WO2007094354A1 - CAPTEUR D'HÉMOGLOBINE A1c - Google Patents

CAPTEUR D'HÉMOGLOBINE A1c Download PDF

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Publication number
WO2007094354A1
WO2007094354A1 PCT/JP2007/052602 JP2007052602W WO2007094354A1 WO 2007094354 A1 WO2007094354 A1 WO 2007094354A1 JP 2007052602 W JP2007052602 W JP 2007052602W WO 2007094354 A1 WO2007094354 A1 WO 2007094354A1
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WIPO (PCT)
Prior art keywords
hemoglobin
hemoglobin ale
sensor
electrode
sample
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PCT/JP2007/052602
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English (en)
Japanese (ja)
Inventor
Masao Gotoh
Fumiyo Kurusu
Isao Karube
Haruki Tsunoda
Akira Tsukada
Asami Saito
Akira Kadoda
Akihito Tomita
Norihiko Kayahara
Hideki Tanigaki
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National Institute Of Advanced Industrial Science And Technology
Kyowa Medex Co., Ltd.
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Application filed by National Institute Of Advanced Industrial Science And Technology, Kyowa Medex Co., Ltd. filed Critical National Institute Of Advanced Industrial Science And Technology
Priority to JP2008500520A priority Critical patent/JPWO2007094354A1/ja
Publication of WO2007094354A1 publication Critical patent/WO2007094354A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/795—Porphyrin- or corrin-ring-containing peptides
    • G01N2333/805—Haemoglobins; Myoglobins

Definitions

  • the present invention relates to a hemoglobin Ale sensor. More specifically, the present invention relates to a hemoglobin Ale sensor that enables accurate measurement of hemoglobin Ale with a simple operation.
  • glycoprotein is produced by a 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.
  • Glycoprotein is also called a so-called Amadori compound because a Schiff base generated as a reaction intermediate undergoes Amadori rearrangement.
  • Powerful 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.
  • concentration of glycoprotein present in the blood strongly depends on the concentration of reducing sugars such as glucose in the blood.
  • glucose reducing sugars
  • the production of glycoprotein is enhanced, and the concentration of glycated hemoglobin contained in erythrocytes and glycated albumin in serum reflects the average blood glucose level over a certain period in the past.
  • Glucose shows the current blood glucose level
  • 1,5-anhydroglucitol shows blood sugar levels about several days ago
  • glycated albumin about 1-2 weeks ago
  • glycated hemoglobin about 1-2 months ago.
  • measurement of these glycated proteins is important for the time-lapse diagnosis or symptom management of diabetic symptoms in that the blood dalcose concentration can be determined over time.
  • Glycated proteins currently used as an index for diagnosis of diabetes include glycated albumin in which the ⁇ -amino group of lysine residues in proteins such as albumin is glycated, and ⁇ -terminal of proteins such as hemoglobin Various glycated proteins such as glycated hemoglobin in which the ⁇ -amino group of the amino acid is glycated are known.
  • Hemoglobin Ale is one of the glycated hemoglobins. After gnolecose is non-enzymatically bound to the ⁇ terminal amino acid of hemoglobin ⁇ -subunit to form a Schiff base, It is a glycoprotein having a structure in which fructose is bound as a result of rearrangement. Hemoglobin Ale is important as an index for diabetes management because it reflects clinical mean blood glucose levels in the past 1 to 2 months, and a rapid and accurate quantitative method is required.
  • a glycated protein is decomposed by a proteolytic enzyme, and a fructosyl amino acid oxidase (hereinafter abbreviated as FAOX), which is a kind of glycated amino acid oxidase, is allowed to act on the released glycated amino acid.
  • FAOX fructosyl amino acid oxidase
  • a method for measuring the generated hydrogen peroxide by absorptiometry has been proposed.
  • a hemoglobin Ale sensor that responds to glycated amino acids released from glycated proteins by proteolytic enzymes and has a FAOX immobilized electrode as a working electrode has also been proposed.
  • Patent Document 1 JP-A-5-192193
  • Patent Document 2 JP 2003-274976
  • fructosyl amino acid oxidase currently used has high reactivity to free glycamino acids, there are glycated peptides obtained by degrading glycated proteins with proteases. Since the enzyme method hardly acts, the accuracy of the enzyme method is not necessarily the same as the method.
  • An object of the present invention is to provide a hemoglobin Ale sensor that enables easy, rapid and accurate measurement of hemoglobin Ale.
  • An object of the present invention is a sensor for measuring hemoglobin Ale in a sample, which has an electrode composed of at least a counter electrode and a working electrode, and is characterized in that fructosyl peptide oxidase is immobilized on the working electrode.
  • the present invention relates to the following (1) to (: 12).
  • Hemoglobin Ale in a sample having at least an electrode composed of a counter electrode and a working electrode A hemoglobin Ale sensor characterized in that fructosyl peptide oxidase (hereinafter abbreviated as FPOX) is immobilized on the working electrode.
  • FPOX fructosyl peptide oxidase
  • hemoglobin Ale sensor 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 Ale is immobilized.
  • the hemoglobin Ale measuring device according to (10), further comprising a hemoglobin measuring unit and a hemoglobin Ale concentration calculating unit
  • the hemoglobin Ale measuring device according to (10) or (11), further comprising a measuring unit for detecting a diabetes marker other than hemoglobin Ale
  • the concentration of hemoglobin Ale contained in the sample can be easily measured.
  • the concentration of hemoglobin Ale contained in the sample can be easily measured.
  • there is no need for special pretreatment of the sample there is no need for special pretreatment of the sample.
  • better sensitivity can be achieved by treating the sample with a surfactant. Enables accurate measurement.
  • proteolytic enzyme When proteolytic enzyme is immobilized in addition to FPOX or FPOX and a mediator on the working electrode, it can be directly measured for hemoglobin Ale without pretreatment of the sample.
  • FIG. 1 shows an example of a measuring apparatus equipped with the batch type hemoglobin Ale sensor of the present invention.
  • FIG. 2 shows an example of a measuring apparatus equipped with the flow type hemoglobin Ale sensor of the present invention.
  • FIG. 3 shows another example of a measuring apparatus including the flow type hemoglobin Ale sensor of the present invention.
  • FIG. 4 A graph showing the response of the hemoglobin Ale sensor to hemoglobin Ale.
  • FIG. 5 is a graph showing the responsiveness of a glucose sensor attached to this hemoglobin Ale sensor to glucose.
  • FIG. 6 is a graph showing the measurement results of glycated amino acids by spectrophotometry using FPOX or FAOX.
  • FIG. 7 is a graph showing responsiveness to glycated amino acid by a hemoglobin Ale sensor in which FP0X or FA0X is immobilized on the working electrode.
  • FIG. 8 is a graph showing the responsiveness to glycoin amino acids in the presence of blood cells by a hemoglobin Ale sensor with FPOX or FAOX immobilized on the working electrode.
  • FIG. 9 is a graph showing the measurement results of glycated amino acid in the presence of blood cells by spectrophotometry using FPOX or FAOX.
  • FIG. 10 is a graph showing the effect of surfactants on the response to glycated amino acids in the presence of blood cells by the hemoglobin Ale sensor.
  • FIG. 11 is a graph showing the effect of a surfactant on the responsiveness to glycohexapeptide by the hemoglobin Ale sensor.
  • FIG. 12 Draft showing responsiveness to glycated hexapeptide in the presence of blood cells and surfactants by hemoglobin Ale sensor with FPX or FAOX immobilized on the working electrode.
  • 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.
  • 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.
  • 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 it is a material that can stably hold an enzyme, such as platinum, platinum black, gold, silver, palladium, carbon, carbon paste, carbon nanotube, iridium, and diamond.
  • platinum black include platinum formed by sputtering, vapor deposition, and screen printing. Carbon nanotubes are manufactured by methods such as arc discharge, vapor deposition, and laser evaporation.
  • 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.
  • the material for the insulating substrate include ceramic, glass, plastic, biodegradable material, non-woven fabric or paper, and electrodes are formed on the substrate by screen printing, vapor deposition, sputtering, etc. Is called.
  • the substrate on which the electrodes are formed is used as a flow injection analysis (FIA) sensor unit and as a disposable simple sensor.
  • FIA flow injection analysis
  • FPOX an enzyme that acts on a glycated peptide and generates hydrogen peroxide
  • Fru-Va ⁇ His fructosyl valine
  • Fru-Val fructosyl valine
  • FPOX-CE manufactured by Kikkoman
  • FPOX-EE manufactured by KK
  • Patent Document 3 JP 2004-275013
  • the proteolytic enzyme is not particularly limited as long as it is an enzyme capable of degrading the glycoprotein in the sample into a glycated peptide, and is suitable depending on the type of glycated protein or glycopeptide that undergoes cleavage. Can be appropriately selected and used.
  • proteolytic enzymes include proteinase, pronase, thermolysin, subtilisin (subtilisin), carboxypeptidase B, pancreatin, cathebsin, carboxypeptidase, endoproteinase Glu-C, papain, ficin,
  • proteolytic enzymes including peptide degrading enzymes such as bromelain and aminopeptidase.
  • proteolytic enzyme that efficiently produces a glycated dipeptide.
  • Aspergillus-derived proteolytic enzyme Rhizopus-derived proteolytic enzyme, Bacillus-derived proteolytic enzyme, Streptomyces-derived proteolytic enzyme, Tritylatium-derived proteolytic enzyme, Staphylococcus-derived proteolytic enzyme, Plant-derived proteolytic enzyme Examples thereof include enzymes and animal-derived proteolytic enzymes.
  • Aspergillus-derived proteolytic enzymes include, for example, “IP enzyme”, “A0 protease”, “peptidase”, “Morcin” (manufactured by Kikkoman), “Protea Aze A5" ”(Kyowa Kasei Co., Ltd.),“ Ummamizyme ”,“ Protease A ”,“ Protease M ”,“ Protea Ize?
  • lysopath-derived proteolytic enzymes include “peptidase R” (manufactured by Amano Enzyme) and the like.
  • Bacillus-derived proteolytic enzymes include, for example, “Dispase” (Roche), “Satilaisin” (Boehringer), “Protinase ⁇ (manufactured by Full Riki),” Proteinase Type VII.
  • Streptomyces-derived proteolytic enzymes include, for example, “Pronase” (manufactured by Boehringer Mannheim), “Protinase TypeXIV” (manufactured by Sigma), “Alkaline protease” (manufactured by Toyobo Co., Ltd.), etc. Is given.
  • tritylatium-derived protease examples include “Protinase 1" (Roche, Wako Pure Chemical Industries).
  • Staphylococcus-derived proteolytic enzymes include "Glu_C” (manufactured by Behringer).
  • plant-derived proteolytic enzymes include, for example, papain (Roche, Wako Pure Chemical Industries, Sigma, Amano Enzaim, Asahi), Huisin (Sigma) ), Bromelain (manufactured by Amano Enzyme, Sigma), and the like.
  • animal-derived proteolytic enzyme examples include “pancreatin” (manufactured by Wako Pure Chemical Industries, Ltd.), cathebsin B (manufactured by Sigma), and the like.
  • the proteolytic enzymes may be used alone or in combination of two or more.
  • hemoglobin Ale has been shown to produce the glycated hexapeptide Fru-Va ⁇ 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 above glycated dipeptide is an extremely effective method for producing a glycated dipeptide from hemoglobin Ale (Clin. Chem. 1997, 43 1944-1951). ).
  • the method for immobilizing FPOX or FPOX and a proteolytic enzyme on the working electrode is not particularly limited as long as the method can stably hold the enzyme on the working electrode, but the enzyme solution is dropped on the electrode.
  • Drying method, crosslinking method using albumin-gnoretalaldehyde, inclusion method using water-soluble photocrosslinkable resin, mixing method in mineral oil, bromoform, nudiol, tetrafluoroethylene or naphthion, reagent Examples include the covalent bond method used.
  • a mediator is preferably used on the working electrode.
  • the mediator is not particularly limited as long as it is a substance that is involved in the reaction that generates electrons in the presence of FPOX, and that improves the efficiency of transfer between the electrons and the electrodes.
  • potassium ferricyanide Methoxyphenazine methosulfate, quinone derivatives, furcene or derivatives thereof, osmium complexes, and the like. Since a larger output value can be obtained by using a mediator, the hemoglobin Ale concentration in the measurement sample can be measured more accurately.
  • Examples of the configuration of the working electrode include the following embodiments.
  • 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. When forming multiple layers, it is preferable that FPOX is directly fixed on the working electrode and proteolytic enzyme is laminated on the FPOX layer. If mediators are used, the working electrode It is preferable to form a mediator layer between the FPOX layer.
  • a polymer material layer can be provided on the electrode.
  • the polymer material include synthetic polymer materials, polysaccharides, and porous materials.
  • a hydrogen peroxide electrode which may be either a hydrogen peroxide electrode or an oxygen electrode
  • the amount of hydrogen peroxide generated from hemoglobin Ale by an enzymatic reaction is electrochemically measured.
  • the concentration of hemoglobin Alc is measured by electrochemically measuring the amount of oxygen consumed in the enzymatic reaction of hemoglobin Ale.
  • H 0 generated when FPOX further acts on Fru-Va ⁇ His produced by the action of proteolytic enzyme on hemoglobin Ale is as follows.
  • the amount of hydrogen peroxide generated from hemoglobin Ale is measured electrochemically by measuring the electrons generated or consumed according to the reaction formula in the case of oxidation by electrodes: H 0 ⁇ 0 + 2H + + 2e
  • the amount of oxygen consumed in the enzymatic reaction of hemoglobin Ale is measured electrochemically by measuring electrons consumed by the following reaction formula as a current value.
  • the method for measuring the electrons generated or consumed by the enzymatic reaction is not particularly limited as long as it is a method capable of measuring electrons.
  • chrono-amperometry cyclic voltammetry.
  • coulometry or the like is used.
  • the sample for measuring hemoglobin Ale is not particularly limited as long as it contains hemoglobin Ale, and examples thereof include a sample containing whole blood and blood cells.
  • the sample can be subjected to processing such as centrifugation, washing, dilution, concentration, addition of a surfactant, and heating.
  • a sample that has been previously treated with the proteolytic enzyme can be used.
  • a nonionic surfactant for example, a nonionic surfactant, a cationic surfactant, an anionic surfactant, and an amphoteric surfactant are used, and preferably an amphoteric surfactant is used. It is done.
  • Nonionic surfactants include, for example, polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether, polyoxyethylene polycyclic phenyl ether, polyoxyethylene polyoxypropylene condensate, polyoxyethylene alkylamine. , Alkylenediamine polyoxyethylene 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 alcohol amide, sucrose fatty acid Examples include esters.
  • Examples of the cationic surfactant include aliphatic amine salts and aliphatic quaternary ammonium salts.
  • anionic surfactant examples include carboxylate, sulfonate, ester sulfate, phosphate ester, cholate and the like.
  • amphoteric surfactants include betaines, glycines, alanines, 2-alkylimidazoline derivatives, amin oxides, and alkylamino salts.
  • the form of the hemoglobin Ale sensor according to the present invention is not particularly limited as long as the user can use the sensor. This sensor may also be thrown away with force that can be used repeatedly.
  • the method for measuring hemoglobin Ale using the hemoglobin Ale sensor according to the present invention is not particularly limited as long as it can measure hemoglobin Ale in the sample.
  • the sample reaches the electrode through the channel.
  • the sample and electrode are in contact in a reaction vessel. Examples include batch type to touch or chip type to connect terminal at one end of electrode to output measuring instrument.
  • the hemoglobin Ale measuring method of the present invention is a method for measuring hemoglobin Ale in a sample using the hemoglobin Ale sensor of the present invention.
  • the measurement can be performed, for example, including the following steps.
  • the ratio of hemoglobin Alc to the total hemoglobin can be calculated.
  • the total hemoglobin concentration can be any method as long as it is a known method for measuring hemoglobin, such as the methemoglobin method, cyanmethemoglobin method, azaid methemoglobin method, sodium dodecyl sulfonate (SLS) method, alkali Examples include the hematin method, the green chromophore formation method, and the oxyhemoglobin method.
  • the standard products used for the preparation of the calibration curve include hemoglobin Alc with a known concentration, Arre, Fru-Val-His-Leu-Thr-Pro-Glu, Fru-Vato His, etc.
  • Glycated amino acids such as glycated peptides and Fru-Val can be used.
  • the hemoglobin Ale concentration is measured by a hemoglobin Ale measuring device.
  • the measuring device has a mechanism for detecting a current value generated at least when the hemoglobin Ale sensor comes into contact with the sample.
  • the hemoglobin measuring unit and a concentration calculation for calculating hemoglobin Alc (%).
  • Fig. 1 shows a measurement device that uses a batch type hemoglobin Ale sensor
  • Fig. 2 and Fig. 3 show examples of measurement devices that use a flow type hemoglobin Ale sensor. Show. In either case, the sample supplied from the sample addition unit 1 is connected to the voltage application unit 2.
  • the hemoglobin Ale sensor 3 measures the current value corresponding to the amount of hemoglobin Ale in the sample at the current value measurement unit 4, while the hemoglobin measurement unit 5 measures the hemoglobin concentration, and finally calculates the concentration from these values.
  • the hemoglobin Ale concentration is calculated in Part 6, this is the mode.
  • 1 and 2 show a device in which the hemoglobin measurement unit and the hemoglobin Ale sensor are in parallel
  • FIG. 3 shows a device in which the hemoglobin measurement unit and the hemoglobin Ale sensor are in series.
  • the current value measurement unit 4 is a potentiostat that generates or consumes electrons generated in the reaction between hemoglobin Ale supplied from the sample addition unit 1 and an enzyme immobilized on the working electrode of the hemoglobin A1 c sensor 3. This is the part that is measured and displayed as a current value when a voltage is applied by the voltage application unit 2 such as a shoe.
  • the hemoglobin measuring unit 5 measures the amount of hemoglobin. This is necessary in order to measure the concentration of hemoglobin Ale by calculating the ratio of the amount of hemoglobin Ale supplied from the sample addition unit 1 to the amount of hemoglobin.
  • a well-known method can be used, and preferably absorbance measurement is used.
  • the hemoglobin measurement unit can be the same as the sensor, especially in the case of a batch type.
  • the concentration calculation unit 6 is a part that calculates the ratio of the hemoglobin Ale amount to the hemoglobin amount from the hemoglobin Ale amount measured by the hemoglobin Ale sensor 3 and the hemoglobin amount measured by the hemoglobin measurement unit 5. .
  • the carrier 10 in the case of using a hemoglobin Ale measuring apparatus equipped with a flow type hemoglobin Ale sensor is not particularly limited as long as it is an aqueous medium for feeding a sample to a detection site.
  • examples include deionized water, distilled water, and a buffer solution, and a buffer solution is preferably used.
  • the buffer used in the buffer is not particularly limited as long as it has a buffering capacity.
  • a lactate buffer for example, a lactate buffer, a citrate buffer, an acetate buffer, a succinate buffer, a phthalate buffer, a phosphate buffer, Ethanolamine buffer, diethanolamine buffer, lysine buffer, barbitur buffer, tris (hydroxymethyl) aminomethane buffer, imidazole buffer, malate buffer, oxalate buffer, glycine buffer, borate buffer Agent, carbonate buffer, glycine buffer, Good buffer, etc., and the pH is preferably 1. To 11, more preferably 4 to 10.
  • the concentration of the buffer solution is not particularly limited as long as it is suitable for 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. Used in L.
  • Such carriers can be supplemented with surfactants, polymer substances, salts, saccharides, pH adjusters, preservatives and the like as necessary.
  • the pump 11 is not particularly limited as long as the sample can be delivered to the hemoglobin Ale sensor 3 through the tube 9 by the carrier 10 as described above, and the waste liquid discharge unit 8 includes the hemoglobin Ale sensor 3 or hemoglobin. This is the part where the reaction solution that has passed through the measuring section 5 is discharged.
  • the hemoglobin Ale sensor has a working electrode to which an enzyme for detecting a diabetes marker other than hemoglobin Ale is fixed separately from the working electrode to which FPOX is fixed, or as a configuration of a measuring apparatus, other than hemoglobin Ale.
  • diabetes markers other than hemoglobin Ale examples include glucose, glycoalbumine, 1,5-anhydrognoreitol and the like.
  • a measurement unit for diabetes markers other than hemoglobin Ale is provided as a configuration of the measurement device, a sensor or a reagent for measuring the target substance with a spectrophotometer is used as the measurement unit.
  • the equipped spectrophotometer is used.
  • a measuring method using a spectrophotometer for example, hydrogen peroxide generated by an oxidoreductase reaction is used for peroxidation such as an oxidative coloring chromogen and peroxidase.
  • the method of measuring the dye as a detection substance with an active substance, the amount of change in oxidized coenzymes such as NAD in the dehydrogenase reaction, and the reduced coenzyme produced by the reaction of the coenzyme near its maximum absorption wavelength range Direct quantification using a known technique such as measurement with a colorimeter at a wavelength of, and the resulting reduced coenzyme can be selected from various diaphorases, electron carriers such as phenazine methosulfate, and nitrotetrazolium, WST-1 (Manufactured by Dojindo Laboratories Inc.), WST-8 (manufactured by the same company) and various other methods such as indirect measurement using reducing color developing reagents such as tetrazolium salts or other known methods. And indirectly measuring methods.
  • the platinum electrodes consisting of a counter electrode and the working electrode by a screen printing method, both produced as area is 1 mm 2, dropping the dope 5 mu L of the following composition on the working electrode Then, it was dried at 40 ° C for 30 minutes to produce a hemoglobin Ale sensor in which a film containing 0.5 units of fructosyl peptide oxidase was formed on the working electrode.
  • FPOX-CE manufactured by Kikkoman
  • Bovine serum albumin (A-4503: Sigma) 10 mg
  • a sensor was prepared in the same manner using glucose oxidase (G-7016: Sigma) 2000 units instead of FP ⁇ X, and a membrane with 10 units of gnolecose oxidase immobilized was formed on the working electrode.
  • a gnole course sensor was fabricated.
  • Carrier flow path consisting of / 16 inch tube, injector, hemoglobin Ale sensor Cell, glucose sensor cell, and hemostat Ale sensor cell and glucose sensor cell of a measuring apparatus equipped with potentiostat (manufactured by BAS).
  • hemoglobin Ale and glucose were measured.
  • a carrier a 0.1 mol / L Tris-HCl buffer having a pH of 7.5 was used 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.
  • the substrate sample 20 samples were used respectively.
  • fructosyl dipeptide Fru-Va ⁇ ⁇ His prepared by reacting protease solution (proteinase TypeXXin) to 1 unit / mL in a solution dissolved in 0.1 mol / L Tris-HCl buffer solution It was.
  • a sample prepared by dissolving glucose (manufactured by Sigma) in a 0.1 mol / L Tris-HCl buffer solution at pH 7.5 so as to have a predetermined concentration was used as a sample.
  • hemoglobin Ale and glucose can be measured simultaneously by the measuring apparatus.
  • a platinum electrode consisting of a counter electrode and working electrode with an area of 7 mm 2 , and add 10 ⁇ L of a dope solution of the following composition on the working electrode, dry at 40 ° C for 30 minutes, and 1 unit FPOX on the working electrode.
  • a hemoglobin Ale sensor in which a film containing selenium was formed was produced.
  • the hemoglobin Ale sensor was set in a batch-type measuring apparatus connected to an electrochemical analyzer (manufactured by BAS). Using this measuring device, a substrate sample was measured in 4 mL of PBS containing a substrate sample of a predetermined concentration under the conditions of an applied voltage of 0.6 V to the working electrode and room temperature. Here, the same substrate sample was used as in Reference Example 1.
  • Example 2 a hemoglobin Ale sensor was prepared in the same manner as in Example 2 except that FAOX (FAODL) 100 units was used instead of FPOX in preparation of the dope solution, and measurement using the sensor was performed.
  • FAOX FAODL
  • Example 2 The results obtained in Example 2 and Comparative Example 1 are shown in the graph of FIG. Example 2 ( It was confirmed that a higher current value was obtained in (ii) than in Comparative Example 1 (mouth).
  • Example 2 the measurement was performed in the same manner as in Example 2 except that 1% of washed blood cells were present in the PBS containing the substrate sample instead of PBS containing the substrate sample.
  • 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.
  • Example 2 instead of FPOX instead of FPOX, a hemoglobin Ale sensor prepared in the same manner as in Example 2 except that FAOX (FAODL) 100 units was used, and instead of PBS containing the substrate sample, The measurement was performed in the same manner as in Example 2 except that the PBS containing the substrate sample contained 1% of washed blood cells.
  • the same washed blood cells as in Example 3 were used.
  • Example 3 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 can be measured with extremely high sensitivity compared to Comparative Example 2 (mouth).
  • Example 2 the measurement was performed in the same manner as in Reference Example 2 except that a substrate sample-containing FPOX solution containing 1% of washed blood cells was used instead of the substrate sample-containing FPOX solution.
  • a substrate sample-containing FPOX solution containing 1% of washed blood cells was used instead of the substrate sample-containing FPOX solution.
  • the same washed blood cells as in Example 3 were used.
  • Reference Example 1 instead of the FAOX solution containing the substrate sample, further washed blood cells were added 1 The measurement was performed in the same manner as in Reference Example 1 except that the substrate sample-containing FAOX solution was used. Here, the same washed blood cells as in Example 3 were used.
  • Example 3 in place of the substrate sample-containing PBS solution, a substrate sample-containing PBS solution containing 0.5% of a surfactant, dodecyldimethylamine oxide (Amphithol 20N: manufactured by Kao Corporation) was used. Measurement was performed in the same manner as in Example 3. The obtained results are shown in the graph of FIG. 10 together with the results of Example 3. Compared to the absence of surfactant (Example 3: mouth), it was confirmed that the current value increased due to the presence of surfactant in the measurement of sugar amino acids (Example 4 :). .
  • a surfactant Amphithol 20N: manufactured by Kao Corporation
  • Example 2 instead of the PBS solution containing the substrate sample, a predetermined concentration of glycated hexapeptide (Fru-6P: Fru-Val-His-Leu-Thr-Pro-Glu; manufactured by Peptide Laboratories) and 100 units Example 2 and Example 2 except that a PBS solution containing 10 mL / mL proteolytic enzyme was heated at 37 ° C for 10 minutes, and then the protease was inactivated by heating at 95 ° C for 5 minutes. Measurement was carried out by the same method.
  • glycated hexapeptide Fru-6P: Fru-Val-His-Leu-Thr-Pro-Glu; manufactured by Peptide Laboratories
  • Example 2 instead of the substrate sample-containing PBS solution, glycated hexapeptide (Fru-6P: Fru-Va ⁇ His-Leu-Thr-Pro-Glu; manufactured by Peptide Laboratories), 1000 un its Protein solution is obtained by warming a PBS solution containing 10 mL / mL proteolytic enzyme and 0.5% dodecyldimethylamine oxide (Ammitol 20N) at 37 ° C for 10 minutes and then at 95 ° C for 5 minutes. The measurement was performed in the same manner as in Example 2 except that the enzyme deactivating the degrading enzyme was used. I did it.
  • glycated hexapeptide Fru-6P: Fru-Va ⁇ His-Leu-Thr-Pro-Glu; manufactured by Peptide Laboratories
  • 1000 un its Protein solution is obtained by warming a PBS solution containing 10 mL / mL proteolytic enzyme and 0.5% dodecyldimethylamine oxide (Ammitol 20N)
  • Example 2 instead of PBS solution containing substrate sample, glycated hexapeptide (Fru-6P: Fru-Va ⁇ His- Leu-Thr-Pro-Glu; manufactured by Peptide Laboratories, Inc.), PBS solution containing 1000 un its / mL proteolytic enzyme, 1% washed blood cells and 0.5% dodecyldimethylamine oxide (Amphitol 20N) at 37 ° C The measurement was carried out in the same manner as in Example 2 except that the sample was heated for 10 minutes and then used at 95 ° C for 5 minutes to inactivate the proteolytic enzyme. Here, the same washed blood cells as in Example 3 were used.
  • glycated hexapeptide Fru-6P: Fru-Va ⁇ His- Leu-Thr-Pro-Glu; manufactured by Peptide Laboratories, Inc.
  • PBS solution containing 1000 un its / mL proteolytic enzyme 1% washed blood cells and 0.5% dodecyldimethyl
  • Comparative Example 1 instead of the substrate sample-containing PBS solution, glycated hexapeptide (Fru-6P: Fm-Va ⁇ His-Leu-Thr-Pro-Glu; manufactured by Peptide Laboratories), 1000 un its A PBS solution containing 1 / mL proteolytic enzyme, 1% washed blood cells and 0.5% dodecyldimethylamine oxide (Amphitol 20N) should be warmed at 37 ° C for 10 minutes and then at 95 ° C for 5 minutes. The measurement was carried out in the same manner as in Comparative Example 1 except that a proteinase inactivated by was used. Here, the same washed blood cells as in Example 3 were used.
  • Example 7 and Comparative Example 5 are shown in the graph of FIG.
  • Example 7 ( ⁇ ) using a sensor prepared using FPOX is FA It was confirmed that glycated peptides can be measured with extremely high sensitivity compared to Comparative Example 5 (mouth) using a sensor prepared using X.

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Abstract

L'invention concerne un capteur de mesure de l'hémoglobine A1c dans un échantillon, possédant des électrodes qui comprennent au moins une contre-électrode et une électrode de travail, dans lequel de la fructosyle peptide oxydase est immobilisée sur l'électrode de travail, de préférence associée à un médiateur et de façon plus préférentielle à une protéase. Ce capteur d'hémoglobine A1c permet une mesure aisée et rapide de l'hémoglobine A1c. En prévoyant une autre électrode de travail sur laquelle est immobilisée une enzyme permettant de détecter un marqueur diabétique autre que l'hémoglobine A1c, séparée de l'électrode sur laquelle la FPOX est immobilisée, il devient possible de mesurer simultanément une pluralité de marqueurs diabétiques ayant des fonctions différentes dans un seul et même échantillon.
PCT/JP2007/052602 2006-02-14 2007-02-14 CAPTEUR D'HÉMOGLOBINE A1c WO2007094354A1 (fr)

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009150686A (ja) * 2007-12-19 2009-07-09 Sekisui Chem Co Ltd ヘモグロビン類の測定システム
JP2009171874A (ja) * 2008-01-23 2009-08-06 Citizen Holdings Co Ltd 糖化タンパク質濃度測定方法及びバイオセンサ
WO2009140343A1 (fr) * 2008-05-13 2009-11-19 General Atomics Biodétecteur électrochimique pour une détermination directe du pourcentage d'hémoglobine glyquée
EP2281900A1 (fr) * 2009-08-03 2011-02-09 Roche Diagnostics GmbH Fructosyl-oxydase peptidique et capteur pour la détermination des protéines glycées
US7943385B2 (en) 2006-07-25 2011-05-17 General Atomics Methods for assaying percentage of glycated hemoglobin
WO2011125750A1 (fr) * 2010-03-31 2011-10-13 シーシーアイ株式会社 Biocapteur
US8318501B2 (en) 2006-07-25 2012-11-27 General Atomics Methods for assaying percentage of glycated hemoglobin
JP2013079809A (ja) * 2011-09-30 2013-05-02 Oji Keisoku Kiki Kk 糖化タンパク質の分析装置及び分析方法
JP6126325B1 (ja) * 2016-02-25 2017-05-10 パナソニックヘルスケアホールディングス株式会社 バイオセンサ
WO2017082253A1 (fr) * 2015-11-09 2017-05-18 東レ株式会社 Capteur
WO2017145420A1 (fr) * 2016-02-25 2017-08-31 パナソニックヘルスケアホールディングス株式会社 Biocapteur
CN108562751A (zh) * 2018-05-04 2018-09-21 深圳市西尔曼科技有限公司 酶膜及其制备方法、糖化血红蛋白的检测方法
WO2019221264A1 (fr) * 2018-05-18 2019-11-21 株式会社Ｐｒｏｖｉｇａｔｅ Capteur de protéine glyquée, procédé de mesure, programme et procédé de fabrication de capteur
US11634745B2 (en) * 2017-03-03 2023-04-25 Polymer Technology Systems, Inc. Systems and methods for sample preparation for enzymatic A1C detection and quantification

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001264336A (ja) * 2000-03-22 2001-09-26 Internatl Reagents Corp 自己血糖検査手段
WO2002025262A1 (fr) * 2000-09-25 2002-03-28 Asahi Kasei Kabushiki Kaisha Electrode a enzyme
JP2005114359A (ja) * 2003-10-02 2005-04-28 Matsushita Electric Ind Co Ltd 血液中のグルコースの測定方法およびそれに用いるセンサ

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001264336A (ja) * 2000-03-22 2001-09-26 Internatl Reagents Corp 自己血糖検査手段
WO2002025262A1 (fr) * 2000-09-25 2002-03-28 Asahi Kasei Kabushiki Kaisha Electrode a enzyme
JP2005114359A (ja) * 2003-10-02 2005-04-28 Matsushita Electric Ind Co Ltd 血液中のグルコースの測定方法およびそれに用いるセンサ

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
HIROKAWA K. ET AL.: "HbA1c Sokutei ni Tekishita Shinki Fructosyl Peptide Oxidase na Tansaku", JAPAN SOCIETY FOR BIOSCIENCE, BIOTECHNOLOGY, AND AGROCHEMISTRY 2003 NENDO (HEISEI 15 NEN) TAIKAI KOEN YOSHISHU, 2003 NEN 3 GATSU 5 NICHI HAKKO, JAPAN SOCIETY FOR BIOSCIENCE, BIOTECHNOLOGY, AND AGROCHEMISTRY HAKKO, pages 105, 2D03A10, XP003016667 *
KOZO HIROKAWA, KAZUHIKO SHIMOJI, NAOKI KAJIYAMA: "An enyzmatic method for the determination of hemoglobin-A1c", BIOTECHNOLOGY LETTERS, vol. 27, no. 14, July 2005 (2005-07-01), pages 963 - 968, XP003016668 *
KOZO HIROKAWA, KAZUO NAKAMURA NAOKI KAJIYAMA: "Enzymes used for the determination of Hba1c", FEMS MICROBIOLOGY LETTERS, vol. 235, no. 1, 1 June 2004 (2004-06-01), pages 157 - 162, XP002903440 *
KOZO HIROKAWA, KEIKO GOMI, NAOKI KAJIYAMA: "Molecular cloning and expression of novel fructosyl peptide oxidases and their application for the measurement of glycated protein", BIOCHEM. BIOPHYS. RES. COMMUN., vol. 311, no. 1, 7 November 2003 (2003-11-07), pages 104 - 111, XP003016669 *

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US7943385B2 (en) 2006-07-25 2011-05-17 General Atomics Methods for assaying percentage of glycated hemoglobin
US8318501B2 (en) 2006-07-25 2012-11-27 General Atomics Methods for assaying percentage of glycated hemoglobin
US8338184B2 (en) 2006-07-25 2012-12-25 General Atomics Methods for assaying percentage of glycated hemoglobin
US8557591B2 (en) 2006-07-25 2013-10-15 General Atomics Methods for assaying percentage of glycated hemoglobin
JP2009150686A (ja) * 2007-12-19 2009-07-09 Sekisui Chem Co Ltd ヘモグロビン類の測定システム
JP2009171874A (ja) * 2008-01-23 2009-08-06 Citizen Holdings Co Ltd 糖化タンパク質濃度測定方法及びバイオセンサ
US8673646B2 (en) 2008-05-13 2014-03-18 General Atomics Electrochemical biosensor for direct determination of percentage of glycated hemoglobin
WO2009140343A1 (fr) * 2008-05-13 2009-11-19 General Atomics Biodétecteur électrochimique pour une détermination directe du pourcentage d'hémoglobine glyquée
EP2281900A1 (fr) * 2009-08-03 2011-02-09 Roche Diagnostics GmbH Fructosyl-oxydase peptidique et capteur pour la détermination des protéines glycées
WO2011015325A1 (fr) * 2009-08-03 2011-02-10 Roche Diagnostics Gmbh Nouvelle fructosyl pepsidyl oxydase
US8962271B2 (en) 2009-08-03 2015-02-24 Roche Diagnostics Operations, Inc. Fructosyl peptidyl oxidase
US8721853B2 (en) 2009-08-03 2014-05-13 Roche Diagnostics Operations, Inc. Fructosyl peptidyl oxidase
US8852413B2 (en) 2010-03-31 2014-10-07 Cci Corporation Biosensor
CN102959392A (zh) * 2010-03-31 2013-03-06 Cci株式会社 生物传感器
CN102959392B (zh) * 2010-03-31 2015-02-18 Cci株式会社 生物传感器
WO2011125750A1 (fr) * 2010-03-31 2011-10-13 シーシーアイ株式会社 Biocapteur
JP5950816B2 (ja) * 2010-03-31 2016-07-13 シーシーアイ株式会社 バイオセンサ
KR101727630B1 (ko) 2010-03-31 2017-04-17 씨씨아이 가부시키가이샤 바이오센서
JP2013079809A (ja) * 2011-09-30 2013-05-02 Oji Keisoku Kiki Kk 糖化タンパク質の分析装置及び分析方法
WO2017082253A1 (fr) * 2015-11-09 2017-05-18 東レ株式会社 Capteur
JP6126325B1 (ja) * 2016-02-25 2017-05-10 パナソニックヘルスケアホールディングス株式会社 バイオセンサ
WO2017145420A1 (fr) * 2016-02-25 2017-08-31 パナソニックヘルスケアホールディングス株式会社 Biocapteur
US11634745B2 (en) * 2017-03-03 2023-04-25 Polymer Technology Systems, Inc. Systems and methods for sample preparation for enzymatic A1C detection and quantification
CN108562751A (zh) * 2018-05-04 2018-09-21 深圳市西尔曼科技有限公司 酶膜及其制备方法、糖化血红蛋白的检测方法
WO2019221264A1 (fr) * 2018-05-18 2019-11-21 株式会社Ｐｒｏｖｉｇａｔｅ Capteur de protéine glyquée, procédé de mesure, programme et procédé de fabrication de capteur
CN112292446A (zh) * 2018-05-18 2021-01-29 株式会社普欧威盖特 糖化蛋白传感器、测定方法、程序和传感器的制造方法
JPWO2019221264A1 (ja) * 2018-05-18 2021-06-17 株式会社Ｐｒｏｖｉｇａｔｅ 糖化タンパク質センサ、測定方法、プログラム及びセンサの製造方法
JP7307959B2 (ja) 2018-05-18 2023-07-13 株式会社Provigate 糖化タンパク質センサ、測定方法、プログラム及びセンサの製造方法

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