JPH01262461A - Concentration measuring apparatus - Google Patents

Abstract

PURPOSE: To enhance response and stability by employing a working electrode in which a porous three-dimensional structure composed of carbon particles coated with a platinum group element and a polymer binder is employed as a conductive support and an organic catalyst is fixed thereto. CONSTITUTION: The concentration measuring apparatus is provided with a working electrode 2, a reference electrode 3 and a counter electrode 4 connected with a potentiostat and the electrodes 2, 3, 4 are immersed into a liquid to be examined in a container 5, e.g. a beaker. The electrode 2 employs a porous three-dimensional structure composed of carbon particles and graphite particles fixed through a polymer binder as a conductive support wherein the particles are coated uniformly with a platinum group element. An organic catalyst, e.g. oxygen, is fixed in the pore of a porous carbon substrate to take conformation. This arrangement enhances response and stability.

Description

DETAILED DESCRIPTION OF THE INVENTION A. Field of Industrial Application The present invention relates to a concentration measuring device for measuring the concentration of a test substance using a current flowing between a working electrode and a counter electrode immersed in a test liquid. This invention relates to a concentration measuring device in which a catalyst acts on a test substance to generate a flow of electrons.

B. Conventional technology For example, the analysis of glucose, a reducing substrate, has been actively researched due to its clinical, industrial, and commercial importance.In fact, in the fields of research and control, In many places, this is performed as frequently as mm-based analytical measurement methods such as pH and oxygen quantification.

-1, glucose analysis uses a reaction in which glucose oxidase acts on glucose to produce hydrogen peroxide; for example, a colorimetric method that uses a secondary reaction of hydrogen peroxide, or a Physicochemical measurement methods such as conductivity measurement methods that utilize the fact that gluconic acid is rapidly hydrolyzed into gluconic acid have been used.

However, these methods often have slow response times and have the disadvantage of being sensitive to the dissolved oxygen concentration in the sample, which can vary over a considerable range in some cases.

Therefore, in recent years, electric measuring instruments using organic catalysts such as enzymes have been proposed. - As an example, to explain the case where glucose oxidase is applied to the detection of glucose, first hydrogen peroxide is oxidized on the surface of an electrode set at a constant potential and releases electrons, and as the electrons move, an electrical signal is generated. is generated. Under appropriate conditions, the response-based current value is proportional to the concentration of the test substance.

However, if the above reaction is used as it is, the response speed is slow, so it has been considered to utilize the by-product of hydrogen peroxide due to the direct transfer of electrons from the enzyme to the electrode. In this case, since a direct mechanism is difficult to achieve, it is said that the presence of electron carriers in the reaction system is necessary.

This electron carrier acts as a substitute for oxygen, which is an electron acceptor, and takes electrons from the enzyme when it is in the oxidized form, transfers the electron to TLi when it is in the reduced form, and returns to its oxidized form. As a career,
It has been found that it is difficult to reduce the influence of dissolved oxygen during test t4.

However, those using the above-mentioned electron carriers have problems such as that the electron carriers themselves are unstable and that it is difficult to stably hold the electron carriers in the electrodes.

C8 Problems to be Solved by the Invention As mentioned above, many of the conventionally known concentration measuring devices have limited reproducibility, responsiveness, stability, 111 constant concentration range, etc. , the emergence of a versatile analytical device is awaited.

The present invention has been proposed in view of the conventional situation, and an object of the present invention is to provide a concentration measuring device that is excellent in high-speed response and long-term stability, and can be easily handled even by beginners.

A further object of the present invention is to provide a concentration measuring device that is hardly affected by the oxygen concentration in a sample.

Means for Solving the D0 Problem The inventors of the present invention have conducted intensive research over a long period of time in order to achieve the above-mentioned objective, and have developed a carbon electrode coated with a platinum group element with high catalytic activity as a working electrode. By using i,
We have come to the knowledge that rapid electron transfer can be achieved by using oxygen as a co-reactant without involving electron carriers.

The present invention was completed based on such findings, and consists of a working electrode, a counter electrode, and a reference bridge connected to a potentiometer stand, and the working electrode is made of carbon coated with a platinum group element. It is characterized in that a porous three-dimensional structure consisting of particles and a polymeric binder is used as a conductive support, and an organic catalyst is immobilized thereon.

In addition, it is preferable that a protective film is formed on the working electrode, and a polycarbonate film having micropores through which the test substance can pass is suitable as such a protective film.

81 Effect In the concentration measuring device of the present invention, the electrode substrate material of the working electrode in particular has extremely non-uniform properties. This brings diversity to the crosslinked structure and increases the possibility that various orientations will appear in the three-dimensional structure. Even when no crosslinking agent is used, surface adsorption appears strongly.

This point is that oxygen can enter into the vacancies of such carbonaceous substrates, which increases the surface area of the organic catalyst (enzyme) and helps it adopt a conformation that does not interfere with its stability and activity. This is different from traditional binding modes in which the enzyme is attached to a relatively flat surface with a much smaller surface area, such as platinum, glassy carbon, or graphite, which limits the possible conformations of the enzyme. That's a big difference. Furthermore, the responsiveness of the action TL according to the present invention is extremely fast at 1 to 2 seconds, and not only is the enzyme activity high, but also the electrode itself has many electron accepting sites, so electron transfer occurs extremely quickly. It shows that there is. This means that platinum-coated carbon particles exist at a high density over a very wide area in the microstructure.
This is because the platinum on the surface and the active site of the enzyme can successfully approach each other.

F. Examples The present invention will be explained below based on specific examples. It goes without saying that the present invention is not limited to these Examples.

The 74 degree measuring device basically consists of a working electrode (2) connected to a potentiometer (1), as shown in FIG.
It consists of a reference electrode (3) and a counter electrode (4),
Add each of these U* to the test liquid (6) in the container (5)
By immersing (2), (3), and (4), the concentration of the test substance is detected.

As the reference electrode (3) and the counter electrode (4), any conventional electrode can be used, for example, the reference electrode (3) is a silver chloride electrode, and the counter electrode (4) is a platinum foil.

In addition, here, the working electrode (2), the reference electrode (3), and the counter electrode (4) are all immersed in the test liquid (6) in the same container (5), but especially regarding the counter electrode (4), The reference electrode (3) may be immersed in a buffer solution or the like in another container connected to the container (5) via a salt bridge. Hold, the opposite pole (
4) may be omitted and a two-electrode type device may be used.

On the other hand, the working electrode (2) used here uses an extremely non-uniform porous three-dimensional structure consisting of carbon particles or graphite particles bonded by a polymeric binder as an R-electrode support. Since platinum group elements are adhered to the carbon particles or graphite particles (hereinafter simply referred to as carbon particles), the platinum group elements are extremely uniformly dispersed in this conductive support. ing.

Any carbon particles may be used as the carbon particles as long as they do not interfere with the subsequent immobilization of the organic catalyst (enzyme). things are used. Such carbon particles are in stark contrast to glassy carbon, which cannot adsorb enzymes well.
nm, more preferably 5 to 30 nm.

A platinum group element (e.g. platinum) is added onto the carbon particles.

Not only palladium, but also other platinum group elements such as ruthenium and rhodium can be substituted for these. ) can be deposited by vapor deposition, electrochemical deposition, or simple adsorption from a colloidal suspension, and the amount of precipitation ranges from 1 to 20% by weight based on the simple weight, and Preferably 5-15%
It is. However, the above range is not absolute (it is determined from a practical point of view). If the amount of precipitated platinum group elements is less than 1% by weight, the signal intensity will decrease and measurement will be difficult. .

On the other hand, when the amount of precipitation is greater than 20 weight, the improvement in response speed and sensitivity is small in spite of the high cost, and in fact, when the amount of precipitation is extremely large, the sensitivity actually decreases. Therefore, in order to form a platinum film or a palladium film on carbon particles, for example, by oxidative decomposition of a compound such as chloroplatinic acid, or by using, for example, a platinum or palladium complex having a ligand that is easily oxidized, colloidal It is preferable to directly deposit platinum, palladium, etc. on the surface of carbon particles.

The carbon particles thus coated with a platinum group element such as platinum or palladium are then molded using a suitable polymeric binder.

In this case, a completely self-supporting porous three-dimensional structure mainly composed of the above-mentioned platinum-coated or palladium-coated carbon particles may be formed using a polymeric material as a binder, or a porous three-dimensional structure layer may be formed. may be formed on the surface of a conductive substrate made of metal, carbon, graphite, etc. In the latter case, particularly preferable materials for the conductive substrate include, for example, carbon paper and flower-shaped carbon cloth.

In order to maximize the porosity, the amount of polymeric material used as a binder is minimized to ensure the structural integrity and stability of the working electrode.

The thickness of the working electrode at this time is 0.1 to 0, although there are exceptions.
．． It is usually about 5+n or less. From the viewpoint of optimizing structural properties, mechanical strength, and porosity, the amount of polymeric binder should be at least 5 to IO%, or more, based on the weight of the platinum-coated or palladium-coated carbon particles. Both were selected to be 80% by weight,
It is usually in the range of 30 to 70j1% by weight.

All kinds of polymeric binders can be used, including conductive and semiconductive ones, but synthetic fluororesins, especially polytetrafluoroethylene, are preferred. It is necessary that the agent be permeable to oxygen.

Therefore, the above polymer binder must have the minimum oxygen solubility under atmospheric pressure, and that value is the same as that of a polymer binder at room temperature and pressure. At least 2×10−'− of oxygen per d.

Therefore, usable polymeric binders include fluororesins such as polytetrafluoroethylene, polyethyl methacrylate, polystyrene, polyvinyl acetate, polyvinyl chloride, polycarbonate, poly(4-methylpentene-1), polyisoprene, Polychloroprene, poly1.
3-butadiene, silicone rubber, etc.

It is necessary to immobilize an organic catalyst such as an enzyme on the conductive support described above, and methods for immobilization include covalent bonding via carbodiimide or carbodiimidazole, and 1,6-sinitroin. 3゜4-difluorobenzene (
Any conventionally known method can be employed, including a covalent bonding method via DFDNB), a crosslinking method using glutaraldehyde, and the like. -For example, to immobilize glucose oxidase, ■-cyclohexyl-3
- (2-morpholino) carbodiimide treatment by immersing p-methyltoluenesulfonate in an acetate buffer solution and glucose oxidase in an acetate buffer solution, or an anhydrous dimethylformamide solution of N,N'-carbonyldiimidazole. Carbonyldiimidazole treatment by sequential immersion in glucose oxidase solution, 1,6-
DFD sequentially immersed in a methanol solution of Sinitro 3.4-difluorobenzene and a glucose oxidase solution
NB processing or the like may be used.

Other types of polymeric binders containing bifunctional groups with variable chain lengths such as cimidate groups such as dimethylmalonimidad and dimethylshemidad can also be used in this immobilization step.

Furthermore, depending on the type of enzyme, simple adsorption in which the enzyme is physically adsorbed and immobilized on a conductive support without using a crosslinking method or the like may be effective.

The surface of the working electrode (2) can usually be physically protected by providing a suitable porous protective layer or protective film such as polycarbonate. However, the above-mentioned protective thin film and protective film are used to protect the fixed enzyme substrate such as glucose.
It goes without saying that these guarantees must be able to give you three benefits! ! ! Providing a film usually slows down the response speed, which can be considered a disadvantage, but the concentration measuring device according to the present invention, even with such a protective film, is still inferior to conventional measuring devices. In fact, it can be said that it is actually superior in many cases.

Next, an example of the configuration of a concentration measuring device actually assembled by the present inventors and measurement results obtained using the device will be described in detail.

The base electrode used here is made of conductive carbon paper containing platinum-coated carbon blank particles.

Here, the platinum-coated carbon particles (Palcan XC-72) used in the platinum-coated carbon paper are as follows:
Colloidal platinum (particle size 1.5 to 2.5 nm) is precipitated on the surface of carbon particles (nominal particle size 30 nm) by oxidative decomposition of platinum CI+) sulfite complex with hydrogen peroxide. was further formed and bonded to the surface of commercially available graphitized carbon paper using about 50 ft 1% polytetrafluoroethylene.

The final platinum content is 0.24 oz/-, this carbon material has a non-uniform three-dimensional structure, which together with oxidized surface functional groups helps to incorporate enzymes with the help of cross-linking agents. The base electrode was cut out into a disk shape with a diameter of 6.5 fi, and processed according to the method described below.

Enzymes can be immobilized on carbon surfaces using various crosslinking agents. There is generally no significant difference in the effectiveness of the crosslinking agents used; The II summit seems to have less significance than the nature of the Hess Denmei. As a crosslinking agent, 1-cyclohexyl-3-(2-morpholino)carbodiimide p-methyltoluenesulfonic acid (manufactured by Sigma) is convenient, and is used in this example as well. Glucose oxidase (EC1,1,3°4, derived from Aspergillus niger, Anderson
Laboratories) used a standard product with a specific activity of 2601 U, -1. Enzyme stock solutions were prepared in each case in A11 using Q, 1M potassium acetate buffer (pH 5,6);
Stored at 4°C.

To create the ri electrode, a carbon paper cut into a disk shape was first immersed in ethanol for 5 minutes to sufficiently wet the electrode surface, and then thoroughly washed with distilled water. In order to modify the electrode surface with carbodiimide, it was immersed for 90 minutes at room temperature in an O, 1 M carbodiimide solution using the above-mentioned pH 4.5 sodium acetate buffer. After thoroughly washing the above disc with distilled water, glucose oxidase'a
f! Lc sodium acetate! 5. To street liquid. Q tgm 1
2-' concentration, pH 5.6), and immersed at room temperature for 90 minutes. Finally, add 0.1M sodium acetate buffer (
It was thoroughly washed at pH 5, 6> and stored at 4°C while immersed in the same buffer.

Next, a concentration measuring device was assembled using this as a working electrode, but the central part of this device was an enzyme 1 scraping device (rank
Brothers, Bowtisham, Cambridge). This structure is shown in FIG. 2, where the enzyme electrode (11) is fixed on the platinum contacts at the bottom of the sample chamber by rubber rings (12) and (13). Action depending on purpose': A polycarbonate membrane is inserted between the 4 electrodes (11) and the test solution.
1), which serves as an effective screen against interfering substances, has a shelf in the sample chamber for a magnetic stirrer, which allows the test solution to be stirred above the enzyme electrode. , the entire sample chamber rests on a magnetic stirrer base (Rank Brothers). platinum lamp)i(
14) and a silver-silver chloride reference electrode (15) are incorporated into the lid, which serves as a stopper.

Connect these three electrodes to a small potentiostat (16)
connected to. The working electrode (11) is kept at a constant potential with respect to the reference electrode (15) by means of the 3-stand (16), which typically has a value of 0.4 to 0.6V.

For routine analysis, a simple device π may be used in a two-electrode system in which reference 7M, 1aj (15) also serves as a counter electrode.

On the other hand, the sample chamber is roughly composed of a substrate (17) and an annular jacket (19) containing an annular water reservoir (18). These base plate (17) and water shaker 7 (18) are connected to each other by a threaded tightening ring (20). - The reservoir i [(21) located in the center of (18) has a diameter of 1.6 cm in this example, and is designed to be able to contain 1 to 4 m/m of the test solution. being done.

A platinum contact (22) is provided in the center of the substrate (17), and a working hole (11) provided opposite to the platinum contact (22) is a rubber O-ring seal (12), (
13) is sealed. Of these, the upper O-ring seal (12) is cut in half so that the bottom surface is flat. The contact area between the working electrode (11) and the test solution was 0.14 ci.

Further, the upper part of the liquid storage tank (21) is closed by a stop bar (23). In the liquid storage tank (20), a platinum counter electrode (4) and a reference electrode (I5) are inserted into the stopper (23) and lead wires (24), (2)
5). The stopper (23) is provided with an adjustable coupling ring (26), and the vertical movement of the stopper (23) can be adjusted by rotating the adjustable coupling ring (26). There is.

In addition, the platinum counter electrode (14) and the reference voltage 1 (15) are connected to the potentiometer stand (16) via the lead wires (24) and (25), respectively, and the two-legged platinum contact (22) is also It is likewise connected to this potentiostat (16) via a lead wire (27). This is to set the working electrode (11) at a constant potential and measure the current output.

The measurement method is quite simple. First, a 11-well solution of 0.1M potassium phosphate containing 0.1M or 1.OM potassium chloride, pH
7,0) 2m7! into the sample chamber and pair it with the reference electrode (15))! A stopper (23) containing ij (+4) is placed over the sample chamber, and the magnetic stirring device is turned on. Next, turn on the potentiostat and set it to the desired potential. In this state, wait 1 to 2 minutes until the background current decreases to a nearly constant value (several μA).

Next, the test solution containing glucose was added to an appropriate tube (
(100 μl Hamilton, etc.) into the buffer solution in the sample chamber. Then, the current flowing between the 0-acting type 44 (11) and the pair 1 (14), where the enzyme acts on glucose in the test solution and electron flow occurs, is the potentiostat.
Monitored by a meter and recorded by an attached chart recorder.

FIG. 3 shows an example of the electrode response when the glucose level is increased by 1 degree. In the example shown here, C
roomM? 50 μl of a glucose solution of 50 μl was added. The increase in current value following the addition of the glucose-containing test solution is extremely rapid;
It usually takes 1 to 2 seconds when a protective film is not used, and 5 to 10 seconds when a protective film is used. When the solution is stirred, the signal waveform and magnitude of the electrode response hardly change even if a protective film is used.The amplitude of the signal measured at the point where the signal waveform becomes flat after 10 to 20 seconds is As seen in Figure 4, the glucose concentration increases almost linearly up to about 20mM. This linearity deteriorates when the concentration of glucose added exceeds 20 mM.

Since the current response strength in this measurement is very large, the measurable glucose concentration range extends to the μM level (see Figure 5). However, when the glucose concentration is low, the reaction rate is In this device, the effective diameter of the tffi in contact with the test solution is only about 2 L (therefore the area is 0.14 cm'); By slightly modifying the design and using a slightly larger electrode, it is possible to measure glucose solutions with lower concentrations.

'Wffi created as described above always showed good responsiveness to glucose. A two-electrode system fabricated using a similar method and having the same size also yielded very similar results (within a few percent of the current response when tested under the same conditions). Even with repeated use of a single electrode, accuracy and reproducibility were good (see Table 1).

Table 1 shows the reproducibility of the electrode response in a glucose solution with a constant concentration.
00mM) 100μ#2 potassium phosphate buffer (
(pH 7.0) and the current value was read. The final concentration of the solution in the cell was 4.76mM, and each test was performed on the same day over a 6-hour period. l
The 11 and 12 tests were conducted the next day.

Table 1 It should be noted that these data were obtained using very simple laboratory-level techniques, which can improve accuracy by more tightly controlling glucose concentrations. Electrical equipment only needs to be calibrated once in a while if it is used for a short period of time (Hq degrees per day). Furthermore, this electric scraper is extremely stable when stored in a humid state at 4°C, but even when stored at room temperature, it showed good responsiveness for one year (see Table 2).

Table 2 shows the response stability of the electrodes stored at 4°C, and the current value was 4.76mM in each test shown in Table 1.
Glucose) volume; S sold at night.

(Left below) What about the second surface electrode? Will? ! If stored in this condition, it can withstand intermittent use over a long period of time as described above, but if dried, the enzyme activity is lost.

Although specific embodiments of the present invention have been described above, the present invention is not limited to these embodiments, and good results can also be obtained in other applications such as probes.

As an example, by connecting the working electrode to an electric wire and enclosing it in a glass tube to create a probe with a diameter of 2 ml, and inserting it into a container together with a reference electrode and a countershape, a highly reliable probe can be obtained without removing dissolved oxygen. Concentration measurements can be made.

If it is further miniaturized, it can be incorporated into a catheter needle or the like and used as a probe type sensor. With this configuration, the electrode is protected by the catheter needle during insertion, and the catheter needle can also be kept clean.

Further, the organic catalyst (enzyme) to be immobilized is not limited to the above-mentioned glucose oxidase, and it is of course possible to use other oxidoreductases. A wide variety of suitable oxidoreductases include lactate oxidase, galactose oxidase, cholesterol oxidase and other peroxide-producing enzymes. Furthermore, combinations of oxidase and enzymes other than oxidase are also possible. Examples of such combinations include combinations of β-galactosidase and glucose oxidase for quantifying lactose, or combinations of β-glucan depolymerase, β-glucosidase, and glucose oxidase for quantifying β-glucan.

Further, although the concentration measuring device of the present invention is intended for routine analysis in research or quality control, it is of course suitable for purposes such as clinical sample analysis and food analysis. Furthermore, it is expected to be used in household biosensors for diabetic patients, advanced control systems for fermenters, and various other industrial processes that require glucose monitoring.

G1 Effects of the Invention As is clear from the above explanation, in the concentration measuring device of the present invention, a novel carbon substrate is used as a conductive support for the working electrode, and organic catalysts such as enzymes are effectively immobilized. This makes it possible to measure concentration with excellent responsiveness and stability.

Further, the device of the present invention does not particularly require an electron carrier and functions even in the presence of dissolved oxygen.

Furthermore, the concentration measuring device of the present invention has excellent current responsiveness, and the electrode area can be easily reduced.

Furthermore, especially by providing a protective film made of polycarbonate on the surface of the working electrode, it is possible to further improve the φn reliability, and the provision of the protective film does not deteriorate the response characteristics.

Needless to say, it has a wide applicable concentration range and excellent storage stability.

[Brief explanation of the drawing]

FIG. 1 is a perspective view schematically showing an example of the positional configuration of a concentration measuring device to which the present invention is applied, and FIG. 2 is a schematic cross-sectional view showing the configuration of the concentration measuring device used in the experiment. FIG. 3 is a characteristic diagram showing the current response characteristics of the concentration measuring device to which the present invention is applied, FIG. 4 is a characteristic diagram showing the relationship between glucose concentration and electrode current value, and FIG. 5 is a characteristic diagram showing the relationship between glucose concentration and electrode current value. FIG. 3 is a characteristic diagram showing the relationship between glucose concentration and electrode current value in the ill region. Patent applicant Cambridge Life Sciences PLC Agent Patent attorney Akira Koike (and 2 others) Figure 1 Figure 2 Staff member (LIA)

Claims (2)

[Claims] (1) A working electrode, a counter electrode, and a reference electrode are connected to a potentiostat. A concentration measuring device characterized by having a support and an organic catalyst fixed thereon. (2) The concentration measuring device according to claim 1, wherein the surface of the working electrode is protected by a polycarbonate film having micropores.