JP2003227811A - Fructose concentration sensor and fructose concentration-measuring method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fructose sensor that can be easily operated, is less expensive and is practical, and has high selectivity and sensitivity. <P>SOLUTION: A gold electrode (12) where fructose dehydrogenases (FDH) is immobilized by utilizing the combination of gold and cysteamine (Au-S), a platinum counter electrode (11), and cobalt phenanthroline complex (Co(phen)<SB>3</SB><SP>2+</SP>) solution 5 are used as a fructose concentration sensor. Preferably, further as a reference electrode, an Ag/AgCl electrode 13 is used. The fructose concentration sensor has an advantage where the Co(phen)<SB>3</SB><SP>2+</SP>can be easily adjusted, reversible oxidation reduction is enabled, and oxidation reduction potential is the lowest among other metal complexes with phenanthroline as ligand at relatively low potential. When an oxidation reduction voltage is low, there is less influence of interfering substances such as an ancorbic acid that coexists in an actual sample, and the background current of a capacity current is small, thus manufacturing the fructose concentration sensor having high selectivity and sensitivity. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a fructose concentration sensor. More specifically, the present invention relates to a fructose concentration sensor and a fructose concentration measuring method, which can measure fructose concentration with high selectivity and high sensitivity, are low in price, and are easy to operate.

[0002]

BACKGROUND OF THE INVENTION Fructose (fructose) is
Since it has a stronger sweetness than glucose and sucrose and is a main component of sugar contained in many vegetables and fruits, the concentration of fructose in food is measured in food industry from the viewpoint of food quality control. Important in the field. In addition, measurement of fructose concentration in serum, urine, and serum is clinically valuable.

As a technique for detecting the fructose concentration, redox titration method, gas chromatography method, HPLC method and the like are known. However, the fructose concentration sensor using the redox titration method, the gas chromatography method, and the HPLC method has encountered a problem that the device price is expensive and the measurement method is complicated.

In addition, hexokinase, glucose phosphate isolase and glucose hexaphosphate dehydrogenase
Kits of enzymatic methods using one enzyme and two coenzymes of ATP and NADP are already on the market. However, since the enzyme analysis kit also reacts with glucose at the same time, the glucose concentration is measured by another method, and the glucose concentration is reduced by another method from the result measured by the enzyme analysis kit to reduce the fructose concentration. I'm running into problems that need to be sought, work is complicated and laborious.

Further, as a fructose concentration sensor, Gluconobacter sp. More isolated enzyme fructose dehydrogenase (FDH: fructose deh)
Ydrogenase) -based biosensors are being studied.

[0006] FDH is one of the few enzymes reported to have direct electron transfer between the enzyme and the electrode. FDH is 3
It is a membrane-bound pyrroloquinoline quinone (PQQ) -dependent oxidoreductase with a molecular weight of 140,000 Da consisting of two subunits. FDH is strongly bound to PQQ. FDH is D-fructose 5-keto
-Oxidized to fructose, PQQ is PQQ
Reduced to H ₂ .

[0007] PQQ-dependent oxidoreductase has the advantage that it is hardly affected by the enzyme concentration and does not require the addition of coenzyme. Therefore, by using FDH, a simple and inexpensive fructose concentration sensor can be produced. You can So far, various forms of fructose concentration sensors using FDH have been reported.

For example, there has been proposed a fructose concentration sensor in which a polypyrrole film is polymerized on a platinum electrode and FDH is immobilized therein, and the oxidation reaction of fructose is detected by the oxidation reaction of polypyrrole as the oxidation current. To measure. However, since the oxidation voltage of polypyrrole is high, it is necessary to operate at a voltage of + 0.4V or higher. In this way, if the redox voltage is high, it is easily affected by interfering substances such as ascorbic acid (vitamin C), and if ascorbic acid or the like is contained in the target substance for measuring the fructose concentration, it reacts with them and becomes accurate. We have encountered a problem that we cannot measure fructose concentration.

An amberometric fructose concentration sensor is
It is classified into two types, a direct electron transfer type and an indirect electron transfer type.

In the direct electron transfer type fructose concentration sensor, PQQH ₂ produced by fructose oxidation is directly oxidized on the electrode, and the current response according to the fructose concentration is measured. The charge transfer is greatly affected by the orientation of FDH on the electrode.
However, at present, it is very difficult to solidify the enzyme with a high orientation.

In the indirect electron transfer type fructose concentration sensor, PQQH ₂ is oxidized by a mediator (arbitration means) and the reduced type mediator is oxidized on the electrode to measure a current response proportional to the fructose concentration of the sample. . However, until now, no suitable mediator has been proposed for application to a fructose concentration sensor.

[0012]

As described above, various fructose concentration sensors have been proposed so far, but they are easy to operate as a fructose concentration sensor, the sensor price is low, the selectivity is high, and the selectivity is high. A sensitive fructose sensor has not been realized.

The object of the present invention is to quickly, simply and
An object of the present invention is to provide a fructose concentration sensor capable of measuring (measuring) the concentration of an object to be measured, for example, the fructose concentration with high selectivity and high sensitivity at low cost.

[0014]

As a result of earnest research on a fructose concentration sensor which achieves the above object, the present inventor has obtained the following findings.

Cobalt phenane as a mediator
Trollin Complex The present inventor oxidizes fructose with FDH and converts it into an electric signal as a fructose sensor, and as a mediator (arbitration means or electrochemically active species), a cobalt-phenanthroline complex or a phenanthroline-cobalt complex (Co (phen)) is used. ₃ ²⁺ ): phenanthroline cobalt (I
I) found it desirable to use complex). Co (phen) ₃ ²⁺ is easy to adjust, is capable of reversible redox, and has the lowest redox potential among other metal complexes having phenanthroline as a ligand. Has the advantage of.

In the fructose concentration sensor, if the redox voltage is low, the influence of interfering substances such as ascorbic acid coexisting in the actual sample is small, and the background current of the capacitive current becomes small, so that high selectivity and high It is possible to manufacture a sensitive fructose concentration sensor.

Based on such knowledge, the present inventor used Co (phen) ₃ ²⁺ as an electrochemically active species (mediator) in a fructose concentration sensor.

Utilizes a gold-cysteamine (Au-S) bond
It is the electrode further present inventors, a suitable fructose dehydrogenase using the fructose concentration sensor (FDH: fructose dehyd
ro-genase), gold-cysteamine (Au
It has been found that it is desirable to accurately measure the fructose concentration by using an electrode on which FDH is immobilized by using -S) bond as a working electrode of the fructose concentration sensor. The thiol compound reacts with the gold surface to form an Au-S bond. Among them, it has been found that the use of alkanethiol compounds also allows van der Waals interaction between alkyl chains to act, so that a defect-free monomolecular film having high orientation can be produced. Such a monolayer is called a self-assembled monolayer.

Since the self-assembled monolayer can be prepared simply by immersing the gold substrate in the solution of the thiol compound, a dense monolayer can be easily prepared. Therefore,
When an FDH-solidified gold electrode using a gold-cysteamine (Au-S) bond is used as a working electrode of a fructose concentration sensor, the fructose concentration sensor can be manufactured at low cost in addition to the accuracy of the fructose concentration measurement. .

The inventor of the present application has further found that by utilizing the Au—S bond, a single separation membrane of biopolymer can be easily produced and the thickness of the biometric identification element membrane can be controlled. . The thinning of the biometric identification layer film makes it possible to downsize the fructose concentration sensor, improve the measurement sensitivity and shorten the response time.

Therefore, according to the present invention, the cobalt-phenanthroline complex (Co (ph
en) ₃ ^2+), and gold as a working electrode - cysteamine (Au-S) fructose concentration sensor using immobilized electrode fructose dehydrogenase utilizing conjugation (FDH) is provided.

Preferably, the apparatus further comprises potential applying means for applying a redox potential to the working electrode, and computing means for detecting a current flowing between the working electrode and the counter electrode to calculate a corresponding fructose concentration. Have.

Also preferably, further comprising a reference electrode,
The potential applying means applies a predetermined potential to the working electrode with respect to the potential of the reference electrode.

Further, according to the present invention, a fructose concentration is obtained by using a working electrode in which fructose dehydrogenase (FDH) is immobilized using a gold-cysteamine (Au-S) bond and a cobalt-phenanthroline complex as a mediator. A method for measuring fructose concentration is provided.

[0025]

BEST MODE FOR CARRYING OUT THE INVENTION First Embodiment FIG. 1 is a configuration diagram of a fructose sensor according to a first embodiment of the present invention. The fructose concentration sensor 1 illustrated in FIG. 1 has a cobalt-phenanthroline complex (Co (p
hen) ₃ ²⁺ ) solution 5 and a measurement vial 3 which is a container containing a measurement object whose fructose concentration is to be measured, and C in the measurement vial 3 fixed to an upper lid 4 of the measurement vial 3.
Platinum wire 11 immersed in o (phen) ₃ ²⁺ solution 5
, FDH-modified gold electrode 12, and silver / silver chloride (Ag / Ag
Cl) electrode 13. The measurement vial 3 contains a measurement object (sample) of fructose concentration and a Co (phen) ₃ ²⁺ solution 5 as a mediator. The fructose concentration sensor 1 further includes a potentio galvanostat device 7 as potential applying means, and a personal computer (PC) 9 as arithmetic processing means.

The potentio-galvanostat device is generally used for electrochemical measurement. Generally, in electrochemical measurement, three types of electrodes, a working electrode (WE), a counter electrode (or counter electrode, CE), and a reference electrode (RE) are used. The potentio-galvanostat device 7
A predetermined potential is applied to the working electrode (WE) with reference to the reference electrode (RE) to cause an electrode reaction at the working electrode (WE). When a reaction occurs in which electrons are given to the substance in the solution at the working electrode (WE), the opposite reaction (reaction of passing electrons from the substance in the solution) is performed by another electrode, the counter electrode (CE), to form a circuit. An electrochemical reaction is measured on the principle that an electric current flows through.

As described above, the general potentio-galvanostat device has the potentiostat function and the galvanostat function, and the potentio-galvanostat device 7 of this embodiment has both of these functions. Although the potentio-galvanostat device which it has can also be used, the potentio-galvanostat device 7 of this Embodiment has only a potentiostat function, and is aiming at cost reduction.

In the fructose concentration sensor of this embodiment, the working electrode corresponds to the FDH-modified gold electrode 12, the counter electrode corresponds to the platinum wire 11, and the reference electrode corresponds to silver / silver chloride (Ag / A).
This corresponds to the gCl) electrode 13.

FIG. 2 is a view showing the concept of the FDH-modified gold electrode 12 in the fructose concentration sensor 1. Figure 3 shows Co (ph
en) F when using ₃ ²⁺ solution 5 as a mediator
It is the figure which illustrated the DH modified gold electrode 12 and the fructose oxidation reaction system in the surface.

The following formula 1 is a reaction formula showing the principle of detection of the fructose concentration and the reaction system illustrated in FIG.

[0031]

[Equation 1]

As illustrated in FIG. 2, the FDH-modified gold electrode 12 as a working electrode is a gold-cysteamine (Au-).
S) A modified gold electrode in which FDH is immobilized using a bond. The thiol compound reacts with the gold surface to form an Au-S bond. Among them, when an alkanethiol compound is used, van der Waals (vander W
aals) interaction also works, so that a dense monolayer without defects with high orientation could be prepared. Such a monolayer is called a self-assembled monolayer. The FDH-modified gold electrode 12 using a self-assembled monolayer can be manufactured simply by immersing a gold substrate in a solution of a thiol compound, and therefore can be easily manufactured at low cost. Therefore, use of such an FDH-modified gold electrode 12 not only contributes to accurate measurement of fructose concentration, but also makes it possible to manufacture a low-cost fructose concentration sensor.

The potentio-galvanostat device 7 of the present embodiment having only the potentiostat function is composed of silver /
By accurately applying a predetermined potential to the FDH-modified gold electrode 12 with reference to the potential applied to the silver chloride (Ag / AgCl) electrode 13, the potential of the FDH-modified gold electrode 12 is maintained, and the potential accompanying the progress of the reaction. The fluctuation of can be eliminated. Thus, the silver / silver chloride (Ag / AgCl) electrode 1
By using 3 as a reference, more accurate measurement of fructose concentration becomes possible.

The potentio-galvanostat device 7 is
Silver / silver chloride (Ag / AgCl) electrode 1 as a reference electrode
3 is given a predetermined potential, and silver / silver chloride (Ag / A
A predetermined potential is applied to the second electric wire W2 connected to the FDH-modified gold electrode 12 serving as a working electrode with reference to the potential applied to the gCl) electrode 13. Therefore, the potentio
The galvanostat device 7 has a power source as a potentiostat function. The potential (oxidation-reduction potential) applied from the potentio-galvanostat device 7 to the FDH-modified gold electrode 12 is, for example, +0.2 to 0.3V.

Such redox potential is determined by the above-mentioned platinum.
Polymerize a polypyrrole film on the electrode and fix FDH in it
+ 0.4V, which is the potential of the converted fructose concentration sensor
Low That is, in the present embodiment, the media
Co (phen) as data ₃ ²⁺Since Solution 5 is used,
Applying such a low redox voltage is enough
Accurate fructose concentration without being affected by interfering substances such as acid
It becomes possible to measure.

On the surface of the FDH-modified gold electrode 12, the reduced form Co (phen) ₃ produced by oxidizing the fructose of the fructose sample in the Co (phen) ₃ ²⁺ solution 5 in the measurement vial 3 by the reaction of the formula 1. ^A current flows due to the oxidation of ^{2+ on} the surface of the FDH-modified gold electrode 12.

The potentio-galvanostat device 7 also receives an electric current generated between the first electric wire W1 connected to the platinum wire 11 and the third electric wire W3 connected to the FDH-modified gold electrode 12 to receive the electric current. 9 is sent. Such an oxidation current indicates the fructose concentration of the measurement target. The potentio-galvanostat device 7
In the above, the received current may be amplified as necessary, converted into a voltage signal and input to the personal computer 9.

The personal computer 9 uses the calibration curve of the fructose concentration, which represents the relational expression between the fructose concentration and the current, which is stored in advance in the memory, and determines the fructose concentration from the measured current value of the unknown fructose sample solution in the measurement vial 3. To decide. The personal computer 9 uses the calculated fructose concentration as, for example,
Display output to the display unit.

In the fructose concentration sensor according to the first embodiment of the present invention described above, Co (phe) is used as a mediator.
n) By using the ₃ ²⁺ solution 5, the potential (oxidation-reduction voltage) applied from the potentio-galvanostat device 7 to the FDH-modified gold electrode 12 is, for example, +0.2 to 0.
Since it may be as low as 3 V, there is no problem that it is easily affected by interfering substances such as ascorbic acid (vitamin C). In addition, in the fructose concentration sensor of the first embodiment, a dense self-assembled monolayer film with high orientation, in which FDH is immobilized using a gold-cysteamine (Au—S) bond as a working electrode, has no defect. FDH modified gold electrode 12
Since fructose is used, the fructose concentration can be accurately measured. Furthermore, the FDH-modified gold electrode 12 having a self-assembled monolayer can be manufactured simply by immersing a gold substrate in a solution of a thiol compound, so that it can be easily manufactured at low cost. Therefore, use of such an FDH-modified gold electrode 12 not only contributes to accurate measurement of fructose concentration, but also makes it possible to manufacture a low-cost fructose concentration sensor. Therefore, Co (phen) ₃
According to the fructose concentration sensor of the embodiment of the present invention, which uses the ²⁺ solution 5 and the FDH-modified gold electrode 12, the fructose concentration can be measured with high selectivity and high sensitivity.

Further, in the fructose concentration sensor according to the first embodiment, since the Ag / AgCl electrode 13 which is the reference electrode is used, the fructose concentration can be accurately determined with higher sensitivity.

Of course, the fructose concentration sensor 1 according to the embodiment of the present invention illustrated in FIG. 1 can automatically and rapidly measure the fructose concentration.

Experimental Example An experiment was carried out using the fructose concentration sensor 1 illustrated in FIG. 1. As a result, Co (p) of 0.1 mM at 37 ° C., pH 7.0 was obtained.
It was possible to measure fructose of 10 to 20 mM in a hen) ₃ ²⁺ solution. The measurement time was 40 to 80 seconds. Most of the measurement time is redox reaction time, and the calculation time by the personal computer 9 is very short. From the experimental results,
It can be seen that the fructose concentration sensor of the present embodiment is suitable for practical use.

Second Embodiment FIG. 4 is a block diagram of a fructose sensor according to a second embodiment of the present invention. The fructose concentration sensor 1A illustrated in FIG. 4 includes Ag / AgC from the components of the fructose concentration sensor 1 illustrated in FIG.
The l-electrode 13 is omitted. Since the fructose concentration sensor 1A of the second embodiment excludes the Ag / AgCl electrode 13 as the reference electrode, it cannot measure using the standard fructose concentration. Therefore, the fructose concentration of various measurement objects can be measured. In this respect, the measurement accuracy may be lower than that of the fructose concentration sensor 1 illustrated in FIG. 1, but it can be used without any problem when the measurement target is already known and the reference is known in advance. . Further, the fructose concentration sensor 1A illustrated in FIG. 4 has an advantage that the structure is simplified and the price of the fructose concentration sensor becomes lower because the Ag / AgCl electrode 13 is not present.

Third and Fourth Embodiments FIG. 5 is a partial configuration diagram of a fructose sensor according to a third embodiment of the present invention. FIG. 6 is a partial configuration diagram of a fructose sensor according to a fourth embodiment of the present invention. 5 and 6, the potentio-galvanostat device 7 and the personal computer 9 which constitute the fructose concentration sensor and are illustrated in FIG.
Is omitted from the illustration.

As described above, the self-assembled monolayer can be prepared simply by immersing the gold substrate in the solution of the thiol compound, so that the dense monolayer can be easily prepared. In addition, the inventor of the present application is further
It was found that the use of the (u-S) bond makes it possible to easily produce a single separation membrane of a biopolymer and also to control the thickness of the biometric identification element membrane. Third and body 4
The embodiment shows an enlarged view of the measurement vial portion of the fructose concentration sensor manufactured by utilizing such an advantage.

[0046] accommodated fructose concentration sensor of the third embodiment as illustrated in the form Figure 5 of a third embodiment, to face the plate-shaped platinum wire 11A in the measuring vial 3A, a flat FDH modified gold electrode 12A It was done. In the measurement vial 3A, of course, Co (phe
n) A ₃ ²⁺ solution 5 and a sample for measuring fructose concentration are contained.

[0047] Fructose concentration sensor of the fourth embodiment as illustrated in the form 6 of the fourth embodiment, a gold electrode 122 deposited on substrate 121 is an electrode obtained by depositing the FDH123 on the gold electrode 122 . Thus, F
When DH123 is used in a thin film, the fructose concentration sensor can be downsized, and the measurement sensitivity can be improved and the response time can be shortened. Such an FDH-modified gold electrode 12B can be used in the fructose concentration sensor illustrated in FIGS. 1 and 4.

Although the exemplary embodiments of the fructose concentration sensor of the present invention have been described above, the embodiments of the present invention are not limited to the above-described embodiments. For example, the platinum wire 11 as the counter electrode may be a platinum mesh,
A platinum plate or the like can be used. The FDH-modified gold electrode 12 as a working electrode may have a plate shape, a linear shape, or the like. Gold is the best working electrode for a fructose concentration sensor. Furthermore, the silver / silver chloride (Ag / AgCl) electrode 13 as a reference electrode is
Although it is used from the viewpoint of low price, a saturated calomel electrode or the like can be used although it is expensive.

The fructose concentration sensor of the present invention, as long as it measures fructose concentration, is used in the field of clinical sciences such as the measurement of the concentration of fructose in food and the field of food industry, the measurement of the concentration of fructose in serum, urine and serum It can be applied to various fields.

[0050]

According to the present invention, it is possible to provide a fructose concentration sensor capable of measuring (measuring) the fructose concentration with high selectivity and high sensitivity, which is quick in measurement, simple in operation, and inexpensive. It was

[Brief description of drawings]

FIG. 1 is a configuration diagram of a first embodiment of a fructose concentration sensor of the present invention.

FIG. 2 is a diagram showing the concept of an FDH-modified gold electrode in the fructose concentration sensor illustrated in FIG.

FIG. 3 is a diagram illustrating an FDH-modified gold electrode and a fructose oxidation reaction system on the surface when a Co (phen) ₃ ²⁺ solution in the fructose concentration sensor illustrated in FIG. 1 is used as a mediator. .

FIG. 4 is a configuration diagram of a second embodiment of the fructose concentration sensor of the present invention.

FIG. 5 is a partial configuration diagram of a fructose concentration sensor according to a third embodiment of the present invention.

FIG. 6 is a partial configuration diagram of a fourth embodiment of the fructose concentration sensor of the present invention.

[Explanation of symbols]

1 ... Fructose concentration sensor 3 ... Measurement vial (container) 4 ... Top lid 5 ... Cobalt-phenanthroline complex (Co (phe
n) ₃ ²⁺ ) solution 7 ... Potentiometer galvanostat device 9 ... Personal computer (computing means) 11 ... Platinum wire 12 ... Fructose dehydrogenase (FDH) modified gold electrode 13 ... Silver / silver chloride (Ag) / AgCl) electrode

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Claims (6)

[Claims] 1. A fructose concentration sensor comprising a working electrode having fructose dehydrogenase (FDH) immobilized using a gold-cysteamine (Au—S) bond, a counter electrode, and a cobalt-phenanthroline complex as a mediator. 2. A potential applying means for applying an oxidation-reduction potential to the working electrode, and a computing means for detecting a current flowing between the working electrode and the counter electrode to calculate a corresponding fructose concentration. The fructose concentration sensor according to claim 1. 3. The fructose concentration sensor according to claim 2, further comprising a reference electrode, wherein the potential applying means applies a predetermined potential to the working electrode with respect to the potential of the reference electrode. 4. The fructose concentration according to claim 1, wherein the working electrode is a self-assembled monolayer formed by reacting an alkanethiol compound on a gold surface to form an Au—S bond. Sensor. 5. The working electrode has a gold electrode deposited on a substrate,
The fructose concentration sensor according to any one of claims 1 to 3, which is an electrode having FDH deposited on the gold electrode. 6. A fructose dehydrogenase (FDH) -immobilized gold electrode utilizing a gold-cysteamine (Au—S) bond and a cobalt-phenanthroline complex as a mediator are used to measure the fructose concentration of an object to be measured. How to measure the fructose concentration measurement method.