WO2013081363A1 - Biosensor for measuring glycosylated haemoglobin using potentiometry - Google Patents

Biosensor for measuring glycosylated haemoglobin using potentiometry 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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French (fr)
Korean (ko)
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임태규
조영식
이효근
황희영
최형길
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에스디 바이오센서 주식회사
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Publication of WO2013081363A1 publication Critical patent/WO2013081363A1/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61BDIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00Measuring for diagnostic purposes; Identification of persons
    • A61B5/145Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue
    • A61B5/14532Measuring 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
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28Electrolytic cell components
    • G01N27/30Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61BDIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00Measuring for diagnostic purposes; Identification of persons
    • A61B5/145Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue
    • A61B5/1486Measuring 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
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28Electrolytic cell components
    • G01N27/30Electrodes, e.g. test electrodes; Half-cells
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416Systems
    • G01N27/4166Systems measuring a particular property of an electrolyte
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483Physical analysis of biological material
    • G01N33/487Physical analysis of biological material of liquid biological material
    • G01N33/49Blood

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

The biosensor for measuring glycosylated haemoglobin according to the present invention comprises: a measuring unit comprising reference electrodes, and comprising a modified electrode having disposed thereon molecules which comprise a site capable of specific bonding to glycosylated haemoglobin and of which the redox reaction potential changes due to glycosylated haemoglobin bonding without supplying voltage and current from the outside, on a current collector; a potential-difference measuring circuit or potentiometric titration device, for measuring the potential difference between the modified electrode and the reference electrode by means of potentiometry; and a display unit for selectively effecting a display of the value of the potential difference between the modified electrode and the reference electrode or of the result when this value is calculated as either the amount or the concentration of glycosylated haemoglobin within the sample. The biosensor for measuring glycosylated haemoglobin according to the present invention has the effect of making it possible to quickly and accurately measure the concentration of glycosylated haemoglobin in a sample, and makes it possible to determine the ratio of haemoglobin and glycosylated haemoglobin by measuring the concentrations of the two substances at the same time without going through a separating step when measuring the concentration of glycosylated haemoglobin.

Description

전위차분석법을 이용한 당화헤모글로빈 측정용 바이오센서Biosensor for Measurement of Glycosylated Hemoglobin Using Potentiometric Analysis
본 발명은 당화헤모글로빈 측정용 바이오센서에 관한 것이다. 보다 상세하게는 기준전극; 및 집전체 상에, 당화헤모글로빈에 특이적 결합가능한 부위를 포함하고 외부로부터 전압 및 전류의 공급없이 당화헤모글로빈 결합에 의해 산화환원 반응전위가 변하는 분자들이 정렬된 변이전극을 구비한 측정부; 및 전위차분석법(Potentiometry)에 의해 변이전극과 기준전극 사이의 전위차를 측정하기 위한, 전위차 측정회로 또는 전위차 적정장치를 구비한 당화헤모글로빈 측정용 바이오센서 및 이를 포함한 당화헤모글로빈 측정키트에 관한 것이다.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.
당뇨병은 체내에 흡수된 포도당을 제대로 사용하지 못하는 부적절한 탄수화물 대사로 인하여 발생하며, 혈액 내에 과다한 혈당을 가지게 되어 다양한 합병증을 유발할 수 있는 질환이다. 이는 크게 세가지로 분류되며, 제1형 당뇨병은 인슐린 의존성 당뇨병으로, 이자 세포의 자가면역반응에 의하여 인슐린을 합성하거나 분비하는 기능을 상실하는 타입이라 할 수 있다. 다음으로 제2형 당뇨병은 인슐린 비의존성 당뇨병으로, 인슐린에 대한 체내 저항성 또는 부적절한 인슐린 분비 등에 의해 발병한다. 그 외에 임신 중 발생할 수 있는 태아 당뇨병이 있다. 그러나, 제1형 당뇨병과 태아 당뇨 형태의 당뇨병은 흔하지 않으며, 당뇨병 중 대부분은 제2형 당뇨병으로서 선진국 당뇨질환 중 90 내지 95%를 차지하고 있는 것으로 알려져 있다.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. This is classified into three types, 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. Next, 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. However, 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.
당뇨병을 진단하는 방법은 요당측정, 혈중 포도당 측정 등 여러 가지가 있지만, 요당측정은 신뢰할 수 없으며, 혈중 포도당 측정은 식사, 운동 등 여러 요인의 영향을 받아 부정확하다. 따라서, 당뇨병을 관리하고 치료하기 위해서는, 2개월 간의 평균 혈당치가 중요시 되고 있으므로, 혈액 중 당화헤모글로빈(HbA1c)을 측정하는 것이 효과적이다.There are many ways to diagnose diabetes, such as urine glucose measurement and blood glucose measurement, but urine glucose measurement is unreliable, and blood glucose measurement is inaccurate under the influence of various factors such as diet and exercise. Therefore, in order to manage and treat diabetes, the average blood sugar level for two months is important, so it is effective to measure glycated hemoglobin (HbA1c) in the blood.
1986년 미국 당뇨 협회에서 모든 형태의 당뇨병을 관리하기 위해 연간 2회씩의 당화헤모글로빈 측정을 제안함으로써 비교적 안정한 지표인 당화 헤모글로빈의 양을 당뇨병 관리지표로 사용하기 시작하였고, 1993년 DCCT(Direct Control and Complication Trial, 당뇨조절과 합병증 연구)에서 당화헤모글로빈의 농도와 당뇨합병증의 관계를 보고하면서 본격적으로 사용하기 시작하였다.In 1986, the American Diabetes Association proposed to measure glycated hemoglobin twice a year to manage all forms of diabetes and began to use the relatively stable amount of glycated hemoglobin as a diabetic management index.In 1993, DCCT (Direct Control and Complication) Trial (diabetes control and complication studies) reported the relationship between glycated hemoglobin concentration and diabetic complications.
당화헤모글로빈의 참고치 설정과 관련하여 ADA(American Diabetes Association, 미국당뇨병학회)에서는 DCCT 및 UKPDS(United Kingdom Prospective Diabetes Study, 영국전향적당뇨병연구)의 보고서를 기초로 하여, 당화헤모글로빈수치를 7% 이내로 관리할 것을 권고하고 있으며, 당화헤모글로빈수치가 8% 이상인 경우 당뇨관리의 재평가 및 적극적인 치료를 권고하고 있다. 2001년 미국 내분비학회에서는 6.5%를 참고치로 제시하였는데 이는 6.5% 이상일 때도 당뇨망막증의 발병율이 증가하는 것으로 UKPDS에서 보고한 결과를 참조한 것이다. 1999년 국제당뇨협회(International Diabetes Federation, IDF)에서도 동일하게 당화헤모글로빈 6.5%를 참고치로 제시하고 있다.Regarding the setting of the glycated hemoglobin reference level, the ADA (American Diabetes Association) manages glycated hemoglobin levels within 7% based on reports from the DCCT and the United Kingdom Prospective Diabetes Study (UKPDS). It is recommended to re-evaluate and actively treat diabetes management if the glycated hemoglobin level is more than 8%. In 2001, the US Endocrinology Society recommended 6.5% as a reference, referring to the results reported by the UKPDS, which increased the incidence of diabetic retinopathy even above 6.5%. In 1999, the International Diabetes Federation (IDF) equally referred to glycated hemoglobin at 6.5%.
성인의 헤모글로빈은 97%의 헤모글로빈 A, 2.5%의 헤모글로빈 A2 및 0.5%의 헤모글로빈 F의 세종류로 구성되어 있다. 이중 헤모글로빈 A는 141개의 아미노산을 가진 두 개의 알파체인과 146개의 아미노산을 가진 두 개의 베타체인으로 이루어진 네 개의 폴리펩타이드 구조이다. 헤모글로빈 A를 크로마토그래피법으로 분석해 보면 약 95%의 일반적인 헤모글로빈과 약 5 내지 6% 정도의 미량 당화헤모글로빈으로 구성되며, 이들 당화헤모글로빈을 통칭하여 헤모글로빈 A1라고 한다. 이러한, 헤모글로빈 A1의 80%는 베타사슬 N-말단의 발린 잔기에 글루코스가 결합된 형태로 헤모글로빈 A1a, 헤모글로빈 A1b, 헤모글로빈 A1c 등이 알려져 있다.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.
단백질의 아미노기에 당잔기가 비효소적인 반응으로 결합하는 것을 당화 과정이라 하며, 이 반응은 매우 점진적인 비가역적인 반응이다. 당화헤모글로빈은 헤모글로빈과 혈중포도당의 결합에 의해 형성되고, 헤모글로빈과 당화헤모글로빈의 비율은 적혈구와 혈중 포도당의 노출 정도에 의해 결정된다. 구체적으로, 당화 과정에서는 헤모글로빈 A의 발린 잔기에 포도당이 결합하여 헤모글로빈 A1c 전구물질을 형성하게 되며, 이는 재배열 반응을 통해 안정적인 케토아민 결합을 가진 헤모글로빈 A1c가 된다. 이때 혈액 내의 포도당 수치가 높아지면 포도당과 헤모글로빈의 접촉빈도가 높아지고, 당화헤모글로빈의 비율도 증가하게 된다. 따라서, 당화헤모글로빈의 비율로써 혈액 내 포도당 수치의 정확한 정량이 가능하다. 또한, 적혈구의 수명은 60 내지 120일 정도이므로 비교적 긴 기간 동안 혈중포도당 농도변화를 모니터링할 수 있다.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. Specifically, in the glycosylation process, 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. In this case, as the glucose level in the blood increases, the contact frequency between glucose and hemoglobin increases, and the ratio of glycated hemoglobin also increases. Thus, accurate quantification of glucose levels in the blood by the ratio of glycated hemoglobin is possible. In addition, 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.
혈액 내의 당화헤모글로빈을 측정하기 위한 다양한 측정법이 개발되어 왔다. 현재 상업적으로 응용되고 있는 방법으로는 이온교환 크로마토그래피법, 친화성 크로마토그래피법, 전기영동법, 복합착색법 등이 있다. 이러한 방법들은 사용법이 어렵고 복잡하여 숙련된 기술을 요구한다. 한편 일회성 임상 분석시스템의 기술개발 동향을 살펴보면, 원격, 재택 또는 현장검사를 위한 장비로 매우 유용하고 다양한 정량방법이 제시되고 있으며, 측정방법으로는 육안판독법, 광학판독법, 전기화학측정법 등이 알려져 있다.Various assays have been developed for measuring glycated hemoglobin in the blood. Current commercially available methods include ion exchange chromatography, affinity chromatography, electrophoresis and complex coloring. These methods are difficult and complex to use and require skill. On the other hand, when looking at the trend of technology development of one-time clinical analysis system, various useful quantitative methods are suggested as equipment for remote, home or on-site inspection, and the measurement methods are visual reading method, optical reading method, and electrochemical measurement method. .
구체적으로, 이러한 혈당측정방법으로서 당화헤모글로빈에 특이적으로 반응하는 면역항체를 고정시킨 패드를 마련하고 시료가 상기 항체를 고정시킨 패드로 전개하도록 한 후 반사광의 강도로 산출하는 방법이 US 5,541,117에 개시되어 있으나, 비싼 항체를 사용해야 하고 다공성 패드의 불균일성에 의해 일정한 품질의 센서를 생산하는 것이 어렵다는 문제점이 있다.Specifically, as a blood glucose measurement method, a method in which a pad having immobilized immune antibodies that specifically react with glycated hemoglobin is prepared, and a sample is developed into a pad having immobilized the antibody, and then calculated by the intensity of reflected light is disclosed in US Pat. No. 5,541,117. However, there is a problem that it is difficult to produce a sensor of a certain quality due to the non-uniformity of the porous pad and the use of expensive antibodies.
또한, US 5,242,842에는 보론산 유도체와 당화헤모글로빈을 결합시킨 후 함께 침전시키거나 분리한 후 분광학적 방법을 사용하여 측정하는 방법이 개시되어 있으나 당화헤모글로빈과 결합하지 않은 보론산 유도체를 세척하는 과정이 필요하고 시료의 양을 정확하게 맞추어야 옳은 결과를 얻을 수 있어 측정이 까다롭다는 문제점이 있다.In addition, 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 6,162,645와 EP0455225B1 및 US 6,174,734에는 면역항체를 고정한 고체상을 사용하여 시료 중의 단백질을 분리한 후 표식자 화합물을 사용하여 당화헤모글로빈의 상대적 양을 결정하는 방법이 제시되어 있으나, 이러한 종래의 전기화학적 당화헤모글로빈 결정방법들은 당화헤모글로빈 및 당화헤모글로빈-표식자들을 전극 표면에 경쟁적으로 모이게 한 후 표식자와 전기화학적반응을 일으키는 기질을 주입하여 신호의 크기를 결정하는 것으로 당화헤모글로빈의 농도측정이 복잡하고 반복측정에서 재현성을 확보하기 어렵다는 문제점이 있다.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.
이러한 문제점을 해결하기 위해 전극에 직접 보론산을 고정하여 표식자 없이 당화헤모글로빈을 측정하는 기술이 개발되었다. US 2010-0089774에는 4-페닐비닐보론산(4-phenyl-vinyl-boronic acid)을 카본페이스트와 혼합하여 제조한 전극을 통해 변화하는 전압을 측정하여 당화헤모글로빈의 비율을 측정하는 기술이 개시되어 있다. 그러나, 보론산을 카본페이스트와 혼합하여 전극을 제조하는 경우 보론산의 첨가량이 많게 되면 카본페이스트 전극을 제조하기 어렵기 때문에 보론산을 소량 첨가해야 하는데, 그러면 당화헤모글로빈의 농도를 측정할 때 고농도의 당화헤모글로빈 측정시 재현성이 떨어진다는 문제점이 있다. 또한 카본페이스 전극을 가지고 생체시료를 측정할 경우 공존 물질에 영향을 받기 쉽다는 문제점을 갖는다.In order to solve this problem, a technique of measuring glycated hemoglobin without an indicator has been developed by fixing boronic acid directly to an electrode. 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. . However, when 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. Then, when the concentration of glycated hemoglobin is measured, There is a problem in that reproducibility is poor when measuring glycated hemoglobin. In addition, when measuring a biological sample with a carbon face electrode has a problem that is susceptible to the coexistence material.
본 발명은 상기와 같이, 당화헤모글로빈의 비율 측정시 재현성이 낮고 제작이 용이하지 않은 문제점을 해결하면서, 간단하고 간편히 측정할 수 있는 point of care 개념의 HbA1c센서를 제안하고자 한다.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.
이에 본 발명자들은 디티오비스-3-부티라미도페닐보론산[Dithiobis-3-butyramidophenylboronic acid; DTB-BAPBA]과 같은 화합물과 반응시켜 상기 분자 내 이황결합을 이용한 결합으로 보론산 함유 분자를 안정적이고 고르게 전극 상에 정렬시킬 수 있고, 이러한 방법을 통해 제조된 변이전극을 사용하여 당화헤모글로빈의 결합량에 따른 변이 전극과 기준전극의 전위차 변화를 측정한 결과, 당화헤모글로빈의 양을 보다 재현성있게 측정할 수 있음을 확인하고, 본 발명을 완성하게 되었다.Accordingly, the inventors of the present invention, Dithiobis-3-butyramidophenylboronic acid; DTB-BAPBA] can be used to align the boronic acid-containing molecules stably and evenly on the electrode by the bond using the disulfide bond in the molecule, and binding of glycosylated hemoglobin using the transition electrode prepared through this method As a result of measuring the potential difference between the transition electrode and the reference electrode according to the amount, it was confirmed that the amount of glycated hemoglobin can be measured more reproducibly, thereby completing the present invention.
본 발명은 기준전극; 및 집전체 상에, 당화헤모글로빈에 특이적 결합가능한 부위를 포함하고 외부로부터 전압 및 전류의 공급없이 당화헤모글로빈 결합에 의해 산화환원 반응전위가 변하는 분자들이 정렬된 변이전극을 구비한 측정부; 및 전위차분석법(Potentiometry)에 의해 변이전극과 기준전극사이의 전위차를 측정하기 위한, 전위차 측정회로 또는 전위차 적정장치를 구비한 당화헤모글로빈 측정용 바이오센서; 및 상기 바이오센서를 구비한 당화헤모글로빈 측정키트를 제공한다.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.
또한, 본 발명은 기준전극; 및 집전체 상에, 황원자를 통해 3-(4-머캅토부탄아미도)페닐보론산(3-(4-mercaptobutanamido)phenylboronic acid)이 정렬된 변이전극을 구비한 당화헤모글로빈 측정 바이오센서용 스트립을 제공한다.In addition, 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.
본 발명에 따르면, 헤모글로빈과 당화헤모글로빈의 분리단계를 거치지 않고 전위차를 이용하여 총헤모글로빈(total Hb)과 HbA1c의 농도를 측정하기 때문에, 소형이면서 미량의 시료라도 시료 중의 총헤모글로빈(total Hb)과 HbA1c를 높은 정밀도로 동시에 신속하게 측정할 수 있다.According to the present invention, since 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.
또한, 본 발명의 바이오센서용 스트립은 일회용으로 제작하기에 적합하며, 기존의 전기화학적 방법에 사용되는 3전극을 2전극으로 그 수를 줄임으로써, 일정 면적 이상을 유지 해야 하는 대극이 필요 없게 됨으로 제조공정을 단순화하여 제조비용을 낮출 수 있다.In addition, the 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.
도 1은 당의 존재 하에서 본 발명에 따른 DTBA-PBA 단분자막 수식 변이 전극의 페리시안 이온에 대한 응답을 도시한 모식도이다.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.
도 2은 본 발명의 제1실시형태에 따른 바이오센서용스트립의 구성을 나타낸 모식도이다.2 is a schematic diagram showing the configuration of a strip for a biosensor according to a first embodiment of the present invention.
도 3은 본 발명의 제2실시형태에 따른 바이오센서용스트립의 구성을 나타낸 모식도이다.3 is a schematic diagram showing the configuration of a biosensor strip according to a second embodiment of the present invention.
도 4는 본 발명의 바이오센서를 사용하여 Potentiometry에 의해 측정한 전위차값과 당뇨병 환자의 전혈중의 HbA1c의 농도의 관계를 도시한 그래프이다.4 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 HbA1c in whole blood of diabetic patients.
도 5는 본 발명의 바이오센서를 사용하여 Potentiometry에 의해 측정한 전위차값과 당뇨병 환자의 전혈중의 총 헤모글로빈의 농도의 관계를 도시한 그래프이다.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.
도 6은 미리 작성한 총헤모글로빈의 검량선으로부터 얻어진 결과와 HbA1c를 측정해서 %로 환산한 결과를 도시한 그래프이다. 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%.
도 7은 본 발명에 따른 바이오센서와 액체고속크로마토그래피법을 이용한 당화헤모글로빈 양 측정의 정확도를 비교한 그래프이다.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.
본 발명에 따른 당화헤모글로빈 측정용 바이오센서의 측정부는 기준전극; 집전체상에 당화헤모글로빈에 특이적 결합가능한 부위를 포함하고 외부로부터 전압 및 전류의 공급없이 당화헤모글로빈 결합에 의해 산화환원 반응전위가 변하는 분자들이 정렬된 변이전극; 및 선택적으로 헤모글로빈총량을 측정하기 위한 작용전극; 및/또는 보조전극을 구비한다.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; And optionally a working electrode for measuring the total amount of hemoglobin; And / or an auxiliary electrode.
본 발명자들은 당과 phenylboronic acid와의 관계를 연구하던 중, 합성해서 얻어진 디티오비스-3-부티라미도페닐보론산[Dithiobis-3-butyramidophenylboronic acid; DTB-BAPBA]을 이용해서 전극 표면에 막을 형성시킨 후 페리시안산 이온의 거동(도 1)을 조사한 결과, 디티오비스-3-부티라미도페닐보론산과의 반응으로 도입된 보론산에 당이 결합된 경우와 당이 결합되지 않은 경우와 비교하면 당이 결합된 경우가 페리시안산 이온이 전극에 도달하기가 어렵다는 것을 확인하였다. 또한 전극 상에 정렬시킨 분자 말단의 보론산기에 당이 결합을 하면 pKa가 감소하는 것을 발견하였다.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.
표 1
프락토스(fructose) / mM 단분자막1) 용액2)
0 8.0 ± 0.2 8.6 ± 0.1
50 7.1 ± 0.1 6.0 ± 0.1
100 6.4 ± 0.1 5.7 ± 0.1
Table 1
Fructose / mM Single molecule 1) Solution 2)
0 8.0 ± 0.2 8.6 ± 0.1
50 7.1 ± 0.1 6.0 ± 0.1
100 6.4 ± 0.1 5.7 ± 0.1
1) Cyclic voltammetry에 의해 결정1) Determined by Cyclic voltammetry
2) 메탄올:물 (1:9) 용액 중에서 자외선 가시흡수 스펙트럼으로 결정2) Determined by UV visible absorption spectrum in methanol: water (1: 9) solution
Figure PCTKR2012010145-appb-I000001
Figure PCTKR2012010145-appb-I000001
pKa가 작아진다는 것은 산성화된다는 것을 의미하고 결국 네른스트식에 의해 전압이 변화한다는 것을 의미한다.The smaller pKa means that it is acidified, which in turn means that the voltage changes by the Nernst equation.
Figure PCTKR2012010145-appb-I000002
Figure PCTKR2012010145-appb-I000002
E 0:표준전극전위 E 0 : Standard electrode potential
R:기체상수 R : Gas constant
T:온도 (K) T : Temperature (K)
z:용액이온의 원자가 z : Valence of solution ion
a:환원 및 산화체의 활성도 a : reduction and activity of oxidant
F: 패러데이 상수=96,485 C mol-1 F : Faraday Constant = 96,485 C mol -1
또한 표준상태 (1기압, 25℃)에서는 다음과 같이 계산이 가능하다.In the standard condition (1 atm, 25 ℃), it can be calculated as follows.
따라서, 본 발명은 집전체 상에 당화헤모글로빈에 특이적 결합가능한 부위를 포함하고 외부로부터 전압 및 전류의 공급없이 당화헤모글로빈 결합에 의해 산화환원 반응전위가 변하는 분자들을 정렬시킨 변이전극; 및 기준전극을 준비해서 당화헤모글로빈의 결합 전후의 전압을 측정함으로써, 시료 내 HbA1c의 농도를 측정하는 것이 특징이다.Accordingly, 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.
본 발명에서 용어 "시료"란 헤모글로빈 및/또는 당화헤모글로빈을 포함하고 있는 분석대상을 의미하고, 포유류, 바람직하게는 인간으로부터 분리된 전혈, 혈구, 혈청, 혈장, 골수액, 땀, 오줌, 눈물, 침, 피부, 점막, 모발 등의 모든 생체시료를 포함하며, 예컨대 혈액일 수 있다. 본 발명의 바이오센서는 혈액을 시료로 하여 당화헤모글로빈 농도를 측정함으로써, 혈당측정용도로 적용될 수 있다.As used herein, the term "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.
변이전극에 사용되는 집전체 재질의 비제한적인 예로는 금, 은, 동과 같은 금속이 있으며, 금, 백금 등의 금속성 기판 뿐만 아니라 GaAs, CdS, In 2 O3과 같은 반도체 등의 도전성 기판이 사용될 수 있다.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.
상기 당화헤모글로빈 결합에 의해 산화환원 반응전위가 변하는 분자가 집전체에 용이하게 정렬되기 위해서는 상기 분자는 집전체에 결합할 수 있는 작용기를 포함하는 것이 바람직하다. 금속으로 된 집전체에 분자가 용이하게 정렬되기 위해서는 금속과 결합할 수 있는 티올기, 설파이드(sulfide)기 또는 디설파이드(disulfide) 결합을 포함하는 것이 바람직하다.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.
집전체 표면상에 정렬시 분자 중 집전체에 결합할 수 있는 부위는 집전체 계면에 당화헤모글로빈에 특이적 결합가능한 부위가 집전체로부터 바깥쪽을 향하도록 배향되는 것이 바람직하다.When aligned on the surface of the current collector, 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.
본 발명에서 사용되는 집전체상 정렬된 분자는 당화헤모글로빈의 글리코실기와 결합할 수 있고, 외부로부터 전압, 전류의 공급없이 당화헤모글로빈도입에 의해 산화환원반응전위를 발생하게 하는 물질이다.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.
당화헤모글로빈에 특이적 결합가능한 부위의 예로는 보론산(boronic acid) 작용기(-B(OH)2)가 있다.An example of a site capable of specific binding to glycated hemoglobin is a boronic acid functional group (-B (OH) 2 ).
시료 내에 당화헤모글로빈이 존재하면 당화헤모글로빈과 보론산 작용기와의 결합을 통해 변이전극 표면에 정렬된 분자-당화헤모글로빈 결합체가 형성된다.When glycated hemoglobin is present in the sample, the glycated hemoglobin and the boronic acid functional group combine to form a molecularly glycated hemoglobin conjugate on the surface of the transition electrode.
상기 정렬된 분자는 전기전도성을 갖는 분자인 것이 바람직하며, 예컨대, 폴리아마이드, 폴리아닐린, 폴리페놀, 폴리티오펜, 폴리아세틸렌, 폴리(p-페닐비닐렌), 폴리(p-페닐렌술피드) 등으로 이루어진 군에서 선택된 1종 이상의 것일 수 있으나, 이에 제한되는 것은 아니다. 이들 분자는 우수한 전기전도도를 갖고 다양한 응용이 가능하며, 열적 안정성과 화학적 안정성이 우수하므로, 적용시 바이오센서의 성능 및 수명을 향상시킬 수 있는 장점이 있다.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.
한편, 보론산 작용기를 갖는 전도성 물질의 예로는 디티오비스-3-부티라미도페닐보론산(dithiobis-3-butyramidophenylboronic acid), 3-(4-머캅토부탄아미도)페닐보론산(3-(4-mercaptobutanamido)phenylboronic acid), 디티오비스-4-아미노페닐보론산(dithiobis-4-aminophenyl boronic acid), 4-아미노페닐보론산(4-aminophenylboronic acid), 3-티오펜보론산(3-thiopheneboronic acid), 3-포르밀-4-티오펜보론산(3-formyl-4-thiopheneboronic acid) 등이 있으나, 이에 한정되지 않는다. 바람직하게는, 디티오비스-3-부티라미도페닐보론산 또는 3-(4-머캅토부탄아미도)페닐보론산이다.On the other hand, 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. Preferably, dithiobis-3-butyramidophenylboronic acid or 3- (4-mercaptobutanamido) phenylboronic acid.
<디티오비스-3-부티라미도페닐보론산><Dithiobis-3-butyramidophenylboronic acid>
Figure PCTKR2012010145-appb-I000003
Figure PCTKR2012010145-appb-I000003
티오펜보론산은 물에 녹지 않고 에탄올 등에 녹기 때문에, 직접 혈액 샘플과 반응시키기 어려운 물질이고, 이를 사용하기 위해서는 카본페이스트 등에 섞어서 사용한다.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.
변이전극의 집전체 상에 정렬된 분자가 산(acid)일 경우, 분자의 화학적 변화가 야기되어 변이전극 표면에 정렬되어 있는 분자의 pKa값이 달라져 전위변화가 야기된다.When a molecule aligned on the current collector of the transition electrode is an acid, chemical change of the molecule is caused, thereby changing the pKa value of the molecule aligned on the surface of the transition electrode, thereby causing a potential change.
변이전극의 집전체 상에 정렬된 분자의 pKa를 변이전극으로 측정하기 위해서는, 집전체 위에 페닐보론산과 같은 당화헤모글로빈에 특이적으로 결합하는 분자가 많이 분포되어 있는 것이 적게 분포되어 있는 것보다 당화헤모글로빈의 농도차이에 대한 전압변화폭(예, pKa 민감도)을 높일 수 있으며, 분자가 집전체 상에 정렬되는 것이 무작위로 배향된 것에 의한 pKa에 영향을 주는 인자를 감소시켜 정확한 pKa 변화를 측정할 수 있다.In order to measure pKa of a molecule aligned on a current collector of a transition electrode with a transition electrode, 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. .
본 발명에 따라, 당화헤모글로빈의 당과 특이적으로 결합가능한 분자는 집전체 표면 상에서 자가조립할 수 있는 것이 바람직하며, 자가조립 단일층을 형성하는 것이 더욱 바람직하다.According to the present invention, 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.
금전극에 티올기에 의한 자가조립 단일층을 형성하였을 때 STM 관찰에 의해 티올기 간의 거리는 5 옹스트롬인 것을 확인했다. 또한, 수식 전극의 표면분자밀도는 5.1×10-10 mM/cm2가 계산되어 변이전극에 수식된 단분자막은 최대로 밀집될 수 있음을 확인하였다.When a self-assembled monolayer formed of a thiol group was formed on a gold electrode, STM observation confirmed that the distance between thiol groups was 5 angstroms. In addition, the surface molecular density of the modified electrode was calculated to 5.1 × 10 -10 mM / cm 2 it was confirmed that the modified monomolecular film can be densely packed.
이에 반해, 보론산 함유 분자를 전도성 페이스트와 혼합하여 스크린 코팅하는 경우는 페이스트의 특성상 도입되는 보론산량이 정해져 있어 고농도의 당화헤모글로빈 측정에 높은 재현성을 얻을 수 없다.On the other hand, in the case of screen coating a mixture of boronic acid-containing molecules with a conductive paste, 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.
한편, 상기 분자를 집전체 상에 정렬시켜 변이전극을 형성하는 방법의 일 구체예로, 금전극을 0.5 M 황산 중에 0.2 내지 1.5 V, scan rate 100 mV/s로 20분간 연속 scan해서 청결화한 다음, tetrahyrofuran:methanol=9:1용액으로 조제한 0.5 mg/ml 디티오비스-3-부티라미도페닐보론산 용액에 8시간 동안 담가 두어 단일막 수식전극을 제조할 수 있다.On the other hand, in one embodiment of the method of forming a transition electrode by aligning the molecules on the current collector, the gold electrode was cleaned by continuously scanning for 20 minutes at 0.2 to 1.5 V, scan rate 100 mV / s in 0.5 M sulfuric acid Next, a single membrane modified electrode may be prepared by soaking in a 0.5 mg / ml dithiobis-3-butyramidophenylboronic acid solution prepared with tetrahyrofuran: methanol = 9: 1 solution for 8 hours.
집전체 상에 분자를 정렬시키기 위해 사용되는 용매의 비제한적인 예로는 테트라하이드로퓨란, 메탄올, 이소프로필알코올, 에탄올, 프로판올, 아세톤, 또는 이의 혼합물 등이 있다.Non-limiting examples of solvents used to align molecules on the current collector include tetrahydrofuran, methanol, isopropyl alcohol, ethanol, propanol, acetone, or mixtures thereof.
한편, 변이전극의 정렬된 분자와 산화환원반응하는 산화-환원물질은 페리시안산(ferricyanic acid), 페로센(ferrocene), 페로센유도체, 퀴논(quinones), 퀴논유도체, 유기전도성염(organic conducting salt), 비오로겐(viologen), 헥사아민루세늄(III) 클로라이드(hexaammineruthenium(III) chloride), 디메틸페로센(dimethylferrocene; DMF), 페리시니움(ferricinium), 페로센모노카르복실산(ferocene monocarboxylic acid; FCOOH), 7,7,8,8,-테트라시아노퀴노디메탄(7,7,8,8-tetracyanoquino-dimethane; TCNQ), 테트라티아풀발렌(tetrathiafulvalene; TTF), 니켈로센(nickelocene; Nc), N-메틸아시디니움(N-methyl acidinium; NMA+), 테트라티아테트라센(tetrathiatetracene; TTT), N-메틸페나지니움(N-methylphenazinium; NMP+), 히드로퀴논(hydroquinone), 3-디메틸아미노벤조산(3-dimethylaminobenzoic acid; MBTHDMAB), 3-메틸-2-벤조티오조리논히드라존(3-methyl-2-benzothiozolinone hydrazone), 2-메톡시-4-아릴페놀(2-methoxy-4-allylphenol), 4-아미노안티피린(4-aminoantipyrin; AAP), 디메틸아닐린(dimethylaniline), 4-아미노안티피렌(4-aminoantipyrene), 4-메톡시나프톨(4-methoxynaphthol), 3,3',5,5'-테트라메틸벤지딘(3,3',5,5'-tetramethyl benzidine; TMB), 2,2-아지노-디-[3-에틸-벤즈티아졸린술포네이트](2,2-azino-di-[3-ethyl-benzthiazoline sulfonate]), o-디아니지딘(o-dianisidine), o-톨루이딘(o-toluidine), 2,4-디클로로페놀(2,4-dichlorophenol), 4-아미노페나논(4-aminophenazone), 벤지딘(benzidine) 등이 있다.On the other hand, 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 hydrazone (3-methyl-2-benzothiozolinone hydraz one), 2-methoxy-4-allylphenol, 4-aminoantipyrin (AAP), dimethylaniline, 4-aminoantipyrene , 4-methoxynaphthol, 3,3 ', 5,5'-tetramethylbenzidine (3,3', 5,5'-tetramethyl benzidine; TMB), 2,2-azino-di- [3-ethyl-benzthiazolinesulfonate] (2,2-azino-di- [3-ethyl-benzthiazoline sulfonate]), o-dianisidine, o-toluidine, 2,4-dichlorophenol, 4-aminophenazone, 4-benzidine, and the like.
이와 같이 본 발명에 따라 준비된 변이전극과 시료의 계면(界面)에서, 변이전극의 집전체 상에 정렬된 분자들과 시료 중 산화환원가능물질 사이에 전자의 수수(授受)가 이루어지면, 즉 산화환원반응이 이루어지면 변이전극과 기준전극 사이에는 전위(電位)가 발생한다.As such, at the interface between 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.
이러한 계면반응(界面反應)이 화학평형(化學平衡) 상태가 되었을 때, 변이전극의 전극전위(電極電位)는 안정되며, 본 발명에서는 변이전극의 이러한 평형전극(平衡電極)을 측정하는 것이다.When such an interfacial reaction is in a chemical equilibrium state, the electrode potential of the transition electrode is stabilized, and in the present invention, such an equilibrium electrode of the transition electrode is measured.
한편, 변이전극상 정렬된 분자들은 당화헤모글로빈에 특이적 결합가능한 부위를 갖고 있어서, 당화헤모글로빈이 상기 정렬된 분자에 결합하면, 상기 정렬된 분자의 화학적 변화를 야기하여 변이전극표면의 평형전극전위가 변한다.On the other hand, 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. .
상기 정렬된 분자들이 시료 중에 있는 당화헤모글로빈의 당과 얼마나 결합되었는지에 따라 변이전극의 평형전극전위가 달라지므로, 시료 중의 변이전극의 평형전극전위를 측정하여 변이전극 상의 정렬된 분자들이 당과 결합된 정도를 산출하고 이로부터 시료상의 당화헤모글로빈의 농도를 분석할 수 있다.Since 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.
이에 반해, 당 결합 여부에 따른 전극의 임피던스를 측정하는 경우는 변이전극과 기준전극 간에 전압 혹은 전류를 인가하여 그때의 변화량을 측정하는 것이다. 본 발명은 외부에서 전압 혹은 전류의 공급없이 변이전극 내 물질의 산화환원전위와 기준전극과의 전위의 차이에 의해 전압이 발생되고, 변이전극 내 물질이 당과 결합함에 따라 산화환원전위(예, pKa)가 변함에 따라 변이전극과 기준전극 사이의 전압이 변하게 되고 이를 측정하는 것으로, 특별한 외부 전원 공급이 필요없다는 잇점이 있다.On the other hand, in the case of measuring the impedance of the electrode according to whether or not sugar coupling, the change amount at that time is measured by applying a voltage or a current between the transition electrode and the reference electrode. In the present invention, 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.
이와 같은 변이전극의 전위차 변화는, 전위차분석법(Potentiometry)에 의해 변이전극과 기준전극 사이의 전위차를 측정하여 얻을 수 있으며, 이를 위해 전위차 측정회로 또는 전위차 적정장치가 사용될 수 있다.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.
전위의 기준으로 표준 수소전극의 전위를 편의상 0 V라 하고, 일반적으로 칼로멜(calomel) 전극, 은-염화은 전극 등의 단극전위가 기준전극으로 사용된다. 기준전극은 일정전위를 유지할 수 있는 은/염화은 또는 유사 기준전극으로 사용할 수 있는 전극들이 바람직하다. 본 발명의 실시양태에서, 기준전극은 시료와 접촉하는 부분에 은/염화은을 코팅하여 제조할 수 있다.As a reference for the potential, the potential of the standard hydrogen electrode is referred to as 0 V for convenience. In general, 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. In an embodiment of the present invention, the reference electrode may be prepared by coating silver / silver chloride on a portion in contact with a sample.
한편, 당화헤모글로빈은 혈액 중 총헤모글로빈의 양에 대한 상대적인 당화헤모글로빈의 양으로 나타낸다. 따라서 당화헤모글로빈의 수치를 구하려면 총헤모글로빈의 양을 함께 측정하는 것이 바람직하다.Meanwhile, 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.
따라서, 본 발명에 따른 측정부는 헤모글로빈의 양 또한 상기 시료 내에 포함된 헤모글로빈의 산화-환원반응을 통해 전위차법으로 측정할 수 있는 것이 바람직하다. 따라서, 당화헤모글로빈을 측정하거나, 총헤모글로빈을 측정하기 위해서는 두가지 전극, 즉, 변이전극과 기준전극만 있으면 된다. Therefore, 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.
특히, 헤모글로빈의 산화-환원반응을 통한 전위차를 측정하기 위한 변이전극을 이하 작용전극이라 지칭한다.In particular, the transition electrode for measuring the potential difference through the redox reaction of hemoglobin is referred to as working electrode hereinafter.
당화헤모글로빈의 측정은 디티오비스-3-부티라미도페닐보론산과 같이 당화헤모글로빈에 특이적 결합가능한 분자를 수식한 변이전극에 당화헤모글로빈이 결합함으로써 기준전극과 전압차가 발생하기 때문에 측정이 가능한 것이다.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.
총헤모글로빈의 경우는 아무것도 수식하지 않은 변이 전극(즉, 작용전극)과 기준전극을 사용하고, 햄(HEME)기에 포함된 Fe2+와 완충용액에 혼합된 Fe3+와의 가역적 산화, 환원 반응에 의해 두 전극 사이에 전위차가 발생하기 때문에 총헤모글로빈을 측정할 수 있다.In the case of total hemoglobin, a transition electrode (that is, a working electrode) and a reference electrode that do not modify anything are used, and 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.
작용전극의 재료는 예를 들면, 동, 백금, 은, 금, 팔라듐, 루테늄, 로듐, 및 이리듐 등의 금속, 카본, 또는 각각의 재료에 표면처리를 한 재료를 들 수있다. 가장 좋은 것은 금전극이다. 또한 시료 중의 단백질 등의 흡착을 방지할 목적으로 표면 친수성처리를 해도 좋다. 기준전극으로는 Ag/AgCl전극을 사용할 수 있다.Examples of the material of the working electrode 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.
종래에는 헤모글로빈과 당화헤모글로빈을 분리하는 단계를 거쳐 측정하는 방법이 사용되었다. 이에 반해 본 발명에서 제시하는 당화헤모글로빈 측정용 바이오센서는 Fe2+를 헤모글로빈과 당화헤모글로빈의 반응지표물질로 사용하여 분리과정 없이 동시에 측정할 수 있다.In the related art, a method of measuring the separation of hemoglobin and glycated hemoglobin has been used. On the contrary, 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.
따라서, 본 발명의 경우 기존의 당화단백질의 측정방법과는 달리 헤모글로빈과 당화단백질의 분리과정 없이 전압 측정만으로도, 헤모글로빈 및 당화헤모글로빈 양을 동시 측정할 수 있다.Therefore, in the case of the present invention, unlike the conventional method for measuring glycated proteins, hemoglobin and glycated hemoglobin can be simultaneously measured by voltage measurement without separating the hemoglobin and glycated proteins.
또한, 종래에는 헤모글로빈의 환원성을 이용하여 산화환원방식에 의해 발생한 전류를 전극으로 검지해서 전류치로부터 헤모글로빈 농도를 정량하는 방식을 제시하였다. 전류계측에 의한 측정방식은 크로마토그래피나 흡광광도계를 사용하는 방법에 비교해서 소형화된 장치로 간편하게 헤모글로빈의 측정이 가능하나, 측정결과 나타내는 전류치는 전극의 면적이나 시료량에 비례하기 때문에 고정밀도로 측정을 하기 위해서는 전극의 면적을 넓게 하거나, 시료량을 많이 사용해야 문제점을 가지고 있기 때문에 point-of-care 의미의 소형화 측정장치나 시료량의 미량화에는 어려운 부분이 있다. 그러나, 본 발명은 두 전극 사이에 전위차를 통해 총헤모글로빈을 측정하기 때문에 상기 문제점을 해결할 수 있다.In addition, conventionally, a method of quantifying hemoglobin concentration from an electric current value by detecting an electric current generated by a redox method using an electrode using the reducibility of hemoglobin has been proposed. 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. However, the present invention solves the above problem because the total hemoglobin is measured through the potential difference between the two electrodes.
헤모글로빈 총량을 정확히 측정하기 위해 헤모글로빈으로부터 햄을 분리하는 것이 바람직하고, 이를 위해 시료에 계면활성제(surfactant) 용액을 처리할 수 있다.It is desirable to separate the ham from hemoglobin in order to accurately measure the total amount of hemoglobin, for which the sample can be treated with a surfactant solution.
총헤모글로빈을 측정하기 위해서는 혈액에 계면활성제와 페리시안화칼륨을 섞어 처리한 시료를 상기 두 전극에 첨가하는 것이 바람직하다. 본 발명의 일구체예에서는, 혈액에 계면활성제와 페리시안화칼륨을 따로 처리하는 것을 피하기 위해 두 전극 사이에 부직포를 설치하고 미리 계면활성제와 페리시안화칼륨을 동결건조시켜 설치해 놓으면 단지 전혈을 전극 위에 첨가하게 되면 동결건조된 시약이 전혈을 용혈시켜서 산화환원반응을 발생케 해서 두 전극의 전위차를 측정할 수 있다.In order to measure total hemoglobin, it is preferable to add a sample treated with a mixture of surfactant and potassium ferricyanide to the blood to the two electrodes. In one embodiment of the present invention, in order to avoid treating the surfactant and potassium ferricyanide separately in the blood, 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. In this case, the lyophilized reagent can hemolyze whole blood to generate a redox reaction, thereby measuring the potential difference between the two electrodes.
온도나 참조전극의 노후 등, 조건의 변화에 따라 측정전위가 시료에 의해 발생하는 전압으로부터 약간 변화한다. 따라서, 미리 산화환원전위를 알고 있는 기준용액을 측정함으로 조건변화에 따른 측정오차를 보정할 수 있다.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.
본 발명의 측정부는 측정시료 이외의 요인에 의한 전위변화에 따른 오차보정을 위하여 보조전극을 더 구비할 수 있으며, 이로부터 당화헤모글로빈 또는 총헤모글로빈 측정에 방해되는 요인들의 영향을 제거할 수 있다.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.
보조전극은 총헤모글로빈측정용 변위전극 또는 작용전극과 같은 재질을 사용할 수 있다.The auxiliary electrode may be made of a material such as a displacement electrode or a working electrode for measuring total hemoglobin.
또한, 보조전극은 작용전극과 같은 공정, 조성으로 제작되어 작용전극과 같은 전기적 응답을 나타낸다. 기준용액으로는 일정의 조건하에서 산화환원전위가 알려진 것으로 안정한 전위가 얻어지는 시료라면 문제가 없다. 예를들면 금속염, 금속착체, 키논계화합물, 벤죠페논과 이러한 물질과의 혼합용액이 사용가능하다.In addition, 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. As the reference solution, the redox potential is known under a certain condition, and there is no problem as long as the sample has a stable potential. For example, metal salts, metal complexes, quinonic compounds, benjophenones and mixed solutions of these substances can be used.
한편, 본 발명에 따라 기준전극; 변이전극; 선택적으로 작용전극; 및 선택적으로 보조전극을 구비한 측정부는 교체가능한 스트립형태일 수 있다(도 2 및 3 참조).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).
본 발명의 측정부는 변이전극, 기준전극, 선택적으로 작용전극 및 선택적으로 보조전극과 각 전극을 측정장비와 연결하는 전기 연결선으로 구성되어있다. 상기 전극을 제외한 나머지 부분은 절연물질을 이용해 절연층을 형성한다.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.
도 2 및 3에 도시된 바와 같이 본 발명에 따른 바이오센서용 스트립은 비전도절연물질로 이루어진 지지체상에 각각의 전극들이 구성되어지는 것이 바람직하다. 이러한 지지체는 20 내지 60 마이크론의 두께를 갖도록 제조되는 것이 바람직하고, 더욱 바람직하게는 30 마이크론의 두께를 갖는 것이다.As shown in FIGS. 2 and 3, 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. Such a support is preferably made to have a thickness of 20 to 60 microns, more preferably one having a thickness of 30 microns.
상기 비전도 절연물질로 이루어진 지지체의 재료로는 어떠한 절연체도 사용할 수 있지만, 동시에 대량으로 제조하기 위해서는 어느 정도의 유연성과 지지체로서의 강성을 지닌 것이 적합하다. 일반적으로 지지체의 표면은 매우 고르게 형성되어 있어야 한다. 왜냐하면, 고르지않은 표면은 대량생산시 각 센서 스트립간의 전극표면적의 불균일성의 원인이 되고 결과적으로 센서 출력신호의 불균일성을 초래하기 때문이다.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. In general, 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.
가장 고른 표면을 갖는 물질은 반도체제조에 사용되는 실리콘웨이퍼를 들 수 있다. 다음으로는 투명하고 가공성이 용이한 석영 유리기판이나 일반 유리기판을 사용할 수 있다. 한편, 일반적인 음악용 컴팩트 디스크는 그용도의 특성상 표면이 매우 고르게 형성되고 평탄도도 우수하며, 원형으로 반도체 웨이퍼와 비슷한 형상을 하고 있어, 별도의 장비를 제작하지 않고 반도체 제조공정장비를 그대로 사용할 수 있으면서도, 가격이 저렴하고 손쉽게 구할 수 있는 장점이 있다. 이외에도, 일반적인 플라스틱필름을 사용할 수 있다.Materials having the most even surface include silicon wafers used in semiconductor manufacturing. Next, a quartz glass substrate or a general glass substrate that is transparent and easy to work may be used. On the other hand, 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. In addition, a general plastic film can be used.
컴팩트디스크 또는 플라스틱 필름 재료의 예로서, 폴리에스테르(poly ester), 폴리카보네이트(poly carbonate), 폴리스틸렌(poly stylene), 폴리이미드(poly imide), 폴리비닐클로라이드(poly vinyl chloride), 폴리에틸렌(poly ethylene), 폴리에틸렌테레프탈레이트(poly ethylene telephthalate) 등이 사용될 수 있다.Examples of compact disc or plastic film materials include polyester, polycarbonate, poly stylene, polyimide, poly vinyl chloride, polyethylene ), Polyethylene terephthalate (polyethylene telephthalate) and the like can be used.
작용전극은 14 ㎜ 내지 19 ㎜의 길이, 0.5 ㎜ 내지 2 ㎜의 너비 및 20 내지 150 마이크론의 두께로 이루어지는 것이 바람직하고, 더욱 바람직하게는 14 ㎜ 길이, 1 ㎜ 너비 및 60 마이크론의 두께로 이루어진 것이다.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. .
변이전극, 기준전극 및 보조전극은 각각 독립적으로 15 ㎜ 내지 20 ㎜의 길이, 0.5 ㎜ 내지 2 ㎜의 너비 및 20 내지 150 마이크론의 두께로 이루어지는 것이 바람직하고, 더욱 바람직하게는 15 ㎜ 길이, 1 ㎜ 너비 및 60 마이크론의 두께로 이루어진 것이다.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.
본 발명의 전극들은 시료에 직접 접촉하는 부분과 검출기에 신호를 전달하는 부분 사이를 절연피복을 통해 구획하는 것이 바람직하나 이에 한정되지 않는다.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.
한편, 바이오센서는 상기 변이전극과 기준전극 사이의 전위차 값 또는 이를 시료 내 당화헤모글로빈의 양 또는 농도로 환산시켜 표시하는 표시부를 더 구비할 수 있다.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.
나아가, 본 발명은 상기 본 발명에 따른 바이오센서를 구비한 당화헤모글로빈 측정 키트를 제공한다.Furthermore, the present invention provides a glycated hemoglobin measuring kit having the biosensor according to the present invention.
당화헤모글로빈 측정키트는 용해용액(lysis solution), 계면활성제용액, 또는 둘 다를 더 포함할 수 있다.The glycated hemoglobin measurement kit may further comprise a lysis solution, a surfactant solution, or both.
두 전극 사이의 전위차를 측정하기 위한 전압계는 높은 임피던스를 가지는 것이 좋다.The voltmeter for measuring the potential difference between the two electrodes preferably has a high impedance.
검출되는 전압은 헤모글로빈과의 반응에 의해 환원되는 메디에타의 양에 의존하기 때문에 미리 검량선을 작성하여 검량선을 이용하여 헤모글로빈농도를 정량할 수도 있다.Since the detected voltage depends on the amount of mediaetta reduced by the reaction with hemoglobin, a calibration curve may be prepared in advance and the hemoglobin concentration may be quantified using the calibration curve.
예로서, 헤모글로빈 농도를 측정하는 경우 적혈구를 포함한 시료가 반응용기내에 도입되면 용혈제에 의해 적혈구 중의 헤모글로빈이 방출되어 메디에타와 산화환원반응을 일으켜 메디에타가 환원된다. 결과적으로 생긴 산화형/환원형의 농도 비에 대해 작용 전극에 전압이 발생, 그 전압으로부터 미리 작성한 검량선을 이용하여 전혈 중의 헤모글로빈의 농도를 산출한다.For example, 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.
측정시료는 항응고제나 용혈시약에 의해 처리된 전혈일 수 있다. 반응용기에 도입되는 시료의 부피는 1 마이크로리터 이상이 적당하다. 바람직한 부피는 5 마이크로리터 이상이다.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.
반응용기는 측정시료 등이 전극의 변이전극/작용전극과 이의 각 기준전극이 접촉되도록 구성되어 있어, 액체시료가 도입되면 2개의 전극이 통전되어 회로가 형성된다. 측정시료가 액체가 아닐 경우에는 물 등의 용매에 용해한 후에 반응용기에 도입할 수 있다.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.
반응용기의 형태는 시료 및 측정시약을 유지하고, 시료 도입 후에 2개의 전극 사이를 통전시키는 것이 가능한 구조로서 변이전극/작용전극과 이의 각 기준전극을 수용할 수 있는 크기의 용기이면 된다. 반응용기의 재료는 여과지 등의 섬유집합체, 부직포, 다공질재료, 젤 등, 전기적으로 불활성 및/또는 시료나 전극에 대해서도 불활성인 한 제한없이 사용될 수 있다. 그 중에서도 폴리염화비닐, 폴리이미도, 젤라틴, 유리섬유 등을 들 수 있다.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. Among them, polyvinyl chloride, polyimido, gelatin, glass fibers and the like can be mentioned.
반응용기에는 측정시약으로서 용혈제와 메디에타(redox mediator), pH 완충시약이 포함될 수 있다. 또한, 반응용기 안에 전기화학적 측정을 방해하는 방해성분을 제거하기 위해 방해제거시약을 포함시킬 수도 있다.The reaction vessel may include a hemolytic agent, a redox mediator, and a pH buffer reagent as measurement reagents. In addition, an interference elimination reagent may be included in the reaction vessel to remove the interference component that interferes with the electrochemical measurement.
용혈제로서는, 이온성 또는 비이온성의 계면활성제, 유기용매, 염, 효소 등을 사용할 수 있다. 계면활성제로는 폴리옥시에틸렌옥틸페닐에테르 (polyoxyethyleneoctylphenyl ether), 라우릴유산나트륨, 사포닌 등을 사용할 수 있다. 유기용매로는 포름알데이드, 핵산, 아세톤 등이 사용가능하다. 염으로는 염화암모늄, 염화알루미늄 등이 사용가능하다. 바람직한 예로는 폴리옥시에틸렌옥틸페닐에테르(polyoxyethyleneoctylphenyl ether)가 있다. 이 경우 농도는 1 내지 20%(v/v)가 사용가능하다. 또한, 증류수에 의해 희석되어 염농도의 변화에 의한 용혈을 유도할 수도 있다.As the hemolytic agent, ionic or nonionic surfactants, organic solvents, salts, enzymes and the like can be used. As the 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. As the 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.
메디에타(redox mediator)로서는 헤모글로빈과 산화환원반응을 일으키는 것이라면 사용가능하다. 비제한적인 예로는 금속염, 금속착제, 키논계 화합물이나 벤조페논 등이 있다. 가장 안정된 메디에타로는 페리시안화물이 있으며, 사용 농도는 예상되는 헤모글로빈농도의 2배 이상의 농도로 사용하는 것이 안정된 결과를 얻을 수 있고 10 내지 500 mM 범위이다.As a mediae (redox mediator) 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.
pH 완충액으로서는 시료 첨가 후에 pH 4 내지 8을 유지하면서 반응용기나 전극이나 시료에 반응하지 않는 것이라면 제한없이 사용가능하다. 최종적인사용농도는 5 내지 500 mM이고, 보다 좋은 조건은 50 내지 200 mM이다. 예를 들면 pH 6.5 내지 7.0의 인산완충액을 사용할 수 있다.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. For example, a phosphate buffer of pH 6.5 to 7.0 can be used.
이하, 첨부된 도 2 및 3을 참조하여 본 발명의 바이오센서를 상세히 설명한다.Hereinafter, the biosensor of the present invention will be described in detail with reference to FIGS. 2 and 3.
본 발명의 제1실시형태에 대해서 헤모글로빈 및 HbA1c을 측정하기 위한 장치의 개략도인 도 2를 참조하여 설명한다.EMBODIMENT OF THE INVENTION The 1st Embodiment of this invention is described with reference to FIG. 2 which is schematic of the apparatus for measuring hemoglobin and HbA1c.
전극은 HbA1c 측정용 변이전극과 제1기준전극; 그리고 총헤모글로빈 측정용 작용전극과 제2기준전극으로 구성되어 있다. 기준전극은 Ag/AgCl 페이스트가 도포되어 고정화되어 있다.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.
변이전극과 제1기준전극은 근접하여 배치되어 있고, 변이전극과 제1기준전극 모두와 접촉할 수 있는 형상으로 반응용기가 배치되어 있다. 반응용기에 시료를 도입하면 변이전극과 제1기준전극은 시료를 통해 통전할 수 있다.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. When the sample is introduced into the reaction vessel, the transition electrode and the first reference electrode can be energized through the sample.
작용전극과 제2기준전극도 근접하여 배치되어 있고, 작용전극과 제2기준전극 모두와 접촉할 수 있는 형상으로 반응용기가 배치되어 있다. 반응용기에 시료를 도입하면 작용전극과 제2기준전극은 시료를 통해 통전할 수 있다.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. When the sample is introduced into the reaction vessel, the working electrode and the second reference electrode can be energized through the sample.
반응용기는 시료를 그 내부에 흡수해서 보지(保持)할 수 있고 한번 사용후 버릴 수 있도록 설치되어 있다. 반응용기 안에는 부직포가 들어있는 것이 바람직하다.The reaction vessel is installed so that the sample can be absorbed and retained therein and discarded after one use. Preferably, the reaction vessel contains a nonwoven fabric.
HbA1c 측정을 위한 반응용기에는 인산완충액, Triton X-10, 디티오비스-3-부티라미도페닐보론산 또는 페닐보론산 류가 건조상태로 보지되어 있고 총헤모글로빈 측정을 위한 반응용기에는 측정시약으로 인산완충액, Triton X-100, potassium ferricyanide가 건조상태로 보지되어 있어서 10마이크로 리터 정도의 전혈시료의 도입으로 시료 중의 수분이 용해되고 외부로부터 특별한 전압, 전류의 공급이 없어도 산화환원전위가 발생한다.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.
변이전극과 제1기준전극, 그리고 작용전극과 제2기준전극은 각각 전압계에 접속되어 이들 두 전극 쌍 사이에 발생하는 전압을 측정할 수 있다. 따라서, 전압계에서 계측된 전압을 바탕으로 시료 중의 총헤모글로빈 농도와 HbA1c 농도의 산출이 가능하다. 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.
전술한 바와 같이, 2개의 전극에 시료를 접촉시켜 산화환원반응에 의해 두 전극 간에 발생한 산화환원전위를 계측하여 측정한 전위를 바탕으로 시료 중의 헤모글로빈과 HbA1c 농도를 산출할 수 있도록 구성하였기 때문에 2개의 전극 간에 전력을 공급하기 위한 전원이 불필요하고 미량의 시료라도 측정가능하기 때문에 소형으로 미량의 시료라도 고정밀도의 헤모글로빈과 HbA1c 농도가 측정가능하다.As described above, since the sample was contacted with two electrodes to measure the redox potential generated between the two electrodes by the redox reaction, 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.
본 발명의 제2실시형태에는 대해서 또 다른 헤모글로빈 및 HbA1c을 측정하기 위한 장치의 개략도인 도 3을 참조하여 설명한다.A second embodiment of the present invention will be described with reference to FIG. 3, which is a schematic diagram of another device for measuring hemoglobin and HbA1c.
본 발명의 제2실시형태는 측정시료 이외의 요인에 의한 전위변화에 따른 오차보정을 위하여 보조전극이 설치되어있다. 온도나 기준전극의 노후 등, 조건의 변화에 따라 측정전위가 시료에 의해 발생하는 전압으로부터 약간 변화한다. 따라서, 미리 산화환원전위를 알고 있는 기준용액을 측정함으로 조건변화에 따른 측정오차를 보정할 수 있다.In the second embodiment of the present invention, 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.
도 3에 대해 전극2는 전극1과 전극3 사이에 배치되어, 반응용기는 전극1과 전극2, 전극2와 전극3에 접촉하도록 배치한다.3, 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.
전압계는 전극1과 전극2 사이에 발생한 전압을 측정함과 동시에 전극2와 전극3 사이에 발생한 전압을 기준전압으로 측정한다.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.
반응용기a는 제1실시형태에서 반응용기와 같은 측정시료 및 측정시약이 수용되어 있고, 반응용기b는 기준용액이 수용되어 있다. 기준용액이 반응용기b에 도입되어, 전극2와 전극3의 양전극에 접촉되면 전극2와 전극3 사이의 기준이 되는 전압이 발생한다. 또한 기준용액은 반응용기b에 측정 개시할 때 도입해도 좋고, 미리 도입해 두어도 좋다. 전극1과 전극2 간에 발생하는 전압과 전극2와 전극3 사이에 발생하는 전압을 전압계로 측정해서 표시하고 전극1과 전극2 간에 발생한 전압을 전극2와 전극3 사이에 발생한 전압으로 보정한다. 측정시료 이외의 요인으로 전압이 변동하는 것도 생각되어질 수 있지만, 전극2와 전극3 간의 발생하는 전압을 이용하여 상기 요인에 의한 오차를 감소시키는 것이 가능하다.In the first embodiment, the reaction vessel a contains a measurement sample and a reagent such as the reaction vessel, and the reaction vessel b contains a reference solution. When 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. In addition, 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.
예를들어 전극1과 전극2 간에 발생한 전압으로부터 전극2와 전극3 사이에 발생한 전압을 감산해서 얻어진 전압을 이용해서 측정시료 중의 헤모글로빈농도나 HbA1c 농도를 산출할 수 있다.For example, 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.
본 발명의 제2실시형태에 의하면 제1실시형태와 같은 효과를 얻을 수 있을 뿐만아니라 보조전극을 추가함으로 측정시료 이외의 요인에 의한 전압변화를 제외하는 것이 가능하게 되어 측정정밀도를 향상시킬 수 있다.According to 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. .
이하, 본 발명을 실시예를 통하여 보다 상세하게 설명한다. 그러나 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.
<실시예 1><Example 1>
도 3에 도시된 바와 같은 바이오센서용 스트립을 제조하였다.A strip for biosensor as shown in Figure 3 was prepared.
당화헤모글로빈 선택적인 변이전극(1)을 제조하기 위해 15 ㎜ 길이, 1 ㎜ 너비 및 60 마이크론 두께의 금전극을 5분간 초음파로 2회 세정한 후, 0.5 M의 황산 용액 내에서 0.2 내지 0.5 V 사이의 전압을 조사하는 전기화학적 방법으로 표면을 처리하여 준비하였다. 준비된 금 전극을 0.5 ㎎/㎖ 농도로 디티오비스-3-부티라미도페닐보론산(Dithiobis-3-butyramidophenyl boronic acid)이 함유된 9 : 1 비율의 테트라하이드로퓨란과 메탄올 혼합 용액에 8시간 반응시켜 금 전극 표면에 5.1 × 10-10 mM/㎠ 밀도의 페닐보론산 단분자막을 분자 내 이황화결합을 이용하여 고정시켜 변이전극(1)을 제조하였다. To prepare the glycated hemoglobin selective transition electrode 1, 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.
당뇨병환자의 전혈을 샘플로 사용했다. Lysis buffer(1% KCl) 중에 전혈 100㎕(마이크로리터)를 첨가한 후 단분자막을 고정시킨 변이전극과 기준전극을 이용해 전압을 측정했다.Whole blood from diabetic patients was used as a sample. After 100 μl (microliter) of whole blood was added to Lysis buffer (1% KCl), voltage was measured using a transition electrode and a reference electrode to which monolayers were fixed.
바이오센서를 이용한 헤모글로빈 및 당화헤모글로빈 양 측정의 정확성을 확인하기 위해 4%, 7%, 10%, 14%의 표준 당화헤모글로빈(HbA1c) 용액을 이용하여 상기 제조된 스트립을 측정부로 갖는 바이오센서와 액체고속크로마토 그래피법으로 측정하여 그 결과를 도 7에 나타내었다.Biosensor and liquid having the prepared strip as a measuring unit using standard glycated hemoglobin (HbA1c) solution of 4%, 7%, 10%, 14% to confirm the accuracy of the measurement of hemoglobin and glycated hemoglobin amount using a biosensor The result of the measurement by high speed chromatography was shown in FIG. 7.
도 7에 나타난 바와 같이 기존에 당화헤모글로빈 양의 측정방법들 중에서 높은 정확도를 갖는 액체고속크로마토그래피법으로 측정한 결과와의 상관관계를 나타내는 R2값이 0.9856으로 확인되었다.As shown in FIG. 7, a value of R 2 indicating a correlation with a result measured by a liquid high-speed chromatography method with high accuracy among the conventional methods of measuring glycated hemoglobin was confirmed as 0.9856.
이러한 결과는 본 발명에 따른 바이오센서를 이용한 당화헤모글로빈 양의 측정방법이 실제 적용되기에 충분한 정확도를 가짐을 나타낸다.These results indicate that the method for measuring the amount of glycated hemoglobin using the biosensor according to the present invention has sufficient accuracy to be actually applied.
[부호의 설명][Description of the code]
1: 변이전극1: transition electrode
2, 5: 보조전극2, 5: auxiliary electrode
3, 4: 기준전극3, 4: reference electrode
6: 작용전극6: working electrode

Claims (17)

  1. 기준전극; 및 집전체 상에, 당화헤모글로빈에 특이적 결합가능한 부위를 포함하고 외부로부터 전압 및 전류의 공급없이 당화헤모글로빈 결합에 의해 산화환원 반응전위가 변하는 분자들이 정렬된 변이전극을 구비한 측정부; 및Reference 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
    전위차분석법(Potentiometry)에 의해 변이전극과 기준전극 사이의 전위차를 측정하기 위한, 전위차 측정회로 또는 전위차 적정장치Potential difference measuring circuit or potentiometric titration device for measuring the potential difference between the transition electrode and the reference electrode by potentiometry
    를 구비한 당화헤모글로빈 측정용 바이오센서.Biosensor for measuring glycated hemoglobin provided with.
  2. 제1항에 있어서,The method of claim 1,
    상기 변이전극과 기준전극 사이의 전위차 값 또는 이를 시료 내 당화헤모글로빈의 양 또는 농도로 환산시켜 표시하는 표시부를 더 구비하는 것인 바이오센서.And a display unit for converting the value of the potential difference between the transition electrode and the reference electrode or the amount or concentration of glycated hemoglobin into a sample.
  3. 제1항에 있어서,The method of claim 1,
    상기 정렬된 분자는 집전체에 결합할 수 있는 작용기를 포함하는 것이 특징인 바이오센서.The sorted molecule biosensor characterized in that it comprises a functional group capable of binding to the current collector.
  4. 제1항에 있어서,The method of claim 1,
    상기 정렬된 분자는 당화헤모글로빈에 특이적 결합가능한 부위로 보론산 작용기를 포함하는 것이 특징인 바이오센서.Wherein said aligned molecule comprises a boronic acid functional group as a site capable of specific binding to glycated hemoglobin.
  5. 제1항에 있어서,The method of claim 1,
    상기 정렬된 분자는 집전체에 결합할 수 있는 작용기로 티올(-SH)기, 설파이드(sulfide)기 또는 디설파이드(disulfide) 결합을 포함하는 것이 특징인 바이오센서.The ordered molecule is a biosensor characterized in that it comprises a thiol (-SH) group, a sulfide (sulfide) group or disulfide (disulfide) bond as a functional group capable of binding to the current collector.
  6. 제1항에 있어서,The method of claim 1,
    상기 정렬된 분자는 3-(4-머캅토부탄아미도)페닐보론산(3-(4-mercaptobutanamido)phenylboronic acid)인 것이 특징인 바이오센서.The aligned molecules are 3- (4-mercaptobutanamido) phenylboronic acid (3- (4-mercaptobutanamido) phenylboronic acid), characterized in that the biosensor.
  7. 제1항에 있어서,The method of claim 1,
    정렬된 분자와 산화환원반응하는 물질은 페리시안산(ferricyanic acid), 페로센(ferrocene), 페로센유도체, 퀴논(quinones), 퀴논유도체, 유기전도성염(organic conducting salt), 비오로겐(viologen), 헥사아민루세늄(III)클로라이드(hexaammineruthenium(III) chloride), 디메틸페로센(dimethylferrocene; DMF), 페리시니움(ferricinium), 페로센모노카르복실산(ferocene monocarboxylic acid; FCOOH), 7,7,8,8,-테트라시아노퀴노디메탄(7,7,8,8-tetracyanoquino-dimethane; TCNQ), 테트라티아풀발렌(tetrathiafulvalene; TTF), 니켈로센(nickelocene; Nc), N-메틸아시디니움(N-methyl acidinium; NMA+), 테트라티아테트라센(tetrathiatetracene; TTT), N-메틸페나지니움(N-methylphenazinium; NMP+), 히드로퀴논(hydroquinone), 3-디메틸아미노벤조산(3-dimethylaminobenzoic acid; MBTHDMAB), 3-메틸-2-벤조티오조리논히드라존(3-methyl-2-benzothiozolinone hydrazone), 2-메톡시-4-아릴페놀(2-methoxy-4-allylphenol), 4-아미노안티피린(4-aminoantipyrin; AAP), 디메틸아닐린(dimethylaniline), 4-아미노안티피렌(4-aminoantipyrene), 4-메톡시나프톨(4-methoxynaphthol), 3,3',5,5'-테트라메틸벤지딘(3,3',5,5'-tetramethyl benzidine; TMB), 2,2-아지노-디-[3-에틸-벤즈티아졸린술포네이트](2,2-azino-di-[3-ethyl-benzthiazoline sulfonate]), o-디아니지딘(o-dianisidine), o-톨루이딘(o-toluidine), 2,4-디클로로페놀(2,4-dichlorophenol), 4-아미노페나논(4-aminophenazone), 벤지딘(benzidine)으로 구성된 군에서 선택된 것이 특징인 바이오센서.Redox reactions with aligned molecules include ferricyanic acid, ferrocene, ferrocene derivatives, quinones, quinone derivatives, organic conducting salts, viologens, Hexaamineruthenium (III) chloride, dimethylferrocene (DMF), ferricinium, ferocene monocarboxylic acid (FCOOH), 7,7,8, 8, -tetracyanoquinodimethane (7,7,8,8-tetracyanoquino-dimethane (TCNQ), tetrathiafulvalene (TTF), nickelocene (Nc), N-methylasidinium (N-methyl acidinium; NMA +), tetrathiatetracene (TTT), N-methylphenazinium (NMP +), hydroquinone, 3-dimethylaminobenzoic acid (3-dimethylaminobenzoic acid; MBTHDMAB ), 3-methyl-2-benzothiozolinone hydrazone (3-methyl-2-benzothiozolinone hydrazone), 2-methoxy-4-arylphenol (2-met hoxy-4-allylphenol, 4-aminoantipyrin (AAP), dimethylaniline, 4-aminoantipyrene, 4-methoxynaphthol, 3,3 ', 5,5'-tetramethylbenzidine (3,3', 5,5'-tetramethyl benzidine; TMB), 2,2-azino-di- [3-ethyl-benzthiazolinesulfonate] (2,2 -azino-di- [3-ethyl-benzthiazoline sulfonate]), o-dianisidine, o-toluidine, 2,4-dichlorophenol, 4 Biosensor, characterized in that selected from the group consisting of 4-aminophenazone, benzine (benzidine).
  8. 제1항에 있어서,The method of claim 1,
    측정부는 헤모글로빈 총량을 측정하기 위한 작용전극 및 기준전극을 더 구비하고, 상기 작용전극과 기준전극 사이의 전위차를 측정하여, 시료 내 포함된 헤모글로빈 및 당화헤모글로빈을 동시에 측정하는 것이 특징인 바이오센서.The measurement unit further comprises a working electrode and a reference electrode for measuring the total amount of hemoglobin, by measuring the potential difference between the working electrode and the reference electrode, characterized in that simultaneously measuring the hemoglobin and glycated hemoglobin contained in the sample.
  9. 제8항에 있어서,The method of claim 8,
    헤모글로빈의 햄(HEME)기에 포함된 Fe2+의 가역적 산화환원 반응에 의해 형성된 작용전극과 기준전극 사이의 전위차를 통해 헤모글로빈 총량을 측정하는 것이 특징인 바이오센서.A biosensor characterized by measuring the total amount of hemoglobin through the potential difference between the working electrode and the reference electrode formed by the reversible redox reaction of Fe 2+ contained in the ham (HEME) group of hemoglobin.
  10. 제1항에 있어서,The method of claim 1,
    측정부는 보조전극을 추가로 포함하는 바이오센서.A biosensor further comprising a measuring unit.
  11. 기준전극; 및 집전체 상에, 황원자를 통해 3-(4-머캅토부탄아미도)페닐보론산이 정렬된 변이전극을 구비한 당화헤모글로빈 측정바이오센서용 스트립.Reference electrode; And a glycosylated hemoglobin measuring biosensor strip having a transition electrode in which 3- (4-mercaptobutaneamido) phenylboronic acid is aligned on a current collector through sulfur atoms.
  12. 제11항에 있어서,The method of claim 11,
    작용전극과 기준전극 사이의 전위차를 측정하여 헤모글로빈 총량을 측정하기 위한 작용전극 및 기준전극을 더 구비하여, 시료내 포함된 헤모글로빈 및 당화헤모글로빈을 동시에 측정하게 하는 바이오센서용 스트립.A biosensor strip further comprising a working electrode and a reference electrode for measuring the total amount of hemoglobin by measuring the potential difference between the working electrode and the reference electrode, thereby simultaneously measuring the hemoglobin and glycated hemoglobin contained in the sample.
  13. 제11항에 있어서,The method of claim 11,
    상기 스트립은 분리교환될 수 있어 일회 또는 복수의 사용이 가능한 것이 특징인 바이오센서용 스트립.The strip may be separated and exchanged, the strip for biosensor, characterized in that one or a plurality of uses.
  14. 제1항 내지 제10항 중 어느 한 항에 기재된 바이오센서를 구비한 당화헤모글로빈 측정 키트.A glycosylated hemoglobin measurement kit comprising the biosensor according to any one of claims 1 to 10.
  15. 제14항에 있어서,The method of claim 14,
    용해용액(lysis solution), 계면활성제용액, 또는 둘다를 더 포함하는 것이 특징인 키트.Kit comprising a lysis solution, a surfactant solution, or both.
  16. 제14항에 있어서,The method of claim 14,
    상기 바이오센서의 측정부는 분리교환될 수 있는 스트립형태인 것이 특징인 키트.The measuring unit of the biosensor characterized in that the strip form that can be separated and exchanged.
  17. 제14항에 있어서,The method of claim 14,
    변이전극과 기준전극 모두와 접촉할 수 있는 반응용기 또는, 추가로 작용전극과 기준전극 모두와 접촉할 수 반응용기를 더 구비하는 키트.And a reaction vessel capable of contacting with both the transition electrode and the reference electrode, or further comprising a reaction vessel capable of contacting with both the working electrode and the reference electrode.
PCT/KR2012/010145 2011-11-28 2012-11-28 Biosensor for measuring glycosylated haemoglobin using potentiometry WO2013081363A1 (en)

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