WO2013081363A1 - Biocapteur de mesure d'hémoglobine glycosylée à l'aide d'une potentiométrie - Google Patents

Biocapteur de mesure d'hémoglobine glycosylée à l'aide d'une potentiométrie Download PDF

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WO2013081363A1
WO2013081363A1 PCT/KR2012/010145 KR2012010145W WO2013081363A1 WO 2013081363 A1 WO2013081363 A1 WO 2013081363A1 KR 2012010145 W KR2012010145 W KR 2012010145W WO 2013081363 A1 WO2013081363 A1 WO 2013081363A1
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electrode
measuring
hemoglobin
biosensor
reference electrode
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PCT/KR2012/010145
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English (en)
Korean (ko)
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임태규
조영식
이효근
황희영
최형길
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에스디 바이오센서 주식회사
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue
    • A61B5/14532—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue for measuring glucose, e.g. by tissue impedance measurement
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue
    • A61B5/1486—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue using enzyme electrodes, e.g. with immobilised oxidase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/4166—Systems measuring a particular property of an electrolyte
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/49—Blood

Definitions

  • the present invention relates to a biosensor for measuring glycated hemoglobin. More specifically, the reference electrode; And on the current collector, molecules comprising a site capable of specific binding to glycated hemoglobin, and in which molecules whose redox reaction potential is changed by glycated hemoglobin binding without supply of voltage and current from outside are arranged.
  • a measuring unit having a transition electrode; And a glycosylated hemoglobin measuring kit including the same, and a glycosylated hemoglobin measuring kit including a potentiometric measuring circuit or a potentiometric titration device for measuring a potential difference between a transition electrode and a reference electrode by potentiometry.
  • Diabetes is caused by inadequate carbohydrate metabolism, which does not properly use glucose absorbed into the body, and is a disease that can cause various complications due to excessive blood sugar in the blood.
  • type 1 diabetes mellitus is insulin-dependent diabetes mellitus, a type that loses the function of synthesizing or secreting insulin by the autoimmune response of interest cells.
  • type 2 diabetes is insulin-independent diabetes, which is caused by body resistance to insulin or inappropriate insulin secretion. Other fetal diabetes may occur during pregnancy.
  • diabetes mellitus of type 1 and fetal diabetes is not common, and most of the diabetes is type 2 diabetes, which accounts for 90% to 95% of developed diabetes.
  • HbA1c glycated hemoglobin
  • the ADA American Diabetes Association
  • UPF United Kingdom Prospective Diabetes Study
  • Hemoglobin in adults consists of three types: 97% hemoglobin A, 2.5% hemoglobin A 2 and 0.5% hemoglobin F.
  • Hemoglobin A is a four polypeptide structure consisting of two alpha chains with 141 amino acids and two beta chains with 146 amino acids. Chromatographic analysis of hemoglobin A consists of about 95% of normal hemoglobin and about 5 to 6% of microglycosylated hemoglobin. These glycosylated hemoglobins are collectively called hemoglobin A1.
  • Such hemoglobin A1 is known that hemoglobin A1a, hemoglobin A1b, hemoglobin A1c, and the like, in which glucose is bound to a beta chain N-terminal valine residue.
  • glycosylation The binding of sugar residues to non-enzymatic reactions of amino groups in proteins is called glycosylation, which is a very gradual irreversible reaction.
  • Glycosylated hemoglobin is formed by the combination of hemoglobin and blood glucose, and the ratio of hemoglobin and glycated hemoglobin is determined by the degree of exposure of red blood cells and blood glucose.
  • glucose binds to the valine residue of hemoglobin A to form a hemoglobin A1c precursor, which is a hemoglobin A1c having a stable ketoamine bond through a rearrangement reaction.
  • the contact frequency between glucose and hemoglobin increases, and the ratio of glycated hemoglobin also increases.
  • accurate quantification of glucose levels in the blood by the ratio of glycated hemoglobin is possible.
  • since the life of red blood cells is about 60 to 120 days, blood glucose concentration changes can be monitored for a relatively long period of time.
  • US 5,242,842 discloses a method in which boronic acid derivatives and glycated hemoglobin are combined and then precipitated or separated and measured using spectroscopic methods, but a process of washing boronic acid derivatives not bound to glycated hemoglobin is required.
  • the problem is that the measurement is difficult because the correct amount of sample can be accurately obtained.
  • US Pat. No. 6,162,645 and EP0455225B1 and US Pat. No. 6,174,734 disclose methods for determining the relative amounts of glycated hemoglobin using a marker compound after separating proteins in a sample using a solid phase immobilized with an immuno antibody. Methods are to collect glycated hemoglobin and glycated hemoglobin-markers competitively on the electrode surface, and then to determine the magnitude of the signal by injecting a substrate that causes an electrochemical reaction with the marker to determine the concentration of glycated hemoglobin and ensure reproducibility in repeat measurements. There is a problem that is difficult to do.
  • US 2010-0089774 discloses a technique for measuring the proportion of glycated hemoglobin by measuring a voltage change through an electrode prepared by mixing 4-phenyl-vinyl-boronic acid with carbon paste. .
  • the electrode is prepared by mixing boronic acid with carbon paste, a small amount of boronic acid should be added when the amount of boronic acid is increased, which makes it difficult to prepare the carbon paste electrode.
  • concentration of glycated hemoglobin is measured, There is a problem in that reproducibility is poor when measuring glycated hemoglobin.
  • when measuring a biological sample with a carbon face electrode has a problem that is susceptible to the coexistence material.
  • the present invention is to propose a HbA1c sensor of the point of care concept that can be measured simply and simply, while solving the problem of low reproducibility and not easy to manufacture when measuring the ratio of glycated hemoglobin as described above.
  • Dithiobis-3-butyramidophenylboronic acid DTB-BAPBA
  • DTB-BAPBA Dithiobis-3-butyramidophenylboronic acid
  • the present invention is a reference electrode; And a measurement unit including a transition electrode on the current collector, the transition electrode including a site capable of specific binding to glycated hemoglobin and arranged with molecules whose redox reaction potential changes by glycated hemoglobin binding without supply of voltage and current from the outside; And a biosensor for measuring glycated hemoglobin provided with a potentiometric measuring circuit or a potentiometric titrator for measuring the potential difference between the transition electrode and the reference electrode by potentiometry; And it provides a glycated hemoglobin measurement kit having the biosensor.
  • the present invention is a reference electrode; And a glycosylated hemoglobin measuring biosensor strip having a transition electrode on which a 3- (4-mercaptobutanamido) phenylboronic acid (3- (4-mercaptobutanamido) phenylboronic acid) is aligned on a current collector. to provide.
  • the concentration of total Hb and HbA1c is measured by using a potential difference without going through the separation step of hemoglobin and glycated hemoglobin, the total hemoglobin (Htal) and HbA1c in the sample are small and even a small amount of the sample. Can be measured quickly and simultaneously with high precision.
  • biosensor strip of the present invention is suitable for manufacturing disposable, and by reducing the number of three electrodes used in the existing electrochemical method to two electrodes, there is no need for the counter electrode to maintain a certain area or more.
  • the manufacturing process can be simplified to lower the manufacturing cost.
  • Figure 1 is a schematic diagram showing the response to the ferricyan ion of the DTBA-PBA monomolecular film modified electrode according to the present invention in the presence of sugar.
  • FIG. 2 is a schematic diagram showing the configuration of a strip for a biosensor according to a first embodiment of the present invention.
  • FIG. 3 is a schematic diagram showing the configuration of a biosensor strip according to a second embodiment of the present invention.
  • 5 is a graph showing the relationship between the potential difference value measured by potentiometry using the biosensor of the present invention and the concentration of total hemoglobin in whole blood of diabetic patients.
  • Fig. 6 is a graph showing the results obtained from the calibration curve of total hemoglobin prepared in advance and the results obtained by measuring HbA1c and converting it into%.
  • FIG. 7 is a graph comparing the accuracy of the measurement of glycated hemoglobin amount using a biosensor according to the present invention and a liquid high-speed chromatography method.
  • the measuring unit of the glycosylated hemoglobin measurement biosensor according to the present invention;
  • a transition electrode comprising a site capable of specific binding to glycated hemoglobin on the current collector and arranged with molecules whose redox reaction potential is changed by glycated hemoglobin binding without supply of voltage and current from the outside;
  • a working electrode for measuring the total amount of hemoglobin;
  • / or an auxiliary electrode optionally a working electrode for measuring the total amount of hemoglobin.
  • the present inventors studied the relationship between sugars and phenylboronic acid, and synthesized dithiobis-3-butyramidophenylboronic acid [Dithiobis-3-butyramidophenylboronic acid; DTB-BAPBA] was used to form a film on the surface of the electrode and examined the behavior of ferricyanate ions (Fig. 1). As a result, sugars were bound to boronic acid introduced by reaction with dithiobis-3-butyramidophenylboronic acid. Compared with the case where the sugar is not bonded to the case that the sugar is not bonded, it was confirmed that the ferricyanate ion is difficult to reach the electrode. In addition, it was found that pKa decreases when sugar is bonded to boronic acid groups at the terminal ends of molecules aligned on electrodes.
  • the smaller pKa means that it is acidified, which in turn means that the voltage changes by the Nernst equation.
  • the present invention includes a transition electrode comprising a site capable of specific binding to glycated hemoglobin on the current collector and aligning molecules whose redox reaction potential is changed by glycated hemoglobin binding without supply of voltage and current from the outside; And measuring the voltage before and after the glycosylated hemoglobin binding by preparing a reference electrode to measure the concentration of HbA1c in the sample.
  • sample refers to an analyte comprising hemoglobin and / or glycated hemoglobin, and is isolated from mammals, preferably humans, whole blood, blood cells, serum, plasma, bone marrow fluid, sweat, urine, tears, It includes all biological samples such as saliva, skin, mucous membranes, hair, and the like, and may be, for example, blood.
  • the biosensor of the present invention can be applied to blood glucose measurement by measuring glycated hemoglobin concentration using blood as a sample.
  • Non-limiting examples of current collector materials used in the transition electrode include metals such as gold, silver, and copper, and conductive substrates such as semiconductors such as GaAs, CdS, and In 2 O 3 as well as metallic substrates such as gold and platinum. Can be used.
  • the molecule In order for the molecule whose redox reaction potential is changed by the glycated hemoglobin bond to be easily aligned with the current collector, the molecule preferably includes a functional group capable of binding to the current collector. In order for the molecules to be easily aligned in the current collector made of metal, it is preferable to include a thiol group, a sulfide group or a disulfide bond capable of bonding with the metal.
  • the sites capable of binding to the current collector in the molecule are preferably oriented such that the site specifically binding to glycated hemoglobin at the current collector interface faces outward from the current collector.
  • the molecules aligned on the current collector used in the present invention can bind to glycosyl groups of glycated hemoglobin, and are substances that generate a redox reaction potential by introducing glycated hemoglobin without supplying voltage and current from the outside.
  • An example of a site capable of specific binding to glycated hemoglobin is a boronic acid functional group (-B (OH) 2 ).
  • the glycated hemoglobin and the boronic acid functional group combine to form a molecularly glycated hemoglobin conjugate on the surface of the transition electrode.
  • the aligned molecules are preferably molecules having electrical conductivity, for example, polyamide, polyaniline, polyphenol, polythiophene, polyacetylene, poly (p-phenylvinylene), poly (p-phenylene sulfide), and the like. It may be one or more selected from the group consisting of, but is not limited thereto. These molecules have excellent electrical conductivity and can be used for various applications, and have excellent thermal and chemical stability, and thus have advantages in improving performance and lifespan of biosensors.
  • examples of the conductive material having a boronic acid functional group include dithiobis-3-butyramidophenylboronic acid, 3- (4-mercaptobutaneamido) phenylboronic acid (3- ( 4-mercaptobutanamido) phenylboronic acid), dithiobis-4-aminophenyl boronic acid, 4-aminophenylboronic acid, 3-thiopheneboronic acid (3-thiopheneboronic acid) acid), 3-formyl-4-thiopheneboronic acid, and the like, but are not limited thereto.
  • dithiobis-3-butyramidophenylboronic acid or 3- (4-mercaptobutanamido) phenylboronic acid are not limited thereto.
  • thiophenboronic acid Since thiophenboronic acid is not dissolved in water but is dissolved in ethanol or the like, it is a substance that is difficult to react directly with a blood sample. In order to use it, it is mixed with carbon paste or the like.
  • a glycosylated hemoglobin is present in the current collector rather than a smaller number of molecules that specifically bind to glycated hemoglobin such as phenylboronic acid. It is possible to increase the voltage change range (e.g. pKa sensitivity) for the difference in concentration of, and to accurately measure the change of pKa by reducing the factors affecting pKa due to randomly oriented molecules being aligned on the current collector. .
  • molecules capable of specifically binding to sugars of glycated hemoglobin are preferably self-assembled on the surface of the current collector, more preferably forming a self-assembled monolayer.
  • the amount of boronic acid to be introduced is determined due to the characteristics of the paste, and thus high reproducibility cannot be obtained for high concentration of glycated hemoglobin.
  • Non-limiting examples of solvents used to align molecules on the current collector include tetrahydrofuran, methanol, isopropyl alcohol, ethanol, propanol, acetone, or mixtures thereof.
  • the redox material reacting with the ordered molecule of the transition electrode is ferricyanic acid, ferrocene, ferrocene derivative, quinones, quinone derivative, organic conducting salt. , Viologen, hexaamineruthenium (III) chloride, dimethylferrocene (DMF), ferricinium, ferocene monocarboxylic acid (FCOOH) ), 7,7,8,8, -tetracyanoquinodimethane (7,7,8,8-tetracyanoquino-dimethane (TCNQ), tetrathiafulvalene (TTF), nickellocene (Nc) ), N-methyl acidinium (NMA +), tetrathiatetracene (TTT), N-methylphenazinium (NMP +), hydroquinone, 3-dimethylamino Benzoic acid (3-dimethylaminobenzoic acid; MBTHDMAB), 3-methyl-2-benzothiozolinone
  • the transition electrode and the sample prepared according to the present invention if the electron transfer is made between the molecules arranged on the current collector of the transition electrode and the redox material in the sample, that is, oxidation When the reduction reaction occurs, a potential is generated between the transition electrode and the reference electrode.
  • the electrode potential of the transition electrode is stabilized, and in the present invention, such an equilibrium electrode of the transition electrode is measured.
  • the molecules aligned on the transition electrode have a site capable of specific binding to glycated hemoglobin, so that when the glycated hemoglobin binds to the aligned molecule, a chemical change of the aligned molecule is caused to change the equilibrium electrode potential on the surface of the transition electrode. .
  • the equilibrium electrode potential of the transition electrode varies depending on how much of the aligned molecules are combined with the sugars of the glycated hemoglobin in the sample
  • the equilibrium electrode potential of the transition electrode in the sample is measured so that the aligned molecules on the transition electrode are combined with the sugar. The extent can be calculated and the concentration of glycated hemoglobin on the sample can be analyzed.
  • the change amount at that time is measured by applying a voltage or a current between the transition electrode and the reference electrode.
  • the voltage is generated by the difference between the redox potential of the material in the transition electrode and the reference electrode without supplying a voltage or current from the outside, and the redox potential (eg, As pKa is changed, the voltage between the transition electrode and the reference electrode is changed, and the measurement is made, and there is an advantage that no special external power supply is required.
  • Such a potential difference change of the transition electrode can be obtained by measuring the potential difference between the transition electrode and the reference electrode by potentiometry, and for this purpose, a potential difference measurement circuit or a potential difference titration device can be used.
  • the potential of the standard hydrogen electrode is referred to as 0 V for convenience.
  • a monopolar potential such as a calomel electrode and a silver-silver chloride electrode is used as the reference electrode.
  • the reference electrode is preferably an electrode that can be used as a silver / silver chloride or similar reference electrode capable of maintaining a constant potential.
  • the reference electrode may be prepared by coating silver / silver chloride on a portion in contact with a sample.
  • glycated hemoglobin is expressed as the amount of glycated hemoglobin relative to the amount of total hemoglobin in the blood. Therefore, to obtain the glycated hemoglobin level, it is desirable to measure the total amount of hemoglobin together.
  • the measuring unit according to the present invention preferably can be measured by the potentiometric method through the amount of hemoglobin and also the oxidation-reduction reaction of hemoglobin contained in the sample. Therefore, in order to measure glycated hemoglobin or to measure total hemoglobin, only two electrodes, that is, a transition electrode and a reference electrode, are required.
  • the transition electrode for measuring the potential difference through the redox reaction of hemoglobin is referred to as working electrode hereinafter.
  • the glycated hemoglobin can be measured because the glycated hemoglobin binds to a transition electrode that modifies a glycosylated hemoglobin-specific molecule, such as dithiobis-3-butyramidophenylboronic acid, to generate a voltage difference with the reference electrode.
  • a transition electrode that modifies a glycosylated hemoglobin-specific molecule, such as dithiobis-3-butyramidophenylboronic acid
  • a transition electrode that is, a working electrode
  • a reference electrode that do not modify anything
  • a reversible oxidation and reduction reaction between Fe 2+ in a ham (HEME) group and Fe 3+ mixed in a buffer solution is performed. This results in a potential difference between the two electrodes, so that the total hemoglobin can be measured.
  • the material of the working electrode examples include metals such as copper, platinum, silver, gold, palladium, ruthenium, rhodium, and iridium, carbon or materials subjected to surface treatment. The best is the gold electrode. Further, the surface hydrophilic treatment may be performed for the purpose of preventing adsorption of proteins and the like in the sample. Ag / AgCl electrode may be used as the reference electrode.
  • the biosensor for measuring glycated hemoglobin according to the present invention can be simultaneously measured without separation using Fe 2+ as a reaction indicator of hemoglobin and glycated hemoglobin.
  • hemoglobin and glycated hemoglobin can be simultaneously measured by voltage measurement without separating the hemoglobin and glycated proteins.
  • the measuring method by amperometric method can measure hemoglobin easily with a miniaturized device in comparison with the method using chromatography or absorbance photometer.However, since the current value shown in the measurement result is proportional to the electrode area or the sample amount, In order to increase the area of the electrode or to use a large amount of sample, there is a problem in miniaturization of a measuring device or a small amount of sample due to the point-of-care meaning.
  • the present invention solves the above problem because the total hemoglobin is measured through the potential difference between the two electrodes.
  • a sample treated with a mixture of surfactant and potassium ferricyanide to the blood to the two electrodes.
  • a nonwoven fabric is provided between the two electrodes, and if the surfactant and the potassium ferricyanide are lyophilized beforehand, only whole blood is added on the electrode.
  • the lyophilized reagent can hemolyze whole blood to generate a redox reaction, thereby measuring the potential difference between the two electrodes.
  • the measurement potential changes slightly from the voltage generated by the sample in accordance with a change in conditions such as temperature and age of the reference electrode. Therefore, the measurement error according to the change of condition can be corrected by measuring the reference solution which knows the redox potential in advance.
  • the measurement unit of the present invention may further include an auxiliary electrode for error correction due to a potential change due to factors other than the measurement sample, thereby eliminating the influence of factors that interfere with the measurement of glycated hemoglobin or total hemoglobin.
  • the auxiliary electrode may be made of a material such as a displacement electrode or a working electrode for measuring total hemoglobin.
  • the auxiliary electrode is manufactured in the same process and composition as the working electrode to exhibit the same electrical response as the working electrode.
  • the redox potential is known under a certain condition, and there is no problem as long as the sample has a stable potential.
  • metal salts, metal complexes, quinonic compounds, benjophenones and mixed solutions of these substances can be used.
  • Transition electrode On the other hand, according to the present invention. Transition electrode; Optionally a working electrode; And optionally the measuring part with the auxiliary electrode may be in the form of a replaceable strip (see FIGS. 2 and 3).
  • the measuring unit of the present invention is composed of a transition electrode, a reference electrode, optionally a working electrode, and optionally an auxiliary electrode and an electrical connection line connecting each electrode with a measuring device.
  • the remaining part except the electrode forms an insulating layer using an insulating material.
  • the strip for a biosensor according to the present invention preferably has respective electrodes formed on a support made of a non-conductive insulating material.
  • a support is preferably made to have a thickness of 20 to 60 microns, more preferably one having a thickness of 30 microns.
  • any insulator can be used as the material of the support made of the non-conductive insulating material, but at the same time, it is suitable to have a certain degree of flexibility and rigidity as the support to manufacture a large amount.
  • the surface of the support should be very even. This is because an uneven surface causes a nonuniformity of the electrode surface area between the respective sensor strips in mass production and consequently a nonuniformity of the sensor output signal.
  • Materials having the most even surface include silicon wafers used in semiconductor manufacturing.
  • a quartz glass substrate or a general glass substrate that is transparent and easy to work may be used.
  • the general compact disc for music has a very uniform surface, excellent flatness, and has a similar shape to a semiconductor wafer in a circular shape, so that the semiconductor manufacturing process equipment can be used as it is without any separate equipment. Yet, it is inexpensive and easily available.
  • a general plastic film can be used.
  • compact disc or plastic film materials examples include polyester, polycarbonate, poly stylene, polyimide, poly vinyl chloride, polyethylene ), Polyethylene terephthalate (polyethylene telephthalate) and the like can be used.
  • the working electrode is preferably 14 mm to 19 mm long, 0.5 mm to 2 mm wide and 20 to 150 microns thick, more preferably 14 mm long, 1 mm wide and 60 microns thick. .
  • the transition electrode, the reference electrode, and the auxiliary electrode are each independently composed of a length of 15 mm to 20 mm, a width of 0.5 mm to 2 mm, and a thickness of 20 to 150 microns, more preferably 15 mm length, 1 mm. It is composed of a width and a thickness of 60 microns.
  • Electrodes of the present invention preferably partitions between the portion in direct contact with the sample and the portion for transmitting a signal to the detector through an insulating coating, but is not limited thereto.
  • the biosensor may further include a display unit for converting the potential difference value between the transition electrode and the reference electrode or converting it into an amount or concentration of glycated hemoglobin in a sample.
  • the present invention provides a glycated hemoglobin measuring kit having the biosensor according to the present invention.
  • the glycated hemoglobin measurement kit may further comprise a lysis solution, a surfactant solution, or both.
  • the voltmeter for measuring the potential difference between the two electrodes preferably has a high impedance.
  • a calibration curve may be prepared in advance and the hemoglobin concentration may be quantified using the calibration curve.
  • hemoglobin concentration when the hemoglobin concentration is measured, when a sample containing red blood cells is introduced into the reaction vessel, hemoglobin in the red blood cells is released by the hemolytic agent to cause a redox reaction with the mediata, thereby reducing the mediata.
  • a voltage is generated at the working electrode with respect to the resultant oxidized / reduced concentration ratio, and the concentration of hemoglobin in whole blood is calculated using the calibration curve prepared in advance from the voltage.
  • the measurement sample may be whole blood treated with an anticoagulant or hemolytic reagent.
  • the volume of the sample introduced into the reaction vessel is suitably 1 microliter or more. Preferred volumes are at least 5 microliters.
  • the reaction vessel is configured such that a measurement sample or the like is brought into contact with the transition electrode / working electrode of the electrode and each reference electrode thereof, and when a liquid sample is introduced, two electrodes are energized to form a circuit. If the sample is not a liquid, it can be introduced into the reaction vessel after dissolving in a solvent such as water.
  • the reaction vessel may be configured to hold a sample and a measurement reagent, and to conduct electricity between two electrodes after introduction of the sample, and may be a container having a size that can accommodate the transition electrode / working electrode and each reference electrode thereof.
  • the material of the reaction vessel can be used without limitation as long as it is electrically inert and / or inert to a sample or electrode, such as a fiber aggregate such as filter paper, a nonwoven fabric, a porous material, a gel, and the like.
  • a fiber aggregate such as filter paper, a nonwoven fabric, a porous material, a gel, and the like.
  • polyvinyl chloride, polyimido, gelatin, glass fibers and the like can be mentioned.
  • the reaction vessel may include a hemolytic agent, a redox mediator, and a pH buffer reagent as measurement reagents.
  • an interference elimination reagent may be included in the reaction vessel to remove the interference component that interferes with the electrochemical measurement.
  • hemolytic agent ionic or nonionic surfactants, organic solvents, salts, enzymes and the like can be used.
  • surfactant polyoxyethylene octylphenyl ether, sodium lauryl sulfate, saponin and the like can be used. Formaldehyde, nucleic acid, acetone and the like can be used as the organic solvent.
  • salt ammonium chloride, aluminum chloride and the like can be used.
  • Preferred examples include polyoxyethyleneoctylphenyl ether. In this case, the concentration may be 1 to 20% (v / v). It may also be diluted with distilled water to induce hemolysis due to changes in salt concentration.
  • a mediae can be used as long as it causes a redox reaction with hemoglobin.
  • Non-limiting examples include metal salts, metal complexes, quinone compounds and benzophenones.
  • the most stable mediata is ferricyanide, and the use concentration is more than twice the expected hemoglobin concentration, and a stable result is obtained and it is in the range of 10 to 500 mM.
  • the pH buffer may be used without limitation as long as it does not react with the reaction vessel, the electrode or the sample while maintaining pH 4 to 8 after sample addition.
  • the final service concentration is 5 to 500 mM and better conditions are 50 to 200 mM.
  • a phosphate buffer of pH 6.5 to 7.0 can be used.
  • FIG. 2 is schematic of the apparatus for measuring hemoglobin and HbA1c.
  • the electrode includes a HbA1c measuring electrode and a first reference electrode; And a working electrode for measuring total hemoglobin and a second reference electrode.
  • the reference electrode is fixed by applying Ag / AgCl paste.
  • the transition electrode and the first reference electrode are disposed adjacent to each other, and the reaction vessel is disposed in a shape that can be in contact with both the transition electrode and the first reference electrode.
  • the transition electrode and the first reference electrode can be energized through the sample.
  • the working electrode and the second reference electrode are also disposed adjacent to each other, and the reaction vessel is disposed in a shape that can be in contact with both the working electrode and the second reference electrode.
  • the working electrode and the second reference electrode can be energized through the sample.
  • the reaction vessel is installed so that the sample can be absorbed and retained therein and discarded after one use.
  • the reaction vessel contains a nonwoven fabric.
  • Phosphoric acid buffer solution Triton X-10, dithiobis-3-butyramidophenylboronic acid or phenylboronic acid are kept dry in the reaction vessel for measuring HbA1c. Phosphoric acid is used as a reagent in the reaction vessel for total hemoglobin measurement.
  • the buffer, Triton X-100, and potassium ferricyanide are kept dry, and the introduction of a 10 microliter whole blood sample causes the water to dissolve in the sample and the redox potential occurs even when no external voltage or current is supplied.
  • the transition electrode, the first reference electrode, and the working electrode and the second reference electrode are respectively connected to a voltmeter to measure the voltage generated between these two electrode pairs. Therefore, the total hemoglobin concentration and the HbA1c concentration in the sample can be calculated based on the voltage measured by the voltmeter.
  • the hemoglobin and HbA1c concentrations in the sample were calculated based on the measured potential. Since a power source for supplying power between electrodes is not necessary and even a small amount of sample can be measured, even a small amount of sample can measure high-precision hemoglobin and HbA1c concentration.
  • FIG. 3 is a schematic diagram of another device for measuring hemoglobin and HbA1c.
  • an auxiliary electrode is provided for error correction caused by a potential change caused by factors other than the measurement sample.
  • the measurement potential changes slightly from the voltage generated by the sample in accordance with a change in conditions such as temperature and age of the reference electrode. Therefore, the measurement error according to the change of condition can be corrected by measuring the reference solution which knows the redox potential in advance.
  • the electrode 2 is disposed between the electrode 1 and the electrode 3, and the reaction vessel is disposed to contact the electrode 1 and the electrode 2, the electrode 2, and the electrode 3.
  • the voltmeter measures the voltage generated between the electrode 1 and the electrode 2 and simultaneously measures the voltage generated between the electrode 2 and the electrode 3 as a reference voltage.
  • the reaction vessel a contains a measurement sample and a reagent such as the reaction vessel
  • the reaction vessel b contains a reference solution.
  • the reference solution is introduced into the reaction vessel b and contacts the positive electrodes of the electrodes 2 and 3, a reference voltage between the electrodes 2 and 3 is generated.
  • the reference solution may be introduced into the reaction vessel b at the start of measurement, or may be introduced in advance.
  • the voltage generated between the electrode 1 and the electrode 2 and the voltage generated between the electrode 2 and the electrode 3 are measured and displayed by a voltmeter, and the voltage generated between the electrode 1 and the electrode 2 is corrected by the voltage generated between the electrode 2 and the electrode 3. It can also be considered that the voltage fluctuates due to factors other than the measurement sample, but it is possible to reduce the error caused by the above factors by using the voltage generated between the electrodes 2 and 3.
  • the hemoglobin concentration or the HbA1c concentration in the measurement sample can be calculated using the voltage obtained by subtracting the voltage generated between the electrode 2 and the electrode 3 from the voltage generated between the electrode 1 and the electrode 2.
  • the second embodiment of the present invention not only the same effect as in the first embodiment can be obtained but also the addition of the auxiliary electrode makes it possible to exclude voltage changes caused by factors other than the measurement sample, thereby improving the measurement accuracy. .
  • a strip for biosensor as shown in Figure 3 was prepared.
  • a gold electrode of 15 mm long, 1 mm wide and 60 micron thick was washed twice with ultrasonic waves for 5 minutes, and then between 0.2 and 0.5 V in 0.5 M sulfuric acid solution.
  • the surface was prepared by an electrochemical method of irradiating a voltage of.
  • the prepared gold electrode was reacted with a mixed solution of tetrahydrofuran and methanol in a ratio of 9: 1 containing dithiobis-3-butyramidophenyl boronic acid at a concentration of 0.5 mg / ml for 8 hours.
  • a phenylboronic acid monomolecular film having a density of 5.1 ⁇ 10 ⁇ 10 mM / cm 2 was fixed on the surface of the gold electrode using intramolecular disulfide bonds to prepare a transition electrode 1.
  • HbA1c standard glycated hemoglobin

Abstract

L'invention concerne un biocapteur de mesure d'hémoglobine glycosylée comprenant : une unité de mesure comprenant des électrodes de référence, et comprenant une électrode modifiée contenant à sa surface des molécules comprenant un site apte à effectuer une liaison spécifique à l'hémoglobine glycosylée et dont le potentiel de réaction redox change en fonction de la liaison avec l'hémoglobine glycosylée sans apporter de tension et de courant de l'extérieur, sur un collecteur de courant ; un circuit de mesure de différence de potentiel ou un dispositif de titration potentiométrique, destiné à mesurer la différence de potentiel entre l'électrode modifiée et l'électrode de référence au moyen d'une potentiométrie ; et une unité d'affichage destinée à effectuer un affichage sélectif de la valeur de la différence de potentiel entre l'électrode modifiée et l'électrode référence ou du résultat lorsque cette valeur est calculée comme étant la quantité ou la concentration d'hémoglobine glycosylée à l'intérieur de l'échantillon. Le biocapteur de mesure d'hémoglobine glycosylée selon la présente invention a pour effet de rendre possible une mesure rapide et précise de la concentration d'hémoglobine glycosylée dans un échantillon, et de rendre possible la détermination du taux d'hémoglobine et d'hémoglobine glycosylée par mesure des concentrations des deux substances en même temps sans passer par une étape de séparation lors de la mesure d'hémoglobine glycosylée.
PCT/KR2012/010145 2011-11-28 2012-11-28 Biocapteur de mesure d'hémoglobine glycosylée à l'aide d'une potentiométrie WO2013081363A1 (fr)

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