WO2015020149A1 - 電気化学式バイオセンサを用いた物質の測定方法及び測定装置 - Google Patents
電気化学式バイオセンサを用いた物質の測定方法及び測定装置 Download PDFInfo
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- WO2015020149A1 WO2015020149A1 PCT/JP2014/070885 JP2014070885W WO2015020149A1 WO 2015020149 A1 WO2015020149 A1 WO 2015020149A1 JP 2014070885 W JP2014070885 W JP 2014070885W WO 2015020149 A1 WO2015020149 A1 WO 2015020149A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/001—Enzyme electrodes
- C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
- C12Q1/006—Enzyme electrodes involving specific analytes or enzymes for glucose
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
- G01N27/3272—Test elements therefor, i.e. disposable laminated substrates with electrodes, reagent and channels
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/001—Enzyme electrodes
- C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
- G01N27/3273—Devices therefor, e.g. test element readers, circuitry
Definitions
- the present invention relates to a measurement method and a measurement apparatus using an electrochemical biosensor for analyzing a measurement target substance such as a biological component.
- an electrochemical biosensor a method of applying a voltage to an electrode system and measuring a Cottrell current based on the diffusion of a substance is mainly used.
- an oxidizing agent and a buffering agent are contained in a reaction system, and after the reaction is performed until the reaction is substantially completed, a potential is applied between the electrode and the sample to measure the Cottrell current. It is described to do.
- This Cottrell current depends on diffusion and is expressed by the Cottrell equation (the following equation (1)), and is characterized by including a diffusion coefficient (D) of the substance. In reaction kinetics, it can be said to be a diffusion-controlled state.
- Patent Document 2 discloses a measurement condition that uses a microelectrode to measure an analyte in a microfluid and depends on the diffusion coefficient (D) of the analyte.
- Patent Document 3 discloses a Cottrell equation and a diffusion coefficient (D), and discloses an example in which the diffusion coefficient is calculated by experiment.
- Patent Document 4 describes a step of applying a potential between electrodes so that the potential of the working electrode is diffusion-controlled by the redox species.
- an object of the present invention is to provide a method and an apparatus capable of measuring a substance using an electrochemical biosensor in a shorter time and with a simple and accurate system.
- the measuring method of the present invention is A substance is placed in an electrochemical measurement cell, which includes an insulating substrate, two or more electrodes formed on the insulating substrate, and a reagent layer containing an oxidoreductase disposed on at least one of the electrodes.
- Introducing a sample containing Applying a voltage to the electrodes, Detecting a charge transfer limited current generated by movement of electrons derived from the substance in the sample to the electrode, and determining a concentration of the substance contained in the sample based on the charge transfer limited current.
- the charge transfer rate limiting current is preferably a steady current after generation of a transient current due to charging of the electric double layer, and more preferably represented by the following formula (6).
- the oxidoreductase preferably contains pyrroloquinoline quinone or flavin adenine dinucleotide, or has a subunit or domain containing heme. More specifically, it is preferable that the oxidoreductase is an enzyme having glucose oxidation activity, for example, glucose dehydrogenase, and the substance to be measured is glucose.
- the voltage is preferably applied by step application, and the applied voltage is preferably 600 mV or less.
- the measuring device of the present invention comprises: An insulating substrate, two or more electrodes formed on the insulating substrate, and a reagent layer containing an oxidoreductase capable of reacting with a measurement target substance in a sample disposed on at least one of the electrodes Including a biosensor including an electrochemical measurement cell; A control unit for controlling voltage application to the biosensor; A detection unit for detecting a charge transfer rate limiting current based on the movement of electrons derived from the substance to the electrode obtained by applying a voltage to the biosensor; A calculation unit for calculating the concentration of the substance from the current value; And an output unit for outputting the calculated concentration of the substance.
- the control unit is set to perform control so that a voltage is applied by step application.
- the measurement target substance is glucose and the oxidoreductase is an enzyme having glucose oxidation activity, for example, glucose dehydrogenase.
- the concentration of a substance can be measured without being influenced by diffusion, the measurement time can be shortened.
- the cost can be reduced because the electrode system can be simplified. From these effects, it becomes possible to improve the operability of measurement with a smaller amount of sample and a shorter measurement time, which leads to improvement of usability.
- FIG. 1 is a diagram showing the structures of biosensors of examples and comparative examples.
- A Whole perspective view
- B shows an exploded perspective view.
- FIG. 2 is a diagram showing the results of cyclic voltammetry measurement using the biosensors of Examples 1 and 2 and Comparative Example.
- FIG. 3 is a diagram showing the results of chronoamperometry measurement using the biosensors of Example 1 and Comparative Example.
- FIG. 4 is a diagram showing the results of chronoamperometry measurement using the biosensor of Example 1 while changing the voltage parameter.
- FIG. 5 is a diagram showing the results of chronoamperometry measurement using the biosensor of Example 3.
- FIG. 1 is a diagram showing the structures of biosensors of examples and comparative examples.
- A Whole perspective view
- FIG. 3 shows the results of chronoamperometry measurement using the biosensors of Example 1 and Comparative Example.
- FIG. 4 is a diagram showing the results of chronoamperometry measurement using the biosensor of Example 1 while changing the voltage parameter.
- FIG. 6 plots the steady-state current value of charge transfer rate-determined at each glucose concentration measured by the biosensor of Example 1 and the theoretical value of the steady-state current at each glucose concentration calculated by the theoretical formula of Equation (5). It is a graph.
- FIG. 7 is a schematic view showing one embodiment of the measuring apparatus of the present invention.
- FIG. 8 is a flowchart showing one mode of a measurement program using the measurement apparatus of the present invention.
- a method for measuring a substance using a biosensor of the present invention includes an insulating substrate, two or more electrodes formed on the insulating substrate, and an oxidoreductase disposed on at least one of the electrodes. Introducing a sample containing a substance into an electrochemical measurement cell including a reagent layer, applying a voltage to the electrode, and a charge transfer limited current generated by the movement of electrons derived from the substance in the sample to the electrode Detecting and determining a concentration of the substance contained in the sample based on the charge transfer rate limiting current.
- the substance to be measured is not particularly limited as long as it is a substance that can be measured by the measurement method using the biosensor of the present invention, but is a substance derived from a living body and can be an indicator of a disease or a health condition.
- a substance derived from a living body can be an indicator of a disease or a health condition.
- examples thereof include glucose and cholesterol.
- the sample is not particularly limited as long as it contains a substance to be measured, but a biological sample is preferable, and blood, urine and the like can be mentioned.
- the charge transfer rate-limiting current based on the movement of electrons derived from the substance to be measured to the electrode is a current generated when electrons move from the enzyme to the electrode due to the reaction between the oxidoreductase and the substance to be measured. It is a steady current that does not depend on the current, and is preferably a steady current after generation of a transient current due to charging of the electric double layer.
- This charge transfer rate limiting current is preferably represented by the following formula (5). From this equation, it can be seen that the current is proportional to the concentration of the substrate and the enzyme reaction rate constant, and if the constant term is X, it can be developed into equation (6). Although not shown in equations (5) and (6), the constant term X may include a correction coefficient.
- Equation (2) the initial rate equation of the enzyme reaction
- Equation (3) the equation of the electron transfer rate from the enzyme to the electrode
- the fact that the electrode system is charge transfer-controlled can be confirmed by examining the presence or absence of a peak and the increasing tendency of the current depending on the voltage sweep direction by cyclic voltammetry.
- the working electrode can be obtained, for example, by arranging an electrode material on an insulating substrate and arranging a reagent layer containing at least an oxidoreductase in the vicinity of the obtained electrode.
- the electrode is formed using, for example, a carbon material such as carbon.
- a metal material such as gold (Au), platinum (Pt), silver (Ag), or palladium can be used.
- Insulating substrates include, for example, thermoplastic resins such as polyetherimide (PEI), polyethylene terephthalate (PET), polyethylene (PE), various resins (plastics) such as polyimide resin and epoxy resin, glass, ceramic, It is made of an insulating material such as paper. The size and thickness of the electrode and the insulating substrate can be set as appropriate.
- the oxidoreductase may be any enzyme that can oxidize and reduce the substance to be measured, but it may contain at least one of pyrroloquinoline quinone (PQQ) and flavin adenine dinucleotide (FAD) as the catalytic subunit and catalytic domain. It can.
- PQQ glucose dehydrogenase PQQGDH
- CyGDH cytochrome glucose dehydrogenase
- GOD glucose oxidase
- the oxidoreductase can include an electron transfer subunit or an electron transfer domain.
- an electron transfer subunit for example, a subunit having a hem having an electron transfer function can be cited.
- the oxidoreductase containing the heme-containing subunit include those containing cytochrome.
- glucose dehydrogenase or a fusion protein of PQQGDH and cytochrome can be applied.
- Examples of the enzyme containing an electron transfer domain include cholesterol oxidase and quinoheme ethanol dehydrogenase (QHEDH (PQQ Ethanol dh).
- QHEDH quinoheme ethanol dehydrogenase
- QHGDH fusion enzyme; GDH with heme domain of QHGDH
- sorbitol dehydrogenase Sorbitol DH
- D-fructose dehydrogenase Feructose DH
- glucose-3-dehydrogenase derived from Agrobacterium tumefasience Glucose-3-Dehydrogenase
- G3DH from Agrobacterium tumefasience
- cellobiose dehydrogenase cellobiose dehydrogenase.
- an oligomeric enzyme composed of a subunit containing cytochrome having at least a catalytic subunit and a heme having an electron acceptor function.
- the substance to be measured may be a substrate for oxidoreductase.
- cellobiose dehydrogenase oxidizes cellobiose but also oxidizes glucose, so that glucose can also be used as a substance to be measured.
- the working electrode is preferably a “direct electron transfer type enzyme electrode”.
- direct electron transfer type enzyme electrode means that an electron generated by an enzyme reaction in a reagent layer is directly transferred to an electrode without involving a redox substance such as an electron transfer mediator. This is an enzyme electrode of a type in which electrons are transferred between the electrodes.
- the limit distance at which direct electron transfer occurs in a physiological reaction system is said to be 1 to 2 nm. Therefore, it is important to arrange the enzyme so that the electron transfer from the enzyme to the electrode is not impaired.
- an oxidoreductase In order to measure the charge transfer rate limiting current, it is important to place an oxidoreductase in the vicinity of the electrode, but the method for that is not particularly limited. Examples thereof include a method for immobilization, a method for indirectly immobilizing an oxidoreductase on an electrode using a binder, a method for physically adsorbing an oxidoreductase on an electrode, and the like.
- the enzyme reagent layer on the working electrode can contain conductive particles.
- the conductive particles may be metal particles such as gold, platinum, silver, and palladium, or higher-order structures using carbon as a material.
- the higher order structure may include carbon particles or carbon fine particles such as conductive carbon black, carbon nanotubes (CNT), and fullerene. Examples of the conductive carbon black include ketjen black (Degussa) and black pearl (Cabot).
- the enzyme reagent layer on the working electrode can also contain a conductive polymer.
- the conductive polymer is preferably water-soluble, and examples thereof include polyaniline and polyethylenedioxythiophene.
- a typical example is a sulfonated polyaniline aqueous solution (trade name: Aquapass) manufactured by Mitsubishi Rayon.
- the enzyme reagent layer on the working electrode can also contain a binder.
- a binder a water-soluble binder is preferable, and specific examples include a water-soluble polymer containing an oxazoline group.
- the working electrode as described above is produced, for example, as follows. That is, a carbon layer functioning as an electrode is formed on one side of the insulating substrate.
- carbon ink can be screen-printed on one side of a film-like insulating substrate having a predetermined thickness (for example, about 100 ⁇ m) to form a carbon film having a desired thickness (for example, about 10 ⁇ m).
- a metal layer having a desired thickness can be formed by forming a metal material by physical vapor deposition (PVD, for example, sputtering) or chemical vapor deposition (CVD).
- PVD physical vapor deposition
- CVD chemical vapor deposition
- an enzyme reagent layer is formed on the electrode.
- a solution containing an oxidoreductase and conductive particles or a conductive polymer is prepared, and the solution is dropped on the surface of the electrode.
- a working electrode having an enzyme reagent layer formed on the electrode can be obtained.
- the counter electrode may be any electrode that can be generally used as a counter electrode of a biosensor.
- the counter electrode may be formed by a carbon electrode formed by screen printing, physical vapor deposition (PVD, for example, sputtering), or chemical vapor deposition (CVD).
- PVD physical vapor deposition
- CVD chemical vapor deposition
- a filmed metal electrode or a silver / silver chloride electrode formed by screen printing can be used.
- a three-electrode system using a silver / silver chloride electrode as a reference electrode may be used.
- the method of applying a voltage to the electrode is not particularly limited, but step application is preferable in order to efficiently measure the charge transfer limited current.
- the voltage to be applied is preferably 600 mV or less, more preferably 100 mV or less. Although a minimum in particular is not restrict
- the concentration of the substance to be measured can be calculated from the measured current value based on the equation (5). It is also possible to create a calibration curve in advance using a sample with a known concentration and calculate from the measured current value based on the calibration curve. It is also possible to calculate the concentration of the sample by multiplying equation (5) by the correction coefficient found by the test. In this case, the correction coefficient is also included in the constant term X of Equation (6). According to the measurement method of the present invention, both continuous measurement and intermittent measurement are possible.
- FIG. 7 shows a configuration example of main electronic components housed in the measuring apparatus 2. As shown in FIG. 7, a control computer 3, a potentiostat 3A, and a power supply device 21 are provided on a substrate 3a housed in a housing.
- control computer 3 includes a processor such as a CPU (Central Processing Unit), a recording medium such as a memory (RAM (Random Access Memory), ROM (Read Only Memory)), and a communication unit. And the processor functions as an apparatus including the output unit 20, the control unit 22, the calculation unit 23, and the detection unit 24 by loading the program stored in the recording medium (for example, ROM) into the RAM and executing the program. .
- the control computer 3 may include an auxiliary storage device such as a semiconductor memory (EEPROM, flash memory) or a hard disk.
- the controller 22 controls voltage application timing, applied voltage value, and the like.
- the power supply device 21 includes a battery 26 and supplies power for operation to the control unit computer 3 and the potentiostat 3A.
- the power supply device 21 can also be placed outside the housing.
- the potentiostat 3A is a device that makes the potential of the working electrode constant with respect to the reference electrode.
- the potentiostat 3A is controlled by the control unit 22 and uses a terminal CR, W to establish a predetermined gap between the counter electrode and the working electrode of the glucose sensor 4. Is applied by step application, the response current of the working electrode obtained at the terminal W is measured, and the response current measurement result is sent to the detection unit 24.
- the calculation unit 23 calculates the concentration of the substance to be measured from the detected current value and stores it.
- the output unit 20 performs data communication with the display unit 25 and transmits the calculation result of the concentration of the measurement target substance by the calculation unit 23 to the display unit 25.
- the display unit 25 can display the calculation result of the glucose concentration received from the measurement device 2 on the display screen in a predetermined format.
- FIG. 8 is a flowchart showing an example of glucose concentration measurement processing by the control computer 3.
- the control unit 22 controls the potentiostat 3A to apply a predetermined voltage to the working electrode in steps. Application is started and measurement of the response current from the working electrode is started (step S01). It should be noted that detection of sensor mounting on the measuring device may be used as a concentration measurement start instruction.
- the potentiostat 3A has a response current obtained by applying a voltage, that is, a charge transfer rate-limiting current based on the movement of an electron derived from a measurement target substance (here, glucose) in a sample to an electrode, After the generation of the transient current due to the charging of the multilayer, for example, the steady current 1 to 20 seconds after the voltage application is measured and sent to the detection unit 24 (step S02).
- a voltage that is, a charge transfer rate-limiting current based on the movement of an electron derived from a measurement target substance (here, glucose) in a sample to an electrode
- the calculation unit 23 performs calculation processing based on the current value, and calculates the glucose concentration (step S03).
- the computing unit 23 of the control computer 3 calculates the glucose concentration calculation formula (based on the above formula (5) or formula (6)) or the glucose concentration calibration curve data corresponding to the glucose dehydrogenase disposed on the electrode. Is previously stored, and the glucose concentration is calculated using these calculation formulas or calibration curves.
- the output unit 20 transmits the calculation result of the glucose concentration to the display unit 25 through a communication link formed with the display unit 25 (step S04). Thereafter, the control unit 22 detects the presence or absence of a measurement error (step S05). If there is no error, the control unit 22 ends the measurement and displays the glucose concentration on the display unit. If there is an error, an error display is displayed, and then the processing according to the flow of FIG. It is also possible to store the calculation result in the calculation unit 23, call the calculation result later, and display the result on the display unit for confirmation. Here, after the calculation result is transmitted to the display unit 25 (step S04), the measurement error is detected by the control unit 22 (step S05). However, the order of these steps can be changed. .
- the glucose sensor 1 includes a cover plate 10, a spacer 11, and a substrate 12.
- the cover plate 10 is provided with a hole portion 13, and the spacer 11 is provided with a narrow slit 14 that communicates with the hole portion 13 and that has an open end portion 14 a.
- the capillary 15 is defined by the slit 14. The capillary 15 communicates with the outside through the tip opening 14 a and the hole 13 of the slit 14.
- the tip opening portion 14a constitutes a sample solution introduction port 15a, and the sample solution supplied from the sample solution introduction port 15a advances in the capillary 15 toward the hole 13 by capillary action.
- a first electrode 16, a second electrode 17, and a reagent layer 18 are provided on the upper surface 12 a of the substrate 12.
- the first and second electrodes 16 and 17 generally extend in the longitudinal direction of the substrate 12, and their end portions 16 a and 17 a extend in the short direction of the substrate 12.
- the upper surface 12a of the substrate 12 is covered with an insulating film 19 so that the end portions 16a, 16b, 17a, 17b of the first and second electrodes 16, 17 are exposed.
- the reagent layer 18 is provided so as to bridge between the end portions 16 a and 17 a of the first and second electrodes 16 and 17.
- the reagent layer 18 contains glucose dehydrogenase. More specifically, the glucose sensor was produced by the following method.
- ⁇ Base electrode> Conductive carbon ink (FTU series manufactured by Asahi Chemical Research Laboratories) was used as the base electrode material, and this ink was screen-printed with a polyethylene terephthalate substrate (E-22 manufactured by Toray) (length 50 mm, width 5 mm, thickness 250 ⁇ m). ) was printed on one surface to form a two-electrode pattern. Further, in Examples, a silver-silver chloride ink (manufactured by BAS) was applied on one electrode and dried at 80 ° C. for 20 minutes to form a silver-silver chloride electrode, which was used as a counter electrode.
- FTU series manufactured by Asahi Chemical Research Laboratories
- an insulating resin polyester ink (UVF series manufactured by Asahi Chemical Research Laboratory) was screen-printed on the electrode.
- the electrode area formed by the electrode pattern and the insulating pattern was set to 0.5 mm 2 , respectively.
- ⁇ Formation of enzyme reagent layer (Examples 1 and 2)> Enzyme reagent containing cytochrome-containing glucose dehydrogenase (CyGDH), conductive particles (carbon black: Ketjen black KJB), conductive polymer (polyaniline) as a conductive additive and binder (oxazoline group-containing water-soluble polymer) on the electrode was prepared, and 0.04 ⁇ L was dropped on the electrode and dried at 100 ° C. for 30 minutes to form an enzyme reagent layer.
- the final concentration of enzyme reagent is as follows.
- an enzyme reagent layer was formed by dispensing 10 ⁇ L of 5000 U / mL cytochrome-containing glucose dehydrogenase (CyGDH or QHGDH) aqueous solution onto the first reagent layer and drying at 30 ° C. for 10 minutes.
- the base electrode the carbon electrode produced by the same method as in Example 1 is used as the working electrode and the counter electrode.
- a capillary was formed by the following method.
- a glucose sensor was obtained by arranging a spacer having an opening on the insulating layer on the base electrode on which the enzyme reagent layer was formed, and further arranging a cover having a through hole serving as an air hole on the spacer. Since the space of the opening portion of the spacer sandwiched between the cover and the insulating layer has a capillary structure, this was used as a sample supply portion.
- ⁇ Cyclic voltammetry measurement> For Examples 1 and 2 and Comparative Example, the electrode response characteristics of the glucose sensor were evaluated by examining cyclic voltammetry waveforms.
- the cyclic voltammetry waveform is swept so that the applied voltage is -200 mV ⁇ +800 mV ⁇ -200 mV after introducing whole blood with a glucose concentration of 100 mg / dL into the sample supply part of the glucose sensor, setting the sweep rate to 20 mV / sec.
- the response current at the time of sweep was measured.
- FIG. 2 is a cyclic voltammetry waveform obtained by measurement.
- the conductive polymer may or may not be used to detect the charge transfer limited current, but the conductive polymer is higher in Example 1 containing the conductive polymer. It can be seen that has the effect of increasing the response sensitivity. From the above, it can be seen that the charge transfer rate-limiting current is generated in both Examples 1 and 2, and it is understood that there is no inconvenience in the measurement in Example 2. In the following tests, evaluation was performed using the sensor of Example 1 as a representative example.
- Chronoamperometry measurement was conducted by introducing 400 mV of stepped blood into the working electrode after introducing whole blood with a glucose concentration of 100 mg / dL into the sample introduction part of the glucose sensor, and measuring the response current.
- Fig. 3 shows the results of chronoamperometry measurement using the fabricated sensor.
- a transient current flows after voltage application. This is a current generated by charging the electric double layer on the electrode surface.
- the electric double layer is a layer generated by the arrangement of electrolyte ions in order to maintain electrical neutralization on the solution side at the interface between the electrode surface and the solution.
- the biosensor of the comparative example after the charging current was generated, it was confirmed that the observed Cottrell current due to diffusion rate was decreased by 1 / ⁇ t based on the equation (1).
- the biosensor of Example 1 was used, the steady current was detected immediately after the charging current was generated despite the fact that it was not a microelectrode system. By measuring this steady current, The concentration of glucose can be measured. It was confirmed that the measurement current in this example is not diffusion limited but charge transfer limited, and measurement can be performed in a shorter time than measurement by diffusion limited.
- ⁇ Chronoamperometry measurement (voltage parameter)> About Example 1, the electrode response characteristic of the glucose sensor was evaluated by chronoamperometry measurement.
- chronoamperometry measurement after introducing whole blood with a glucose concentration of 0 mg / dL or 336 mg / dL into the sample introduction part of the glucose sensor, a step voltage is applied to the working electrode, and the response current after 10 seconds is measured. It was investigated by. The measurement voltage was changed to 600, 400, 200, 100, and 70 mV, respectively.
- Fig. 4 shows the result of chronoamperometry measurement using the fabricated sensor.
- a steady-state current response due to charge transfer rate limiting at an equivalent level was confirmed at glucose 336 mg / dL.
- the current value was measured 10 seconds after the step voltage was applied. From the results shown in FIG. 4, the steady current can be confirmed in about 1 to 2 seconds after the step voltage is applied. Since a steady current was detected within 2 seconds, it was confirmed that a short-time measurement was possible.
- FIG. 4 by reducing the applied voltage, the background current resulting from the oxidation-reduction reaction of coexisting substances contained in the sample can be reduced. There is also an effect that errors can be suppressed.
- ⁇ Chronoamperometry measurement-Example 3> The electrode response characteristics of glucose sensor using QHGDH were evaluated by chronoamperometry measurement. Chronoamperometry was measured by introducing a whole blood with a glucose concentration of 0 mg / dL or 600 mg / dL into the sample introduction part of the glucose sensor, applying 200 mV to the working electrode in steps, and measuring the response current. . The results are shown in FIG. With glucose 600 mg / dL, a steady current response due to charge transfer rate control was confirmed.
- the glucose concentration can be measured based on the steady-state current response due to the charge transfer limited by glucose. I found it possible.
- the time when the steady current response by charge transfer rate control was obtained was from about 15 seconds, this was considered to be influenced by the purity of the enzyme.
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Abstract
Description
さらに、特許文献3では、コットレル式および拡散係数(D)の記載があり、実験により拡散係数を算出している例が開示されている。
さらに、特許文献4では、作用極の電位をレドックス化学種の拡散律速であるように電極間に電位を加える工程が記載されている。
絶縁性基板と、該絶縁性基板上に形成した2以上の電極と、該電極の少なくとも1つの電極上に配置された酸化還元酵素を含む試薬層とを含む、電気化学測定セル内に物質を含む試料を導入すること、
電極に電圧を印加すること、
試料内の前記物質に由来する電子の電極への移動により生じる電荷移動律速電流を検出すること、および
前記電荷移動律速電流に基づき試料中に含まれる前記物質の濃度を決定すること、を含む。
ここで、前記電荷移動律速電流は、電気二重層の充電による過渡電流発生後の定常電流であることが好ましく、下記式(6)で表されることがより好ましい。
また、酸化還元酵素がピロロキノリンキノンまたはフラビンアデニンジヌクレオチドを含むか、ヘムを含むサブユニットまたはドメインを有することが好ましい。
より具体的には、前記酸化還元酵素がグルコース酸化活性を有する酵素、例えば、グルコースデヒドロゲナーゼであり、測定対象物質がグルコースであることが好ましい。
また、電圧はステップ印加により印加されることが好ましく、印加する電圧は600mV以下であることが好ましい。
絶縁性基板と、該絶縁性基板上に形成した2以上の電極と、該電極の少なくとも1つの電極上に配置された試料中の測定対象物質と反応しうる酸化還元酵素を含む試薬層とを含む、電気化学測定セルを含むバイオセンサと、
バイオセンサへの電圧印加を制御する、制御部と、
バイオセンサへの電圧印加により得られる、前記物質に由来する電子の電極への移動に基づく電荷移動律速電流を検出する、検出部と、
前記電流値から前記物質の濃度を算出する、演算部と、
前記算出された前記物質の濃度を出力する出力部
とから構成される。
ここで、前記制御部は、電圧をステップ印加により印加するように制御を行うように設定されていることが好ましい。また、測定装置は、測定対象物質がグルコースであり、酸化還元酵素がグルコース酸化活性を有する酵素、例えば、グルコースデヒドロゲナーゼであることが好ましい。
前記電荷移動律速電流に基づき試料中に含まれる前記物質の濃度を決定すること、を含む。
試料は測定対象物質を含む試料であれば特に制限されないが、生体試料が好ましく、血液、尿などが挙げられる。
式(5)は、上記式(1)のコットレル電流に含まれる拡散係数(D)を含まない電荷移動律速による電流式である。式(5)からも分かるように、電流は酵素反応速度定数に比例する。本発明の測定方法では、電子受容物質などのメディエーターによる酸化還元反応を介することなく、電極へ電子が移動するため、物質の拡散の影響を受けず、時間の依存性もないことがわかる。
作用電極は例えば、絶縁性基板上に電極材料を配置し、得られた電極の近傍に少なくとも酸化還元酵素を含む試薬層を配置させることによって得ることができる。
電極は、例えば、カーボンのような炭素材料を用いて形成される。或いは、金(Au),白金(Pt),銀(Ag),パラジウムのような金属材料を用いることもできる。
絶縁性基板は、例えば、ポリエーテルイミド(PEI),ポリエチレンテレフタレート(PET),ポリエチレン(PE)のような熱可塑性樹脂、ポリイミド樹脂、エポキシ樹脂のような各種の樹脂(プラスチック),ガラス,セラミック,紙のような絶縁性材料で形成される。
電極及び絶縁性基板の大きさ、厚さは適宜設定可能である。
酸化還元酵素は、測定対象物質を酸化還元しうる酵素であればよいが、触媒サブユニット及び触媒ドメインとして、ピロロキノリンキノン(PQQ)、フラビンアデニンジヌクレオチド(FAD)のうち少なくとも一方を含むことができる。例えば、PQQを含む酸化還元酵素として、PQQグルコースデヒドロゲナーゼ(PQQGDH)が挙げられ、FADを含む酸化還元酵素として、FADを含んだαサブユニットを持つシトクロムグルコースデヒドロゲナーゼ(CyGDH)、グルコースオキシダーゼ(GOD)が挙げられる。
なお、生理学的反応系において直接電子移動が起こる限界距離は1~2nmと云われている。よって、酵素から電極への電子移動が損なわれないように酵素を配置することが重要である。
次に、電極上に酵素試薬層が形成される。まず、酸化還元酵素と導電性粒子や導電性高分子を含む溶液が調製され、該溶液は、電極の表面に滴下される。該溶液が電極上で乾燥により固化することで、電極上に酵素試薬層が形成された作用電極を得ることができる。
また、濃度既知の試料を用いて検量線を予め作成しておき、その検量線に基づいて測定電流値より算出することも可能である。また、試験により見出した補正係数を式(5)に乗じること等により、検体の濃度を算出することも可能である。この場合、式(6)の定数項Xに補正係数も含まれることとなる。
本発明の測定方法によれば、連続的な測定も断続的な測定もいずれも可能である。
図7は、測定装置2内に収容された主な電子部品の構成例を示す。図7に示すような、制御コンピュータ3,ポテンショスタット3A,電力供給装置21が、筐体内に収容された基板3aに設けられている。
電力供給装置21は、バッテリ26を有しており、制御部コンピュータ3やポテンショスタット3Aに動作用の電力を供給する。なお、電力供給装置21は、筐体の外部に置くこともできる。
ポテンショスタット3Aは、作用極の電位を参照電極に対して一定にする装置であり、制御部22によって制御され、端子CR,Wを用いて、グルコースセンサ4の対極と作用極との間に所定の電圧をステップ印加により印加し、端子Wで得られる作用極の応答電流を測定し、応答電流の測定結果を検出部24に送る。
図8において、制御コンピュータ3のCPU(制御部22)は、グルコース濃度測定の開始指示を受け付けると、制御部22は、ポテンショスタット3Aを制御して、作用極への所定の電圧をステップ印加で印加し、作用極からの応答電流の測定を開始する(ステップS01)。なお、測定装置へのセンサの装着の検知を、濃度測定開始指示としてもよい。
〔実施例1〕
以下、バイオセンサの実施例について、グルコースセンサを用いて説明する。
グルコースセンサの構造の一例を図1に示す。
グルコースセンサ1は、図1に示されるように、カバー板10、スペーサー11および基板12を有している。
カバー板10には穴部13が設けられており、スペーサー11には穴部13に連通するとともに先端部14aが開放した細幅なスリット14が設けられている。カバー板10およびスペーサー11が基板12の上面12aに積層された状態では、スリット14によりキャピラリー15が規定されている。このキャピラリー15は、スリット14の先端開口部14aおよび穴部13を介して外部と連通している。先端開口部14aは試料液導入口15aを構成しており、この試料液導入口15aから供給された試料液は、毛細管現象により穴部13に向けてキャピラリー15内を進行する。
基板12の上面12aには、第1電極16、第2電極17、および試薬層18が設けられている。
第1および第2電極16,17は、全体として基板12の長手方向に延びており、それらの端部16a,17aが基板12の短手方向に延びている。基板12の上面12aは、第1および第2電極16,17の端部16a,16b,17a,17bが露出するようにして絶縁膜19により覆われている。
試薬層18は、第1および第2電極16,17の端部16a,17a間を橋渡すようにして設けられている。この試薬層18は、グルコースデヒドロゲナーゼを含んでいる。
より具体的には、グルコースセンサは以下の方法で作製した。
下地電極材料として、導電性カーボンインク(アサヒ化学研究所製FTUシリーズ)を用い、このインクをスクリーン印刷手法にてポリエチレンテレフタレート基材(東レ製E-22)(長さ50mm、幅5mm、厚み250μm)の一方の表面にパターンニング印刷を行い、2電極パターンを形成した。さらに、実施例においては、一方の電極上に銀塩化銀インク(BAS社製)を塗布し、80℃で20分乾燥させ、銀塩化銀電極を形成し、対極とした。
電極上に、シトクロム含有グルコースデヒドロゲナーゼ(CyGDH)、導電性粒子(カーボンブラック:ケッチェンブラックKJB)、導電助剤としての導電性高分子(ポリアニリン)およびバインダー(オキサゾリン基含有水溶性ポリマー)含む酵素試薬を調製し、電極上に0.04μL滴下し、100℃で30分乾燥することで、酵素試薬層を形成した。酵素試薬の最終濃度は以下の通りである。
<酵素試薬の処方>
・KJB:0.4wt%
・酵素(CyGDH):7mg/mL
・リン酸Na緩衝液:10mM pH7
・バインダー(EPOCROS WS-700、日本触媒製)5.0%(w/v)
・ポリアニリン(アクアパス、三菱レーヨン製)0.2%(w/v)
なお、実施例2においては、ポリアニリンは添加せず、蒸留水を添加したが、それ以外の処方は実施例1と同じである。
CyGDHの代わりに、PQQGDHを基にしたシトクロムを含むQHGDH (PQQGDHとシトクロムとの融合蛋白質)を用いた。
以下の処方にて酵素試薬を調製し、電極上に0.08μL滴下し、100℃で2時間乾燥することで、酵素試薬層を形成した。
<酵素試薬の処方>
・ライオンペースト(ケッチェンブラック含有:W-311N)(ライオン製):2.4wt%
・酵素(QHGDH):2.3mg/mL
・HEPES緩衝液:20mM pH7
・バインダー(EPOCROS WS-700、日本触媒製)6.0%(w/v)
・ポリアニリン(アクアパス、三菱レーヨン製)0.4%(w/v)
<酵素試薬層の形成(比較例)>
電極上に、電子受容物質(ルテニウムアンミン錯体)およびバインダーとしての無機ゲル(スメクタイト)含む酵素試薬(第一試薬)を調製し、電極上に0.3μL滴下し、30℃で10分乾燥することで、第一試薬層を形成した。第一試薬の最終濃度は以下の通りである。
<第一試薬の処方>
・スメクタイト(SWN、コープケミカル社製) :0.3%(w/v)
・[Ru(NH3 )6 ]Cl3 (アルドリッチ製):5.0% (w/v)
実施例1,2,3及び比較例ともに、下記の方法でキャピラリーを形成した。
前記、酵素試薬層を形成した下地電極に、開口部を有するスペーサーを絶縁層上に配置し、さらに、前記スペーサー上に空気孔となる貫通孔を有するカバーを配置してグルコースセンサとした。前記カバーと絶縁層とに挟まれたスペーサーの開口部の空間が、キャピラリー構造となるため、これを試料供給部とした。
実施例1,2及び比較例について、サイクリックボルタンメトリー波形を調べることによりグルコースセンサの電極応答特性を評価した。サイクリックボルタンメトリー波形は、グルコースセンサの試料供給部にグルコース濃度が100mg/dLの全血を導入した後に、掃引速度を20mV/secとし、印加電圧が-200mV→+800mV→-200mVとなるように掃引し、掃引時の応答電流を測定することにより調べた。図2は、測定により得られたサイクリックボルタンメトリー波形である。
クロノアンペロメトリー測定によりグルコースセンサの電極応答特性を評価した。クロノアンペロメトリー測定は、グルコースセンサの試料導入部にグルコース濃度が100mg/dLの全血を導入した後に、作用極に400mVをステップ印加し、応答電流を測定することにより調べた。
実施例1について、クロノアンペロメトリー測定によりグルコースセンサの電極応答特性を評価した。クロノアンペロメトリー測定は、グルコースセンサの試料導入部にグルコース濃度が0mg/dLもしくは336mg/dLの全血を導入した後に、作用極にステップ電圧を印加し、10秒後の応答電流を測定することにより調べた。測定電圧は、600、400、200、100、70mVとそれぞれ変えて測定を行った。
クロノアンペロメトリー測定によりQHGDHを用いたグルコースセンサの電極応答特性を評価した。クロノアンペロメトリー測定は、グルコースセンサの試料導入部にグルコース濃度が0mg/dLもしくは600mg/dLの全血を導入した後に、作用極に200mVをステップ印加し、応答電流を測定することにより調べた。
結果を図5に示す。グルコース600mg/dLでは電荷移動律速による定常電流応答が確認された。一方、グルコース0mg/dLの電流値は低く、定常電流応答ではないことから、シトクロムを含むQHGDHを用いて作製したセンサにおいても、グルコースによる電荷移動律速による定常電流応答に基づいてグルコース濃度の測定が可能であることが分かった。なお、電荷移動律速による定常電流応答が得られる時間が15秒あたりからであったが、これは、酵素の精製度等による影響が考えられた。
実施例1のセンサに70mVのステップ電圧を印加し、10秒後に測定した各グルコース濃度における電荷移動律速の定常電流値に対して、式(5)の理論式において、表1の条件で算出した各グルコース濃度での定常電流の理論値の計算結果を比較した(図6)。
その結果、計算値(理論値)と測定結果はよく一致していることがわかる。理論値と測定結果の誤差から補正係数を定めて、式(5)に乗じること等により、計算値(理論値)と測定結果の一致度合いを高めることも可能である。尚、本測定方法では電圧のステップ印加後、10秒後の電流値を測定したが、上記のとおり、ステップ電圧印加後、1~2秒程度で定常電流を測定することが可能であることは、言うまでもない。
10・・・カバー板
11・・・スペーサー
12・・・基板
13・・・穴部
14・・・スリット
15・・・キャピラリー
16・・・第1電極
17・・・第2電極
18・・・試薬層
19・・・絶縁膜
2・・・測定装置
20・・・出力部
21・・・電力供給装置
22・・・制御部
23・・・演算部
24・・・検出部
25・・・表示部ユニット
26・・・バッテリ
3・・・制御コンピュータ
3A・・・ポテンショスタット
3a・・・基板
CR、W・・・端子
4・・・グルコースセンサ
Claims (12)
- 絶縁性基板と、該絶縁性基板上に形成した2以上の電極と、該電極の少なくとも1つの電極上に配置された酸化還元酵素を含む試薬層とを含む、電気化学測定セル内に物質を含む試料を導入すること、
電極に電圧を印加すること、
試料内の前記物質に由来する電子の電極への移動により生じる電荷移動律速電流を検出すること、および
前記電荷移動律速電流に基づき試料中に含まれる前記物質の濃度を決定すること、
を含むバイオセンサを用いた物質の測定方法。 - 前記電荷移動律速電流が、電気二重層の充電による過渡電流発生後の定常電流である請求項1に記載のバイオセンサを用いた物質の測定方法。
- 酸化還元酵素がピロロキノリンキノンまたはフラビンアデニンジヌクレオチドを含む、請求項1~3のいずれか一項に記載のバイオセンサを用いた物質の測定方法。
- 酸化還元酵素がヘムを含むサブユニットまたはドメインを有する、請求項1~4のいずれか一項に記載のバイオセンサを用いた物質の測定方法。
- 酸化還元酵素がグルコース酸化活性を有する、請求項1~5のいずれか一項に記載のバイオセンサを用いた物質の測定方法。
- 酸化還元酵素がグルコースデヒドロゲナーゼである、請求項1~6のいずれか一項に記載のバイオセンサを用いた物質の測定方法。
- 電圧をステップ印加により印加する、請求項1~7のいずれか一項に記載のバイオセンサを用いた物質の測定方法。
- 600mV以下の電圧が印加される、請求項8に記載のバイオセンサを用いた物質の測定方法。
- 絶縁性基板と、該絶縁性基板上に形成した2以上の電極と、該電極の少なくとも1つの電極上に配置された試料中の測定対象物質と反応しうる酸化還元酵素を含む試薬層とを含む、電気化学測定セルを含むバイオセンサと、
バイオセンサへの電圧印加を制御する、制御部と、
バイオセンサへの電圧印加により得られる、前記物質に由来する電子の電極への移動に基づく電荷移動律速電流を検出する、検出部と、
前記電流値から前記物質の濃度を算出する、演算部と、
前記算出された前記物質の濃度を出力する出力部とから構成される測定装置。 - 前記制御部は、電圧をステップ印加により印加するように制御を行う、請求項10に記載の測定装置。
- 前記物質がグルコースであり、酸化還元酵素がグルコースデヒドロゲナーゼである、請求項10または11に記載の測定装置。
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JP2018165729A (ja) | 2018-10-25 |
JPWO2015020149A1 (ja) | 2017-03-02 |
CN105492902A (zh) | 2016-04-13 |
JP6435264B2 (ja) | 2018-12-05 |
TWI637167B (zh) | 2018-10-01 |
US9976168B2 (en) | 2018-05-22 |
CN105492902B (zh) | 2020-07-24 |
EP3032250A1 (en) | 2016-06-15 |
EP3032250A4 (en) | 2017-04-26 |
EP3032250B1 (en) | 2023-10-11 |
US20160177365A1 (en) | 2016-06-23 |
EP3032250C0 (en) | 2023-10-11 |
TW201514486A (zh) | 2015-04-16 |
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