JPH10262695A - Electrochemical measurement of amadori compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for accurately measuring an amadori compound. SOLUTION: This method for measuring amadori compound in a sample comprises a step for adding an electron carrier to an enzyme reaction system of the amadori compound with fructosyl amino acid oxidase and a step for measuring the electron transfer of the electron carrier by an electrochemical method to determine the amadori compound in the sample.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for electrochemically measuring an Amadori compound using fructosyl amino acid oxidase, and more particularly, to a method for measuring the transfer of electrons in an enzymatic reaction via a carrier.

[0002]

2. Description of the Related Art When an Amadori compound contains a substance having an amino group such as protein, peptide and amino acid and a reducing sugar such as aldose, the amino group and the aldehyde group are non-enzymatically and irreversibly. And produced by Amadori rearrangement. The production rate of Amadori compounds depends on the concentration of protein and reducing sugar, the contact time, and the temperature.The larger the amount of protein and reducing sugar, the longer the contact time between the two, the less denaturation of the protein occurs. The higher the temperature, the faster the production rate of glycated protein and the greater the production amount. In the living body, the concentration of the Amadori compound varies depending on the half-life of the protein to be glycated. Therefore, various information can be obtained by measuring the concentration of the Amadori compound. For example, a fructosylamine derivative in which hemoglobin in blood is glycated is called glycohemoglobin, a derivative in which albumin is glycated is called glycoalbumin, and a reducing ability of a derivative in which blood protein is glycated is called fructosamine. These blood levels reflect the average blood glucose level over a period of time in the past,
The establishment of a measuring means is extremely clinically useful because it can be an important indicator for diagnosis and management of symptoms of diabetes. Further, by measuring the Amadori compound in the food, it is possible to know the preservation state and the period of the food after production, which is considered to be useful for quality control. Thus, the analysis of Amadori compounds is useful in a wide range of fields, including medicine and food.

A method of measuring the Amadori compound in a sample is known, for example, by reacting an Amadori compound with an oxidoreductase and measuring the amount of oxygen consumed or the amount of product (eg, hydrogen peroxide) produced. (For example, 5-3399
No. 7, JP-B-6-65300, JP-A-2-195900, JP-A-3-155780, JP-A-4-4874, JP-A-5-192193, JP-A-6-46846 Gazette, JP-A-7-289
253, JP-A-8-154672, JP-A-8-336386). Furthermore, a method for measuring an Amadori compound for diagnosing diabetes is also known (JP-A-2-195899, JP-A-2-195900, JP-A-5-192193 (EP 0 526 15
0 A), JP-A-6-46846 (EP 0 576 838 A), JP-A-7-46846
-289253 JP, JP-A 8-154672, JP-A 8-33638
No. 6).

[0004] Fructosyl amino acid oxidases derived from various microorganisms have been provided as enzymes that catalyze the above reaction.
m ), Gibberella , Penicillium ( Pen
FAODs derived from bacteria belonging to icillium ) and the like were shown to be useful for the measurement of Amadori compounds (JP-A-7-289253, JP-A-8-154672, JP-A-8-154672).
No. 336386.

[0005] The decomposition reaction of an Amadori compound by FAOD can be represented by the following general formula. R ¹ —CO—CH ₂ —NH—R ² + O ₂ + H ₂ O →
R ¹ -CO-CHO + R ² -NH ₂ + H ₂ O ₂ (wherein R ¹ represents an aldose residue and R ² represents an amino acid, protein or peptide residue) In the measurement of an Amadori compound using FAOD, This is done by measuring the amount of oxygen consumed or the amount of product in the enzymatic reaction mixture.

[0006]

Conventionally, as a general method for measuring Amadori compounds, a method based on the amount of hydrogen peroxide produced has been used, but reducing substances such as ascorbic acid and uric acid are present in the test sample. Then, they may not be able to measure accurately because they consume hydrogen peroxide.
As a method for electrochemically measuring hydrogen peroxide, a constant potential (for example, about +600 mV) is applied between a working electrode and a reference electrode, and hydrogen peroxide generated by an enzymatic reaction on the surface of the working electrode is measured. There is a method of measuring a current value flowing at the time of oxidation. However, in this method, since the potential required to oxidize hydrogen peroxide is higher than the potential at which ascorbic acid is oxidized, not only hydrogen peroxide but also ascorbic acid is oxidized, so accurate measurement is performed. No value is obtained.

In addition, the influence of dissolved oxygen in the measurement solution is a problem that cannot be ignored. That is, neither the method of measuring the amount of generated hydrogen peroxide with a hydrogen peroxide electrode nor the method of measuring the amount of consumed oxygen with an oxygen electrode may not be able to accurately measure the amount of dissolved oxygen. . For example, when an oxygen electrode is used, the applied voltage is -400 mV.
And the potential at which dissolved oxygen is reduced on the electrode is also -4.
Since it is below about 00 mV, the dissolved oxygen will affect the measured value as a positive error. Moreover, when oxygen consumption is measured using an oxygen electrode, a large amount of oxygen is required in accordance with the substrate concentration in the enzymatic reaction. There is also the problem that it cannot be done.

Accordingly, the potential applied between the working electrode and the reference electrode is set to about +200 mV where oxidation of ascorbic acid does not occur.
It is desirable to measure the production amount of hydrogen peroxide at -400 mV or more where oxygen decomposition does not occur, but the sensitivity is low at a potential in this range, which is practically unsuitable for the measurement of Amadori compounds. is there. In addition, when using either a hydrogen peroxide electrode or an oxygen electrode, it is necessary to use a noble metal such as platinum or gold as an electrode material, and the cost of the measuring device is high, so that it is difficult to apply it to a wide range of clinical applications. There is also a problem that it becomes. As described above, the conventional method has a problem that it is easily affected by reducing substances such as ascorbic acid and uric acid and various substances such as dissolved oxygen.
Moreover, the former is contained in large amounts in biological samples (serum and urine), and the latter is widely present in all samples, so that it is accurately, efficiently, and not affected by these interfering substances. There has been a need for a method for economically measuring Amadori compounds.

[0009]

SUMMARY OF THE INVENTION The present invention relates to a method for measuring an Amadori compound in a sample, comprising adding an electron carrier to an enzyme reaction system between the Amadori compound and fructosyl amino acid oxidase; It is an object of the present invention to provide a method for measuring an Amadori compound, which comprises quantifying an Amadori compound in a sample by electrochemically measuring the movement. Further, the present invention is an apparatus for measuring the amount of an Amadori compound in a sample based on the transfer of electrons in an enzymatic reaction between the Amadori compound and fructosyl amino acid oxidase, which is close to a reaction system of the enzyme reaction. The present invention provides a working electrode, an electron carrier interposed between the reaction system and the working electrode, and a device for measuring electron transfer in the electron carrier.

[0010]

DETAILED DESCRIPTION OF THE INVENTION In the method of the present invention, any sample containing an Amadori compound can be used. For example, a sample derived from a living body such as blood (whole blood, plasma or serum), urine and soy sauce can be used. Foods can be mentioned. According to the method of the present invention, the Amadori compound can be measured while avoiding the effects of reducing substances such as ascorbic acid and uric acid in the sample. Measurement can be performed accurately. As the FAOD used in the method of the present invention, any one can be used as long as it specifically acts on the Amadori compound. For example, the genus Fusarium (Fusarium), Gibberella genus (Gibbere
lla), Penicillium (Penicillium), there may be mentioned an enzyme derived by culturing bacteria belonging to such genera Aspergillus (Aspergillus) in the presence of fructosyl lysine and / or fructosyl N ^alpha -Z- lysine, They are disclosed, for example, in JP-A-7-289253,
It can be obtained by the methods disclosed in JP-A-8-154672 and JP-A-8-336386.

Although the method of the present invention can be carried out using a sample solution as it is, depending on the target Amadori compound, the treatment is carried out in such a state that the FAOD can easily react. In order to appropriately fragment the Amadori compound, there are a method using a protease (enzymatic method), a method using a chemical substance such as trichloroacetic acid (chemical method), and a method using a physical method such as heat (physical method). . Enzymatic methods are known to those skilled in the art,
Endo- and exo-type proteases can be used alone or in combination. Endo-type proteases are enzymes that degrade from the inside of proteins, such as trypsin, α-chymotrypsin, subtilisin,
Proteinase K, papain, cathepsin B, pepsin, thermolysin, protease XIV, protease XVII, protease XXI, lysyl endopeptidase, proleather, bromelain F and the like. On the other hand, exo-type proteases are enzymes that decompose sequentially from the end of the peptide chain, and include aminopeptidase, carboxypeptidase and the like. Enzyme treatment methods are also known and can be performed, for example, by the method described in the following Examples.

It is preferable that these endo-type and exo-type proteases are used properly in accordance with the saccharification site of the Amadori compound to be measured, utilizing its properties. For example, glycated albumin is an endo-type protease, since the internal lysine residue is glycated, and hemoglobin A1
c is because the valine residue at the N-terminal of the β chain is saccharified,
It can be treated more efficiently with an exo-type protease. Next, FAOD is caused to act on the sample containing the fragmented Amadori compound, electrons generated in the active center of the oxidase are transmitted to the working electrode via an electron mediator, and a current based on the transfer of the electrons is transmitted. By measuring, the amount of the Amadori compound in the sample is measured.

The measurement of an Amadori compound using an electron carrier (electron carrier) according to the method of the present invention will be described in more detail. When FAOD, which is an oxidase, is reacted with an Amadori compound (substrate) in the presence of oxygen, glucosone and hydrogen peroxide are generated as shown in the above formula. Is released. When an oxidized electron carrier is present in the reaction system of this enzymatic reaction, the electron carrier receives electrons emitted during the reaction from the center of the enzyme activity, and supplies electrons to the working electrode as a reduced electron carrier. Itself becomes an oxidized electron carrier. Accordingly, an electric current is generated in the electrode depending on the number of electrons (substrate concentration).
Therefore, by using a standard substance having a known concentration in advance and preparing a calibration equation showing the relationship between the concentration of the substance and the current, the measured current value when an unknown sample is used is applied to the calibration equation to obtain the unknown. The concentration of the Amadori compound in the sample can be determined.

Since the electron carrier has a specific oxidation-reduction potential depending on the type, it is possible to measure even if the potential applied to the working electrode is low depending on the electron carrier used. Therefore, accurate measurement can be performed at a low potential that can avoid the above-described effects of oxidation of ascorbic acid, decomposition of dissolved oxygen, and the like. In addition, the selection of the electron carrier makes it possible to perform measurement with much higher sensitivity than the conventional method of detecting the generation of hydrogen peroxide as a current value using a hydrogen peroxide electrode.

The electron carrier that can be used in the method of the present invention includes potassium ferricyanide, metallocene and metallocene derivatives, octacyanotungsten ion,
Nicotinamide derivatives, flavin derivatives, ferrocene and ferrocene derivatives, quinones and quinone derivatives, hexacyanoferrates, and the like can be used, but potassium ferricyanide, ferrocene, and nickelocene are preferred. These may be used alone or in combination. The nickelocene derivatives, nickelocene also include derivatives of _{((C 5 H 5) Ni} ), for example, 1,1'-dicarboxylate nickel Russia Sen, monocarboxylate nickel Russia Sen, dimethylaminomethyl nickel Russia Sen, hydroxymethyl nickel b Sen etc. Can be exemplified. Nickelocene or its derivatives can also be used alone or in various mixtures with other electron carriers.

Hereinafter, the form of the working electrode used in the present invention will be described. The material of the electrode is not particularly limited as long as it is an electrode that can be used at a low potential, but a readily available carbon electrode material is preferable. However, a noble metal such as gold or platinum may be used if necessary. In the case of carbon electrode material,
Carbon or a combination of carbon and other materials, such as a composition, can be used. Carbon granules and fine granules are preferred because they have the advantage of being highly conductive, being inexpensive to mix with other powders and granular materials, and being easy to mold. The average particle diameter of the carbon particles is preferably about 5 to 20 μm, and more preferably about 10 μm.

The carbon electrode is made of carbon particles (powder).
And an electron carrier, and are formed into a paste in the presence of a pasting agent such as paraffin, nujol, wax, epoxy resin, silicone rubber, and the like, and filled at the tip of a glass tube. It is formed by inserting until it comes into contact with the layer of. The mixing ratio between the carbon particles and the electron carrier ranges from 9: 1 to 5: 5, and 8: 2 to 5: 5.
6: 4 is preferred.

When the paste-like carbon mixture contains platinum particles, particularly fine-particle platinum, a higher response current can be obtained than in the case of using carbon alone, and the peak value of the response current can be reduced at a lower applied potential between the electrodes. It is preferable because it can be obtained. The size of the platinum particles contained in the carbon electrode is preferably about 0.3 to 2.5 μm in average diameter, and particularly preferably smaller than carbon particles. The mixing ratio of carbon particles and platinum particles is 100: 1 to 8
A range of 5:15 is preferred.

When the measurement is performed in the liquid phase, FAOD
It may be mixed with the measurement solution. However, by laminating on the surface of the carbon electrode manufactured as described above, by covering with a porous membrane such as a semipermeable membrane or ultrafiltration membrane so as not to fall off,
It may be integrated with the electrode. FAO formed in this way
In the D-carbon electrode, since the reaction system of the enzyme reaction and the electrode are close to each other, it is possible to accurately detect the transfer of electrons accompanying the enzyme reaction, which is convenient. Moreover, FAOD-
Since the carbon electrode can be prepared in advance and used repeatedly as necessary, not only can the measurement be performed simply and quickly, but also the resources can be effectively used. When the FAOD-carbon electrode is used for measurement in the liquid phase, it is necessary to cover the surface with a porous film or the like, but the sample is dropped directly from above onto the FAOD layer of the electrode, and the An enzymatic reaction can also be performed, in which case it is not necessary to cover with a porous membrane or the like.

Further, the FAOD constructed as described above
-Without contact with the carbon electrode, the FAOD-laminated carbon electrode may be coated with a porous membrane on which protease, ascorbin oxidase, and uricase are immobilized. At this time, it is necessary that the pore size of the porous membrane is large enough to allow a product fragmented by the protease of the Amadori compound or the like to pass through. By using the working electrode configured in this way, even in the case of a sample such as a biological component containing a high concentration of a reducing substance such as ascorbic acid or uric acid, the sample is caused by reduction of the electron carrier by the reducing substance. There is no possibility that a positive error occurs, and an accurate measurement value can be obtained. As described above, if FAOD and other necessary enzymes are immobilized on the carbon working electrode, it is not necessary to add the enzymes every measurement, so that the measurement can be performed quickly and efficiently. Moreover, such electrodes can be used repeatedly and are economical.

The potential applied between the working electrode and the reference electrode varies depending on the electron mediator used, but is usually -400.
To +300 mV, preferably -300 to +200 mV. When the electron carrier is nickelocene or a nickelocene derivative, the potential between the reference electrode and the working electrode is about -3.
A range of 00 mV to +100 mV is preferred, and about -100
When applied to mV, electrons are efficiently transmitted.
According to the method of the present invention, a potential at which oxidation of ascorbic acid hardly occurs and a potential at which oxygen is decomposed (about -4)
Since the measurement can be performed at a potential higher than 00 mV), the measurement can be accurately performed without being affected by ascorbic acid or dissolved oxygen in the sample. Therefore, the method of the present invention is suitable for measuring an Amadori compound in a biological component having a high possibility of containing these substances.

Hereinafter, the present invention will be described in more detail by way of examples. Example 1 Measurement of Saccharified Albumin Concentration (1) Amadori Compound Measuring Apparatus FIG. 1 shows an example of an Amadori compound measuring apparatus, in which 1 is a container, 2 is a measuring solution containing a test substance, and 3 is stirring. Child, 4 working electrode, 5 reference electrode (Ag / AgCl), 6 counter electrode
(Pt). The working electrode 4 can be prepared, for example, as shown in FIG. Carbon powder (average particle size 10
μm) 200 mg, potassium ferricyanide 200 mg and paraffin 250 μl were mixed to form a paste, and the resulting paste-like carbon mixture 8 was placed in a glass tube (with an inner diameter of 5 μm).
mm). Next, the carbon mixture layer 8
, Insert a silver stick 9 into the Fusarium Oxysporum S-
FAOD derived from 1F4 ( Fusarium oxysporum S-1F4; FERM BP-5010) (Japanese Patent Laid-Open No. 7-289253, 70 units / m)
g), and the FAOD layer 10 is covered with a porous membrane 11;
It is created by stopping at the O-ring 12.

(2) Measurement of Glycated Albumin Concentration Glycated human serum albumin (SIGMA) was dissolved in a 0.9% aqueous sodium chloride solution to prepare glycated human serum albumin solutions having different concentrations in the range of 0 to 10 mg / ml.
200 μl of this human serum albumin solution and 12.5 mg / ml
200 μl of protease XIV was mixed and incubated at 37 ° C. for 30 minutes. Thereafter, the protease reaction was stopped by heating at 100 ° C. for 5 minutes to obtain a substrate. 5 μl of 0.1 M Tris-HCl buffer (pH 8.0) was placed in the container 1, and the working electrode 4, the reference electrode 5 and the counter electrode 6 were immersed.
The above-mentioned protease-treated solution (substrate) was added while stirring with the stirrer 3, and the current value was measured at 30 ° C. and 200 mV constant potential. FIG. 3 shows the relationship between the saccharified albumin concentration obtained by this method and the current value. The vertical axis in the figure represents the current value corresponding to the transfer of electrons accompanying the enzyme reaction detected via the electron mediator, and the vertical axis represents the concentration of saccharified albumin. The figure shows that the concentration of glycated albumin and the current value are correlated.

[0024]

According to the method of the present invention, an Amadori compound can be accurately and simply measured without being affected by ascorbic acid or dissolved oxygen. Therefore, the method of the present invention makes it easier to routinely measure an Amadori compound in a biological component likely to contain these substances, and contributes to the management and diagnosis of diabetes symptoms.

[Brief description of the drawings]

FIG. 1 is an explanatory view of an Amadori compound measuring device.

FIG. 2 is an explanatory view showing a method for producing a working electrode according to one embodiment.

FIG. 3 is a graph showing the relationship between glycated human albumin concentration and current.

FIG. 4 is an explanatory view showing a method for producing a working electrode according to another embodiment.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Container 2 Measurement solution 3 Stirrer 4 Working electrode 5 Reference electrode 6 Counter electrode 7 Glass tube 8 Carbon mixture 9 Silver rod 10 FAOD layer 11 Porous film 12 O-ring 13 Porous film

Claims (12)

[Claims] 1. A method for measuring an Amadori compound in a sample, comprising adding an electron carrier to an enzyme reaction system between the Amadori compound and fructosyl amino acid oxidase, and electrochemically measuring the electron transfer of the electron carrier. A method for measuring an Amadori compound, wherein the Amadori compound in the sample is quantified. 2. The method according to claim 1, wherein an electrode comprising a carbon electrode material, an electron carrier and fructosyl amino acid oxidase is used as a working electrode. 3. The method according to claim 2, wherein the carbon electrode material also contains platinum particulates. 4. The method according to claim 1, wherein the electron carrier is potassium ferricyanide,
The method according to any one of claims 1 to 3, wherein the method is one or more selected from ferrocene and nickelocene. 5. A voltage between −400 and + between a working electrode and a reference electrode.
2. The method according to claim 1, wherein a potential of 300 mV is applied.
5. The measuring method according to any one of items 1 to 4. 6. The method according to claim 5, wherein the potential applied between the working electrode and the reference electrode is from −300 to +200 mV. 7. The method according to claim 1, wherein a protease, ascorbate oxidase, and uricase are further included in the reaction system. 8. An apparatus for measuring the amount of an Amadori compound in a sample based on the transfer of electrons in an enzymatic reaction between the Amadori compound and fructosyl amino acid oxidase, wherein the apparatus is located close to a reaction system of the enzyme reaction. A working electrode;
An electron carrier interposed between the reaction system and the working electrode, and an apparatus for measuring electron transfer in the electron carrier. 9. The device according to claim 8, wherein the working electrode comprises a carbon electrode material, an electron carrier and fructosyl amino acid oxidase. 10. The apparatus according to claim 9, wherein the carbon electrode material also contains platinum particulates. 11. The device according to claim 8, wherein the electron carrier is one or more selected from potassium ferricyanide, ferrocene and nickelocene. 12. The apparatus according to claim 8, wherein a porous membrane on which a protease, ascorbate oxidase, and uricase are immobilized is provided near the working electrode without contacting the electrode.