JP4216846B2 - Electrodes for electrochemical measurements and electrochemical measurement methods - Google Patents

Description

  The present invention relates to a microelectrochemical measurement electrode and an electrochemical measurement method, and more particularly, to a microelectrochemical measurement used in a sensor for electrochemically measuring molecules and ions in a biological sample or an environmental sample. The present invention relates to an electrode and an electrochemical measurement method.

  Conventionally, in electrochemical measurements on biological samples and environmental samples, a reference electrode is used as a standard for potential measurement and potential setting. As the reference electrode, a hydrogen electrode, a saturated calomel electrode, or a silver / silver chloride electrode is used. What is common to these electrodes is that an electrolytic solution (electrolyte solution) exists around the metal material used as the electrode. The composition of the electrolytic solution varies depending on the type of the reference electrode, but the concentration of each electrolytic solution needs to be kept constant in order to keep the surface of the metal material.

  For this reason, a saturated concentration electrolyte is used as an electrolyte present around the metal material. In the case of integrating the reference electrode together with other electrodes on a minute chip, Non-Patent Document 1 holds a small amount of electrolyte around the reference electrode on the chip. Further, a gel (electrolyte gel) in which an electrolyte is confined in a polymer may be used for the reference electrode. Non-patent document 2 is an example of a reference electrode using an electrolyte gel.

H. Suzuki, T. Hirakawa, T. Hoshi, H. Toyooka, “Micromachined sensing module for pO2, pCO2, and pH and its design optimization for practical use” Sensors & Actuators B 76, 565-572, 2001 H. Suzuki, H. Shiroishi, S. Sasaki, I. Karube, “Microfabricated Liquid Junction Ag / AgCl Reference Electrode and Its Application to a One-Chip Potentiometric Sensor” Anal. Chem., 71, 5069-5075, 1999

However, as in Non-Patent Document 2, when the electrolyte gel is fixed so as to cover the reference electrode in a minute space and the ion concentration or the like is measured, the potential response may change over time. Usually, potassium chloride at a saturated concentration is used for the internal electrolyte solution (or electrolyte gel) of the reference electrode, but the electrolyte may flow out from the electrolyte gel to the measurement sample side. Therefore, since the electrolyte solution concentration (electrolyte concentration) around the reference electrode decreases with the outflow, the electrode potential of the reference electrode becomes small. Further, the response becomes unstable when other ions are mixed into the internal electrolyte (electrolyte gel).
Therefore, in order to perform measurement stably, a device for keeping the electrolyte concentration, composition, and pH on the surface of the metal for the reference electrode constant is required.

  The present invention has been made in view of such problems, and an object of the present invention is to provide a microelectrochemical measurement electrode and an electrochemical measurement method that can operate stably even for a long time.

In order to achieve such an object, the present invention provides a substrate, a first channel formed on the substrate, through which an electrolyte solution or an acidic solution flows, and the first flow. A reference electrode formed in a channel, formed on the substrate, and is a channel separate from the first channel, and is formed on the substrate, a second channel through which a measurement sample flows, and the substrate A flow path separate from the first and second flow paths, including a third flow path through which the electrolyte solution or acidic solution flows, and a detection electrode formed in the third flow path, The first flow path and the second flow path, and the second flow path and the third flow path are each electrically connected by a liquid junction containing an electrolyte .

According to a second aspect of the present invention, in the first aspect of the present invention, the detection electrode is an electrode for detecting ions.

According to a third aspect of the present invention, in the second aspect of the present invention, the electrode for detecting ions includes a polymer containing an ionophore.

The invention according to claim 4 is the invention according to any one of claims 1 to 3 , wherein the first flow path, the second flow path, and the third flow path are formed from one flow path. The flow path is divided.

The invention according to claim 5 is the invention according to any one of claims 1 to 4 , characterized in that a material of the reference electrode is any one of silver, mercury, platinum, or platinum black.

The invention described in claim 6 is the invention described in any one of claims 1 to 5 , further comprising means for supplying a gas to the reference electrode.

According to a seventh aspect of the present invention, there is provided a substrate, a first channel formed on the substrate, through which an electrolyte solution or an acidic solution flows, a reference electrode formed in the first channel, and formed on the substrate A second flow path through which the measurement sample flows, and a flow path formed on the substrate and separate from the first and second flow paths. A third flow path through which the electrolyte solution or acidic solution flows, and a detection electrode formed in the third flow path, the first flow path and the second flow path, and Each of the second flow path and the third flow path is an electrochemical measurement method in which the electrode potential of the measurement sample is measured by a microelectrochemical measurement electrode electrically connected by a liquid junction containing an electrolyte. And flowing the electrolyte solution or acidic solution through the first channel, and the second channel. A step of flowing the measurement sample and flowing the electrolyte solution or acidic solution through the third flow path; and while the electrolyte solution or acidic solution and the measurement sample are flowing, the measurement sample at the detection electrode And measuring the electrode potential.

  According to the present invention, the flow path for the reference electrode and the flow path for the measurement sample are provided separately, and the electrolyte solution or the acidic solution is allowed to flow through the flow path for the reference electrode during the measurement. But it is possible to measure stably.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. In the drawings described below, components having the same function are denoted by the same reference numerals, and repeated description thereof is omitted.
In one embodiment of the present invention, in order to keep the electrolyte concentration and pH on the surface of the metal material for the reference electrode constant, a metal material as a reference electrode is installed in the microchannel, and the electrolyte solution is placed on the surface of the metal material. Measure while flowing. At this time, depending on the composition of the electrolyte solution, there is a possibility of affecting the detection electrode for detecting a desired substance (molecule, ion, etc.) from the measurement sample. A reference electrode is formed in the flow path for flowing the electrolyte solution, separated from the flow path for flowing the sample, and a detection electrode is formed in the flow path for flowing the measurement sample. In such a configuration, during the measurement, the electrolyte solution and the measurement sample are simultaneously flowed, that is, the electrolyte solution is flowed in the flow path in which the reference electrode is formed, and the measurement sample is flowed in the flow path in which the detection electrode is formed. become. Therefore, the electrolyte solution can be supplied to the reference electrode, and the measurement sample can be supplied to the detection electrode. That is, a necessary solution can be supplied to a desired electrode.

  In one embodiment of the present invention, it is not essential to provide the detection electrode in the flow path through which the measurement sample flows, but it is important to eliminate or reduce the flow of the electrolyte between the electrolyte solution and the measurement sample. is there. Therefore, it is only necessary to separately provide a flow path through which the measurement sample flows and a flow path through which the reference electrode is disposed and the electrolyte solution flows. As described in the third embodiment, the flow path through which the measurement sample flows is provided. The detection electrode may not be provided. Therefore, there may be two or more flow paths.

  As described above, by separately providing a flow path for the reference electrode (flow path for the electrolyte solution in which the reference electrode is formed) and flowing the electrolyte solution through the flow path, the surface of the reference electrode and The surrounding electrolyte concentration can be kept constant. That is, since the reference electrode is arranged in the flow path through which the electrolyte solution flows, the electrolyte solution itself only flows from the upstream side to the downstream side of the reference electrode. Since the solution is supplied, the concentration of the surrounding electrolyte including the surface of the reference electrode can be kept uniform. In addition, since a flow path for the electrolyte solution is provided separately, it is possible to eliminate or reduce the outflow of the electrolyte and the inflow of other ions around the reference electrode.

  Furthermore, by separately providing a reference electrode channel (a channel for the electrolyte solution supplied to the reference electrode), the reference electrode electrolyte solution does not reach the detection electrode or is reduced. The influence of the electrolyte solution for the reference electrode on the detection electrode can be eliminated or reduced. Therefore, measurement accuracy can be improved.

In this embodiment, as described in the first and second examples, a reference electrode flow path and a detection electrode flow path (a flow path for a measurement sample in which a detection electrode is formed) ) May be combined on the downstream side of each electrode. By this merging, the reference electrode and the detection electrode can be electrically connected. Further, even if the reference electrode flow path and the detection electrode flow path are merged, the merge point is downstream of the reference electrode in the reference electrode flow path and is detected in the detection electrode flow path. Since it is on the downstream side of the electrode, the liquid flowing in one channel does not flow to the other channel. Therefore, only the necessary liquid flows (supplied) to the desired electrode.
In the present specification, “upstream” is upstream of the flow of liquid flowing through the flow path, and “downstream” is downstream of the flow.

In one embodiment of the present invention, it is not necessary to join a separately provided reference electrode channel and another channel such as a detection electrode channel. In this case, as described in the third embodiment, the reference electrode channel and the other channel may be electrically connected to each other through a liquid junction. For this liquid junction, an agar layer containing an electrolyte contained in the electrolyte solution to be used may be used.
In the present specification, the “liquid junction” is a portion through which only ions can pass and is formed of a polymer. Due to this liquid junction, the electrical connection between the reference electrode and the detection electrode is maintained. In this case, a discontinuous region such as a through hole may be formed in or near the region where the reference electrode is formed in the reference electrode channel, and the discontinuous region may be filled with a liquid junction.

  In one embodiment of the invention, the substrate is preferably an insulating substrate. The metal material used for the reference electrode is preferably silver, mercury, platinum, or platinum black. The electrolyte solution for the reference electrode may be determined depending on the material used for the reference electrode to be formed. When silver or mercury is used as the reference electrode, for example, a solution containing chloride ions such as a potassium chloride solution should be used. Can do. When platinum or platinum black is used as the reference electrode, for example, a solution containing chloride ions such as hydrochloric acid can be used, and hydrogen gas may be supplied. Such an electrolyte solution desirably has a saturated concentration.

  That is, in one embodiment of the present invention, an electrolyte solution is selected depending on the material of the reference electrode, and gas is supplied if necessary. In one embodiment of the present invention, an electrolyte solution is used as the liquid that flows to the reference electrode (supplied to the reference electrode). However, the purpose is to maintain a constant electrolyte concentration on the surface of the reference electrode and its surroundings. It is to be. Therefore, as long as the object can be achieved, the liquid flowing to the reference electrode is not limited to the electrolyte solution, and an acidic solution may be used.

  Hereinafter, the microelectrochemical measurement electrode and the electrochemical measurement method according to one embodiment of the present invention will be described in detail based on examples, but the present invention is not limited to the following examples, and the gist thereof Needless to say, various modifications can be made without departing from the scope of the present invention.

(First embodiment)
In this embodiment, a case where a reference electrode and a pH sensitive membrane are used in combination will be described. The structure of the flow type pH electrode according to this example is shown in FIG. The flow-type pH electrode according to this example is manufactured by bonding two insulating substrates, but FIG. 1 shows a substrate on which each electrode and a flow path are formed.

  In FIG. 1, a flow-type pH electrode 15 includes a glass substrate 1, and a silver thin film 2, an electrode pad 8a, and a wiring electrode 8b as reference electrodes are formed on the glass substrate 1, and the silver thin film 2 and the electrode pad are formed. 8a is connected via a wiring electrode 8b. Further, the glass substrate 1 is formed with a pH measuring iridium oxide thin film 3, an electrode pad 8c, and a wiring electrode 8d as detection electrodes. The iridium oxide thin film 3 and the electrode pad 8c are connected via the wiring electrode 8d. Connected.

  In addition, an insulating layer 9 is formed on the glass substrate 1 so as to form the flow paths 7a, 7b and 7c. In this embodiment, the widths and depths of the flow paths 7a to 7c are 1 mm and 100 μm, respectively. By forming each flow path in this manner, the silver thin film 2 as the reference electrode is formed in the flow path 7a as the flow path for the reference electrode. On the other hand, an iridium oxide thin film 3 for pH measurement as a detection electrode is formed in the flow path 7b as a detection electrode flow path. In FIG. 1, on the downstream side of the silver thin film 2 and the downstream side of the iridium oxide thin film 3, the flow paths 7a and 7b merge to form a flow path 7c.

  In FIG. 1, reference numeral 4 is a port position for introducing a saturated potassium chloride solution, reference numeral 5 is a port position for introducing a measurement sample, and reference numeral 6 is for discharging a measured sample. The position of the port. In this embodiment, the position 4 is located on the upstream side of the silver thin film 2, and the position 5 is located on the upstream side of the iridium oxide thin film 3. Moreover, the position 6 is located downstream of the junction of the flow path 7a and the flow path 7b.

FIG. 2 is a diagram showing a pH electrode measurement system including the flow-type pH electrode 15 shown in FIG.
In FIG. 2, the glass substrate 1 is formed with a port 17a for introducing a saturated potassium chloride solution, a port 17b for introducing a measurement sample, and a port 17c for discharging a measured sample. Are pasted together. One end of a tube 10a is connected to the port 17a, and the other end of the tube 10a is connected to a syringe 11 in which a saturated potassium chloride solution is stored. On the other hand, one end of a tube 10b is connected to the port 17b, and the other end of the tube 10b is connected to a syringe 12 in which a measurement sample is stored. The syringes 11 and 12 are provided in the syringe pump 13, and when the syringe pump 13 is driven by a pump driving means (not shown), a saturated potassium chloride solution flows from the syringe 11 through the tube 10a to the flow path from the port 17a. The sample to be measured is introduced into the flow path 7b from the port 17b through the tube 10b. One end of the tube 10c is connected to the port 17c, and the sample flowing through the flow path 7c is discharged from the port 17c to the tube 10c.
The electrode pads 8a and 8b are electrically connected to the potentiometer 14, respectively.

  When performing measurement in such a configuration, the syringe pump 13 is driven to introduce the saturated potassium chloride solution and the measurement sample from the syringes 11 and 12 into the flow paths 7a and 7b, respectively. The saturated potassium chloride solution introduced from the port 17a flows through the flow path 7a, passes through the silver thin film 2 as a reference electrode, and travels to the junction with the flow path 7b. The measurement sample introduced from the port 17b flows through the flow path 7b, passes through the iridium oxide thin film 3 as the detection electrode, and travels to the junction with the flow path 7a. The saturated potassium chloride solution and the measurement sample that have flowed to the merge point merge, flow through the flow path 7c, and are discharged from the port 17c. As described above, the liquid corresponding to the flow paths 7a to 7c flows, whereby the detection electrode and the reference electrode are electrically connected. At this time, the potentiometer 14 measures the electrode potential of the detection electrode, thereby measuring the pH of the measurement sample.

  In this embodiment, the saturated potassium chloride solution and the measurement sample are introduced at the same time, but the order of introduction is not limited to this, and saturated potassium chloride flows through the flow path 7a when measuring the potential. The measurement sample only needs to flow through the flow path 7b. Therefore, the saturated potassium chloride solution and the measurement sample may be introduced from either one, and the other may be introduced later.

  In this example, measurement was performed with the solution introduction rate set to 5 μl / min. In the measurement, the saturated potassium chloride solution and the measurement sample were flowed simultaneously. The silver thin film 2 as the reference electrode and the iridium oxide thin film 3 as the pH-sensitive application electrode are separately present in the two flow paths 7a and 7b, and the measurement is performed while flowing the solution, so that the reference electrode is contaminated. The concentration of the potassium chloride solution on the silver thin film is kept constant. A standard solution whose pH value was previously known was introduced, and potential measurement was performed. As a result, a calibration curve having a slope of about 90 mV / pH was obtained. Furthermore, when the measurement was performed for a long time, a stable potential response was obtained after 100 hours or more. This is because the potassium chloride solution concentration on the silver / silver chloride surface is kept constant by performing measurement while flowing a saturated potassium chloride solution.

(Second embodiment)
In the present embodiment, an example of a flow type pH electrode having a configuration different from that of the first embodiment is shown.
The flow type pH electrode according to the present example has the same configuration as the flow type pH electrode according to the first example, except that hydrogen gas is supplied to the reference electrode.
In FIG. 3, in this embodiment, a platinum thin film 19 is used as a reference electrode. Reference numeral 20 denotes a port position for bubbling the platinum thin film 19 as a reference electrode. Accordingly, the bubbling port is formed on the glass substrate 16 to be bonded to the flow type pH electrode 18, and the port is connected to the bubbling generator via a tube. In this embodiment, 1M (M = mol / L) hydrochloric acid is used as the electrolyte solution. Furthermore, the tube connected to each port is a tube made of a fluorine-based material.

  In the measurement in this example, as in the first example, the measurement sample and the electrolyte solution for reference electrode (hydrochloric acid solution) are flowed, and the measurement is performed while bubbling hydrogen gas on the platinum thin film 19. This electrolytic solution is preferably degassed of oxygen. When an experiment similar to that of the first example was performed, a similar measurement result was obtained.

(Third embodiment)
FIG. 4 shows the configuration of an ion measurement electrode using a reference electrode according to this example. Since the electrode for measuring ions also requires an internal electrolyte solution (electrolyte solution), a method of performing measurement while flowing the electrolyte solution can be employed as in the case of the reference electrode described above. However, since the ion electrode can include a polymer film such as polyvinyl chloride having an ion sensitive material (ionophore), leakage of the internal electrolyte does not occur. Therefore, an electrolyte gel in which saturated potassium chloride is held in a polymer such as polyvinylpyrrolidone may be used for the ion electrode.

  4, the ion measurement electrode 21 includes a glass substrate 22, and a silver thin film 23, an electrode pad 27a, and a wiring electrode 27b are formed on the glass substrate 22 as reference electrodes. The silver thin film 23 and the electrode pad 27a is connected via a wiring electrode 27b. The glass substrate 22 is formed with a silver thin film 24 for an ion electrode as a detection electrode, an electrode pad 27c, and a wiring electrode 27d. The silver thin film 24 and the electrode pad 27c are connected via the wiring electrode 27d. Has been. On the silver thin film 24 for ion electrodes, the ion sensitive film | membrane 34 containing a sodium ionophore is formed.

  In this embodiment, since sodium ions are measured, the silver thin film 24 is used as the ion electrode. When a silver thin film is used as the ion electrode, for example, potassium ions can be measured in addition to sodium ions. When measuring the hydrogen ion concentration (pH), for example, iridium oxide may be used as the ion electrode. Thus, the material of the ion electrode may be determined according to the ion to be measured.

  In addition, an insulating layer 28 is formed on such a glass substrate 1 so as to form regions that become the flow paths 25a, 25b, and 25c. In this region, the silver thin films 23 and 24 are exposed. A channel 25a is formed in the region by forming a wall 26a. A silver thin film 23 as a reference electrode is formed in the flow path 25a. Moreover, the flow path 25b and the flow path 25c are formed by forming the wall 26b. A silver thin film 24 as an ion electrode (detection electrode) is formed in the flow path 25c. For the material of the walls 26a and 26b, for example, a polymer material such as a photoresist or polyimide can be used, or glass can be used.

  In the present embodiment, any number of flow paths may be used. For example, the channel 25b may be further divided into two or more channels by a wall. In that case, an electrical connection of each flow path may be established by providing a through hole in a wall for forming the flow path and filling the through hole with a liquid junction.

  A through hole 29a is formed in the wall 26a, and an agar layer containing chloride ions as a liquid junction is formed in the through hole 29a. Similarly, a through hole 29b is formed in the wall 26b, and an agar layer containing chloride ions as a liquid junction is also formed in the through hole 29b.

  In this embodiment, since the silver thin film 23 as the reference electrode is formed in the flow path 25a, the flow path 23 becomes a flow path for the reference electrode, and a saturated potassium chloride solution, which is an electrolyte solution, flows through the flow path 25a. . In addition, a measurement sample is passed through the flow path 25b, and a saturated potassium chloride solution for the ion electrode is also passed through the flow path 25c. Since each flow path is electrically connected by the liquid junction, the reference electrode and the ion electrode can be electrically connected by flowing a corresponding liquid in each flow path.

  In FIG. 4, reference numeral 30 denotes a port position for introducing a saturated potassium chloride solution for a reference electrode, reference numeral 31 denotes a port position for introducing a measurement sample, and reference numeral 32 denotes a saturation for an ion electrode. It is the position of the port for introducing the potassium chloride solution. Reference numeral 33 denotes a port position for discharging a measured sample.

  In the present embodiment, the electrode pads 27a and 27c are electrically connected to the potentiometer 14 as in FIG. The syringe 11 is connected to the flow path 25a via the tube 10a, and the syringe 12 is connected to the flow path 25b via the tube 10b. In the present embodiment, the syringe pump 13 further includes an ion electrode syringe in which a saturated potassium chloride solution is stored, and the ion electrode syringe is connected to the flow path 25c via a tube.

  When performing measurement in such a configuration, the syringe pump 13 is driven to measure each solution from the syringes 11 and 12 and the ion electrode syringe, and to the channels 25a and 25c for the saturated potassium chloride solution. The sample is introduced into the flow path 25b. That is, measurement is performed while flowing the corresponding solution through each channel.

  In this example, the flow rate of each solution introduced into each channel was 5 μl / min. When the potential response when the sodium ion concentration was changed was measured, a good Nernst response was obtained. Moreover, it operated stably for 100 hours or more. On the ion electrode side, when the saturated potassium chloride solution was introduced and the flow path became full, the measurement was performed after the liquid supply was stopped, but the results were the same as when the measurement was performed while the solution was flowing. Obtained.

It is a top view which shows the flow type pH electrode based on the 1st Example of this invention. It is a figure which shows the pH electrode measuring system based on the 1st Example of this invention. It is a top view which shows the flow type pH electrode based on the 2nd Example of this invention. It is a top view of the electrode for ion measurement based on the 3rd Example of this invention.

Explanation of symbols

1, 22 Glass substrate 2, 23, 24 Silver thin film 3 Iridium oxide thin film 4, 5, 6, 30, 31, 32 Port position 7a, 7b, 7c, 25a, 25b, 25c Channel 8a, 8c, 27a, 27c Electrode pad 8b, 8d, 27b, 27d Wiring electrode 9, 28 Insulating layer 15, 18 Flow type pH electrode

Claims (7)

A substrate,
A first flow path formed in the substrate and through which an electrolyte solution or an acidic solution flows;
A reference electrode formed in the first flow path;
A second flow path formed on the substrate and separate from the first flow path, through which the measurement sample flows;
A third flow path formed on the substrate and separate from the first and second flow paths, through which the electrolyte solution or acidic solution flows;
A detection electrode formed in the third flow path,
The first channel and the second channel, and the second channel and the third channel are electrically connected by a liquid junction containing an electrolyte, respectively. Electrode for chemical measurement.   2. The electrode for microelectrochemical measurement according to claim 1, wherein the detection electrode is an electrode for detecting ions. The electrode for microelectrochemical measurement according to claim 2, wherein the electrode for detecting ions includes a polymer containing an ionophore. The said 1st flow path, the 2nd flow path, and the said 3rd flow path are the flow paths divided | segmented from one flow path, The Claim 1 thru | or 3 characterized by the above-mentioned. Electrode for microelectrochemical measurement. The reference electrode material, silver, mercury, platinum or platinum black micro electrochemical measurement electrode according to any of claims 1 to 4, characterized in that either,. The electrode for microelectrochemical measurement according to any one of claims 1 to 5 , further comprising means for supplying a gas to the reference electrode. A substrate, a first flow path formed in the substrate, in which an electrolyte solution or an acidic solution flows, a reference electrode formed in the first flow path, and the first flow path formed in the substrate. A second flow path through which the measurement sample flows and a flow path formed on the substrate, the first flow path and the second flow path being separate from the electrolyte solution or A third flow path through which the acidic solution flows; and a detection electrode formed in the third flow path; the first flow path and the second flow path; and the second flow path and the second flow path. Each of the three flow paths is an electrochemical measurement method in which the electrode potential of the measurement sample is measured by a microelectrochemical measurement electrode electrically connected by a liquid junction containing an electrolyte,
Flowing the electrolyte solution or acidic solution through the first flow path, flowing the measurement sample through the second flow path, and flowing the electrolyte solution or acidic solution through the third flow path;
And a step of measuring an electrode potential of the measurement sample with the detection electrode while the electrolyte solution or the acidic solution and the measurement sample are flowing.

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