JP3440130B2 - Electrode for electrochemical and sensor using the electrode - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an electrochemical electrode capable of catalyzing an in-vivo reaction from a base potential and a sensor using the electrode.

[0002]

Prior art and its problems: Enzymes are indispensable substances for smooth functioning of life activities. Recently, biosensors and fermentation processes focusing on the specific action of enzymes have been actively developed [Shuichi Suzuki, Biosensor, Kodansha (1984), APFTurner, I.Karube and G.
S. Wilson (Eds.): Biosensors-Fundamentals and Applic
ations; Oxford University Press (1987)]. Dehydrogenase, a type of oxidoreductase, is
It is an enzyme that oxidizes organic compounds during metabolic processes in the living body. At this time, the coenzymes nicotinamide adenine dinucleotide (NAD ⁺ ) and nicotinamide adenine dinucleotide phosphate (NADP ⁺ ) are reduced It is known that the reaction proceeds. Therefore, the enzymatic reaction can be quantified by detecting reduced nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) produced by metabolism.

In the body, NADH and NADPH are reoxidized and recycled via the flavin enzyme and cytochrome system, but industrially, it is necessary to measure easily and rapidly, and an electrochemical sensor makes this possible. It is attracting attention as a method. The reaction formula is represented by the following formulas and, where SH ₂ and S are the reduced form and oxidized form of the substrate, respectively, and the formula proceeds with an enzyme and the formula proceeds with an electrode. SH ₂ + NAD ⁺ (or NADP ⁺ ) → S +
NADH (or NADPH) + H ⁺ NADH (or NADPH) → NAD ⁺ (or NA
^{DP +) + H + + 2e} -

However, an electrode capable of reversibly responding to the oxidation reaction of NADH and NADPH has not yet been developed. For example, when a carbon electrode is used as the electrode, the oxidation reaction requires an overvoltage of 1 V or more, and cannot be distinguished from a side reaction such as oxygen generation, so the carbon electrode cannot be used for detecting the oxidation reaction. Further, the platinum electrode adsorbs coexisting organic compounds and cannot be used for quantitative determination of the NADH and the like. Further, in these unmodified electrodes, due to the high overvoltage, it is impossible to distinguish between NADH and NADPH and the oxidation reaction of coexisting biological substances (uric acid, ascorbic acid, dopamine, etc.) that are more easily oxidized.

On the other hand, it has recently been reported that a modified electrode carrying a monomolecular or polymer compound has excellent properties which cannot be obtained by a conventional inorganic electrode surface (L. Gor
don, B. Persson, PD Hale, LI Bogulavsky, HI K
aran, HS Lee, TA Skotheim, HL Lan and Y. Oka
moto, Am. Chem. Soc. Symp. Ser., 487, 56 (1992))
. However, since the detection current is small and the sensitivity is poor in the single molecule modified electrode, application of the polymer modified electrode is now under intense study. It has been reported that the reaction of the formula was carried out using a polythionine modified electrode, and an overvoltage drop of 0.4 V or more as compared with a carbon electrode could be achieved (T. Ohsaka,
K. Tanaka, and K. Tokuda, J. Chem. Soc. Chem. Comm
un., 222 (1993)).

Gorton et al. Reported that an overvoltage reduction of 0.55 V could be achieved on a modified electrode prepared by using N-methylphenandinium sulfate (A. Torstensson a.
nd L. Gorton, J. Electroanal. Chem., 130 , 199 (198
1)). In addition, Albery et al. Have found that compounds having a phenadium ring show excellent catalytic activity (WJ Alber
y, PN Bartlett and AEG Cass, Phil. Trans, R.
Soc. Lond. B, 316, 107 (1987)). Further Persson and Gort
On has found that compounds with a heterocyclic aromatic structure show excellent catalytic activity for the formula, but there is no report as an electrochemical sensor (B. Persson and L. Gorton, J.
Electroanal. Chem., 292 , 115 (1990)).

[0007]

OBJECT OF THE INVENTION Thus, in the prior art, there is no electrode capable of quantifying an enzyme reaction with sufficient sensitivity and an electrochemical sensor using the electrode. It is clear that the cause is mainly due to the electrocatalyst, and as a result of diligent studies, the present inventor developed an electrode carrying an electrocatalyst having a very high activity, particularly against the oxidation of NADH and NADPH, Further, the present invention has found an electrochemical sensor using the same, and an object of the present invention is to provide the electrode and an electrochemical sensor using the electrode.

[0008]

The present invention, in order to solve the problems] is phenosafranine and / or loaded with electrochemical electrode on a conductive substrate and a derivative thereof as port Rimmer, and the electrode or off
Enosafranine and / or its derivatives as monomers
It is an electrochemical sensor that uses an electrochemical electrode supported on a conductive substrate .

The present invention will be described in detail below. As described above, the present inventors have arrived at the present invention as a result of various studies on an electrode catalyst capable of quantitatively detecting an enzymatic reaction mainly including a hydrogen transfer reaction, that is, an in-vivo reaction at a base potential. In the present invention, phenosafranine shown in Chemical formula 1 and / or its derivative is used as a catalyst substance, and R is hydrogen or lower alkyl. This phenosafranine and its derivatives
We carried on a conductive substrate as a substance shown in clogging (Formula 2) in the state of port Rimmer (n = 2 to 100).

[0010]

[Chemical 1]

[Chemical 2]

The electrode carrying the phenosafranine or its derivative shown in (Chemical formula 1) or (Chemical formula 2) has a considerably base potential as compared with a conventionally used carbon electrode or the like, and the raw material represented by the above-mentioned formula, for example. NADH and NAD produced in the body
The PH oxidation reaction (hydrogen transfer reaction) can proceed, and when a minute current generated at this time is detected, it can be distinguished from a current generated by another reaction that occurs only at a noble potential. As a result, by measuring the minute current amount, NADH and NADPH generated in the in-vivo reaction can be quantified.

Although the reason why the electrode of the present invention can detect a current from a base potential is not clear, the electrode surface has a conductive three-dimensional structure, and NADH or NAD reaching the surface is obtained.
It can be inferred from the fact that it forms a reaction field having a configuration with an excellent affinity for incorporating PH and the like. As the substrate of the electrode of the present invention, a metal such as platinum or gold, a metal oxide, or a conductive substance such as carbon is used. Carrying said phenosafranine and / or a derivative thereof to the conductive metal surface, even if the powder port Rimmer of the phenosafranine using a binder supported on the substrate, and the substrate was dissolved phenosafranine The phenosafranine may be immersed in an electrolytic bath to electropolymerize the phenosafranine, and then supported as a polymer on the surface of the substrate. Since the polymerized phenosafranine has low solubility in a solvent and excellent resistance, it is desirable that the polymerized phenosafranine is supported by electrolytic polymerization to provide a stable electrode catalyst.

The amount of phenosafranine supported is not particularly limited, but it may be supported on the surface of the conductive substrate in the range of 10 ^-11 to 10 ^-7 mol / cm ² depending on the number of potential scans and the solvent during electrolytic polymerization. Preferably, the supported amount can be appropriately set by adjusting the phenosafranine concentration and temperature. The electrode of the present invention , or phenosafranine and / or its derivative
Electrode for electrochemical supported on a conductive substrate as a monomer
Since the pole can reversibly proceed in the opposite direction to the in-vivo reaction, it can be used as a sensor for detecting and quantifying the in-vivo reaction by attaching a means for measuring the generated electric current. It is possible to realize an in-vivo reaction system sensor which is impossible with the conventional technique, and particularly when a flow injection type sensor is used, responsiveness and sensitivity are increased. The sensor of the present invention can also be applied to sensors other than in-vivo reaction, such as for detecting the progress of fermentation.

[0014]

EXAMPLES Examples of electrochemical electrodes and sensors using the electrodes according to the present invention will be described below, but the examples do not limit the present invention.

Example 1 Cell 1 in FIG. 1 was filled with 0.1 M perchloric acid aqueous solution in which 1 mM phenosafranine (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved. Union Carbide's BPG (ba
sal-plane pyrolytic graphite) Carbon is used as a working electrode 2, a large-area platinum plate is used as a counter electrode 3, and a silver-silver chloride electrode is used as a reference electrode 4, and these are placed in an aqueous solution of perchloric acid 5 in the cell. Soaked. Next, by repeating the potential scanning of the working electrode from −0.65 V to 1.25 V with respect to the reference electrode, phenosafranine was electrolytically polymerized on the working electrode to form a reddish purple film on the working electrode. The electrode was washed in boiling water to remove the phenosafranine monomer. The amount of phenosafranine supported on this working electrode was 1.1 × 10 ^-8 mol / cm ² .

This polymer-modified electrode is used as it is as a working electrode, and NADH is used in place of the above-mentioned aqueous solution of perchloric acid.
The cell of FIG. 1 was filled with an aqueous solution (phosphate buffer solution) containing 5 mM of pH 7 and the potential was scanned. The results are shown by a in FIG. An oxidation wave of NADH was observed from around -0.2V. In the figure, b is the result of potential scanning using a pH 7 phosphate buffer solution containing no NADH.

[0016]

Comparative Example 1 The BPG carbon electrode of Example 1 was directly used as a working electrode and immersed in the cell of Example 1 to scan the potential. The result is shown by c in FIG. In this comparative example, the oxidation current was observed only at a potential no less than 1 V than in Example 1, and it is understood that the electrode of Example 1 has high activity, that is, detectability at low potential.

[0017]

[Example 2] An electrode prepared in the same manner as in Example 1 except that glassy carbon was used instead of BPG carbon was incorporated into a flow-type cell, and the flow rate was set to 1 ml / min, and an aqueous solution of pH 7 (phosphoric acid was used). Buffer)
Shed. When 20 microliters of a small amount of NADH was injected, no response current was obtained at -0.5 V, but the current peak shown in Fig. 3 was detected when the potential was kept at a potential nobler than -0.4 V [a: -0.50 V]. , B is -0.40
V and c are -0.35V, d is -0.30V (vs. Ag / AgCl)]. The standard redox potential of the formula is -0.515 V, which shows that the quasi-reversible reaction is proceeding.

[0018]

EXAMPLE 3 Methyl phenol safranin (R = CH ₃₎ current peaks in the manufacturing life-and-death -0.3 V the polymer modified electrode in the same manner as in Example 1 using has been detected.

[0019]

INDUSTRIAL APPLICABILITY The present invention is an electrochemical electrode in which phenosafranine and / or its derivative is supported on a conductive substrate. An electrode using phenosafranine or a derivative thereof as a catalyst substance can cause a reverse reaction of an in-vivo reaction to proceed reversibly at a base potential. Furthermore, when the phenosafranine and / or its derivative is supported in a polymerized state, the solubility and durability are improved, the electrode characteristics can be remarkably improved, and a stable solid electrode can be provided.

Further, since the reverse reaction of the above-mentioned in-vivo reaction can be reversibly progressed, when the electrode is used as a sensor, it is not affected by other various reactions which proceed at a noble potential, and therefore unnecessary. Selectively transforming the products of the in-vivo reaction into starting materials without producing
The product of the in-vivo reaction can be quantified by detecting the current generated at this time. As a result, it is possible to provide a sensor for in-vivo reaction capable of detecting a specific substance simply and quickly, which was impossible with the conventional technology.

The sensor can be best applied to current detection when oxidizing reduced NADH and NADPH, which are major coenzymes in the metabolic process in the living body, and the electric current causes NAD ⁺ and NADP accompanying the in-vivo reaction. ^{The in-} vivo reaction itself can be quantified by grasping the progress of the reduction reaction of ⁺ . Further, the sensor of the present invention can be used not only for in-vivo reaction but also for other applications such as a sensor in the fermentation industry.

[Brief description of drawings]

FIG. 1 is a schematic cross-sectional view of a cell used for electrolytic polymerization and potential scanning of Example 1.

2 is a graph showing the results of potential scanning in Example 1 and Comparative Example 1. FIG.

FIG. 3 is a diagram showing current peaks in Example 2.

[Explanation of symbols]

1 ... Cell 2 ... Working electrode 3 ... Counter electrode 4 ...
・ Reference electrode 5: Perchloric acid aqueous solution

─────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP 63-237562 (JP, A) JP 63-237563 (JP, A) JP 7-167824 (JP, A) JP 59- 24243 (JP, A) JP 58-105055 (JP, A) JP 57-69668 (JP, A) JP 57-98853 (JP, A) BENOY B BHOWMIK, S HARMILA ROY & K K ROHATGI -MUKHERJEE, Photoelectrochemical cell With Pheno safranin Coated Electrode, Indian Journal of Chemistry, August 1986, Vol. 25A No. 8,714-718 (58) Fields investigated (Int.Cl. ⁷ , DB name) G01N 27/327 G01N 27/333 C25B 11/06 G01N 27/416 JISC file (JOIS)

Claims (3)

(57) [Claims] 1. An electrochemical electrode in which a polymer of phenosafranine and / or its derivative is supported on a conductive substrate. 2. An electrochemical sensor using an electrochemical electrode in which phenosafranine and / or a derivative thereof is supported as a monomer or polymer on a conductive substrate. 3. The electrochemical sensor according to claim 2 , which is for electrochemical detection of reduced nicotinamide adenine dinucleotide and / or reduced nicotinamide adenine dinucleotide phosphate.