Micro-analysis chip

Abstract

To solve a problem in which: in a micro-analysis chip performing electric potential difference ion concentration measurement, ion crystals arranged to stabilize an electric potential of a reference electrode flow back to a flow path chamber including a working electrode through a specimen, and may affect the electric potential in the working electrode.SOLUTION: A micro-analysis chip has a dispensing part 6 that dispenses a specimen, a first flow path chamber 1, a second flow path chamber 2, a first channel 3, and a second channel 4, which are formed by a channel wall provided inside a porous substrate. A reference electrode 7 is arranged in the first flow path chamber, and ion crystals having specimen solubility are arranged on the surface of the reference electrode. A working electrode 8 is arranged in the second flow path chamber. When the time required for the specimen to reach the ion crystals after the specimen is dispensed by the dispensing part is defined as T1, and the time required for the specimen to reach the working electrode as T2, the first channel and the second channel are configured such that T1 and T2 satisfy a relationship of T1>T2.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 micron-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 reduction of specimens and waste liquids 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 medical care in developing countries, remote areas, and disaster sites where medical facilities are inadequate, and for use in airports where the spread of infectious diseases must be prevented at the border.

[0004] In electrochemical analysis, a stable reference electrode capable of maintaining a constant potential is required for the quantification of electrolyte ions in a solution. Conventionally used glass reference electrodes are expensive and difficult to miniaturize because they require an internal liquid. Furthermore, they require storage in a concentrated ion solution, making them difficult to handle.

[0005] Patent Document 1 proposes a device including a microanalysis chip capable of measuring potential differences using a porous substrate, enabling electrochemical measurements at low cost, with easy handling and disposability. This device includes one or more working electrodes and one reference electrode on a porous substrate, and when measuring potential differences, the two electrodes must be electrically connected by a fluid. It also describes how, during measurement, a highly concentrated aqueous solution of KCl is dispensed as a reference solution into the reference electrode region containing the reference electrode in order to obtain a stable potential at the reference electrode.

[0006] Furthermore, Non-Patent Document 1 proposes a filter paper-based measurement device for measuring the concentrations of Na ions and K ions. This device has a dispensing section for dispensing the sample. The dispensed sample permeates from the dispensing section into the working electrode and reference electrode, electrically connecting the two electrodes and enabling potential difference measurement. To achieve a stable potential at the reference electrode, the device deposits KCl ion crystals on the reference electrode. During measurement, KCl dissolves in 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 ions, allowing measurement to be performed without being affected by other ions. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] US Patent Application Publication No. 2016 / 033438 [Non-patent literature]

[0008] [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]

[0009] However, the above-mentioned configuration may affect the accuracy of analyte concentration measurement. This is because it takes some time for the working electrode to stabilize after the analyte reaches the working electrode before measurement. During this time, analyte-soluble ionic crystals on the reference electrode flow back into the working electrode area through the analyte, affecting the potential of the working electrode. "Analyte-soluble ionic crystals" refers to "ionic crystals that can be dissolved in the analyte," such as KCl ionic crystals. To suppress the effect of backflow, the time it takes for the ions dissolved in the sample to reach the working electrode together with the backflowing sample can be increased by, for example, increasing the distance from the reference electrode to the working electrode. However, increasing the distance from the reference electrode to the working electrode has adverse effects such as increasing the chip size of the microanalytical chip and lengthening the measurement time. The present invention aims to provide a microanalysis chip that can suppress the effect on the working electrode of ion backflow from the reference electrode region to the working electrode region, and that enables stable ion concentration measurement, without increasing the chip size or lengthening the measurement time. [Means for solving the problem]

[0010] In order to achieve the above object, according to one aspect of the present disclosure, A microanalysis chip in which a dispensing section for dispensing a specimen, a first flow path chamber, a second flow path chamber, a first flow path connecting the dispensing section and the first flow path chamber, and a second flow path connecting the dispensing section and the second flow path chamber are formed by flow path walls provided inside a porous substrate, a reference electrode is disposed in the first flow channel chamber, and analyte-soluble ionic crystals are disposed on a surface of the reference electrode; A working electrode is disposed in the second flow chamber, A microanalysis chip is provided, characterized in that, after the sample is dispensed into the dispensing section, the time it takes for the sample to reach the ionic crystal is T1, and the time it takes for the sample to reach the working electrode is T2, and the first flow path and the second flow path are configured so that T1 and T2 satisfy the relationship T1 > T2. [Effects of the Invention]

[0011] According to one aspect of the present disclosure, it is possible to provide a microanalysis chip that can suppress the effect on the working electrode of the backflow of ions from the reference electrode region to the working electrode region, without increasing the chip size or lengthening the measurement time, and that enables stable ion concentration measurement. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 is a simplified top view showing an example of a flow channel configuration of a micro-analysis chip P1 according to Example 1. [Figure 2] A simplified cross-sectional view of the microanalysis chip P1 shown in FIG. [Figure 3] Image showing the behavior of the sample after dispensing in Example 1 [Figure 4] Image showing the behavior of the sample after dispensing in Comparative Example 1-1 [Figure 5] Image showing the behavior of the sample after dispensing in Comparative Example 1-2 [Figure 6] FIG. 10 is a top view showing an example of a flow channel configuration of the micro-analysis chip P2 according to Example 2. [Figure 7]FIG. 10 is a top view showing an example of a flow channel configuration of the micro-analysis chip P3 according to Example 3. [Figure 8] FIG. 10 is a top view showing an example of a flow channel configuration of the micro-analysis chip P4 according to Example 4. [Figure 9] Top view and cross-sectional view for explaining the permeation anisotropy of a porous substrate DETAILED DESCRIPTION OF THE INVENTION

[0013] The microanalysis chip of the present disclosure 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 disclosure. FIG. 1 is a simplified top view of the microanalysis chip P1. FIG. 2 is a simplified cross-sectional view of the micro-analysis chip P1 taken along line AA, in which the lead wire of the reference electrode 7 is omitted. In the micro-analysis chip P1, a dispensing section 6 for dispensing a sample, a flow channel chamber 1 (first flow channel chamber), a flow channel chamber 2 (second flow channel chamber), a flow channel 3 (first flow channel), and a flow channel 4 (second flow channel) are formed by a flow channel wall 5 provided inside the porous substrate S1. The flow channel 3 connects the dispensing section 6 and the flow channel chamber 1, and the flow channel 4 connects the dispensing section 6 and the flow channel chamber 2. A reference electrode 7 is disposed in the flow channel chamber 1, and ion crystals having analyte solubility are disposed on the surface of the reference electrode 7. A working electrode 8 is disposed in the flow channel chamber 2, and the working electrode 8 is covered with an ion selective membrane 9 having ion selectivity. After the sample is dispensed into the dispenser 6, the time it takes for the sample to reach the ionic crystal 10 is defined as T1, and the time it takes for the sample to reach the working electrode 8 is defined as T2. In the flow paths 3 and 4, T1 and T2 satisfy the relationship T1>T2. Hereinafter, examples and comparative examples will be shown to more specifically describe the microanalysis chip of the present disclosure. The fixing belt according to the present disclosure is not limited to only the configuration embodied in the examples. Also, various numerical values such as thickness, length, porosity, etc. are not limited to the following values. Also, in the case of measuring the total amount of ions in a sample, etc., the ion selection membrane 9 is not necessarily required.

[0014] [Example 1] <Configuration of flow path> The microanalysis chip P1 related to Example 1 will be described using FIGS. 1 and 2. In Examples 1 to 3 and Comparative Examples 1 to 3, after disposing a hydrophobic resin on a paper-made porous substrate S1 with a thickness DL1 = 0.1 mm and a porosity P1 of 50%, heat fixing was performed to form a flow path wall 5 through which the sample cannot penetrate and form a flow path pattern.

[0015] The feature of Example 1 is that when the cross-sectional area of the flow path 3 (first flow path) is A1 and the cross-sectional area of the flow path 4 (second flow path) is A2, A1 and A2 satisfy the relationship A1 < A2.

[0016] The flow path pattern has a dispensing part 6 for dispensing the sample, a flow path chamber 1 including a reference electrode 7, and a flow path chamber 2 including a working electrode 8. The dispensing part 6 is connected to the flow path chamber 1 by the flow path 3 and is connected to the flow path chamber 2 by the flow path 4. Here, a flow path was formed so that the volume C1 through which the sample T can penetrate in the flow path chamber 1 is 10 μL. Specifically, the flow path width W1 of the flow path 3 is narrower than the flow path width W2 of the flow path 4, and W1 = 2 mm and W2 = 5 mm, respectively. The depth DP1 of the flow path 3 and the depth DP2 of the flow path 4 were made equal to the thickness DL1 of the porous substrate S1. That is, the cross-sectional area A1 of the flow path 3 is DL1 × W1 = 0.2 mm 2 , the cross-sectional area A2 of the flow path 4 is DL1 × W2 = 0.5 mm 2 and The relationship is A1 < A2.

[0017] In this embodiment, the cross-sectional area of the flow path is adjusted by the flow path width, but it is not limited to this. As long as the relationship of the cross-sectional area is A1 < A2, for example, the flow path widths W1 and W2 may be the same, and the depth DP1 of the flow path 3 may be set to be shallower than the depth DP2 of the flow path 4. Also, the cross-sectional shape of the flow path is not limited to a rectangle or a square, and may be a semi-circle, a semi-ellipse, a triangle, or a trapezoid. Furthermore, it may be a shape formed by a polygon or a curved surface without limitation. In the case of such a shape, the cross-sectional area of the flow path is obtained by measuring the area of the cross-section when cut in a direction orthogonal to the direction in which the flow path extends. In this embodiment, a paper-made porous base material is used, but the porous base material is not limited to paper-made ones. It is only necessary to generate capillary action with respect to the liquid, and it may also be one having a porous structure such as a continuous foam and nanofibers inside, a mesh-like structure, etc., and resin, glass, an inorganic substrate, a fabric, a metal paper, etc. may also be used. In this embodiment, the hydrophobic resin disposed on the porous base material is heated, and the melted hydrophobic resin is infiltrated and fixed inside the porous base material to form a flow path pattern, but the method for forming the flow path pattern is not limited to this. In addition to the method of cutting the paper-made porous base material S1 to leave only the flow path shape, the flow path wall may be formed by a wax printer or the like.

[0018] <Prescription of the electrode> The prescription of the electrode related to Example 1 will be described. The flow path chamber 1 has a reference electrode 7 mainly composed of Ag / AgCl at a position where the distance L3 from the flow path connection portion 12, which is the connection portion with the flow path 3, is 1 mm. The reference electrode 7 has a lead wire that continuously extends from inside the flow path chamber 1 to the flow path wall 5 as a contact point during measurement. Also, the entire surface area of the reference electrode 7 is arranged almost uniformly by 103.5 mg of KCl ionic crystals.

[0019] On the other hand, a working electrode 8 mainly composed of PEDOT:PSS (a dispersion of polyethylenedioxythiophene and polystyrene sulfonic acid) was provided in the flow channel chamber 2. The working electrode 8 also had a lead wire that continuously extended from inside the flow channel chamber 2 onto the flow channel wall 5. A K ion-selective membrane 9 was formed so as to cover the entire area of ​​the top and side surfaces of the working electrode 8. The ion selective membrane 9 was made of the following material. Valinomycin 1.0 wt% is an ion-selective material Potassium tetrakis (4-chlorophenyl) borate 0.5wt% as an anion scavenger o-nitrophenyl octyl ether 65.0wt% Polyvinyl chloride 33.5wt%

[0020] 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. The material of the ionic crystal 10 is not limited to KCl as long as it contains Cl ions. Furthermore, the mass of the ionic crystal 10 to be placed is not limited to 3.5 mg, and is only required to be within a mass range that results in a saturated solution when the ionic crystal is dissolved in pure water having a volume equivalent to the volume C1.

[0021] <Sample penetration> The penetration of the specimen will now be described. After being dispensed into the dispensing unit 6, the specimen permeates the paper porous substrate S1 by capillary action according to the flow path pattern. Here, the time t (Sec) required for the sample to penetrate is Considering the gaps between the fibers that make up the paper porous substrate S1 as tubular pores, the pore radius is a (cm), the viscosity of the solution is η (P), the pore length is h (cm), the surface tension of the solution is γ (dyne / cm), and the contact angle is Θ (rad), Based on the Lucas-Washburn equation, it is expressed as the following equation (1).

[0022]

number

[0023] Furthermore, when an analyte permeates along a channel, the narrower the cross-sectional area of ​​the channel, the slower it permeates. This is because the narrower the cross-sectional area of ​​the channel, the more it is affected by the hydrophobic channel walls. In other words, as shown in Equation (1), the permeation rate of the analyte is slower near the hydrophobic channel walls with a large contact angle Θ. Therefore, after the sample is dispensed into the dispensing section 6, the relationship between the time T1 it takes to reach the flow channel chamber 1 through the flow channel 3 and the time T2 it takes to reach the flow channel chamber 2 through the flow channel 4 is T1>T2.

[0024] <Measurement of sample concentration> Measurement of the sample concentration will be explained with reference to FIG. FIG. 3 is an image diagram showing the behavior of the sample after dispensing in Example 1. After being dispensed into the dispensing unit 6, the sample permeates the paper porous substrate S1 by capillary action according to the flow path pattern. As described above, in Example 1, the flow path 3 connected to the flow path chamber 1 containing the reference electrode 7 and the flow path 4 connected to the flow path chamber 2 containing the working electrode 8 have different configurations, and therefore the time it takes for the sample to reach each flow path chamber after being dispensed differs. In other words, the relationship between the reference electrode arrival time T1 and the working electrode arrival time T2 is T1>T2.

[0025] Furthermore, after the sample arrives at the working electrode 8, a certain amount of time is required for the measured potential of the working electrode 8 to stabilize. Furthermore, once the specimen reaches the reference electrode 7 and the measurement potential of the working electrode 8 is stable, the specimen concentration can be measured, and measurement of the specimen concentration is completed after a predetermined measurement time has elapsed. Here, when the sample reaches the reference electrode 7, the KCl ion crystals 10 begin to dissolve in the sample, and high-concentration KCl begins to flow backward through the flow path. 3, in Example 1, by setting the relationship between the reference electrode arrival time T1 and the working electrode arrival time T2 such that T1>T2, the backflow arrival time could be made later than the measurement completion time, allowing the analyte concentration measurement to be completed without the influence of the backflowing KCl.

[0026] [Comparative Example 1] To explain the effects of Example 1 in more detail, Comparative Examples 1-1 and 1-2 will be given. In Comparative Example 1-1, the cross-sectional area A1 of the flow path 3 and the cross-sectional area A2 of the flow path 4 are equal, and A1 = A2 = 0.5 mm 2 It was decided. In Comparative Example 1-2, the cross-sectional area A1 of the flow path 3 and the cross-sectional area A2 of the flow path 4 are equal, but A1=A2=0.2 mm 2 It was decided.

[0027] Here, the measurement of the specimen concentration in Comparative Example 1-1 will be described with reference to FIG. Figure 4 is an image diagram showing the behavior of the sample after dispensing in Comparative Example 1-1. In Comparative Example 1-1, the sample concentration may not be accurately measured due to the influence of backflowing KCl. This is because flow path 3 and flow path 4 have the same configuration, so the relationship between the reference electrode arrival time T1 and the working electrode arrival time T2 is T1 = T2. Compared to Example 1, the reference electrode arrival time T1 is faster, and as a result, the backflow arrival time is also faster.

[0028] Next, the measurement of the specimen concentration in Comparative Example 1-2 will be described with reference to FIG. Figure 5 is an image diagram showing the behavior of the sample after dispensing in Comparative Example 1-2. In Comparative Example 1-2, even though the relationship between the reference electrode arrival time T1 and the working electrode arrival time T2 was T1 = T2, it was possible to complete the measurement of the sample concentration before the backflow arrival time. However, the end time of the measurement of the sample concentration was delayed. This is because the start time of the measurement of the sample concentration was delayed because the sample flow rate was reduced, delaying the reference electrode arrival time T1 and the working electrode arrival time T2.

[0029] [Effects of Example 1] [Advantages of Example 1 over Comparative Example 1] The advantages of Example 1 over Comparative Examples 1-1 and 1-2 will be described. The time it takes for the sample to reach the flow channel chamber after being dispensed depends on the cross-sectional area of ​​the flow channel. The volume of each flow channel was as follows:

[0030] Example 1: The cross-sectional area of ​​the flow path between the dispensing part and the reference electrode is 0.2 mm 2 The cross-sectional area of ​​the flow path between the dispensing part and the working electrode is 0.5 mm 2 Comparative Example 1-1: The cross-sectional area of ​​the flow path between the dispensing part and the reference electrode is 0.5 mm 2 The cross-sectional area of ​​the flow path between the dispensing part and the working electrode is 0.5 mm 2 Comparative Example 1-2: The cross-sectional area of ​​the flow path between the dispensing part and the reference electrode is 0.2 mm 2 The cross-sectional area of ​​the flow path between the dispensing part and the working electrode is 0.2 mm 2

[0031] After dispensing, the flow of the sample to the working electrode and the reference electrode was measured in Example 1 and Comparative Example 1. 2 If so, it takes about 50 seconds, and the cross-sectional area is 0.5 mm 2 In that case, approximately 20 seconds would be required. Furthermore, after the sample reached the ion selective membrane 9, it took about 45 seconds for the measured potential of the working electrode 8 to stabilize. Furthermore, once the specimen reaches the reference electrode 7 and the measurement potential of the working electrode 8 is stable, the specimen concentration can be measured, and it takes 50 seconds from the start to the end of the measurement. The time required for KCl to flow backward is 2 It takes about 75 seconds, and the cross-sectional area of ​​the flow path is 0.5 mm 2In this case, it takes about 30 seconds.

[0032] Therefore, in Example 1, the time it takes for the liquid to reach the reference electrode 7 from the dispensing section 6 is 0.2 mm 2 After reaching the reference electrode 7, it took about 50 seconds for the KCl to flow back to the dispensing part 6. 2 The time required for the solution to flow back from the dispensing section to the working electrode 8 was about 75 seconds. 2 Therefore, it took about 30 seconds. That is, in Example 1, after the sample was dispensed, it took about 155 seconds for KCl to flow back to the working electrode 8. Similarly, the time required for the backflow to reach the working electrode 8 in Comparative Example 1-1 was about 80 seconds, and in Comparative Example 1-2 it was about 200 seconds. Table 1 shows the measurement end time, backflow arrival time, etc. in Example 1 and Comparative Examples 1-1 and 1-2.

[0033] [Table 1]

[0034] Table 1 shows the results of dispensing the specimen in the configurations of Example 1 and Comparative Example 1. The time it takes for the sample to reach each electrode ("reference electrode arrival time" and "working electrode arrival time"), The time until the sample concentration measurement can begin ("measurement start time"), The time until the measurement can be completed ("measurement completion time"), The time it takes for the ionic crystal KCl to reach the working electrode through the backflow of the sample ("backflow arrival time"); The time elapsed from the "measurement start time" to the "backflow arrival time" ("measurable time") is expressed in units of seconds.

[0035] In Example 1, the measurement was completed in 115 seconds before the ionic crystal KCl flowed back to the working electrode. On the other hand, in Comparative Example 1-1, KCl reached the working electrode before the measurement was completed, and therefore the ion concentration of the sample could not be measured accurately. In Comparative Example 1-2, the measurement could be completed before KCl reached the working electrode, but it took 30 seconds longer than in Example 1 to complete the measurement. The above evaluation confirmed the superiority of Example 1 over Comparative Examples 1-1 and 1-2.

[0036] [Example 2] The micro-analysis chip P2 according to Example 2 will be described. Here, the dispensing section 6, flow channel chamber 1, flow channel chamber 2, reference electrode 7, working electrode 8, ion selective membrane 9, and ion crystal 10 have the same configuration as the micro-analysis chip P1, and therefore their description will be omitted. Hereinafter, parts common to the micro-analysis chip P1 will be described using the common part names.

[0037] <Flow path configuration> The flow path pattern of the micro-analysis chip P2 will be described. FIG. 6 is a simplified top view of the micro-analysis chip P2. The features of the second embodiment are: The flow channel volume of flow channel 3 is V1, When the flow channel volume of flow channel 4 is V2, V1 and V2 satisfy the relationship V1>V2.

[0038] for example, The channel width of channel 3 is W1, the channel depth is DP1, the channel length is L1, and the channel volume is V1. When the channel width of channel 4 is W2, the channel depth is DP2, the channel length is L2, and the channel volume is V2, V1=W1×DP1×L1 V2=W2×DP2×L2 The relationship is established, W1×DP1×L1>W2×DP2×L2 The following relationship is established.

[0039] An example of the micro-analysis chip P2 will be specifically described with reference to FIG. The channel length L1 of channel 3 was longer than the channel length L2 of channel 4, L1 = 12 mm and L2 = 6 mm, respectively. The channel widths W1 and W2 were equal, W1 = W2 = 5 mm, respectively. That is, the flow path volume V1 of the flow path 3 is DP1 × W1 × L1 = 6 mm 3 , the flow channel volume V2 of flow channel 4 is DP2 × W2 × L2 = 3 mm 3 This results in a relationship of V1>V2. In this embodiment, the flow path volume is adjusted by the flow path length, but this is not limiting. The relationship of the flow path volumes may be V1>V2, for example, by making the flow path lengths L1 and L2 equal, and the flow path widths W1 and W2 equal, and setting the depth DP1 of flow path 3 to be deeper than the depth DP2 of flow path 4, thereby making V1>V2.

[0040] Comparative Example 2 To explain the effects of Example 2 in more detail, Comparative Examples 2-1 and 2-2 will be given. Both Comparative Example 2-1 and Comparative Example 2-2 were the same as Example 2 except for the following points. In Comparative Example 2-1, the flow channel volume V1 of flow channel 3 and the flow channel volume V2 of flow channel 4 are equal, and V1 = V2 = 3 mm 3 It was decided. In Comparative Example 2-2, the flow path volume V1 of flow path 3 and the flow path volume V2 of flow path 4 are equal, but V1 = V2 = 6 mm 3 It was decided. [Effects of Example 2] [Advantages of Example 2 over Comparative Example 2] The advantages of Example 2 over Comparative Examples 2-1 and 2-2 will be described. The time it takes for the sample to reach the flow channel chamber after being dispensed depends on the flow channel volume. The volume of each flow channel was as follows:

[0041] Example 2: The flow path volume between the dispensing part and the reference electrode is 12 mm 3 The flow path volume between the dispensing part and the working electrode is 6 mm 3 Comparative Example 2-1: The flow path volume between the dispensing part and the reference electrode is 6 mm 3 The flow path volume between the dispensing part and the working electrode is 6 mm 3 Comparative Example 2-2: The flow path volume between the dispensing part and the reference electrode is 12 mm 3 The flow path volume between the dispensing part and the working electrode is 12 mm 3

[0042] After dispensing, the flow of the sample to the working electrode and reference electrode was measured with a flow channel volume of 6 mm 3 If so, it takes about 20 seconds, and the flow path volume is 12 mm 3 In that case, approximately 40 seconds would be required. After the sample reached the working electrode, it took 45 seconds for the measured potential to stabilize, and the time required for measurement was 50 seconds. The time required for KCl to flow backward is 3 It takes about 30 seconds, and the flow path volume is 12 mm 3 In this case, it takes about 60 seconds.

[0043] Therefore, in Example 2, the backflow arrival time was 40 seconds from the dispensing part to the reference electrode, 60 seconds from the reference electrode to the dispensing part, and 30 seconds from the dispensing part to the working electrode. The total is 130 seconds. Table 2 shows the measurement end time, backflow arrival time, etc. in Example 2 and Comparative Examples 2-1 to 2-2.

[0044] [Table 2]

[0045] Table 2 shows the results of dispensing the specimen in the configurations of Example 2 and Comparative Example 2. The time it takes for the sample to reach each electrode ("reference electrode arrival time" and "working electrode arrival time"), The time until the sample concentration measurement can begin ("measurement start time"), The time until the measurement can be completed ("measurement completion time"), The time it takes for the ionic crystal KCl to reach the working electrode through the backflow of the sample ("backflow arrival time"); The time elapsed from the "measurement start time" to the "backflow arrival time" ("measurable time") is expressed in units of seconds.

[0046] In Example 2, the measurement could be completed in 115 seconds before the ionic crystal KCl flowed back to the working electrode. On the other hand, in Comparative Example 2-1, KCl reached the working electrode before the measurement was completed, and therefore the ion concentration of the sample could not be measured accurately. In addition, in Comparative Example 2-2, the measurement could be completed before KCl reached the working electrode, but it took 20 seconds longer to complete the measurement than in Example 2, and the size of the microanalysis chip was larger to ensure the flow path volume. The above evaluation confirmed the superiority of Example 2 over Comparative Examples 2-1 and 2-2.

[0047] [Example 3] A micro-analysis chip P3 according to Example 3 will be described. Here, the dispensing section 6, flow channel chamber 1, flow channel chamber 2, reference electrode 7, working electrode 8, ion selective membrane 9, and ion crystal 10 have the same configuration as the micro-analysis chip P1, and therefore their description will be omitted. Hereinafter, parts that are common to the micro-analysis chip P1 will be described using the common part names.

[0048] <Flow path configuration> The flow path pattern of the micro-analysis chip P3 will be described. The third embodiment is characterized in that minute hydrophobic resin regions are formed in the shape of dots inside the flow channel 3, and the formed hydrophobic resin regions inhibit the penetration of the specimen. Formed in a dot pattern means that a plurality of hydrophobic resin regions are arranged apart from one another, rather than being concentrated in one place. In the third embodiment, no hydrophobic resin region is formed inside the flow channel 4, but a hydrophobic resin region may be formed inside the flow channel 4.

[0049] The shape of the hydrophobic resin region is not limited to a sphere, but may be a rectangular parallelepiped, a cube, or the like.

[0050] FIG. 7 is a simplified top view of a micro-analysis chip P3 in which no hydrophobic resin region is formed inside the flow channel 4. In FIG.

[0051] An example of a micro-analysis chip P3 in which no hydrophobic resin region is formed inside the flow channel 4 will be specifically described with reference to FIG. A hydrophobic resin cube was formed as a hydrophobic resin region inside the flow channel 3. The hydrophobic resin cube (width 0.1 mm × length 0.1 mm × depth 0.1 mm = volume 0.001 mm) 3 ) was taken as one unit (piece), and such hydrophobic resin cubes 8 (pieces) were arranged in a staggered pattern as shown in FIG. On the other hand, no hydrophobic resin region was formed inside the flow channel 4.

[0052] As described above, in this embodiment, the hydrophobic resin region is formed only in the flow channel 3, but this is not limiting. The hydrophobic resin region may also be formed in the flow channel 4. In this embodiment, when forming the flow path walls 5, the hydrophobic resin is placed on the paper porous substrate S1 at the locations corresponding to the flow paths, and then thermally fixed to form the hydrophobic resin regions, but this is not limiting. For example, after forming the flow path walls 5, part or all of the hydrophobic resin regions may be formed using a wax printer or the like.

[0053] Comparative Example 3 To explain the effects of Example 3 in more detail, Comparative Examples 3-1 and 3-2 will be given. Both Comparative Examples 3-1 and 3-2 were the same as Example 3 except for the following points. In Comparative Example 3-1, no hydrophobic resin region is formed inside the flow channels 3 and 4. In addition, in the flow channel 3 and the flow channel 4 in Comparative Example 3-2, hydrophobic resin cubes 8 (pieces) are formed in the same manner as in the flow channel 3 in Example 3.

[0054] [Effects of Example 3] [Advantages of Example 3 over Comparative Example 3] The advantages of Example 3 over Comparative Examples 3-1 and 3-2 will be described. The hydrophobic resin region formed inside the channel inhibits the permeation of the sample, that is, the speed of the sample flowing through the channel can be adjusted by the hydrophobic resin region inside the channel. After dispensing, the flow of the sample to the working electrode and reference electrode took approximately 20 seconds when there were 0 hydrophobic resin cubes, and approximately 80 seconds when there were 8 hydrophobic resin cubes.

[0055] After the sample reached the working electrode, it took 45 seconds for the measured potential to stabilize, and the time required for measurement was 50 seconds. In Example 3 and Comparative Example 3-2, measurement became possible when the sample reached the reference electrode. Furthermore, the time required for KCl to flow backward through the flow path was approximately 30 seconds if there were no hydrophobic resin cubes, and approximately 120 seconds if there were eight hydrophobic resin cubes.

[0056] Therefore, in Example 3, the backflow arrival time was 80 seconds from the dispensing part to the reference electrode, 120 seconds from the reference electrode to the dispensing part, and 30 seconds from the dispensing part to the working electrode. The total is 230 seconds. Table 3 shows the measurement end time, backflow arrival time, etc. in Example 3 and Comparative Examples 3-1 and 3-2.

[0057] [Table 3]

[0058] Table 3 shows the results of dispensing the specimen in the configurations of Example 3 and Comparative Example 3. The time it takes for the sample to reach each electrode ("reference electrode arrival time" and "working electrode arrival time"), The time required for the measured potential of the working electrode 8 to stabilize, The time until the sample concentration measurement can begin ("measurement start time"), The time until the measurement can be completed ("measurement completion time"), The time it takes for the ionic crystal KCl to reach the working electrode through the backflow of the sample ("backflow arrival time"); The time elapsed from the "measurement start time" to the "backflow arrival time" ("measurable time") is expressed in units of seconds.

[0059] In Example 3, the measurement could be completed in 130 seconds before the ionic crystal KCl flowed back to the working electrode. On the other hand, in Comparative Example 3-1, KCl reached the working electrode before the measurement was completed, and therefore the ion concentration of the sample could not be measured accurately. In Comparative Example 3-2, the measurement could be completed before KCl reached the working electrode, but it took 45 seconds longer than in Example 3 to complete the measurement. The above evaluation confirmed the superiority of Example 3 over Comparative Examples 3-1 and 3-2.

[0060] [Example 4] A micro-analysis chip P4 according to Example 4 will be described. Here, the dispensing unit 6, flow channel chamber 1, flow channel chamber 2, reference electrode 7, working electrode 8, ion selective membrane 9, and ion crystal 10 have the same configuration as those of the micro-analysis chip P1, and therefore their description will be omitted. "Similar configuration" means that the individual components such as the dispensing unit 6, flow channel chamber 1, and flow channel chamber 2 are the same, but does not mean that the relative positional relationships of the individual components are also the same. Hereinafter, parts common to the micro-analysis chip P1 will be explained using the common part names.

[0061] <Anisotropic penetration> The microanalysis chip P4 was formed on a porous paper substrate S2 having the following permeation anisotropy. The permeation anisotropy of the paper porous substrate S2 will be described with reference to FIG. In FIGS. 9(a) to 9(c), the X-axis, Y-axis, and Z-axis intersect perpendicularly to one another. Figure 9(a) shows the XY plane parallel to the top surface of the paper porous substrate S2, Figure 9(b) shows the XZ plane (BB cross section) perpendicular to the XY plane, and Figure 9(c) shows the YZ plane (CC cross section) perpendicular to the XY plane. As shown in FIG. 9, when a sample is dispensed into a porous substrate on which no flow path walls are formed, the dispensed sample forms an elliptical penetration shape 91 on the surface of the porous substrate. The long axis direction (X axis direction) of the ellipse coincides with the fiber direction F1.

[0062] When a predetermined time has elapsed since the sample T was dispensed at the point (X, Y) = (0, 0) shown in Figure 9(a), the penetration shape 91 of the sample T became a shape that approximated a shape obtained by cutting an ellipsoid in half along its major axis, as shown in Figures 9(a) to 9(c). An ellipsoid is a body of revolution obtained by rotating an ellipse around its major axis.

[0063] <Direction of specimen movement> When a predetermined time has elapsed since the sample T was dispensed at the point (X, Y) = (0, 0) shown in Figure 9(a), the maximum value in the X-axis direction of the outer periphery of the penetration shape 91 of the sample T is set to X1, the maximum value in the Y-axis direction is set to Y1, and the maximum value in the Z-axis direction is set to Z1, as shown in Figures 9(a) to (c). Then, the "direction of the vector" extending from the dispensing point (X,Y,Z)=(0,0,0) to the point (X,Y,Z)=(X1,Y1,Z1) in the XYZ space can be defined as the "direction of travel of the specimen T."

[0064] However, the maximum value that Z1 can take is the flow path depth DP. If the channel depth DP is equal to the thickness DP1 of the porous substrate, the maximum value that Z1 can have is the thickness DL1 of the porous substrate. Therefore, when the thickness of the porous substrate is thin compared to the length or width of the flow path, several seconds after the sample T is dispensed, the "direction of travel of the sample T" will mean the "direction in which the sample T travels parallel to the XY plane." Thereafter, the specimen T reaches the wall surface of the channel and begins to spread along the wall surface of the channel, and the "direction of travel of the specimen T" becomes parallel to the wall surface of the channel.

[0065] The features of the fourth embodiment will be described with reference to Fig. 8. The features of the fourth embodiment are as follows. an angle formed by a major axis direction of the ellipse and a flow path wall direction parallel to a first flow path first wall surface 3 a that defines the flow path 3 (first flow path); The arithmetic mean value of the angle between the major axis direction of the ellipse and the flow path wall direction parallel to the first flow path second wall surface 3b that defines the flow path 3 (first flow path) is defined as the average angle r1 (0°≦r1≦90°). an angle formed by a major axis direction of the ellipse and a flow path wall direction parallel to a second flow path first wall surface 4a that defines the flow path 4 (second flow path); The arithmetic mean value of the angle between the major axis direction of the ellipse and the flow path wall direction parallel to the second flow path second wall surface 4b that defines the flow path 4 (second flow path) is defined as the average angle r2 (0°≦r2≦90°). The fourth embodiment is characterized in that r1 and r2 satisfy the relationship r1>r2.

[0066] <Porous substrate used in Example 4> The penetration speed in the long axis (X axis) direction shown in Figure 9 is V X The penetration speed in the first minor axis (Y axis) direction is V Y The penetration velocity in the second minor axis (Z axis) direction is V Z year, The first permeability ratio E1 in the direction of the major and minor axes is E1 = (V X / V Y )year, The second permeability ratio E2 in the direction of the major and minor axes is E2 = (V X / V Z ), The primary permeability ratio E1 in the direction of the major and minor axes is 2, The second permeability ratio E2 in the direction of the major and minor axes was also 2. The thickness DL1 was 0.1 mm and the porosity ε1 was 50%.

[0067] However, the material of the paper porous substrate S2 is not limited to paper, and resin, glass, inorganic substrate, fabric, metal paper, etc. may also be used. It is sufficient if the porous substrate has a mesh structure such as open cells or nanofibers inside, generates capillary action in liquid, and has the permeation anisotropy described above. Furthermore, the permeation ratio E1, thickness DL1, and porosity ε1 of the paper porous substrate S2 are not limited to the values ​​described above.

[0068] <Flow path configuration> The flow path pattern of the microanalysis chip P4 will be described. FIG. 8 is a simplified top view of an example of the microanalysis chip P4. The feature of Example 4 is that when the fiber direction F1 is 0°, the average angle of flow path 3 is r1 (0°≦r1≦90°) and the average angle of flow path 4 is r2 (0°≦r2≦90°), then r1>r2. As described above, the fiber direction F1 is the same as the long axis direction of the elliptical penetration shape formed on the surface of the porous substrate by the sample dispensed into the porous substrate. As an example of Example 4, a flow path pattern was created with the average angle of flow path 3 set to r1=90° and the average angle of flow path 4 set to r2=0°.

[0069] Comparative Example 4 To explain the effects of Example 4 in more detail, Comparative Examples 4-1 and 4-2 will be given. Both Comparative Examples 4-1 and 4-2 were the same as Example 4 except for the following points. In Comparative Example 4-1, the average angle r1 of the flow channel 3 and the average angle r2 of the flow channel 4 were equal, with r1=r2=0°. In Comparative Example 4-2, r1 and r2 were also equal, but r1=r2=90°.

[0070] [Effects of Example 4] [Advantages of Example 4 over Comparative Example 4] The advantages of Example 4 over Comparative Examples 4-1 and 4-2 will be described. The time it takes for a sample to reach the flow channel chamber after being dispensed depends on the average angle of the flow channel relative to the fiber direction, even if the flow channel volume is the same. As mentioned above, in this example, a flow channel pattern was formed on a porous paper substrate S2 with permeation anisotropy, with a permeation ratio E1 in the major axis and minor axis directions being twice as large. In this case, if the fiber direction F1 is set to 0°, the time it takes for the sample to travel the same distance in the 90° direction will be twice as long as the time it takes to travel in the 0° direction.

[0071] Example 4: The average angle of the flow path between the dispensing part and the reference electrode is 90°. The average angle of the flow path between the dispensing part and the working electrode is 0°. Comparative Example 3-1: The average angle of the flow path between the dispensing part and the reference electrode is 0°. The average angle of the flow path between the dispensing part and the working electrode is 0°. Comparative Example 3-2: The average angle of the flow path between the dispensing part and the reference electrode is 90°. The average angle of the flow path between the dispensing part and the working electrode is 90°.

[0072] After dispensing, the flow of the sample to the working electrode and reference electrode took approximately 20 seconds from the time the sample was dispensed until it reached the flow channel chamber if the average angle of the flow channel was 0°, and approximately 40 seconds if the average angle of the flow channel was 90°. After the sample reached the working electrode, it took 45 seconds for the measured potential to stabilize, and the time required for measurement was 50 seconds. Furthermore, the time required for KCl to flow backward through the flow channel is approximately 30 seconds if the average angle of the flow channel is 0°, and approximately 60 seconds if the average angle of the flow channel is 90°.

[0073] Therefore, in Example 4, the backflow arrival time was 40 seconds from the dispensing part to the reference electrode, 60 seconds from the reference electrode to the dispensing part, and 30 seconds from the dispensing part to the working electrode. The total is 130 seconds. Table 4 shows the measurement end time, backflow arrival time, etc. in Example 4 and Comparative Examples 4-1 and 4-2.

[0074] [Table 4]

[0075] Table 4 shows the results of dispensing the specimen in the configurations of Example 4 and Comparative Example 4. The time it takes for the sample to reach each electrode ("reference electrode arrival time" and "working electrode arrival time"), The time required for the measured potential of the working electrode 8 to stabilize, The time until the sample concentration measurement can begin ("measurement start time"), The time until the measurement can be completed ("measurement completion time"), The time it takes for the ionic crystal KCl to reach the working electrode through the backflow of the sample ("backflow arrival time"); The time elapsed from the "measurement start time" to the "backflow arrival time" ("measurable time") is expressed in units of seconds.

[0076] In Example 4, the measurement could be completed in 115 seconds before the ionic crystal KCl flowed back to the working electrode. On the other hand, in Comparative Example 4-1, KCl reached the working electrode before the measurement was completed, so that the ion concentration of the sample could not be measured accurately. In Comparative Example 4-2, the measurement could be completed before KCl reached the working electrode, but it took a longer time, 20 seconds, to complete the measurement than in Example 1. The above evaluation confirmed the superiority of Example 4 over Comparative Examples 4-1 and 4-2. [Explanation of symbols]

[0077] 1. Flow path chamber including reference electrode 7 2. Flow path chamber including working electrode 8 3... A flow path connecting the dispensing unit 6 to the flow path chamber 1 4... A flow path connecting the dispensing unit 6 to the flow path chamber 2 5. Channel wall 6. Dispensing section 7‥Reference electrode 8‥Working electrode 9. Ion-selective membrane 10. Ionic crystals containing Cl ions 11. Hydrophobic resin pattern 12...Connection between flow channel chamber 1 and flow channel 3 S1,S2‥Porous base material F1: Long axis direction of pores in anisotropic porous substrate DL1: Thickness of porous substrate

Claims

1. A microanalysis chip in which a dispensing section for dispensing a specimen, a first flow path chamber, a second flow path chamber, a first flow path connecting the dispensing section and the first flow path chamber, and a second flow path connecting the dispensing section and the second flow path chamber are formed by flow path walls provided inside a porous substrate, a reference electrode is disposed in the first flow channel chamber, and analyte-soluble ionic crystals are disposed on a surface of the reference electrode; A working electrode is disposed in the second flow channel chamber, A microanalysis chip characterized in that, when the time it takes for the sample to reach the ionic crystal after being dispensed into the dispenser is T1 and the time it takes for the sample to reach the working electrode is T2, the first flow path and the second flow path are configured so that T1 and T2 satisfy the relationship T1 > T2.

2. A flow path cross-sectional area of ​​the first flow path is A1, When the flow path cross-sectional area of ​​the second flow path is A2, 2. The microanalysis chip according to claim 1, wherein the relationship between A1 and A2 is A1<A2.

3. The flow path volume of the first flow path is V1, When the flow path volume of the second flow path is V2, 2. The microanalysis chip according to claim 1, wherein the relationship between V1 and V2 is V1>V2.

4. 2. The microchip according to claim 1, wherein a hydrophobic resin region is formed in a dot pattern inside the first flow channel.

5. The porous substrate has permeation anisotropy, when a sample is dispensed into the porous substrate on which the flow path wall is not formed, a penetration shape formed on the surface of the porous substrate by the dispensed sample is elliptical; an angle formed by a major axis direction of the ellipse and a flow path wall surface direction parallel to a first flow path first wall surface defining the first flow path; The arithmetic mean value of the angle between the major axis direction of the ellipse and the flow path wall surface direction parallel to the first flow path second wall surface defining the first flow path is defined as an average angle r1 (0°≦r1≦90°), an angle formed by a major axis direction of the ellipse and a flow path wall direction parallel to a second flow path first wall surface defining the second flow path; When the arithmetic mean value of the angle between the major axis direction of the ellipse and the flow path wall surface direction parallel to the second flow path second wall surface defining the second flow path is defined as an average angle r2 (0°≦r2≦90°), 2. The microanalysis chip according to claim 1, wherein the relationship between r1 and r2 is r1>r2.

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

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