JPS6243068A - Modified electrode - Google Patents

Abstract

PURPOSE:To increase stability and efficiency of an oxidation-reduction electrode serving as oxygen electrode of fuel cell by modifying the surface of a conductive substrate with a thin film of polymer having an acidic functional group, and fixing cytochrome c3 through the thin film. CONSTITUTION:The surface of a conductive solid substrate comprising carbon substrate such as graphite or metal substrate such as gold and silver is modified with a thin film of polymer compound having acidic functional group such as acidic amino acid and alginic acid, and cytochrome c3 whose isoelectric point is in a neutral or basic region is fixed through the thin film, or cyto chrome c3 is included with an including agent such as agar and agarose and the surface of substrate is covered with cytochrome c3. Thereby, a modified electrode is formed. Cytochrome c3 is strongly bonded on the surface of sub strate electrostatically, therefore, when it is used in an oxygen electrode of fuel cell, low oxygen reduction overvoltage and high current density can effec tively be obtained.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention relates to an electrode in which cytochrome C3 is immobilized on the surface of a conductive solid substrate with an organic polymer compound, and more specifically, the surface of the conductive solid substrate is The present invention relates to an electrode in which cytochrome C3 is stably immobilized by modification with a specific polymer compound containing a group, or an electrode in which cytochrome C5 is stably immobilized by an entrapping immobilization method. Such electrodes have applications such as being used for oxygen reduction and as oxygen electrodes in fuel cells.

(Prior art and problems) Recently, a lot of research has been conducted on modified electrodes, but among them, research on catalysts for oxygen electrodes that carry out reactions according to the following reaction equation: This has become an extremely important issue.

Conventionally, M, R, Tarasev, Chi (M, R, Tarasev
It is known to use heme protein based on the research of ich et al., or to use case, but the materials used in these cases are of animal and plant origin and cannot be obtained in large quantities. It is difficult [Bloe
electrochemistry and bioan
ergs-ticm) Volume 2, pages 69-78 (1975)
and Volume 6, pp. 393-403 (1979)).

In addition, oxygen electrodes in which metal (F'@, Co, Mn) phthalocyanine, I-lufilin, or their derivatives are immobilized on the electrode are well known; It merely reduces to hydrogen peroxide, that is, with two electrons. When using an electrode with immobilized copaltoporphyrin dimer, a strong acid (perchloric acid or trifluoroacetic acid) solution is required, and Co, Mn
When an electrode in which a phthalocyanine complex such as r Fe or Fe-protoporphyrin is immobilized is used, a strong alkali (potassium hydroxide or sodium hydroxide) is required. In these cases, oxygen is directly reduced to water, but since the electrolyte solution is diluted with water produced by the reaction, there is a disadvantage that a step is required to regenerate the strong acid or strong alkaline solution by concentration [Journal. Op Electroanalytical Chemistry (J.

Electroanal, Chem, ) Vol. 101, pp. 117-122 (1979) and The Journal of the American Chemical Society (J, A.
m.

Chem, Soc, ) Volume 102 6027-603
6 (1980), Bioelectrochemistry and Bioenergetics Vol. 4, pp. 18-29 (
(1977)].

(Objective of the present invention) The present invention aims to solve these problems.
By stably fixing cytochrome C3 to a nuclear substrate via a compound, or by stably fixing cytochrome C3 to the substrate by an entrapping immobilization method, it can be used as an efficient oxygen reduction catalyst. The purpose is to

(Means and Actions for Solving Problems) As mentioned above, many oxygen reduction catalysts have been reported, mainly phthalocyanine and Iluphyrin #lI conductors. However, a stable catalyst that efficiently reduces oxygen to water and can draw a large current density when used as an oxygen electrode in a fuel cell has not yet been found.

Japanese Patent Publication No. 57-205970 shows that an electrode in which cytochrome c3 is adsorbed and immobilized on the surface of a conductive solid substrate efficiently reduces oxygen to water. However, when cytochrome c3 is strongly and stably adsorbed and immobilized on the surface of a substrate, the range of its use is limited because the adsorption stability of cytochrome c3 and its oxygen reduction catalytic ability vary depending on the type of conductive solid substrate used. was.

Therefore, as a result of extensive research in the present invention, we have discovered that cytochrome C5, which behaves as a cationic protein in its isoelectric point and acidic voice region, can be synthesized using an organic polymer compound containing acidic functional groups on the surface of a conductive solid substrate. It has been found that by chemically modifying and stably immobilizing or comprehensively immobilizing the cytochrome C3, the cytochrome C3 immobilized electrode becomes a stable oxygen reduction electrode.

That is, the electrodes of the present invention can be broadly classified into the following types.

(a) An electrode in which high molecular weight acidic amino acids, broken acrylic acid, alginic acid, Nafion, etc., and cytochrome CSF are simultaneously immobilized on the surface of a conductive solid substrate.

(b) An electrode in which cytochrome C3 is comprehensively immobilized with agar, agarose, polyacrylamide gel, etc.

The cytochrome C3 used in the present invention is a Desulfovibrio vulg whose isoelectric point is in the neutral or basic region.
arisMiyazaki, D, vulgari
s Hlldenborough, D.

desulfur [cana Norway 1 D
, aalexlgens , D.

Cytochrome c3 such as G. baculatum, D, GLGAs, etc. is used, preferably Glycine, Vulgaria M.
lyazaki F (D, v, M, F), D, vulg
arls Hlldenborough (D4.H)
, D. desul-furleana Norway
y (D, d, N) is used. The numbers in parentheses are abbreviations.

These sulfate-reducing bacteria are ubiquitous and easily available (e.g. NCIMB Ltd, TorryRe
seareh 5tation (Scotland)
, available from]. By the way, sulfate-reducing bacteria can be easily isolated from the natural world (Science No. 51).
Vol. 6, 369-376 (1981)).

Further, examples of the high molecular weight acidic amino acids used for immobilization (a) include polyglutamic acid and polyasno-4-lagic acid, and esters thereof may also be used as long as they are partially hydrolyzed.

The agar or agarose used in (b) may have a sulfurization temperature of 35 to 70°C, and polyacrylamide gel may be used, for example, with a degree of crosslinking C = 2 or more and a total concentration T = 54 or more. However, ding=5Q/.

It is preferable to use ViC=2 to 5%, or more.

Examples of conductive solid substrates used in (a) and middle) include carbonaceous substrates such as glassy carbon, mesocarbon, and graphite; metal substrates such as gold, silver, and platinum; transparent electrodes such as tin oxide and indium oxide; Examples include copper conductor substrates such as titanium dioxide and titanium dioxide.

The detonation of the electrode of the present invention can be carried out, for example, as follows: first, a conductive solid substrate, fll, t
A thin film of an immobilizing agent, such as polyglutamic acid, is applied to the surface of H-stress annealed pyrolytic graphite (SAPG) using a spin-coding method (patent application No. 6O-4077).
2) etc., the electrode is 10-3M to 10-
8M, preferably 10-5 to 10-'M cytochrome c
A modified electrode can be obtained by immersing the electrode in a solution of 5, or by dissolving cytochrome c3 in a solution of polyglutamic acid, coating the electrode surface, drying, and washing with water to obtain a modified electrode.

Generally speaking, the surface of a conductive solid substrate is first modified with a thin film of a polymeric compound having an acidic functional group by an appropriate method, and then cytochrome c3 is added via the acidic functional group of this organic polymeric compound. fixed on the surface of a conductive solid substrate, or
Alternatively, after cytochrome c3 is immobilized on the organic polymer compound via the acidic functional group of the organic polymer compound, the immobilized product is coated and fixed on the surface of the conductive solid substrate by an appropriate method.

Moreover, comprehensive immobilization can be carried out as follows, for example. 7 in an entrapping fixative, e.g. a 1-3% solution of agar.
A modified electrode can be obtained by uniformly dissolving cytochrome c3 at a temperature of 0° C. or lower, quickly coating the electrode surface before gluing, cooling and washing with water after moleization.

Furthermore, alginic acid having an acidic functional group can be glued by adding calcium chloride or the like, and can also be used as an entrapping fixative.

That is, after dissolving cytochrome c3 in a 1 to 3 s solution of alginic acid, the mixed solution is applied onto the electrode, and then a 0.1M calcium chloride aqueous solution is added to form a gel, and after washing with water, a modified electrode can be obtained.

Generally speaking, entrapping immobilization occurs when an organic polymer compound is changed from soluble to insoluble conditions.
This is a method to identify cytochrome c3 in a sample.

Yojie: The reduction of oxygen molecules at the above electrode can be carried out as follows. That is, the modified electrode of the present invention is placed in an electrolysis cell filled with an air-saturated phosphate buffer solution, and the reduction of oxygen molecules is measured by cyclic deltametry at 25°C.

The modified electrode of the present invention can reduce oxygen more efficiently in the neutral region than an unmodified electrode.

Next, the stability of the modified electrode of the present invention will be outlined. In this modified electrode, cytochrome c3 is electrostatically strongly and stably bonded to the acidic functional group on the surface of the modified electrode, so it is superior in reducing oxygen compared to an electrode in which cytochrome c5 is directly adsorbed on an unmodified electrode. Shows stable performance.

Furthermore, in recent years, power generation using fuel cells has been attracting attention from the viewpoint of effective use of energy. Three types of batteries are currently known, and among these, a first-generation phosphoric acid type battery that uses hydrogen gas and oxygen gas is being studied. The structure of this battery consists of a hydrogen electrode that oxidizes hydrogen and an oxygen electrode that reduces oxygen.From the point of view of catalytic activity, there is a strong demand for a catalyst that is superior to the oxygen electrode compared to the hydrogen electrode. There is a need for high output power that can handle overvoltages as well as large current densities. It has been found that by using the electrode of the present invention as an oxygen electrode of a fuel cell, a large current density can be extracted in a neutral region where material corrosion is low.

The invention will now be illustrated with examples.

Example 1 Polyglutamic acid modified electrode PG (Polyglutamic acid M!
5AP
G (geometric surface area: 0.2 cm"), air-dried, and then dried under reduced pressure at room temperature of 1 wHg for 3 hours.

This is 3.5 x 10-5M. Mu c3 solution (D
, M, M, F) solution (pH 7,0) for 1 minute, and after thorough washing with water, a cytochrome c3-modified electrode was obtained.

Example 2 SPG (Sodium polyglutamate
Mw = 6,200)
After coating on APG (5waφ) and drying, it was neutralized with an aqueous IN sulfuric acid solution, washed with water, and dried to obtain a polyglutamine-modified electrode.

Thereafter, in the same manner as in Example 1, a cytochrome c3 modified electrode was obtained.

Example 3 Using GC (Glassy Carbon, 3Wφ), PC was applied in the same manner as in Example 1, and cytochrome c3 was immobilized to form a modified electrode.

Example 4 (Fixation with sodium alginate) Sodium alginate 3Qq was added to 1- water and dissolved at 100°C.
After dissolving, cool to room temperature and dissolve cytochrome c, (D, v, M,
F) Add 111F and dissolve it uniformly, and mix this solution with 5AP
After applying on G. The electrode was immersed in an IM calcium chloride aqueous solution for 12 hours to obtain a cytochrome C5 immobilized electrode.

The electrode was immersed in a pH7, 4, 10mM T') buffer solution, degassed with argon gas, and the cyclic virtammogram was completely measured at a sweep rate of 200 mV/S. 3 Figure 1 shows the measurement results. From the peak area, the amount of cytochrome c3 fixed on the electrode surface is 1. IXIQ-”mol
/cm”.

Example 5 Immobilization of cytochrome C5 with Naflon Naflon 117 (manufactured by Aldrlch) R-fluoroalkyl sulfonic acid resin, Equiv wt, 1
10 μl of 100.5 wt% alcohol solution) and 50 μl of cytochrome C5 solution (3×10 M) were mixed well, 1 μl of the mixed solution was applied onto 5APG using a microsyringe, dried, and thoroughly washed with water to obtain a cytochrome C3 modified electrode. .

The amount of immobilization was calculated from the cyclic gel tamogram.1.
2×10 mol/cm”.

Example 6 - Immobilization of cytochrome c3 with lyacrylamide Acrylamide 475rn9, Bisacrylamide 25
■, Cytochrome e3 (D, v, M, F) 40 W
Cytochrome C with a degree of crosslinking C=5T and a total concentration T=5%
3 solutions exploded. After applying 1 μl of this aqueous solution on 5APG and air drying, ammonium persulfate of O,OS*,
It was immersed in an aqueous solution of tetramethylethylenesoamine for crosslinking.

After thoroughly washing the electrode with water, the amount of cytochrome c3 fixed on the electrode surface was measured by cyclic soltammetry.

The amount fixed on the electrode surface is 1. OX was 10-10-12%.

Example q Fixation of cytochrome C5 with agar Agar powder (D
ifco Laboratories) 15■ in water 0
．． 5d and dissolved under heating and boiling. After cooling to 50℃, cytochrome e3 (D, v, M, F) 0.1■
Add and stir until uniform, then apply on 5APG and cool.
After drying, a cytochrome C3 agar-immobilized electrode was obtained.

The amount fixed on the electrode surface was determined by cyclic sartanmetry.
) 2.7 x 10 mol/CR".

Example 8 Reduction of oxygen (1) The electrode of Example 2 was saturated with air at 25°C, pH 7,
It is installed in an electrolytic cell filled with 0,0,03M phosphate buffer solution, and the potential is set to + with respect to the reference electrode (Ag/AgCt).
〇, 3v to -1,0■ 100 mV/S (7)
The cyclic voltammodalum was measured by sweeping at speed.

On the other hand, measurements were performed under similar conditions using unmodified 5APG.

As shown in FIG. 2, the electrode decorated with cytochrome C3 (solid line) exhibited extremely superior catalytic ability for the oxygen reduction reaction compared to the unmodified electrode (broken line).

Example 9 Oxygen Reduction (If) A cyclic portamogram was measured using the electrode of Example 3 under the same conditions as Example 8.

As a result, as shown in FIG. 3, the cytochrome C3-modified electrode (solid line) showed superior catalytic ability compared to the unmodified electrode (broken line).

Actual mf'No Reduction of oxygen by sodium alginate-immobilized cytochrome cs (D, v, M, F) Using the electrode of Example 4, -7,0110 mM saturated with air
The sample was placed in an electrolytic cell filled with a Tris buffer solution, and the cyclic limit/v tamogram was measured under the same conditions as in Example 8.

As a result, as shown in FIG. 4, the cytochrome C3-modified electrode showed excellent catalytic ability.

Example 11 Naflon-immobilized cytochrome e! ,!
Reduction of oxygen by electrode The electrode of Example 5 was saturated with air at pH 7,0,30m.
M! The sample was placed in an electrolytic cell filled with a J-acid buffer solution, and a cyclic biltamogram was measured under the same conditions as in Example 8.

As a result, the electrode shown in FIG. 5 showed excellent catalytic ability for oxygen reduction.

Example 12 Sodium alginate immobilized cytochrome e
3 Reduction of oxygen using (D, d, N) An electrode in which cytochrome c3 of D and dR was immobilized with sodium alginate was obtained in the same manner as in Example 4. The amount of cytochrome c3 immobilized on the electrode surface was determined by cyclic soltammetry to be 5.2 x 10-'' mo 1//Crr?.

The electrode was placed in an electrolytic cell filled with p)l 7.010mM Tris buffer solution saturated with air and
Cyclic measured the lutamogram under similar conditions.

As a result, as shown in Figure 6, cytochrome c s (D, d
R) The modified electrode showed excellent catalytic ability.

Example 13 Sodium alginate immobilized cytochrome e
3 Reduction of oxygen using (D, v, H) By the same method as in Example 4, an electrode in which cytochrome c3 of j) D, v, and H was immobilized with sodium alginate was obtained.

The amount of cytochrome C3 immobilized on the electrode surface was determined by cyclic soltammetry to be 7.7 x 10-10-l2/
(/Met.

The electrode was placed in an electrolytic cell filled with an air-saturated -7.0.10mM) Lith buffer solution, and cyclic cod? Lutamogram was measured.

As a result, as shown in Figure 7, cytochrome c3 (D, v, H)
The modified electrode showed excellent catalytic ability.

Example 14 Reduction of oxygen using cytochrome C3 agar-immobilized electrode The electrode of Example 7 was used to conduct air-saturated, - (cyclic birutamme in 70,30 phosphate buffer solution) IJ-
Oxygen reduction was measured by

As a result, the cytochrome C3 (D,
v, M, F) The modified electrode showed excellent catalytic ability.

Example 1 (stability) The electrode of Example 1 was immersed in an air-saturated pH 7,0,0,03M phosphate buffer solution, and cyclic birutammetry was measured every 30 minutes to measure the peak ml of oxygen reduction. .

On the other hand, an unmodified 5APG electrode was immersed in a 3.5×10 M cytochrome c3 solution for 30 seconds to obtain a cytochrome C3 modified electrode, and the oxygen reduction peak current was measured under the same conditions as the above electrode.

The electrode of Example 1 shown in FIG. 9 showed stable performance in oxygen reduction.

Example 16 (Polarization curve of oxygen electrode) The electrode of Example 2 was used as a cathode, and the air saturation was -7.0,
Polarization curves were measured in 0.03M phosphate buffer solution at a sweep rate of 3 mV/S.

As shown in FIG. 10, very excellent current-voltage characteristics were exhibited.

[Brief explanation of the drawing]

Figures 1.2, 3.4.5, 6.7 and 8 are, respectively,
Examples 4 and 8. 9.10.1+, 12°13 and 1
Cyclic gyrtammogram obtained in step 4 (solid line...
present invention, dashed line...control example), and FIG. 9 shows Example 10.
This shows the stability of the cathode in oxygen reduction (see Example 15), and FIG. 10 shows the polarization curve obtained in Example 16.

Claims (1)

[Claims] An electrode with cytochrome c_3 fixed on the surface of a conductive solid substrate using an organic polymer compound.