstream of the reference electrode, and the Cl of the sample - By stably contacting the reference electrode in a state where the concentration is saturated, the potential of the reference electrode is stabilized.

[0035] That is, in the microanalysis chip of the present invention, the upstream end of the reference electrode exists at a position downstream of the position where the sample flowing through the flow path dissolves the electrolyte and the electrolyte concentration in the sample becomes a concentration close to the saturated concentration (88% of the saturated concentration). It is preferable to have a configuration in which a desired amount of electrolyte is arranged at a position where it will be. Note that "88% of the saturated concentration" corresponds to about 4.6 mol / L in a NaCl solution with a saturation of 5.2 mol / L.

[0036] Further, the microanalysis chip of the present invention may have a third flow path chamber in which a working electrode is arranged, etc., in addition to the first flow path chamber and the second flow path chamber, and they may be connected by a flow path. The number of flow path chambers arranged on the microanalysis chip is not particularly limited.

Example

[0037] Hereinafter, exemplary embodiments of the present invention will be described with reference to the drawings. Note that the following embodiments are examples, and the present invention is not limited to the contents of the embodiments. Also, in the following figures, components not necessary for the description of the embodiments are omitted from the figures.

[0038] <Example 1> Example 1 will be described with reference to FIGS. 1 to 6.

[0039] Fig. 5(a) shows the microanalysis chip of Example 1. It has a flow channel region 1 surrounded by a flow channel wall 2 provided inside a porous substrate. The flow channel region 1 has a first flow channel chamber 12, a second flow channel chamber 13, and a flow channel connecting the first flow channel chamber 12 and the second flow channel chamber 13, and a dispensing section 11 exists in this flow channel. A reference electrode 3 is arranged in the first flow channel chamber 12. Taking the advancing direction of the sample as a reference (i.e., the side of the dispensing section 11 is the upstream), an electrolyte layer 3a is arranged on the upstream side of the reference electrode 3. By arranging the electrolyte layer 3a on the upstream side of the reference electrode, the electrolyte in the sample reaches the reference electrode stably in a saturated state. An operating electrode 6 is arranged in the second flow channel chamber 13, and the operating electrode 6 is composed of a base electrode 6b and an ion-selective membrane 6a provided to cover it.

[0040] Regarding the formation of the flow channels, the method described in (Japanese Unexamined Patent Application Publication No. 2021-37612) was used. Specifically, toner, which is particles for forming flow channels and has characteristics of fusibility, was used to form a desired flow channel pattern in an unfixed state on filter paper by an electrophotographic method, and then the flow channel pattern was formed by infiltrating the flow channel pattern into the interior of the paper using an oven or a heater. The formed flow channel pattern is as shown in Fig. 1(b), where reference numeral 1 is the flow channel and reference numeral 2 is the flow channel wall formed by the infiltration of the particles for forming flow channels.

[0041] Next, the reference electrode and the operating electrode are formed on the flow channel pattern by screen printing, an inkjet device (IJ), or a dispenser, etc. Since this patent is an invention related to the reference electrode, its configuration will be described centering on the reference electrode.

[0042] As shown in Fig. 1(a), an Ag / AgCl electrode was printed as a reference electrode on the flow path pattern by screen printing. Further, as shown in Fig. 1(a) and Fig. 1(c) (the cross-sectional view along the dotted line in Fig. 1(a) is Fig. 1(c)), an electrolyte (NaCl or KCl) was printed by an IJ or a dispenser at a position upstream of the Ag / AgCl electrode on the flow path and in the advancing direction of the specimen. By disposing an electrolyte layer 3a (NaCl or KCl) at such a position, the advancing specimen will surely pass through the electrolyte layer 3a. At that time, by dissolving the electrolyte, the concentration of the electrolyte in the specimen (Cl - concentration) becomes saturated, and in that state, the specimen reaches the reference electrode (Ag / AgCl electrode). As a result, the potential of the reference electrode (Ag / AgCl electrode) is stabilized.

[0043] As the electrolyte, a chloride is preferable, and in particular, sodium chloride (NaCl) or potassium chloride (KCl) which is easy to handle is preferable. Hereinafter, NaCl with more stable temperature dependence of solubility will be described.

[0044] The electrolyte (NaCl) was arranged in layers on the flow path in front of the reference electrode so as to be 3.0×10 2 g / L or more, preferably 3.4×10 2 g / L or more per liter of the specimen amount supplied to the reference electrode. This is because the saturation concentration of the electrolyte also depends on the specimen amount, and it is the value obtained by dividing the amount of NaCl required for saturation by the specimen amount supplied to the reference electrode. If 3.0×10 2 g / L or more of NaCl is arranged per liter of the specimen amount supplied to the reference electrode, the concentration of the specimen passing through the NaCl layer becomes 4.6 mol / L or more, which is a concentration close to saturation at 25°C, so that stable measurement becomes possible. Preferably, it is to arrange 3.4×10 2 g / L or more of NaCl. In this case, the concentration of the specimen passing through the NaCl layer becomes almost the saturation concentration (5.2 mol / L), and it becomes a concentration sufficient to maintain a state where the concentration of Cl - is almost saturated. The upper limit is not particularly defined, but when applying NaCl, NaCl may overflow from the flow path, so 5.6×10 2It is appropriate to set it to g / L or less.

[0045] In addition, even when the electrolyte is KCl, per 1 L of the sample amount supplied to the reference electrode, 3.0×10 2 g / L or more, preferably 3.4×10 2 g / L or more, and the electrolyte can be used accordingly. In this case, the concentrations of the samples are 3.6 mol / L or more and about 4.2 mol / L (saturated concentration), respectively, and become the saturated concentration of the KCl solution or a value close thereto.

[0046] In addition, the appropriate amount of the sample supplied to the dispensing section varies depending on the size and performance of each device. Generally, it is about 10 μL to 50 μL. For example, as an example of a small device, when the size of the reference electrode is 3 mm × 3 mm, the size of the working electrode is 3 mm × 3 mm, and the paper thickness is 200 μm, including the volume of the flow path therebetween, the total volume of the flow path is 3.6×10 -9 m 3 (3.6 μL), approximately (1.8 μL each for the reference electrode and the working electrode). Therefore, if there is about 10 μL of the sample, the sample can be sufficiently supplied to the reference electrode and the working electrode.

[0047] If the size of the device is increased, the required sample amount will increase. However, since the sample is based on human blood or urine, it is preferably less, and generally 50 μL or less. Although the present invention does not depend on the size of the device or the required sample amount, for the above reasons, the description will be made within the range of about 10 μL to 50 μL as the sample amount.

[0048] In the case of the microanalysis chip of Example 1, 10 μL of the sample is dispensed and supplied to the dispensing section 11 at the center of the flow channel region. Approximately half of it is supplied to the reference electrode 3 side (S portion in Fig. 1(a)), and the other half is supplied to the working electrode side. Therefore, the amount of the sample supplied to the reference electrode 3 is about 5 μL. In this example, an electrolyte layer 3a with a saturation concentration is arranged in the S portion of Fig. 1. In this case, when all the arranged electrolyte is dissolved, the electrolyte concentration in the sample becomes 88% or more of the saturation concentration. Therefore, between the electrolyte layer 3a and the base electrode 3 in Fig. 1, the electrolyte concentration in the sample is 88% or more of the saturation concentration. Thus, this configuration satisfies the configuration that "an electrolyte is arranged at a position where the upstream end of the reference electrode exists at a position downstream of the position where the electrolyte concentration in the sample becomes 88% or more of the saturation concentration."

[0049] For example, when the amount of the sample supplied to the reference electrode is 5 μL, if 1.5 mg of NaCl is arranged, an NaCl concentration of 4.6 mol / L or more can be obtained. Therefore, when NaCl is arranged in a 3 mm × 3 mm region with a thickness of 200 μm, 8.3×10 5 g / m 3 or more of NaCl arranged, the NaCl concentration of the sample passing through the NaCl layer becomes 4.6 mol / L or more, which is a value close to saturation. Further, if 9.4×10 5 g / m 3 or more of NaCl is arranged, the concentration of the sample passing through the NaCl layer becomes close to the saturation concentration (about 5.2 mol / L), which is a sufficient concentration to bring the Ag / AgCl electrode to an equilibrium state. In specifying the concentration of the solution in which NaCl is dissolved in the sample, it is necessary to consider the specific gravity of the solution, and the above NaCl concentration is a value considering this.

[0050] When the amount of the sample supplied to the reference electrode is 25 μL and NaCl is arranged in a 3 mm × 3 mm region with a thickness of 200 μm in the same manner as in the above case, 4.2×10 6 g / m 3If the above NaCl is arranged, the NaCl concentration of the specimen passing through the NaCl layer will be 4.6 mol / L or more. In order to make the NaCl concentration of the specimen passing through the NaCl layer approximately the saturation concentration (about 5.2 mol / L), 4.7×10 6 g / m 3 or more of NaCl may be arranged.

[0051] Also, when the amount of the specimen supplied to the reference electrode is 5 μL and NaCl is arranged in a region of 6.7 mm × 6.7 mm with a thickness of 200 μm, 1.7×10 5 g / m 3 or more of NaCl may be arranged. If the above NaCl is arranged, the NaCl concentration of the specimen passing through the NaCl layer will be 4.6 mol / L or more. In order to make the NaCl concentration of the specimen passing through the NaCl layer approximately the saturation concentration (about 5.2 mol / L), 1.9×10 5 g / m 3 or more of NaCl may be arranged.

[0052] When the specimen reaches the arranged electrolyte (NaCl) layer 3a, it advances in the direction of the reference electrode (Ag / AgCl electrode) 3 while dissolving the arranged electrolyte (NaCl). At this time, the concentration of Cl - in the specimen passing through the electrolyte (NaCl) layer is always maintained at a saturated or nearly saturated state, and since this saturated specimen is continuously supplied to the reference electrode (Ag / AgCl electrode), a stable potential can be obtained.

[0053] In this configuration (Fig. 1(a)), the reference electrode 3 and the electrolyte layer 3a are not in contact, but the electrolyte layer 3a may be in contact with the reference electrode 3 or may overlap the reference electrode 3. The important point is that the specimen reaches the reference electrode 3 after the concentration of the electrolyte in the specimen becomes saturated.

[0054] The concentration of the electrolyte (NaCl) is 10 -5The results of measuring the reference electrode potential using specimens with concentrations varying from 0 mol / L to 1 mol / L are shown in Fig. 3. To focus only on the performance of the reference electrode, originally, on the side of the microanalysis chip where the working electrode is formed, similar to the case of Fig. 2, a commercially available electrode 4 was placed instead of the working electrode, the commercially available electrode and the reference electrode were connected with the specimen, and the potential difference between the two electrodes was measured.

[0055] As a result of the measurement, it was confirmed that the obtained potential was constant when using specimens with different concentrations. The variation when using specimens with different concentrations was within about 0.3 mV (Fig. 3). Furthermore, it was confirmed that even when measuring at the same concentration (140 mmol / L, ○ in Fig. 3) using a plurality of microanalysis chips prepared in the same manner, the variation was within about 0.2 mV (Fig. 4).

[0056] Furthermore, as shown in Fig. 5(a), a working electrode which is an ion-selective electrode was arranged on one side, and with both electrodes arranged, the specimen was dispensed and the potential between the reference electrode was measured. As the specimen, a solution with an electrolyte (Na + ) concentration of 10 -5 mol / L to 1 mol / L and with interfering ions added (about 5 mmol / L of KCl in the case of K + ) was used. The results are shown in Fig. 5(b).

[0057] As the working electrode (ion-selective electrode), a solid-contact type ion-selective electrode with an ion-selective membrane having selectivity for the target ion laminated on the base electrode was used. In this example, an ion-selective electrode with Na + as the target ion was used.

[0058] Regarding the base electrode, attempts using Ag / AgCl, carbon, and PEDOT / PASS have been proposed, and in the present invention, it can be used without particular limitation. A base electrode can be selected according to the required characteristics of the device such as cost and performance. In this example, an Ag / AgCl electrode was used as the base electrode.

[0059] As the ion-selective membrane, any commonly used one may be employed, and a membrane that is sensitive to the target ion and has sufficient selectivity against interfering ions may be used. Examples of materials used for the ion-selective membrane include 12-crown-4-ether having a crown ether structure as the ionophore, sodium tetraphenylborate (NaTPB) as the anion scavenger, NPOE and DOS as the plasticizer, and PVC alone or a copolymer of vinyl chloride and vinyl acetate as the polymer agent.

[0060] Then, appropriate amounts of each component are mixed, dissolved or dispersed in solvents such as THF or cyclohexanone, and the resulting solution is applied onto a base electrode (Ag / AgCl electrode) with an intermediate layer such as NaCl laminated thereon by an inkjet method, whereby it can be fabricated. Also, the coating method does not have to be limited to the inkjet method. The viscosity of the solution is adjusted according to each printing method such as a dispenser or screen printing, and then the ion-selective membrane may be laminated onto the base electrode.

[0061] In this test, a potential profile between the electrodes can be obtained according to the concentration of Na + in the sample, and it can be seen that results showing sufficient sensitivity (the slope of the graph) and selectivity have been obtained.

[0062] Furthermore, using this device, a commercially available electrode is set in the dispensing part between the reference electrode and the working electrode (ion-selective electrode), the potential between the reference electrode and the commercially available electrode is measured (□ plot in FIG. 6), and the potential between the working electrode (ion-selective electrode) and the commercially available electrode (〇 plot in FIG. 6) is also measured.

[0063] The potential on the reference electrode side (□) hardly changes even when the concentration of Na + in the sample changes, and it can be seen that the potential on the ion-selective electrode side (〇) changes according to the change in the concentration of Na + The difference is the potential difference between the reference electrode and the working electrode, which is shown by the ● plot in FIG. 6, and it was confirmed that the reference electrode exhibits sufficient stability.

[0064] Furthermore, the present invention is not limited to a microanalysis chip using a single Ag / AgCl electrode as a reference electrode. As described in Non-Patent Document 1, a configuration may be adopted in which Ag / AgCl is used as a base electrode and a supporting electrolyte layer is provided thereon to stabilize the interfacial potential with the sample and reduce the influence of interfering ions. For example, as described in Non-Patent Document 1, an appropriate amount of TBA-TBB (tetrabutylammonium tetrabutylborate), TDMACl (tridodecylmethylammonium chloride), a plasticizer, and PVC are mixed together, and a solution prepared by mixing these in a solvent such as THF or cyclohexanone is applied and dried to form a supporting electrolyte layer.

[0065] <Example 2> In this example, as shown in Fig. 11(a), an ion-selective membrane 6a was also provided on the reference electrode. An ion-selective membrane in which the selected ion is Na + is used, and a configuration in which NaCl is arranged as an electrolyte upstream of the reference electrode can be considered. In this case, when the sample dissolves NaCl, which is an electrolyte, the sample contains Cl - at a saturated concentration, and at the same concentration, Na + whose concentration no longer changes because the dissolution of NaCl no longer occurs. When this sample contacts the reference electrode (ion-selective electrode) provided with the ion-selective membrane, since the concentration of Na + does not change, a stable potential can be obtained (Fig. 11(b)).

[0066] In the case of this configuration, the electrolyte is not limited to chlorides. When the ion-selective membrane used for the reference electrode is a Na + selective membrane, hydroxides such as sodium hydroxide (NaOH) may be used, and an electrolyte suitable for the target ion may be selected.

[0067] <Example 3> A configuration for making the operation in Example 1 more effective will be described.

[0068] In the configuration of the reference electrode in which the electrolyte layer (NaCl or KCl) 3a is disposed upstream of the Ag / AgCl electrode, a laminate layer 14 is provided on the front and back surfaces of the reference electrode (FIG. 10).

[0069] Normally, the Ag / AgCl electrode is printed on paper by screen printing. However, since the Ag / AgCl paste ink for screen printing has a certain viscosity, it is not printed entirely in the thickness direction of the filter paper. Instead, printing is performed while leaving a certain portion of the filter paper on the back side. Also, considering the shift due to the printing of the Ag / AgCl electrode with respect to the flow path, printing is performed with a gap of at least about 0.2 mm from the flow path wall. Therefore, filter paper portions acting as flow paths remain on the left and right and lower sides of the reference electrode. When the front surface of the reference electrode is covered with the laminate layer, the region where the saturated specimen advances is restricted, and the specimen preferentially passes through the filter paper portions remaining on the left and right and lower sides of the Ag / AgCl electrode.

[0070] By adopting such a configuration, only the specimen that has passed through the restricted flow path comes into contact with the reference electrode, and it becomes easy to restrict the specimen in contact with the reference electrode to a saturated specimen. Therefore, the output potential becomes more stable.

[0071] Note that the relationship between the electrolyte 3a and the reference electrode 3 according to the present invention is not limited to the arrangement configuration shown in FIG. 1. For example, arrangements as shown in FIGS. 12(a) and (b) may also be used. As long as the electrolyte layer (NaCl or KCl) is disposed upstream of the reference electrode (Ag / AgCl electrode) with respect to the advancing direction of the specimen. Also, even if the contact area between the reference electrode and the specimen is narrow, if a saturated specimen is provided, it can act as a stable reference electrode (FIG. 12(b)).

Explanation of Reference Numerals

[0072] 1 Flow path (filter paper portion) 2 Flow path wall (image forming portion = toner penetration portion) 3 Reference electrode 3a Electrolyte layer 4,7 Commercially available reference electrodes 5 Specimen 6 Function (Ion Selection) Electrode 6a Ion Selection Membrane 6b Ion Selection Side Base Electrode

Claims

1. A microanalysis chip having a flow path region surrounded by a flow path wall provided inside a porous substrate, The porous substrate is filter paper; The flow path region is formed in a single porous substrate, and 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 provided inside the porous substrate of the first flow chamber, A working electrode is disposed in the second flow chamber, A microanalysis chip, characterized in that an electrolyte is disposed inside the porous substrate and upstream of the reference electrode when the direction of sample flow in the flow path region is taken as a reference.

2. 2. The microanalysis chip according to claim 1, wherein the electrolyte is disposed at a position such that the upstream end of the reference electrode is located downstream of a position where a specimen flowing through the flow path dissolves the electrolyte and the electrolyte concentration in the specimen becomes 88% or more of a saturated concentration.

3. 3. The microanalysis chip according to claim 1, wherein the electrolyte is a chloride.

4. 4. The microanalytical chip according to claim 3, wherein the chloride is sodium chloride or potassium chloride.

5. 3. The microanalysis chip according to claim 1, wherein the electrolyte is a hydroxide.

6. The amount of the electrolyte is 3.0 x 10 per 1 L of the sample volume supplied to the reference electrode. ２ The microanalysis chip according to claim 4, wherein the concentration is 100 g / L or more.

7. The amount of the electrolyte is 3.4 × 10 per 1 L of the sample volume supplied to the reference electrode. ２ The microanalysis chip according to claim 6, wherein the concentration is 100 g / L or more.

8. 3. The microanalytical chip according to claim 1, wherein the base electrode of the reference electrode is an Ag / AgCl electrode.

9. 3. The microanalysis chip according to claim 1, wherein the working electrode is an ion-selective electrode having an ion-selective membrane laminated on a base electrode.

10. 3. The microanalysis chip according to claim 1, wherein the reference electrode is an ion-selective electrode having an ion-selective membrane laminated on a base electrode.

11. 3. The microanalysis chip according to claim 1, further comprising a laminate layer on the surface of the reference electrode.

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