JPH0623732B2 - Voltammetric measurement method - Google Patents

Description

TECHNICAL FIELD The present invention relates to a voltammetric measuring method.

(Prior Art) Conventionally, electrochemical analysis has been used as one means for measuring the concentration of ions and redox substances in a solution, and an element utilizing this principle is known. In particular, a voltammetric method and device using two conductive electrodes and one reference electrode using a potentiostat device are known. For example, when measuring hydrogen peroxide, an electric potential is applied between the working electrode and the counter electrode, which is the other metal electrode, so that the electric potential of one conductive electrode, called the working electrode, is around 0.6 V with respect to the reference electrode. It is possible to know the concentration by the flowing current (Shuichi Suzuki, “Ion and Enzyme Electrodes” p.80 Kodansha Scientific 198
1). In this case, the reference electrode is a saturated sweet koh electrode (SCE).
An electrode having a liquid junction such as a silver-silver oxide electrode having saturated KC1 as an internal liquid is generally used.

On the other hand, as a simplification of the above measurement, there is one in which a standard electrode having a liquid junction is abolished and a silver silver chloride electrode is also used as a reference electrode counter electrode and platinum is used as a working electrode,
In this case, a method has been proposed in which 0.65 V is applied between the two poles to measure the current (Fig. 2, "Basics and Applications of Sensors" symposium proceedings p43). Also, as a device using the planar technology, a device formed on a silicon wafer by working with two gold or platinum, each counter electrode and one silver, silver chloride, and reference electrode has been recently reported (W. San.
sen et al. Transducers '85, 1985 International Conference on Solid State Sensors and Actuators, Digest of Technical Papers, pp 344-347).

(Problems to be Solved by the Invention) In the case of the conventionally used three-pole voltammetric measurement method, it was necessary to use an SCE electrode or the like as a reference electrode. The reference electrode including the SCE electrode has an internal solution and a liquid junction to the solution to be measured. Therefore, miniaturization, especially the application of semiconductor planar technology, has been difficult.

Further, in the case of a sensor element having a silver-silver-chloride electrode formed by the planar technology by W. Sansen et al., There is a problem in the stability of the silver-silver-chloride electrode itself, and the potential fluctuates depending on the chlorine concentration in water. An even more important drawback is dissolution in a solution, which means that the reference electrode itself is consumed due to elution by electrolysis, or a substance is deposited on the surface of the electrode, so that it cannot be used for a long period of time. As described above, the existence of the reference electrode in the voltammetric sensor has been a major impediment to miniaturization of conventional voltammetric sensors. The present inventors have proposed a method for realizing the miniaturization of the above three-electrode voltammetry measuring method by using a microelectrode having no liquid junction and not eluting as a standard electrode.

(Means for Solving Problems) The MOS-type transistor ISFET is sensitive to pH and its output follows the Nernst's constant of about 60 mV for one digit change in concentration (edited by Jiri Janata, Robert J. Huber). Solid State Chemical Sensors ”Academic Press, 198
5, pp65-pp.118, Jili Janata Robert J. Hoover, "Solid State Chemical Sensors" Academic Press, 1985, pp65-118)). This also means that the ISFET shows a constant value under a constant pH, and the inventors have used the ISFET conventionally used as a pH measuring element as a standard electrode in a constant pH range. We have come to think that a fixed reference electrode can be provided. That is, a pair of conductive electrodes made of platinum or gold and an ISFET whose potential was measured in advance at a constant pH were prepared, and the working counter electrode was referenced based on the ISFET output so that the working electrode receives a predetermined potential from the standard potential. A voltammetric measurement method can be realized by applying an electric potential between them and measuring the current flowing therethrough.

(Operation) FIG. 1 shows an electrode structure forming the basis of the present invention in comparison with the conventional one. FIG. 1 (a) shows a typical voltammetric measurement electrode and convolution structure which have been conventionally used. In the figure, 1 and 2 are metal electrodes called working and counter electrodes, respectively, and generally the counter electrode 2 has a larger electrode area than the working electrode 1. Further, as a material, for example, when measuring by oxidizing it such as hydrogen peroxide, the counter electrode may be platinum and the working electrode may be silver, or platinum-platinum or gold-gold may be used without any problem. . The reference electrode 3 is used to fix the liquid level by using SCE having a liquid junction. While measuring the working electrode 1 with the potentiometer 5, the value of the variable resistor 7 is changed so that it becomes a constant value, and the partial voltage of the voltage of the constant voltage DC power source 8 is divided into the working electrode 1 and the counter electrode.
Supply between 2 At this time, the reading counter current of the ammeter 6 becomes the measurement value of this measurement system. A device called a potentiostat 9 is constructed by assembling such a measuring instrument. In the figure, 9a is a counter electrode terminal, 96 is a working electrode terminal, and 9c is a reference electrode terminal. Figure 1 (b) shows
It is a voltammetric measurement system using the ISFET according to the present invention as a reference electrode.
FET10 is used and its output is connected to the 9c reference electrode terminal. In the case shown in this figure, drain 1 of ISFET10
Although the source follower circuit 13 that reads the source potential when a constant voltage and a constant flow are applied between the source 1 and the source 12 is used, the present invention is not limited to this and other circuit configurations are also possible.
The present invention will be described based on working examples. The working electrode shown in Fig. 4 is 0.1 mm × 0.4 mm, and the counter electrode and the planar electrode made of gold on the insulator substrate are used together to create the cyclic structure shown in Fig. 1 (b).・ Voltanogram Shown in Figure 3. In addition, the measurement results using SCE
Shown in the figure. The figure shown here is after 30 minutes from the measurement. In the case of FIG. 1 (b), the ISFET in the same chip was used. In both cases, the pH 7.0 phosphate buffer solution (ionic strength μ = 0.1) was used as the buffer solution.
The voltammogram when FT is used as the reference electrode is almost the same as the voltammogram obtained by the normal measurement shown in FIG. ISFE like this
Using T as a reference electrode proved possible,
It was found that it is possible to provide a voltammetric measurement system that is minute and has no liquid junction.

Next, FIG. 4 illustrates the structure of the sensor according to the present invention in which the reference electrode is integrated. (a) is a plan view, and (b), (c), and (d) are sectional views taken along lines AA ', BB', and CC ', respectively. 14 in the figure
Is a sensor chip, 15 is a silicon oxide, 16 is a metal electrode, 17 is chromium, 18 is a silicon nitride film, 19 is a sapphire substrate, 20
Is a p-type ground region, 21 is an n-type source region, 22 is a p-type gate region, 23 is an n-type drain region, 24 is a tantalum oxide film, 25
Is an aluminum electrode. In this sensor element, the ISFET and the metal electrode are formed on the sapphire substrate 19 that can be easily used in water, but the ISFET formed on the silicon substrate is not limited to this.
It is also possible to realize a sensor element by forming a metal electrode on the FET and the element. The size of the tip is
It is 1,4 mm x 2.2 mm, and the working electrode and the counter electrode are patterned by gold together with the ISFET used as the reference electrode. A voltammetric sensor with an ISFET as a reference can be realized with one chip by using the electrode with such a configuration, and the measurement system can be easily oxidized by using the circuit shown in Fig. 1 (b). It is possible to measure reducing substances.

The reference electrode according to the present invention uses ISFET, which is
Since it is sensitive to pH, it is ideal to measure at a constant pH. When used as a hydrogen peroxide sensor,
As shown in Fig. 2, reduction occurs when the voltage rises in the range of 0.3V to 0.65V, and there is no problem in setting it anywhere, but generally 0.60V or 0.65V is used. If 0.6V is used, the stability of the reference potential is at least
Stability within 50 mV is required. This has a pH value of ± 0.8
It means that it is required to be stable within the range, but in general, when used as a transducer of an enzyme sensor, it is common to use a buffer solution and react at a constant pH. This sensor is particularly suitable for use as a sensor.

FIG. 5 shows a plan view of the working electrode portion of the sensor shown in FIG. 4 on which the immobilized enzyme membrane 26 is patterned. The enzyme to be used may be an oxidase-based one that produces hydrogen peroxide by a reaction, and it is possible to detect a substance serving as a substrate for the enzyme. In particular, a glucose sensor that decomposes glucose by using glucose oxidase and measures the generated hydrogen peroxide is important for clinical examination.

(Example) A gold pattern to be a working electrode and a counter electrode in one chip shown in FIG. 4 and an ISFE used as a standard electrode
Experiments were performed using devices with T. The device is immersed in pH 7.0 phosphate buffer solution (ionic strength M = 0.1), and after waiting for the source potential of ISFET to stabilize, the structure shown in Fig. 1 (b) is used for cyclic voltammetry. The electrodes were aged for minutes. The voltanomogram obtained at this time is the one shown in the experiment in Fig. 3, but the one using the SCE shown by the solid line in Fig. 2 as the reference electrode is almost parallel to the negative direction of 460 mV on the X axis. In agreement, it was found that ISFET could be used well as a standard electrode. A voltammogram when 1 mM hydrogen peroxide was added is also shown by a dotted line.
Considering the relationship between Fig. 2 and Fig. 3, + 200mV in Fig. 3
Since it is considered that the current consumption at the time of voltage rise in the range from to -100 mV corresponds to the hydrogen peroxide concentration, the output for various concentrations of hydrogen peroxide was measured at a fixed potential. Figure 6 shows the results of measurements with the working electrode-ISFET potential fixed at +140 mV. The output current showed a very good correlation with the hydrogen peroxide concentration below the experimental concentration of 1.25 mM.

(Example 2) Only the working electrode portion of the sensor element shown in FIG. 4 was covered with a glucose oxidase-immobilized film and aged by the cyclic voltammetry method as in Example 1. In the case of the sensor used here, the source potential of the ISFET is 430 mV higher than that of the ISFET. Therefore, the cyclic voltammogram almost agrees with the negative 430 mV shift compared to the SCE standard. In this case the working electrode-ISF
FIG. 7 shows the relationship between glucose and output when the potential between the ETs was set to 170 mV and the potentiostat shown in FIG. 1 (b) was measured for constant potential. The concentration and output showed a very good correlation within the measured concentration range within 100 mg / dl.

(Effects of the Invention) As described above, the voltammetric measurement using the output of the ISFET according to the present invention as the reference potential has the same accuracy as that using the normal reference electrode, but is miniaturized,
It is an epoch-making measurement method that can be solidified, and it has been found that the effect is maximized when a voltammetric sensor element having a conductive electrode integrated in the same chip as ISFET is used. .

[Brief description of drawings]

FIGS. 1 (a) and (b) show a conventional voltammetric measurement system and a system using ISFET according to the present invention as a standard, and FIGS. 2 and 3 show voltammetric measurement systems. Figure showing an example, FIG. 4 is a view showing an example of a sensor element according to the present invention, FIG. 5 is a plan view of an element in which an immobilized enzyme membrane is formed only on the working electrode portion of the element shown in FIG. 4, FIG. Is the relationship diagram between the hydrogen peroxide concentration in the measured solution and the output current,
The figure shows the relationship between glucose concentration and output current. In the figure, 1: Working electrode, 2: Counter electrode 3: Reference electrode, 4: Reaction tank 5 potentiometer, 6: Current measuring device 7: Resistance, 8: DC constant current 9: Potentiostat 9a: Potentiostat counter electrode input 9b: Potentiostat counter working electrode input 9b: Potentiostat reference electrode input 10: ISFET 11: ISFET drain 12: ISFET source 13: ISFET measuring device (source follower) 14: Sensor chip, 15: Silicon oxide 16: Gold electrode, 17: Chromium 18: Silicon nitride film, 19: Sapphire substrate 20: p-type ground region, 21: n-type source region 22: p-type gate region 23: n-type drain region, 24: tantalum oxide film 25: aluminum electrode, 26 : Immobilized enzyme membrane.

Continuation of front page (51) Int.Cl. ⁵ Identification code Office reference number FI Technical display location 7235-2J G01N 27/30 301 R 7235-2J 301 L

Claims (3)

[Claims] 1. Using two conductive electrodes and one reference electrode,
In order to keep the potential between one of the two metal electrodes and the reference electrode constant, a potential is applied between the two conductive electrodes, and in the voltammetric measurement method of measuring the current flowing at this time, Ion-sensitive field-effect transistor (ISFET) is used for the reference electrode, and a voltammetric sensor element in which two conductive electrode patterns and one ion-sensitive field-effect transistor are formed in one semiconductor chip. A voltammetric measuring method characterized by being used. 2. Using two conductive electrodes and one reference electrode,
In order to keep the potential between one of the two metal electrodes and the reference electrode constant, a potential is applied between the two conductive electrodes, and in the voltammetric measurement method of measuring the current flowing at this time, Ion-sensitive field effect transistor (ISFET) is used as the reference electrode, and one of the two conductive electrodes is a voltammetric one characterized by using an immobilized enzyme membrane. Measuring method. 3. A voltammetric sensor element in which two conductive electrode patterns and one ion-sensitive field effect transistor are formed in one semiconductor chip is used. Voltammetric measuring method described in the item.