JP4608696B2 - Electrode for electrochemical measurement, and sensor for electrochemical measurement using the same - Google Patents

Description

  The present invention relates to an electrode used for detecting an electrochemical reaction of a test solution, and a two-electrode sensor for measuring an electrochemical reaction using the electrode.

Generally known is a method of applying an Ag / AgCl paste acting as a reference electrode on a conductive material of a counter electrode on a conductive material of a counter electrode in a two-electrode sensor composed of a working electrode and an electrode serving as a reference electrode and a counter electrode. ing.
Patent publication gazette JP, 2001-242115, A Analyca Chimica Acta 433 (2001) pp. 81-88 Surface Science Vol. 24, no. 11.2003.9-14 pages

  As seen in the above document, electrochemical measurement of a test solution using a sensor in which a paste material of Ag / AgCl is applied to the entire surface of a conductive material as a prototype of the counter electrode and a working electrode combined therewith. However, when measurement is performed using such an electrode or sensor, a large variation occurs in output, and it is difficult to obtain a stable measurement result.

In electrochemical measurements, the amount of test solution is often very small, and the response is weak, and it is easily affected by electrical noise from the external environment. Remarkably impair the performance.
In addition, for example, in the detection of a DNA reaction, it is said that a plurality of measurements cannot be performed with the same electrode and the sensor, but in this case, variations in output value have a fatal effect on the reliability of the detection result. give.

  Electrochemical reaction detection electrodes or sensors using the same are often used as disposable types, and it is desirable that the amount of highly corrosive Ag / AgCl used be as small as possible from the viewpoint of environmental impact.

  The present invention provides a counter electrode in an electrode having an ohmic contact with an Ag / AgCl mixture acting as a reference electrode on a conductive material acting as a counter electrode, and a two-electrode sensor using the same. The above-mentioned problem is to be solved by disposing Ag / AgCl in ohmic contact with a smaller area than the surface of the conductive material.

By arranging Ag / AgCl so that the area where the counter electrode and the counter electrode form an ohmic contact is smaller than the area of the counter electrode, Ag / AgCl is arranged over the entire surface of the conductive material as the prototype of the counter electrode. Compared with the case, the variation in output is reduced, and the output size is not affected.
In addition, the amount of relatively expensive Ag / AgCl used can be reduced, so that the production cost can be reduced.
Moreover, this kind of electrode and a sensor using the same are often used in a disposable type, and the fact that the amount of highly corrosive Ag / AgCl used can be suppressed has an effect of preventing pollution.
Furthermore, experimental results have been obtained that the electrode or sensor of the present invention can withstand multiple uses.

A general sensor used for electrochemical measurement and a measurement method using the same will be described with reference to FIG.
In FIG. 7, reference numeral 71 denotes a reaction cell, and the reaction cell 71 includes a container 72 and a sensor 73. The sensor 73 is formed by forming a working electrode 73b and a counter electrode 73c on an insulating substrate 73a. The sensor 73 is inserted into the socket 74 at the base, and is connected to an external measuring device by a lead wire 75 connected to the socket 74.
When the container 72 is now filled with the test solution 76 and the sensor 73 is immersed in the test solution 76 in the direction of the arrow, an electrical signal corresponding to the state of the test solution 76 can be detected.

FIG. 1 is a plan view showing an example of a sensor using the electrode of the present invention.
Reference numeral 11 denotes a substrate which is made of a material that does not elute into the solution and has an insulating property, such as glass epoxy, Teflon, polyimide, BT resin, mylar, PET, and ceramic.
A working electrode 12 made of a conductive material is formed on the surface of the substrate 11 by a known method such as etching or printing.
A counter electrode 13 is formed on the outside of the working electrode 12 by the same method so as to surround the working electrode 12.
The counter electrode 13 does not necessarily have to have a shape surrounding the working electrode 12, and may be, for example, a parallel electrode or a comb-shaped electrode. However, as shown in the illustrated example, It is known that if the pole is circular, forming it in an arc shape surrounding the pole can provide a stable output with a high resistance to electrical noise.
Depending on the test solution, from the viewpoint of noise resistance, the arrangement of the working electrode and the counter electrode may be reversed from FIG.

14 is an external connection wiring for the working electrode 12, and 15 is an external connection wiring for the counter electrode 13, which is formed of a conductive material on the substrate 11 in the same manner as the working electrode 12 and the counter electrode 13.
Each of the external connection wires 14 and 15 has one end connected to the working electrode 12 and the counter electrode 13, and the other end extended to the end portions 14 a and 15 a of the substrate. The end portions 14 a and 15 a are connected to sockets (not shown). ) Can be connected to an external circuit by being respectively inserted into the positive electrode and the negative electrode.

  The entire surface of the substrate 11 or the surface on which the electrodes are formed, except for designated portions such as the working electrode 12, the counter electrode 13, and the end portions 14 a and 15 a of the external connection wiring, and the like. Part), when immersed in the test solution, only the working electrode 12 and the counter electrode 13 are exposed to the liquid.

In the sensor of the present invention, the reference electrode 17 is formed on a part of the surface of the counter electrode 13 with an Ag / AgCl paste so as to have an ohmic contact.
The position where the reference electrode 17 is formed may be anywhere on the counter electrode as long as it is exposed to the surface of the counter electrode, but it is important that the area of the portion having the ohmic contact is smaller than the area of the counter electrode.
The shape of the ohmic contact portion may be any shape, and may be a square shape, a circular shape, or a lattice shape.

FIG. 6 is a diagram showing an example in which an Ag / AgCl reference electrode is formed on a part of the surface of the counter electrode so as to have an ohmic contact.
6A to 6C, reference numeral 64 denotes an insulating substrate, 63 denotes a counter electrode of a conductive material formed on the substrate 64, 61 denotes an insulating film, and 62 denotes an Ag / AgCl reference electrode.

The example of FIG. 6 a is an example in which Ag / AgCl is directly applied to a portion of the counter electrode 63 that is not covered with the insulating film 61.
In this case, the area of the portion where the reference electrode (Ag / AgCl) 62 is in contact with the counter electrode 63 forms an ohmic contact as it is.

  In the example of FIG. 6b, since the reference electrode (Ag / AgCl) 62 is applied so as to partially overlap the insulating film 61, the reference electrode (Ag / AgCl) 62 forms an ohmic contact with the counter electrode 63. The area of the portion in direct contact with the counter electrode 63 is obtained.

The example of FIG. 6C is an example in which a gap is provided in advance in the insulating film 61 and a reference electrode (Ag / AgCl) is applied thereon.
Also in this case, the reason why the reference electrode (Ag / AgCl) 62 forms an ohmic contact with the counter electrode 63 is limited to the area in contact with the counter electrode 13.

In the sensor of the present invention shown in FIG. 1, the characteristics of the present invention are best represented by the Ag / AgCl so that an ohmic contact is formed on the counter electrode 13, that is, on the surface thereof with an area smaller than the area of the counter electrode 13. This is a portion of the counter electrode 13 provided with the reference electrode 17.
Therefore, the working electrode and the counter electrode do not necessarily have to be formed on the same substrate, and it is natural that the effect is sufficiently exhibited even if the substrate is separated into the working electrode portion and the counter electrode portion.
It is also clear that the working electrode and the counter electrode need not be flat as long as the area of the counter electrode and the area of the ohmic contact of the reference electrode of Ag / AgCl formed thereon satisfy the above conditions.

FIG. 2 shows an example of a counter electrode formed in a form separated from the working electrode.
In FIG. 2, 21 indicates a counter electrode. The counter electrode 21 is made by applying a conductive material such as carbon or silver or a conductive material such as carbon or silver on an insulator.
A socket or handle 22 is provided on one end side of the counter electrode 21 and holds the counter electrode, and its output is connected to an external measuring device (not shown) by a lead wire 23.
Reference numeral 24 denotes an insulating layer for the connection boundary, which serves as an indicator that it is appropriate to immerse the test solution liquid surface up to this portion.

In the electrode of the present invention, an Ag / AgCl reference electrode 25 is applied to the surface of the counter electrode 21 so as to form an ohmic contact with an area smaller than the surface area of the counter electrode 21.
FIG. 2 shows an example in which the reference electrode 25 is attached to the tip of the counter electrode, but the position is not limited to the tip.

  The same effect as that of the sensor described in Example 1 can be obtained by immersing the counter electrode having the above-described configuration in the test solution while simultaneously immersing the working electrode having an appropriate shape in the test solution.

The reason why it is important that the area of the reference electrode having the ohmic contact on the counter electrode is smaller than the area of the counter electrode will be described with reference to the drawings.
FIG. 3 shows a sensor having an electrode as shown in FIG. 1, which uses an electrode sensor in which the ratio of the ohmic contact area of the Ag / AgCl reference electrode to the counter electrode area is changed stepwise from 0% to 100%. This is an example in which the peak value of current is obtained by linear sweep voltammetry (hereinafter referred to as LSV) measurement.
However, if the reference electrode and the counter electrode do not have ohmic contact in a two-electrode system, contact with water molecules present in the test solution for several tens of seconds makes it impossible to obtain an appropriate measurement output. It has been removed from.
From this figure, it can be seen that the ohmic contact area ratio (hereinafter referred to as area ratio) does not affect the output value.
Further, since there are variations due to individual electrodes, FIG. 3 shows a plurality of samples (N = 50) for each area ratio, which are represented by average values of the area ratio outputs.

  FIG. 4 shows the variation of the peak current value due to the area ratio of Ag / AgCl under the same conditions as FIG. From this figure, it can be seen that the correlation function is 0.58 and the variation in output tends to decrease as the area decreases.

  FIG. 8 shows the results of measurement of enzyme activity using the electrode A having an area ratio of ohmic contact to the counter electrode of 25%. FIG. 9 shows the electrode A used for the measurement. The electrode A is a type provided on the surface of the substrate 91 in parallel with the counter electrode 92 and the working electrode 93, and a reference electrode 94 is provided on the surface of the counter electrode 92 along one side in the parallel direction. The area ratio of the ohmic contact between the counter electrode 92 and the reference electrode 94 is 25% of the area of the counter electrode 92. The enzyme used was alkaline phosphatase (ALP) (derived from human placenta) 3800 KA / mL, the substrate was p-phenyl phosphate 1 mg / mL, and the buffer was 50 mM Tris-HCl, pH 8.0. The measurement temperature was 21 ° C. and 36 ° C., and the peak current value was measured every 2 minutes from the mixing of ALP and the substrate solution at each measurement temperature. Taking into account variations due to individual electrodes, a plurality of samples (n = 3) are prepared and measured for each of the measurement temperatures of 21 ° C. and 36 ° C., and the average value is taken as the peak current value. As can be seen from FIG. 8, the maximum variation in peak current value at each measurement point (X axis: time) is about 5.7%, and a good calibration curve is obtained at both measurement temperatures of 21 ° C. and 36 ° C. ing.

  For example, when the same measurement is performed using an electrode (conventional electrode) in which the area ratio of the ohmic contact is 100%, the electrode causes a measurement error of about 8% from the result of FIG. It is considered difficult to obtain a 21 ° C. calibration curve. Since the measurement error of the electrode of the present invention is smaller than that of a conventional electrode, the measurement error is also effective in measurement in a low temperature region where the measurement error greatly affects. Reducing the variation in peak current value in this way is very important in the measurement of biosensors and the like, and it is possible to measure substances that have been difficult to perform by the present invention.

  Next, performance evaluation was performed using the electrode B having an area ratio of the ohmic contact to the counter electrode of 3%. FIG. 10 shows an example of a sensor using the electrode B. The sensor is of the same type as that shown in FIG. 1, and an Ag / AgCl reference electrode 17 is printed on the surface of the counter electrode 13 so that the ohmic contact area ratio is 3%. The reference numerals in FIG. 10 are the same as those in FIG. Electrochemical measurement was performed by a linear sweep voltammetry (LSV) method using 60 μM-Hoechst 33258 and a sweep rate of 100 mV / sec.

FIG. 11 and Table 1 show the measurement results. When the electrode B in which the area ratio of the ohmic contact to the counter electrode was 3% was used, the peak current value was high (see FIG. 11), and a good electrochemical response was obtained. Further, when the variation of the peak current value was calculated (n = 11), the variation was very small as 2.7% (see Table 1). From the above, it can be seen that the electrode B with an area ratio of 3% shows a good value for both the peak current value and the variation, and is a very good electrode. When an electrode having a surface area ratio of Ag / AgCl of 0% was used, a good peak current value could not be obtained. Therefore, even if the reference electrode of Ag / AgCl has a very small area on the counter electrode, it can be measured sufficiently if it is in ohmic contact, and the entire surface of the counter electrode as in the conventional case can be measured with Ag / AgCl. It can be said that the performance (resolution or sensitivity) is improved compared to the type of electrode covered.

  Next, a plurality of electrodes having the same type as in FIG. 10 and having an area ratio of the ohmic contact to the counter electrode varied stepwise within a range of 0% to 6% are prepared, and the area of each ohmic contact using each electrode is prepared. The output current and peak potential at the ratio were measured. The reference electrode was formed by printing Ag / AgCl on the counter electrode surface. The measurement was performed by a linear sweep voltammetry (LSV) method using 60 μM-Hoechst 33258 and a sweep rate of 100 mV / sec.

  The measurement results are shown in FIGS. In any area ratio electrode, a sufficient output current value was exhibited, the potential tended to be stable at the peak potential, and a sufficient electrical response reaction was obtained. Further, it can be seen that an electrode having an area ratio of 1% or more is preferable because the output current value is high and the potential tends to be stable at the peak potential as compared with an electrode having an area ratio of less than 1%. Furthermore, an electrode with an area ratio of 2% or more had a higher output current value, a stable potential at the peak potential, and a good electrical response reaction compared to an electrode with an area ratio of less than 2%. . When an electrode having a surface area ratio of Ag / AgCl of 0% was used, a good peak current value could not be obtained. From the above measurement results, when the area ratio of the ohmic contact is larger than 0, the performance (resolution or sensitivity) is improved as compared with the conventional electrode in which the entire surface of the counter electrode is covered with Ag / AgCl. It can be said that. Furthermore, in the electrode of the same type as FIG. 10 used this time, it can be said that it is 1% or more, more preferably 2% or more.

  From these results, even if the reference electrode of Ag / AgCl has a very small area on the target electrode, it can be measured sufficiently if it has an ohmic contact, and the entire surface of the counter electrode as in the conventional case can be measured with Ag / AgCl. It can be said that the performance (resolution or sensitivity) is better than the electrode covered with the reference electrode or the electrode using Ag / AgCl as the counter electrode. Furthermore, the performance improves as the area ratio of the ohmic contact to the counter electrode decreases. However, when the area ratio of the ohmic contact to the counter electrode is 2% or less, a slight decrease in performance was observed. In other words, the area ratio at which the printed Ag / AgCl can sufficiently function as a reference electrode is up to about 2%. Considering the above results and the yield of electrode manufacturing, it can be seen that it is preferable to form a smaller area ratio in the range of 2% or more. In addition, when the area ratio is greater than 0% and less than 2%, the performance is inferior to that of the area ratio of 2%, but the performance is better than the conventional electrodes with an area ratio of 100%. Yes, it can be said that the effect of reducing the production cost and preventing pollution accompanying the reduction of the amount of Ag / AgCl used is high.

  In addition, when Ag / AgCl is not present at all (the area ratio of the ohmic contact is 0%, that is, Ag / AgCl is not printed from the beginning), it means a measurement system without a reference electrode. I can't get the value.

In addition, it goes without saying that the optimum area ratio of the ohmic contact varies depending on the measurement object and conditions, and the present invention does not limit the area.
For example, when it is desired to obtain a calibration curve at 36 ° C. in FIG. 8, even a conventional electrode (area ratio of ohmic contact 100%) or an electrode having a high surface area ratio can be measured. FIG. 8 shows an embodiment using an ohmic contact area ratio of 25%. However, when actually developing and selling a biosensor or the like, the number of steps and cost associated with electrode fabrication, and required performance ( Therefore, it is difficult to say that an electrode having a small Ag / AgCl surface area ratio is always effective. In other words, the present application is an invention that improves the performance (resolution or sensitivity) of the electrode as the Ag / AgCl surface area ratio decreases, and does not limit the area ratio.

The graph of FIG. 5a is an example of an LSV measurement output waveform when the heat history during processing is somewhat excessive, and the horizontal axis represents the sweep voltage (V) and the vertical axis represents the output current (μA).
FIG. 5b is an enlarged view of the rising portion (elliptical portion) in the graph of FIG. 5a.

In general, in this type of measurement, the slope of the rising portion of the output change continuously changes. However, in the example of FIG. It shows that there is an unstable region in the range of .4 to 0.55v.
At the same time, the situation changes depending on the area ratio.
That is, 100% (solid line) shows a slow and fluctuating rising edge, but 10% (dotted line) becomes steep and the inclination of the rising part has a continuous tendency.
This indicates that the potential noise factor of the electrode tends to be reduced by reducing the area of Ag / AgCl due to distortion, diffusion, etc. due to the thermal history during processing of the printed paste material. Yes.
This graph shows that the area of the ohmic contact portion of Ag / AgCl is preferably small, but it can be seen that even in the case of 80%, it is far superior to the case of 100%. Moreover, it turns out that about 20% or less is preferable.

When the ohmic contact area of Ag / AgCl constituting the reference electrode is the same (100%) with the counter electrode, the output current varies greatly, and the reason why it is not preferable is not necessarily clear. However, the reason is as follows. It seems to be due to.
Originally Ag / AgCl acts as a reference electrode to give a reference potential, but besides that, it is affected by diffusion / dispersion in Ag / AgCl, diffusion with the counter electrode, etc. due to thermal history during processing, and similarly The counter electrode and the working electrode are also affected by the thermal history and have a potential noise source, which affects the movement of electrons when a voltage is applied.
This noise-like effect cannot be ignored when handling minute currents as in electrochemical measurements.
The tendency shown in FIG. 4 shows that a large factor that increases output variation can be reduced by reducing the ohmic contact area of Ag / AgCl.
From the following results, the smaller the area ratio, the better.
From FIG. 5, the area ratio is preferably about 20% or less, but is selected within a range that takes into consideration the electrode forming process and the reproducibility of the completed electrode.
For example, when the counter electrode area is small, there is a limit to printing even smaller Ag / AgCl. In this case, the position of the Ag / AgCl pattern having a printable size is shifted as shown in FIG. It is also possible to print so as to have an area ratio.
Further, as shown in FIG. 6c, it is also possible to create a fine gap in the counter electrode using an insulating film, and to provide an ohmic contact of Ag / AgCl with the counter electrode.
In general, in detecting a DNA reaction, the electrode and the sensor can withstand only one measurement. However, the electrode and the sensor of the present invention have also been found to withstand a plurality of uses.

The top view which shows the example of the sensor of this invention. The schematic diagram which shows the example of the electrode of this invention. The graph showing the output of the electrode. The figure showing the relationship between the area of an ohmic contact and output. The figure showing an example of an output waveform when LSV measurement is performed. Fig. 5a (Ellipse part) enlarged and displayed The figure which shows the example which forms ohmic contact. The figure showing the general structure of the sensor for electrochemical measurements. The figure which graphed the result of having performed performance evaluation using the electrode which made the area ratio of ohmic contact 25%. The figure which shows the electrode whose area ratio of the ohmic contact used for the said measurement is 25%. The figure which shows the electrode utilized in the performance evaluation of an electrode when the area ratio of an ohmic contact is 3%. The figure which plotted the result of the performance evaluation of the electrode of the said area ratio 3%. The figure which plotted the relationship between the area ratio of an ohmic contact, and output current. The figure which plotted the relationship between the area ratio of an ohmic contact and peak potential.

Explanation of symbols

DESCRIPTION OF SYMBOLS 11 Board | substrate 12 Working electrode 13 Counter electrode 14 Wiring for external connection of working electrode 14a End of substrate 15 Wiring for external connection of counter electrode 15a End of substrate 16 Insulating film 17 Reference electrode 71 Reaction cell 72 Container 73 Sensor 73a Substrate 73b Working electrode 73c Counter electrode 74 Socket 75 Lead wire 76 Test solution 92 Counter electrode 93 Working electrode 94 Reference electrode

Claims (2)

An electrode used as a counter electrode for electrochemical measurement,
A conductive material, or a counter electrode formed of a conductive material on an insulator substrate;
An Ag / AgCl reference electrode formed on the counter electrode,
The counter electrode and a part or all of the reference electrode form an ohmic contact,
The electrode for electrochemical measurements, wherein an area of the ohmic contact is smaller than an area of a counter electrode. Insulator substrate,
A working electrode formed of a conductive material on the substrate;
A counter electrode formed of a conductive material on the substrate in proximity to the working electrode;
On the counter electrode, an electrochemical measurement sensor comprising: a reference electrode formed by Ag / AgCl so as to form an ohmic contact with the counter electrode;
The sensor for electrochemical measurements, wherein the area of the ohmic contact is smaller than the area of the counter electrode.

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