JPWO2007094354A1 - Hemoglobin A1c sensor - Google Patents

Abstract

A sensor for measuring hemoglobin A1c in a sample having at least an electrode composed of a counter electrode and a working electrode, wherein fructosyl peptide oxidase, preferably further a mediator, more preferably a proteolytic enzyme is immobilized on the working electrode Hemoglobin A1c sensor. This hemoglobin A1c sensor enables easy and rapid measurement of hemoglobin A1c, and by providing a working electrode on which an enzyme for detecting a diabetes marker other than hemoglobin A1c is fixed separately from the working electrode on which FPOX is fixed, A plurality of diabetes markers having different functions can be simultaneously measured from the same sample.

Description

  The present invention relates to a hemoglobin A1c sensor. More specifically, the present invention relates to a hemoglobin A1c sensor that can accurately measure hemoglobin A1c by a simple operation.

  In diagnosing, managing and preventing diseases such as diabetes, measurement of glycated protein is important along with measurement of glucose. This glycated protein is produced by non-enzymatic reaction between the amino group of the protein main chain or side chain and the reducing end of a reducing sugar such as glucose. The glycated protein is also called a so-called Amadori compound because a Schiff base generated as a reaction intermediate undergoes Amadori rearrangement.

  Such glycated proteins are contained in body fluids such as blood and saliva in living bodies, biological samples such as hair, and foods such as juices, candy, seasonings, and powdered foods. The concentration of glycated protein present in blood strongly depends on the concentration of reducing sugar such as glucose in blood. In diabetes, the production of glycated protein is enhanced, and the concentration of glycated hemoglobin contained in erythrocytes and the concentration of glycated albumin in serum reflect the average blood glucose level over a certain period in the past. Glucose shows the current glycemic state, whereas 1,5-anhydroglucitol is about several days ago, glycated albumin is about 1-2 weeks ago, and glycated hemoglobin is about 1-2 months ago In order to reflect the blood glucose level, measurement of these glycated proteins is important for the time-dependent diagnosis or symptom management of diabetes symptoms in that the blood glucose concentration can be determined over time.

  Currently, glycated proteins used as an index for diagnosis of diabetes include glycated albumin in which the ε-amino group of lysine residues in proteins such as albumin is glycated, and α-amino acid of the N-terminal amino acid of proteins such as hemoglobin. Various glycated proteins such as glycated hemoglobin in which an amino group is glycated are known.

  Hemoglobin A1c is one of the glycated hemoglobins. Glucose non-enzymatically binds to the N-terminal amino acid of hemoglobin β-subunit, forms a Schiff base, and then has a structure in which fructose is bound by Amadori rearrangement. It is a glycated protein. Since hemoglobin A1c clinically reflects the average blood glucose level in the past 1 to 2 months, it is important as an index for diabetes management, and a rapid and accurate quantitative method is required.

  Currently, methods for quantifying hemoglobin A1c include, for example, chromatographic methods such as HPLC, immunoassay methods using antibodies such as electrophoresis and latex immunoagglutination, and enzyme methods using enzymes that act on glycated proteins. ing.

Among these methods, the enzymatic method is a process in which glycated proteins are decomposed with proteolytic enzymes, and fructosyl amino acid oxidase (hereinafter abbreviated as FAOX), which is a type of glycated amino acid oxidase, acts on the released glycated amino acids. A method for measuring hydrogen oxide by absorptiometry has been proposed. A hemoglobin A1c sensor that responds to glycated amino acids released from glycated proteins by proteolytic enzymes and that uses a FAOX-immobilized electrode as a working electrode has also been proposed.
JP-A-5-192193 JP2003-274976

  However, although the fructosyl amino acid oxidase currently used has high reactivity with a free glycated amino acid, it has little effect on a glycated peptide obtained by degrading a glycated protein with a protease. Enzymatic methods are not necessarily accurate methods.

  An object of the present invention is to provide a hemoglobin A1c sensor that enables simple, rapid and accurate measurement of hemoglobin A1c.

An object of the present invention is a sensor for measuring hemoglobin A1c in a sample, which has an electrode composed of at least a counter electrode and a working electrode, wherein the fructosyl peptide oxidase is immobilized on the working electrode. Achieved by sensor.
The present invention relates to the following (1) to (12).
(1) A sensor for measuring hemoglobin A1c in a sample having at least a counter electrode and a working electrode, wherein fructosyl peptide oxidase (hereinafter abbreviated as FPOX) is fixed on the working electrode. The hemoglobin A1c sensor (2) is a sample containing blood cells. The hemoglobin A1c sensor (3) described in (1) is a sample treated with a surfactant (1) or (2) The hemoglobin A1c sensor (4) The mediator is further immobilized on the working electrode. The hemoglobin A1c sensor (5) according to any one of (1) to (3), and a proteolytic enzyme is further immobilized on the working electrode. The hemoglobin A1c sensor according to any one of (1) to (4) (6), wherein the electrode is a hydrogen peroxide electrode or an oxygen electrode (1) to (5) The hemoglobin A1c sensor according to (7), further comprising a reference electrode, the hemoglobin A1c sensor according to any one of (1) to (6), wherein the electrode is formed on an insulating substrate (1) to (7 The hemoglobin A1c sensor according to any one of (1) (9) and the hemoglobin according to any one of (1) to (8), further comprising a working electrode on which an enzyme for detecting a diabetes marker other than hemoglobin A1c is immobilized. A1c sensor (10) The hemoglobin A1c sensor according to any one of (1) to (9), a hemoglobin A1c measurement device (11) having a voltage application unit and a current value measurement unit, and further comprising a hemoglobin measurement unit and a hemoglobin A1c concentration calculation unit. The provided hemoglobin A1c measuring device according to (10) (12) and further provided with a measuring unit for detecting a diabetes marker other than hemoglobin A1c. And (10) or (11) hemoglobin A1c measurement apparatus according

  Since the hemoglobin A1c sensor according to the present invention has FPOX fixed on the working electrode, the concentration of hemoglobin A1c contained in the sample can be easily measured. In addition, there is an excellent effect that no special pretreatment of the sample is required even in the presence of blood cells. Furthermore, better measurement sensitivity and accurate measurement are possible by treating the sample with a surfactant.

  Here, when a mediator is fixed on the working electrode in addition to FPOX, a larger output value can be obtained, so that the hemoglobin A1c concentration of the measurement sample can be measured more accurately.

  When proteolytic enzyme is immobilized on the working electrode in addition to FPOX or FPOX and a mediator, it can be directly used for measurement of hemoglobin A1c without pretreatment of the sample.

  On the other hand, by using a device that can measure diabetes markers other than hemoglobin A1c at the same time, it is possible to measure multiple diabetes markers with different functions from the same sample at the same time, and grasp changes in blood glucose status of the subject This makes it possible to achieve an excellent effect that blood glucose control can be made possible.

An example of the measuring apparatus which comprises the batch type hemoglobin A1c sensor of this invention is shown. An example of the measuring apparatus which comprises the flow type hemoglobin A1c sensor of this invention is shown. The other example of the measuring apparatus which comprises the flow type hemoglobin A1c sensor of this invention is shown. It is a graph which shows the responsiveness of this hemoglobin A1c sensor with respect to hemoglobin A1c. It is a graph which shows the responsiveness of the glucose sensor attached to this hemoglobin A1c sensor with respect to glucose. It is a graph which shows the measurement result of the glycated amino acid by the absorptiometry using FPOX or FAOX. It is a graph which shows the response with respect to glycated amino acid by the hemoglobin A1c sensor which fixed FPOX or FAOX on the working pole. It is a graph which shows the response with respect to the glycated amino acid in the presence of a blood cell by the hemoglobin A1c sensor which fixed FPOX or FAOX on the working pole. It is a graph which shows the measurement result of the glycated amino acid in the presence of a blood cell by the absorptiometry using FPOX or FAOX. It is a graph which shows the effect of surfactant in the responsiveness with respect to glycated amino acid in the presence of a blood cell by this hemoglobin A1c sensor. It is a graph which shows the effect of surfactant in the response with respect to glycated hexapeptide by this hemoglobin A1c sensor. It is a graph which shows the response with respect to the glycated hexapeptide in presence of a blood cell and surfactant by the hemoglobin A1c sensor which fixed FPOX or FAOX on the working pole.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Sample addition part 2 Voltage application part 3 Hemoglobin A1c sensor 4 Current value measurement part 5 Hemoglobin measurement part 6 Concentration calculation part 7 Wiring 8 Waste liquid discharge part 9 Tube 10 Carrier 11 Pump

Hemoglobin A1c sensor The sensor of the present invention has at least two electrodes of a counter electrode and a working electrode, and FPOX, preferably a mediator and / or a proteolytic enzyme is immobilized on the working electrode. The sensor of the present invention preferably has three electrodes provided with a reference electrode.

  Here, a sensor having two electrodes, a working electrode and a counter electrode, is suitable for downsizing, and a sensor having three electrodes, a working electrode, a counter electrode, and a reference electrode, is suitable for measurement with high measurement accuracy.

  The electrode material is not particularly limited as long as the enzyme can be stably held, and examples thereof include platinum, platinum black, gold, silver, palladium, carbon, carbon paste, carbon nanotube, iridium, and diamond. Examples of platinum black include platinum formed by sputtering, vapor deposition, and screen printing, and carbon nanotubes are manufactured by methods such as arc discharge, vapor deposition, and laser evaporation. A multilayer structure or a single-layer structure is also used. The counter electrode, the reference electrode, and the working electrode may be the same material or different materials, but preferably the working electrode and the counter electrode are made of the same material, and the reference electrode is made of a material different from these.

  The electrode can be used by immersing it in a measurement sample as it is, but it can also be formed on an insulating substrate. Examples of the material for the insulating substrate include ceramic, glass, plastic, biodegradable material, non-woven fabric, or paper. The electrode is formed on the substrate by a screen printing method, a vapor deposition method, a sputtering method, or the like. The substrate on which the electrodes are formed is used as a flow injection analysis (FIA) type sensor unit and as a disposable simple sensor.

Examples of FPOX, which is an enzyme that acts on a glycated peptide and generates hydrogen peroxide, include, for example, fructosyl valyl histidine (hereinafter abbreviated as Fru-Val-His), fructosyl valine (hereinafter abbreviated as Fru-Val). Examples include commercially available products such as FPOX-CE (manufactured by Kikkoman), FPOX-EE (manufactured by the same company), and the like.
JP 2004-275013 A

  The proteolytic enzyme is not particularly limited as long as it is an enzyme capable of degrading a glycated protein in a sample into a glycated peptide, and a suitable one is appropriately selected depending on the type of glycated protein or glycated peptide to be cleaved. Can do.

  Examples of the proteolytic enzyme include proteinase K, pronase, thermolysin, subtilisin (subtilisin), carboxypeptidase B, pancreatin, cathepsin, carboxypeptidase, endoproteinase Glu-C, papain, ficin, bromelain, aminopeptidase and the like. Examples include proteolytic enzymes including enzymes.

  In the present invention, particularly as a proteolytic enzyme that efficiently generates a glycated dipeptide, Aspergillus-derived proteolytic enzyme, lysopath-derived proteolytic enzyme, Bacillus-derived proteolytic enzyme, Streptomyces-derived proteolytic enzyme, Trityratiium-derived proteolytic enzyme, Staphylococcus-derived proteolytic enzymes, plant-derived proteolytic enzymes, animal-derived proteolytic enzymes and the like can be mentioned.

  Specific examples of Aspergillus derived proteolytic enzymes include, for example, "IP enzyme", "AO protease", "peptidase", "Morcine" (manufactured by Kikkoman), "Protease A5" (manufactured by Kyowa Kasei), "Umamizyme" , `` Protease A '', `` Protease M '', `` Protease P '' (manufactured by Amano Enzyme), `` Sumiteam MP '', `` Sumiteam LP-20 '', `` Sumiteam LPL '', `` Sumiteam AP '' , “Proteinase 6” (Fluka), “Proteinase TypeXXIII” (Sigma) and the like.

  Specific examples of the lysopath-derived proteolytic enzyme include “peptidase R” (manufactured by Amano Enzyme) and the like.

  Specific examples of proteolytic enzymes derived from Bacillus include, for example, “dispase” (manufactured by Roche), “subtilisin” (manufactured by Boehringer), “proteinase N” (manufactured by Fluka), “proteinase Type VII” (manufactured by Sigma), “Proteinase, Bacterial” (Fluka), “Protease N”, “Protease S”, “Protease N“ Amano ”G”, “Pro Leather FG-F”, “Protease S“ Amano ”G” (above Amano Enzyme) ), Proteinase TypeX (Sigma), Thermolysin (Daiwa Kasei), Pronase E (Kaken Chemical), Neutral Protease (Toyobo), etc. .

  Specific examples of the Streptomyces-derived proteolytic enzyme include “pronase” (manufactured by Boehringer Mannheim), “proteinase TypeXIV” (manufactured by Sigma), “alkaline protease” (manufactured by Toyobo Co., Ltd.), and the like.

  Examples of the tritylatium-derived protease include “proteinase K” (Roche, Wako Pure Chemical Industries).

  Specific examples of Staphylococcus-derived proteolytic enzymes include, for example, “Glu-C” (manufactured by Boehringer).

  Specific examples of plant-derived proteolytic enzymes include, for example, papain (Roche, Wako Pure Chemical Industries, Sigma, Amano Enzyme, Asahi), Ficin (Sigma), Bromelain (Amano Enzyme). And Sigma).

  Specific examples of animal-derived proteolytic enzymes include “pancreatin” (manufactured by Wako Pure Chemical Industries, Ltd.), cathepsin B (manufactured by Sigma), and the like.

The above proteolytic enzymes may be used alone or in combination of two or more. For example, hemoglobin A1c has been shown to produce the glycated hexapeptide Fru-Val-His-Leu-Thr-Pro-Glu (hereinafter abbreviated as Fru-6P) by endoproteinase Glu-C. Therefore, combining Glu-C with the proteolytic enzyme that produces the glycated dipeptide is an extremely effective method for producing a glycated dipeptide from hemoglobin A1c (Clin. Chem. 1997, 43 1944-1951).

  The method for immobilizing FPOX or FPOX and proteolytic enzyme on the working electrode is not particularly limited as long as the enzyme can be stably held on the working electrode, but the enzyme solution is dropped on the electrode and dried. Method, crosslinking method using albumin-glutaraldehyde, inclusion method using water-soluble photocrosslinkable resin, method of mixing in mineral oil, bromoform, nujol, tetrafluoroethylene or Nafion, covalent bonding method using reagents, etc. Is mentioned.

  In addition to these enzymes, a mediator is preferably used on the working electrode. The mediator is not particularly limited as long as it is a substance that participates in the reaction that generates electrons in the presence of FPOX and makes the transfer between the electron and the electrode efficient, but for example, potassium ferricyanide, methoxyphenazine methosulfate, Examples include quinone derivatives, ferrocene or derivatives thereof, and osmium complexes. Since a larger output value can be obtained by using the mediator, the hemoglobin A1c concentration in the measurement sample can be measured more accurately.

As a structure of a working electrode, the following aspect is mentioned, for example.
(1) Only FPOX is fixed to the same working electrode
(2) FPOX and mediator are fixed to the same working electrode
(3) FPOX and proteolytic enzyme are immobilized on the same working electrode
(4) FPOX, mediator and proteolytic enzyme are immobilized on the same working electrode

  The FPOX, mediator, and proteolytic enzyme on the working electrode can be formed as at least two layers as the same layer, but can also be formed as a multilayer with different layers. In the case of forming a multilayer, it is preferable that FPOX is directly fixed on the working electrode, and that the proteolytic enzyme is laminated on the FPOX layer. In the case where a mediator is used, the working electrode and the FPOX layer are used. It is preferable to form a mediator layer between them.

  Furthermore, a polymer material layer can be provided on the electrode for the purpose of efficiently fixing the enzyme on the electrode while maintaining the enzyme activity. Examples of the polymer material include synthetic polymer materials, polysaccharides, and porous materials. When a polymer material layer is provided, it is preferable to fix FPOX on the polymer layer.

  The electrode may be either a hydrogen peroxide electrode or an oxygen electrode, and in the case of a hydrogen peroxide electrode, by electrochemically measuring the amount of hydrogen peroxide generated from hemoglobin A1c by an enzymatic reaction, In the case of an oxygen electrode, the concentration of hemoglobin A1c is measured by electrochemically measuring the amount of oxygen consumed in the enzymatic reaction of hemoglobin A1c.

Specifically, H ₂ O ₂ generated when FPOX is further reacted with Fru-Val-His generated by allowing proteolytic enzyme to act on hemoglobin A1c. The amount of hydrogen peroxide generated from hemoglobin A1c is measured electrochemically by measuring the electrons generated or consumed by the equation as a current value.
For oxidation by the _{electrode: H 2 O 2 → O 2} + 2H + + 2e -
For reduction with ^{_{electrodes: H 2 O 2 + 2H +}} + 2e - → 2H 2 O

In the case of an oxygen electrode, the amount of oxygen consumed in the enzymatic reaction of hemoglobin A1c is electrochemically measured by measuring the electrons consumed by the following reaction equation as a current value.
_{^{O 2 + 2H + + 2e -}} → H 2 O 2

  The method for measuring the electrons generated or consumed by the enzyme reaction is not particularly limited as long as it is a method capable of measuring electrons, and for example, chronoamperometry, cyclic voltammetry, coulometry and the like are used.

  The hemoglobin A1c measurement sample is not particularly limited as long as it contains hemoglobin A1c, and examples thereof include whole blood and blood cell-containing samples. In the measurement, the sample can be subjected to processing such as centrifugation, washing operation, dilution, concentration, addition of a surfactant, and heating. In addition, a sample in which the aforementioned proteolytic enzyme is allowed to act in advance can be used.

  As the surfactant, for example, a nonionic surfactant, a cationic surfactant, an anionic surfactant, or an amphoteric surfactant is used, and an amphoteric surfactant is preferably used.

  Nonionic surfactants include, for example, polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether, polyoxyethylene polycyclic phenyl ether, polyoxyethylene polyoxypropylene condensate, polyoxyethylene alkylamine, alkylenediamine-poly Examples thereof include oxyethylene polyoxypropylene condensate, polyoxyethylene fatty acid ester, polyoxyethylene sorbitan fatty acid ester, sorbitan fatty acid ester, polyoxyethylene sorbitol fatty acid ester, glycerin fatty acid ester, fatty acid alkanolamide, and sucrose fatty acid ester.

  Examples of the cationic surfactant include aliphatic amine salts and aliphatic quaternary ammonium salts.

  Examples of the anionic surfactant include carboxylate, sulfonate, sulfate ester salt, phosphate ester salt, cholate salt and the like.

  Examples of amphoteric surfactants include betaines, glycines, alanines, 2-alkylimidazoline derivatives, amine oxides, and alkylamino salts.

  The form of the hemoglobin A1c sensor according to the present invention is not particularly limited as long as the user can use this sensor. The sensor can be used repeatedly, but may be disposable.

  The method for measuring hemoglobin A1c using the hemoglobin A1c sensor according to the present invention is not particularly limited as long as it can measure hemoglobin A1c in a sample. For example, a flow type in which a sample reaches an electrode through a flow path. Examples thereof include a batch type in which a sample and an electrode are in contact in one reaction vessel, or a chip type in which a terminal at one end of an electrode is connected to a voltage application / output measuring device.

Hemoglobin A1c measurement method of hemoglobin A1c measurement method The present invention uses a hemoglobin A1c sensor of the present invention is a method of measuring hemoglobin A1c in a sample. The measurement can be performed, for example, including the following steps.
(1) Step of creating a calibration curve (2) Step of applying a sample to the hemoglobin A1c sensor of the present invention and measuring the current value of the sample with respect to hemoglobin A1c (3) The calibration curve created in (1) above Step of measuring hemoglobin A1c in the sample from the current value in (2)

  Here, the ratio of hemoglobin A1c to the total hemoglobin can be calculated from the total hemoglobin concentration in the sample. Any method can be used for the concentration of total hemoglobin as long as it is a known method for measuring hemoglobin. For example, the methemoglobin method, the cyanmethemoglobin method, the azaid methemoglobin method, the sodium dodecyl sulfonate (SLS) method, the alkali Examples include the hematin method, the green chromophore formation method, and the oxyhemoglobin method.

  In addition, as a standard product used for preparing a calibration curve, hemoglobin A1c of known concentration, glycated peptide such as Fru-Val-His-Leu-Thr-Pro-Glu, Fru-Val-His, Fru-Val, etc. The glycated amino acid can be used.

Hemoglobin A1c measuring device The hemoglobin A1c concentration is measured by a hemoglobin A1c measuring device. The measuring device has a mechanism for detecting a current value generated when at least the hemoglobin A1c sensor comes into contact with the sample, preferably a hemoglobin measuring unit, a concentration calculating unit for calculating hemoglobin A1c (%), etc. It comprises. For example, FIG. 1 shows a measurement apparatus when a batch type hemoglobin A1c sensor is used, and FIGS. 2 and 3 show an example of the measurement apparatus when a flow type hemoglobin A1c sensor is used. In any case, the current value measurement unit 4 measures the current value corresponding to the amount of hemoglobin A1c in the sample by the hemoglobin A1c sensor 3 connected to the voltage application unit 2 for the sample supplied from the sample addition unit 1. On the other hand, the hemoglobin concentration is measured by the hemoglobin measuring unit 5 and finally the hemoglobin A1c concentration is calculated by the concentration calculating unit 6 from these values. 1 and 2 show an apparatus in which a hemoglobin measurement unit and a hemoglobin A1c sensor are in parallel, and FIG. 3 shows an apparatus in which a hemoglobin measurement unit and a hemoglobin A1c sensor are in series.

  The current value measuring unit 4 is a voltage application unit such as a potentiostat that generates or consumes electrons generated in the reaction between hemoglobin A1c supplied from the sample addition unit 1 and an enzyme fixed on the working electrode of the hemoglobin A1c sensor 3. 2 is a portion that is measured and displayed as a current value by applying a voltage according to 2.

  Here, the hemoglobin measuring unit 5 measures the amount of hemoglobin. This is necessary for measuring the hemoglobin A1c concentration by calculating the ratio of the hemoglobin A1c amount supplied from the sample addition unit 1 to the hemoglobin amount. A known method can be used for the measurement of the amount of hemoglobin, and absorbance measurement is preferably used. The hemoglobin measuring unit may be the same as the sensor, particularly in the case of a batch type.

  The concentration calculation unit 6 is a part that calculates the ratio of the hemoglobin A1c amount to the hemoglobin amount from the hemoglobin A1c amount measured by the hemoglobin A1c sensor 3 and the hemoglobin amount measured by the hemoglobin measurement unit 5.

  The carrier 10 in the case of using a hemoglobin A1c measuring apparatus equipped with a flow type hemoglobin A1c sensor is not particularly limited as long as it is an aqueous medium for feeding a sample to a detection site. For example, deionized water, distilled Water, a buffer solution, etc. are mentioned, Preferably a buffer solution is used. The buffer used for the buffer is not particularly limited as long as it has a buffering capacity, but for example, lactate buffer, citrate buffer, acetate buffer, succinate buffer, phthalate buffer, phosphate buffer, triethanol Amine buffer, diethanolamine buffer, lysine buffer, barbitur buffer, tris (hydroxymethyl) aminomethane buffer, imidazole buffer, malate buffer, oxalate buffer, glycine buffer, borate buffer, A carbonate buffer, a glycine buffer, a Good buffer, etc. are mentioned, The pH becomes like this. Preferably it is 1-11, More preferably, it is used at 4-10. The concentration of the buffer solution is not particularly limited as long as it is suitable for the measurement, but is preferably 0.001 to 2.00 mol / L, more preferably 0.005 to 1.00 mol / L, and particularly preferably 0.01 to 0.50 mol / L. . If necessary, a surfactant, a polymer substance, salts, saccharides, a pH adjuster, a preservative, and the like can be added to such a carrier.

  The pump 11 is not particularly limited as long as the sample can be delivered to the hemoglobin A1c sensor 3 through the tube 9 by the carrier 10, and the waste liquid discharge unit 8 includes the hemoglobin A1c sensor 3 or the hemoglobin measurement unit 5. This is the site where the reaction solution that has passed is discharged.

Hemoglobin A1c and other diabetes marker simultaneous measurement sensor or simultaneous measurement device The hemoglobin A1c sensor according to the present invention is a working electrode fixed with an enzyme for detecting a diabetes marker other than hemoglobin A1c. By providing it separately or as a configuration of the measuring apparatus, a diabetes marker other than hemoglobin A1c can be measured at the same time as hemoglobin A1c by providing a measurement unit for a diabetes marker other than hemoglobin A1c sensor. .

  Examples of diabetes markers other than hemoglobin A1c include glucose, glycoalbumin, 1,5-anhydroglucitol and the like. In the case of providing a measurement unit for diabetes markers other than hemoglobin A1c as a configuration of the measurement device, the measurement unit is a sensor or a spectrophotometer equipped with a reagent for measuring a target substance with a spectrophotometer as necessary. A photometer or the like is used.

  For example, when measuring glucose, the working electrode on which glucose oxidase or glucose dehydrogenase is immobilized is used. To measure glycoalbumin, the working electrode on which proteolytic enzyme and FAOX are immobilized is used. In measuring anhydroglucitol, a sensor having a working electrode to which hexokinase and 1,5-anhydroglucitol-6-phosphate dehydrogenase are immobilized can be used.

  Further, as a measuring method using a spectrophotometer, for example, hydrogen peroxide produced by an oxidoreductase reaction is measured using a dye as a detection substance with a peroxidation active substance such as an oxidative coloring type chromogen and peroxidase. The amount of change in oxidized coenzymes such as NAD in the dehydrogenase reaction is measured with a colorimeter at a wavelength near the maximum absorption wavelength range for the reduced coenzyme produced by the coenzyme reaction. Direct quantification using the above technology, the resulting reduced coenzyme is various diaphorases, electron carriers such as phenazine methosulfate and nitrotetrazolium, WST-1 (manufactured by Dojindo Laboratories), WST-8 (manufactured by the same company) ), A method of measuring indirectly using a reducing coloring reagent such as various tetrazolium salts, or other known methods, directly or indirectly. The measuring method etc. are mentioned.

  EXAMPLES Hereinafter, although this invention is demonstrated about an Example, this invention is not limited to these Examples.

Example 1
Hemoglobin A1c simultaneously measuring ceramic substrate glucose, a platinum electrode consisting of a counter electrode and the working electrode by a screen printing method, both the area is prepared as a 1 mm ^2, the following composition on the working electrode dope 5 μL of the solution was added dropwise and dried at 40 ° C. for 30 minutes to prepare a hemoglobin A1c sensor in which a film containing 0.5 units of fructosyl peptide oxidase was formed on the working electrode.
<Dope solution>
FPOX (FPOX-CE: Kikkoman) 100 units
Bovine serum albumin (A-4503: Sigma) 10 mg
20% glutaraldehyde aqueous solution (Wako Pure Chemical Industries, Ltd.) 10 μL
Pure water 1 mL

  Next, in the production of the hemoglobin A1c sensor, a sensor was similarly produced using 2000 units of glucose oxidase (G-7016: manufactured by Sigma) instead of FPOX used for the preparation of the dope solution. A glucose sensor having a membrane on which 10 units of glucose oxidase was immobilized was prepared.

  The obtained hemoglobin A1c sensor and glucose sensor were equipped with a plunger pump, a carrier flow path consisting of a 1/16 inch tube, an injector, a cell for hemoglobin A1c sensor, a cell for glucose sensor, and a potentiostat (manufactured by BAS). The hemoglobin A1c sensor cell and the glucose sensor cell of the measuring apparatus were set.

Using the measurement apparatus, hemoglobin A1c and glucose were measured. A 0.1 mol / L Tris-HCl buffer at pH 7.5 was used as a carrier, and measurement was performed under conditions of room temperature, a flow rate of 1.4 mL / min, and an applied voltage of 0.6 V to the working electrode. As substrate samples, 20 μL each of the following samples was used.
<Sample for measuring hemoglobin A1c>
A glycated hexapeptide (Fru-6P: Fru-Val-His-Leu-Thr-Pro-Glu; manufactured by Peptide Institute, Inc.) having a β-chain N-terminal 6 amino acid sequence of hemoglobin A1c at a pH of 7. Fructosyl dipeptide Fru-Val-His prepared by reacting 5 units of 0.1 mol / L Tris-HCl buffer solution with 1 unit / mL of proteolytic enzyme (proteinase TypeXXIII) was used as a sample. .
<Sample for glucose measurement>
A sample prepared by dissolving glucose (manufactured by Sigma) in a 0.1 mol / L Tris-HCl buffer solution having a pH of 7.5 to a predetermined concentration was used.

  The obtained results are shown in the graphs of FIGS. As shown in these graphs, it was confirmed that hemoglobin A1c and glucose can be measured at the same time by the measuring apparatus.

Reference example 1
Determination of glycated amino acids using FAOX by spectrophotometry
0.5 mL of a 0.25 units / mL FAOX (FAODL: manufactured by Kikkoman) solution containing a substrate sample of a predetermined concentration prepared using PBS (0.05 mol / L sodium phosphate, pH 7.5, 0.9% sodium chloride) Warm for 30 minutes at 37 ° C. Here, a glycated amino acid at the N-terminal of the β chain of hemoglobin A1c (Fru-Val; manufactured by Peptide Laboratories) was used as a substrate sample. Next, 0.025 mmol / L N- (carboxymethylaminocarbonyl) -4,4′-bis (dimethylamino) diphenylamine sodium salt (DA-64: manufactured by Wako Pure Chemical Industries, Ltd.) and 10 Add 0.5 mL of coloring solution containing U / mL horseradish peroxidase (Toyobo Co., Ltd.), warm at 37 ° C for 5 minutes, measure absorbance at 727 nm, The responsiveness to glycated amino acids was evaluated.

Reference example 2
Measurement of glycated amino acid using FPOX by spectrophotometry In Reference Example 1, the same amount of FPOX (FPOX-CE) solution having the same concentration was used instead of the FAOX solution.

  The results obtained in Reference Examples 1 and 2 are shown in the graph of FIG. As shown in this graph, it was confirmed that glycated amino acids can be measured with the same sensitivity as FAOX (□) even when FPOX is used (●).

Example 2
Using a platinum electrode consisting of a counter electrode and a working electrode with a measurement area of glycated amino acid of 7 mm ² by the electrode method using FPOX, 10 μL of a dope solution having the following composition is dropped on the working electrode and dried at 40 ° C. for 30 minutes. A hemoglobin A1c sensor in which a film containing 1 unit of FPOX was formed on the working electrode was fabricated.
<Dope solution>
FPOX (FPOX-CE) 100 units
Bovine serum albumin (Proliant) 10 mg
20% glutaraldehyde aqueous solution (Wako Pure Chemical Industries, Ltd.) 10 μL
Pure water 1 mL

  Next, the hemoglobin A1c sensor was set in a batch type measuring apparatus connected to an electrochemical analyzer (manufactured by BAS). Using this measuring apparatus, a substrate sample was measured in 4 mL of PBS containing a substrate sample of a predetermined concentration under conditions of an applied voltage to the working electrode of 0.6 V and room temperature. Here, the same substrate sample as in Reference Example 1 was used.

Comparative Example 1
Measurement of glycated amino acid by electrode method using FAOX In Example 2, the hemoglobin A1c sensor was prepared in the same manner as in Example 2, except that 100 units of FAOX (FAODL) was used instead of FPOX for the preparation of the dope solution. The measurement using the sensor was performed.

  The results obtained in Example 2 and Comparative Example 1 are shown in the graph of FIG. It was confirmed that the current value of Example 2 (●) was higher than that of Comparative Example 1 (□).

Example 3
Measurement of glycated amino acid in the presence of blood cells by electrode method using FPOX In Example 2, instead of PBS containing substrate sample, Example 1 except that 1% of washed blood cells were present in PBS containing substrate sample was used. Measurement was carried out by the same method as in 2. For washed blood cells, add 4 times the volume of PBS to the blood collected with the EDTA 2Na blood collection tube, centrifuge at 3000 rpm for 10 minutes, remove the supernatant, and add 4 times the volume to the precipitated blood cells. PBS was added, suspended, centrifuged at 3000 rpm for 10 minutes, and the washing operation except the supernatant was repeated three times. After that, 9 times the volume of distilled water was added to the resulting precipitate and blood cells were added. Was prepared as a 10% washed blood cell solution.

Comparative Example 2
Measurement of glycated amino acid in the presence of blood cells by electrode method using FAOX In Example 2, the same method as in Example 2 was used, except that 100 units of FAOX (FAODL) was used instead of FPOX in preparation of the dope solution. The measurement was performed in the same manner as in Example 2 except that the hemoglobin A1c sensor and a substrate sample-containing PBS containing 1% of washed blood cells were used instead of the substrate sample-containing PBS. Here, the same washed blood cells as in Example 3 were used.

  The results obtained in Example 3 and Comparative Example 2 are shown in the graph of FIG. In Example 3 (●), it was confirmed that glycated amino acids could be measured with extremely high sensitivity compared to Comparative Example 2 (□).

Comparative Example 3
Measurement of glycated amino acid in the presence of blood cells using FPOX by spectrophotometry In Reference Example 2, instead of using a substrate sample-containing FPOX solution, a substrate sample-containing FPOX solution containing 1% of washed blood cells is used as a reference. Measurement was performed in the same manner as in Example 2. Here, the same washed blood cells as in Example 3 were used.

Comparative Example 4
Measurement of glycosylated amino acids in the presence of blood cells using FAOX by spectrophotometry In Reference Example 1, instead of using a FAOX solution containing a substrate sample, a FAOX solution containing a substrate sample containing 1% of washed blood cells is used as a reference. Measurement was performed in the same manner as in Example 1. Here, the same washed blood cells as in Example 3 were used.

  The results obtained in Comparative Example 3 and Comparative Example 4 are shown in the graph of FIG. As shown in this graph, glycated amino acids cannot be measured using either FPOX (Comparative Example 3: ●) or FAOX (Comparative Example 4: □) under conditions where blood cells are present in the enzyme solution. confirmed.

Example 4
Effect of surfactant: measurement of glycated amino acid in the presence of blood cells by the electrode method In Example 3, instead of the substrate sample-containing PBS solution, dodecyldimethylamine oxide (Amphitol 20N: manufactured by Kao Corporation), which is a surfactant, was further used. The measurement was performed in the same manner as in Example 3 except that a PBS solution containing a substrate sample in which 0.5% was present was used. The obtained results are shown in the graph of FIG. 10 together with the results of Example 3. It was confirmed that the current value increased due to the presence of the surfactant in the measurement of the glycated amino acid as compared with the absence of the surfactant (Example 3: □) (Example 4: ●).

Example 5
Measurement of glycated hexapeptide by electrode method In Example 2, instead of the PBS solution containing the substrate sample, glycated hexapeptide (Fru-6P: Fru-Val-His-Leu-Thr-Pro-Glu; Peptide Laboratory) And a PBS solution containing 1000 units / mL of proteolytic enzyme was heated at 37 ° C for 10 minutes, and then the proteinase was inactivated by heating at 95 ° C for 5 minutes. Measurement was performed in the same manner as in Example 2.

Example 6
Effect of surfactant: measurement of glycated hexapeptide by electrode method In Example 2, instead of the PBS solution containing the substrate sample, glycated hexapeptide (Fru-6P: Fru-Val-His-Leu-Thr-Pro) of a predetermined concentration was used. -Glu; manufactured by Peptide Institute Co., Ltd.), PBS unit containing 1000 units / mL proteolytic enzyme and 0.5% dodecyldimethylamine oxide (Amphitol 20N) was heated at 37 ° C for 10 minutes, and then added at 95 ° C for 5 minutes. The measurement was carried out in the same manner as in Example 2 except that a product in which the proteolytic enzyme was inactivated by heating was used.

  The results obtained in Examples 5 and 6 are shown in the graph of FIG. As shown in this graph, also in the measurement of glycated hexapeptide by the electrode method, in the presence of a surfactant (Example 6: ●) compared to the absence of a surfactant (Example 5: □), It was confirmed that the current value increased.

Example 7
Effect of surfactant: measurement of glycated hexapeptide in the presence of blood cells by the electrode method In Example 2, instead of the PBS solution containing the substrate sample, a glycated hexapeptide (Fru-6P: Fru-Val-His-) of a predetermined concentration was used. Leu-Thr-Pro-Glu (manufactured by Peptide Institute, Inc.), PBS unit containing 1000 units / mL proteolytic enzyme, 1% washed blood cells and 0.5% dodecyldimethylamine oxide (Amphithol 20N), added at 37 ° C for 10 minutes After warming, measurement was performed in the same manner as in Example 2 except that a protein inactivated by proteolytic enzyme by heating at 95 ° C. for 5 minutes was used. Here, the same washed blood cells as in Example 3 were used.

Comparative Example 5
In Comparative Example 1, instead of the substrate sample-containing PBS solution, glycated hexapeptide (Fru-6P: Fru-Val-His-Leu-Thr-Pro-Glu; manufactured by Peptide Laboratories), 1000 units / mL Proteolytic enzyme, 1% washed blood cells and 0.5% dodecyldimethylamine oxide (Amphithol 20N) in PBS solution was warmed at 37 ° C for 10 minutes and then at 95 ° C for 5 minutes to lose the proteolytic enzyme. The measurement was performed in the same manner as in Comparative Example 1 except that the activated material was used. Here, the same washed blood cells as in Example 3 were used.

  The results obtained in Example 7 and Comparative Example 5 are shown in the graph of FIG. As shown in this graph, in the measurement of glycated hexapeptide in the presence of blood cells and surfactant by the electrode method, Example 7 (●) using a sensor prepared using FPOX used FAOX. It was confirmed that the glycated peptide can be measured with extremely high sensitivity compared to Comparative Example 5 (□) using the prepared sensor.

Claims (12)

  A hemoglobin A1c sensor having at least a counter electrode and a working electrode for measuring hemoglobin A1c in a sample, wherein a fructosyl peptide oxidase is immobilized on the working electrode.   The hemoglobin A1c sensor according to claim 1, wherein the sample is a sample containing blood cells.   The hemoglobin A1c sensor according to claim 1 or 2, wherein the sample is a sample treated with a surfactant.   The hemoglobin A1c sensor according to any one of claims 1 to 3, wherein a mediator is further fixed on the working electrode.   The hemoglobin A1c sensor according to any one of claims 1 to 4, wherein a proteolytic enzyme is further immobilized on the working electrode.   The hemoglobin A1c sensor according to any one of claims 1 to 5, wherein the electrode is a hydrogen peroxide electrode or an oxygen electrode.   The hemoglobin A1c sensor according to any one of claims 1 to 6, further comprising a reference electrode.   The hemoglobin A1c sensor according to any one of claims 1 to 7, wherein the electrode is formed on an insulating substrate.   The hemoglobin A1c sensor according to any one of claims 1 to 8, further comprising a working electrode on which an enzyme for detecting a diabetes marker other than hemoglobin A1c is immobilized.   A hemoglobin A1c measurement device comprising the hemoglobin A1c sensor according to any one of claims 1 to 9, a voltage application unit, and a current value measurement unit.   The hemoglobin A1c measuring apparatus according to claim 10, further comprising a hemoglobin measuring unit and a hemoglobin A1c concentration calculating unit.   The hemoglobin A1c measurement apparatus according to claim 10 or 11, further comprising a measurement unit for detecting a diabetes marker other than hemoglobin A1c.

Images