Correction of analyte concentration conversion value in electrochemical sensor measurement using electrochemical impedance measurement

Abstract

Provided is a method for correcting a measured value of the concentration of a target substance in an electrochemical sensor which includes: a substrate; an electrode pair including a working electrode and a counter electrode provided on the substrate, the working electrode including an oxidoreductase electrode; and an outer layer film covering the electrode pair, and quantitatively measures the target substance on the basis of an electrical signal generated by an oxidoreductase reaction. The method comprises: measuring electrochemical impedance between the working electrode and the counter electrode; calculating a change in resistance from a measured value of the electrochemical impedance; calculating an electrical signal change from a value of the electrical signal during the measurement of the electrochemical impedance; and correcting a measured value of the concentration of the target substance on the basis of a correction equation using the change in resistance and the electrical signal change.

Description

Correction of converted value of analyte concentration in electrochemical sensor measurement using electrochemical impedance measurement

[0001] The present invention relates to a technique for non-destructively evaluating the characteristics of an electrochemical sensor having a component whose electrochemical characteristics can change over time at any time, predicting the sensor characteristics, and correcting the measured concentration of a target analyte.

[0002] In Patent Document 1, as a real-time self-calibration method for a sensor having a sensor electronic device, a microcontroller, and a working electrode, an electrochemical impedance spectroscopy (EIS) procedure is periodically executed on the working electrode, and a Nyquist plot and values of impedance-based parameters are obtained and calculated, and a sensor calibration coefficient is adjusted based on the variation thereof, thereby performing sensor fault diagnosis and sensor type classification.

[0003] Patent Document 2 discloses a technique related to a sensor provided with a permeation control layer, that is, an outer layer film containing a water-soluble polymer and a water-insoluble polymer on a substrate on which a reaction layer containing a sensing enzyme is placed for the purpose of improving sensing performance.

[0004] Japanese Patent Application Laid-Open No. 2018-47259, Patent No. 7457801

[0005] In Patent Document 1, a technique for performing fault diagnosis and sensor type classification by adjusting the calibration coefficient of a sensor using EIS has been reported. However, when there are significant changes in sensor characteristics due to changes in the membrane quality even though the sensor is operating normally, means for real-time correction of the measured value and measured concentration of the measurement target analyte have not been studied.

[0006] Furthermore, Patent Document 2 reports a technique for coating a sensing substrate with an outer layer film containing water-soluble and water-insoluble polymers in order to improve sensing performance. Generally, when an outer layer film is in an aqueous solution, its structure and electrochemical properties may change during measurement due to changes in hydration state, and this phenomenon is also described in Patent Document 1. Therefore, in order to achieve more accurate quantitative evaluation using a sensor with an outer layer film that may undergo such property changes, it is desirable to have a means to detect these property changes and correct the measured concentration value.

[0007] Therefore, the object of the present invention is to provide a means for detecting changes in the characteristics of an electrochemical sensor having an outer layer film and correcting the measured concentration value in order to achieve more accurate quantitative evaluation using the sensor.

[0008] The inventors diligently conducted research to solve the above problems. As a result, they discovered that in an electrochemical sensor that quantitatively measures a target substance based on an electrical signal generated by an oxidoreductase reaction, which includes a substrate, an electrode pair including a working electrode and a counter electrode provided on the substrate, and an outer layer film covering the electrode pair, the concentration of the target substance can be quantified more accurately by performing electrochemical impedance measurement (EIS) between the working electrode and the counter electrode, calculating the resistance change from the measured electrochemical impedance, calculating the electrical signal change from the value of the electrical signal during electrochemical impedance measurement, and correcting the concentration measurement of the target substance based on the resistance change and the electrical signal change using a correction calculation formula, thus completing the present invention.

[0009] One aspect of the present invention relates to a method for correcting a concentration measurement of a target substance in an electrochemical sensor that quantitatively measures a target substance based on an electrical signal generated by an oxidoreductase reaction, comprising a substrate, an electrode pair comprising a working electrode and a counter electrode provided on the substrate, and an outer layer film covering the electrode pair, the method comprising: measuring electrochemical impedance between the working electrode and the counter electrode; calculating a change in resistance from the measured value of the electrochemical impedance; calculating a change in electrical signal from the value of the electrical signal during the electrochemical impedance measurement; and correcting the concentration measurement of the target substance based on a correction calculation formula using the change in resistance and the change in electrical signal. In one aspect, the electrochemical sensor may be bio-implantable. In one aspect, the value of the electrical signal may be a current value. In one aspect, the oxidoreductase may be a direct electron transfer type oxidoreductase. In one aspect, the outer layer film may contain a hydrophilic polymer and / or a hydrophobic polymer. In one embodiment, the potential in the electrochemical impedance measurement can be a potential that is sufficiently high for the substance to be measured to react, for example, a potential that is sufficiently high than the redox potential of the active site of an oxidoreductase (e.g., +100 to +200 mV (vs Ag / AgCl), preferably +150 mV (vs Ag / AgCl)). In one embodiment, the measurement frequency band in the electrochemical impedance measurement can be a frequency band between 0.1 Hz and 100 kHz. In one embodiment, the resistance change can be the ratio of the resistance value Rt at time t of the electrochemical impedance measurement to the resistance value Rinitial at the reference time, Rt / Rinitial. The electrical signal change can be the ratio of the electrical signal value It at time t of the electrochemical impedance measurement to the electrical signal value Iinitial at the reference time, It / Iinitial (the electrical signal may be a current value). The correction calculation formula is the product of the change in electrical signal value It / Iinitial and the change in resistance value Rt / Rinitial before and after the electrochemical impedance measurement, and the correction can be performed by multiplying this by the concentration measurement value of the target substance.

[0010] According to the present invention, in the quantitative determination of a target substance using an electrochemical sensor having an outer layer membrane and utilizing an oxidoreductase reaction, highly accurate quantitative evaluation can be achieved by correcting the measured concentration value using electrochemical impedance measurement. Specifically, by periodically measuring electrochemical impedance between the working electrode and the counter electrode, calculating the change in resistance and current, and using a correction calculation formula based on these, it is possible to estimate the limiting state of substance permeability of the outer layer membrane, which is intended for biological implantation and limits the amount supplied to the enzyme constituting the electrochemical sensor, and correct the converted concentration value in this sensor. In particular, in the case of an implantable sensor, when the reaction current during measurement changes discontinuously due to external impact, etc., this can be corrected. When a sudden change in impedance (decrease in resistance) is detected due to external impact, compression, membrane damage, etc., it contributes to overestimating the measured current (concentration converted value), but by using a correction calculation formula that combines the current change It / Iinitial with the resistance change Rt / Rinitial, the overestimation of the concentration can be suppressed. Furthermore, the damage and deterioration status of sensor components (e.g., electrodes, enzyme layer, outer film, etc.) can be diagnosed non-destructively and periodically.

[0011] This graph shows the results of chronoamperometry (CA) measurements performed while regularly conducting EIS (Example 1, N1-7). This graph shows the CA measurement results in Comparative Example 1 (Comparative Example 1, N=8-18) where EIS was not performed. This graph shows the Nyquist plot for Example 1. This graph shows the result of plotting the reciprocal value of the R1 value (1 / R1) against the glucose oxidation current value at that time. These graphs show the results calculated based on the respective conversion formulas. Cur. Glu is a graph obtained using equation (2), and Cal. Glu is a graph of the glucose concentration calculation results obtained using equation (3).

[0012] The present invention provides a method for correcting the concentration measurement value of a target substance, which involves measuring the concentration of a target substance using an electrochemical sensor that includes a substrate, an electrode pair including a working electrode and a counter electrode, both of which are provided on the substrate and contain an oxidoreductase electrode, and an outer layer film covering the electrode pair, and which quantitatively measures the target substance based on an electrical signal generated by an oxidoreductase reaction. The method includes measuring electrochemical impedance between the working electrode and the counter electrode, calculating a change in resistance from the measured value of the electrochemical impedance, calculating a change in the electrical signal from the value of the electrical signal during the electrochemical impedance measurement, and correcting the concentration measurement value of the target substance based on a correction calculation formula using the change in resistance and the change in the electrical signal.

[0013] (Electrochemical Sensor) The electrochemical sensor used in the method of the present invention includes a substrate, an electrode pair including a working electrode and a counter electrode, which are provided on the substrate and contain an oxidoreductase electrode, and an outer layer film covering the electrode pair. The electrode pair is an electrode pair including an enzyme electrode (working electrode) containing an oxidoreductase and a counter electrode, but it may also be a three-electrode system comprising an enzyme electrode, a counter electrode, and a reference electrode.

[0014] (Substrate) The electrode, including the electrode pair, is provided on a substrate, and an insulating substrate is used as the substrate. The type of insulating substrate is not particularly limited, but for example, it can be made of various thermoplastic resins such as polyetherimide (PEI), polyethylene terephthalate (PET), and polyethylene (PE), various thermosetting resins such as polyimide resin and epoxy resin, or insulating materials such as glass, ceramic, and paper. The size and thickness of the electrode and substrate can be set as appropriate, but the thickness of the electrode is particularly preferably 1 μm to 100 μm.

[0015] (Enzyme electrode) The enzyme electrode includes an oxidoreductase enzyme arranged on the electrode in a state that allows for direct electron transfer with the electrode. The electrode is formed using metallic materials such as gold (Au), platinum (Pt), silver (Ag), and palladium (Pd), or carbon materials such as graphite, carbon nanotubes, graphene, and mesoporous carbon.

[0016] (Oxidoreductases) Examples of oxidoreductases applicable to the present invention include glucose oxidase (GOD), galactose oxidase, bilirubin oxidase, pyruvate oxidase, D- or L-amino acid oxidase, amine oxidase, cholesterol oxidase, choline oxidase, xanthine oxidase, sarcosine oxidase, L-lactic acid oxidase, ascorbic acid oxidase, alcohol dehydrogenase, glutamate dehydrogenase, cholesterol dehydrogenase, aldehyde dehydrogenase, glucose Examples include sulfate dehydrogenase (GDH), fructose dehydrogenase, sorbitol dehydrogenase, lactate dehydrogenase, malate dehydrogenase, glycerol dehydrogenase, 17B hydroxysteroid dehydrogenase, estradiol 17B dehydrogenase, amino acid dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, 3-hydroxysteroid dehydrogenase, diaphorase, cytochrome oxidoreductase, catalase, peroxidase, and glutathione reductase. In particular, sugar oxidoreductases such as glucose oxidoreductase are preferred, and examples of sugar oxidoreductases include glucose oxidase (GOD), galactose oxidase, glucose dehydrogenase (GDH), fructose dehydrogenase, and sorbitol dehydrogenase. Therefore, the electrochemical sensor of the present invention can be used as a glucose sensor, cholesterol sensor, ethanol sensor, sorbitol sensor, fructose sensor, cellobiose sensor, lactic acid sensor, uric acid sensor, etc., depending on the type of enzyme.

[0017] It is preferable to use an oxidoreductase that can directly transfer electrons to and from the electrode (a direct electron-transfer type oxidoreductase). Using a direct electron-transfer type oxidoreductase has the advantage that electron transfer occurs rapidly and efficiently, making it easier to clearly see the charge transfer resistance component in impedance measurements and to distinguish between changes in the state of the sensor components and changes caused by electrochemical reactions. Examples of direct electron-transfer type oxidoreductases include oxidoreductases that physiologically contain redox molecules involved in electron transfer with the electrode. For example, an oxidoreductase containing an electron transfer subunit or an electron transfer domain as such a redox molecule can be used. Examples of electron transfer subunits include subunits containing heme, and examples of oxidoreductases containing heme subunits include those containing cytochrome such as cytochrome c and cytochrome b. Enzymes containing cytochrome-containing subunits as electron transport subunits include, for example, glucose dehydrogenase (GDH), sorbitol dehydrogenase (Sorbitol DH), D-fructose dehydrogenase (Fructose DH), lactate dehydrogenase, and uric acid oxidase.

[0018] For example, a cytochrome-containing glucose dehydrogenase is cytochrome glucose dehydrogenase (CyGDH) having an α-subunit containing FAD. Examples of CyGDH include FAD-dependent glucose dehydrogenase derived from Burkholderia cepacia or its variants. Examples of variants of FAD-dependent glucose dehydrogenase derived from Burkholderia cepacia include a variant in which the amino acid residues at positions 472 and 475 are substituted (WO 2005 / 103248), a variant in which the amino acid residues at positions 326, 365, and 472 are substituted (JP 2012-090563), and a variant in which positions 365 and 326, 472, 475, and 529 are substituted (WO 2006 / 137283).

[0019] Furthermore, enzymes containing electron transport domains include those containing heme domains and cytochrome domains, specifically, for example, quinohem ethanol dehydrogenase (QHEDH (PQQ Ethanol dh)). In addition, enzymes containing a cytochrome domain as an electron transport domain include, for example, "QHGDH" (fusion enzyme; GDH with heme domain of QHGDH) and cellobiose dehydrogenase. The fusion protein of PQQ glucose dehydrogenase (PQQGDH) and cytochrome disclosed in international publication WO2005 / 030807 can also be used.

[0020] On the other hand, it has been reported that by modifying oxidoreductases with artificial electron acceptors or nanomaterials, or by modifying electrodes with artificial electron acceptors or nanomaterials, direct electron transfer between oxidoreductases and electrodes becomes possible, and enzyme electrodes capable of direct electron transfer can be constructed. Such enzymes can also be used as direct electron-transfer type oxidoreductases. Here, an artificial electron acceptor is any non-catalytic compound that accepts electrons from an oxidoreductase, is reduced, and then reoxidized at the electrode. Examples include quinone compounds (e.g., 1,4-Naphthoquinone, VK3, 9,10-Phenanthrenequinone, 1,2-Naphthoquinone, p-Xyloquinone, Methylbenzoquinone, 2,6-Dimethylbenzoquinone, Sodium 1,2-Naphthoquinone-4-sulfonate, 1,4-Anthraquinone, Tetramethylbenzoquinone, Thymoquinone), and phenylenediamine compounds (e.g., N, Examples include N-Dimethyl-1,4-phenylenediamine, N,N,N',N'-tetramethyl-1,4-phenylenediamine dihydrochloride, 1-Methoxy-PMS (1-Methoxy-5-methylphenazinium methylsulfate), PES (Phenazine ethosulfate), Coenzyme Q0, AZURE A Chloride, Phenosafranin, 6-Aminoquinoxaline, and Tetrathiafulvalene. To modify oxidoreductases with artificial electron acceptors, one method is to chemically bind the artificial electron acceptor to the enzyme. For example, one method involves introducing a functional group such as succinimide into an artificial electron acceptor and then reacting it with the amino group of an enzyme to introduce the electron acceptor.Furthermore, in the case of nanomaterials, conductive materials that can be positioned within a distance of the enzyme's active center to directly exchange electrons are examples such as carbon nanotubes (Analytical Biochemistry, Volume 332, Issue 1, 1 September 2004, Pages 75-83) and metal nanoparticles (Analytical Biochemistry, Volume 331, Issue 1, 1 August 2004, Pages 89-97), but are not limited to these as long as direct electron transfer can be observed in the material.

[0021] A "direct electron transfer type enzyme electrode" is a type of enzyme electrode in which electrons generated by an enzymatic reaction in the reagent layer are directly transferred to the electrode without the need for diffusion of redox substances such as electron transfer mediators, thereby enabling electron transfer between the enzyme and the electrode.

[0022] To create a direct electron transfer type enzyme electrode, it is important to position the oxidoreductase enzyme near the electrode. Since the limiting distance at which direct electron transfer occurs in a physiological reaction system is said to be 10 to 20 Å, it is important to position the enzyme at a distance closer to the electrode than this to avoid impairing electron transfer from the enzyme to the electrode. There are no particular limitations on the methods for achieving this, but examples include chemically immobilizing the oxidoreductase enzyme on the electrode, indirectly immobilizing the oxidoreductase enzyme on the electrode using conductive polymers or crosslinking agents (e.g., WO2014 / 002999 or JP 2016-121989), and immobilizing the enzyme on the electrode via monolayer forming molecules (JP 2017-211383).

[0023] (Other electrode components) Other components in the enzyme electrode may include buffers, sugars, surfactants, and electron mediators (when direct electron transfer type oxidoreductase is not used), as appropriate. In the electrochemical sensor used in the method of the present invention, the insulating film may be formed on the substrate other than on the electrode.

[0024] (Outer film) The outer film covering the electrode pair functions as a diffusion barrier for the substance being measured and also plays a role in enhancing the biocompatibility of the sensor. The state of the outer film (e.g., damage or degradation of the film) is directly reflected in the impedance measurement results, so changes in the state of the outer film can be monitored by impedance measurement. Specifically, when the outer film is damaged, the diffusion resistance of the substance being measured decreases, and the shape of the impedance spectrum changes.

[0025] The outer layer membrane covering the electrode pair is composed of a biocompatible membrane that is permeable to the substance to be measured. Specifically, for example, the membrane is formed from polyurethane, silicone polymer (polysiloxane), cellulose acetate (CA), hydrogel, polyvinyl alcohol, polyvinylpyrrolidone (PVP), HEMA (hydroxyethyl methacrylate), or copolymers containing these (preferably a combination of hydrophilic and hydrophobic polymers, more preferably a combination of CA and PVP; (e.g., CA 70-90 wt%, PVP 10-30 wt%)), creating a structure that allows the substance to be measured to penetrate into the interior. This allows the substance to be measured, upon contact with the surface of the outer layer membrane, to penetrate into the interior of the outer layer membrane, enabling it to reach the enzyme electrode, and also allowing control of the rate at which the substance reaches the electrode.

[0026] (Measurement Method) When a sample containing the substance to be measured is brought into contact with the electrochemical sensor of the present invention, the substance to be measured permeates the outer layer film and reacts with a reagent containing oxidoreductase on the enzyme electrode, causing an oxidation-reduction reaction. After the reaction, an electrical signal based on the reaction is generated by applying a voltage between the electrode pair. By detecting this electrical signal (e.g., current), the substance to be measured can be measured. Specifically, in the case of a sensor containing a direct electron transfer type oxidoreductase, electrons generated by the oxidation reaction of the substance to be measured by the oxidoreductase are directly transferred to the electrode. Then, by applying a voltage between the electrode pair, an oxidation current dependent on the amount of the substance to be measured in the sample is generated. By measuring this current value, the concentration of the substance to be measured in the sample can be measured based on the current value.

[0027] When calculating the concentration of a substance to be measured, the amperometry method is employed. The amperometry method involves applying a constant voltage to an electrode, obtaining the response current value a certain time after the start of the reaction, and calculating the concentration of the substance to be measured based on this response current value.

[0028] (Electrochemical Impedance Measurement) Impedance measurement can be performed based on known methods, but specifically, a method of applying a sine wave to an enzyme electrode at one or more frequencies centered around a constant DC bias voltage, i.e., by changing the frequency, is preferred. The DC bias voltage is preferably higher than the redox potential of the redox molecule (electron transfer subunit, electron transfer domain, or artificial electron acceptor) that directly transfers electrons with the electrode. For example, when using an oxidoreductase containing an electron transfer subunit or electron transfer domain containing heme (cytochrome) as the redox molecule, it can be +100 mV (vs Ag / AgCl) or higher or +300 mV (vs Ag / AgCl) or higher. Also, when using PES as the redox molecule, it can be, for example, +100 mV (vs Ag / AgCl) or higher. There is no particular upper limit, but for example, it is +1000 mV. In one example, the voltage is, for instance, +100 to +200 mV (Ag / AgCl), preferably +150 mV (Ag / AgCl).

[0029] Specifically, for example, first, the DC bias voltage is applied between the working electrode and the counter electrode. Next, an AC voltage is superimposed on the DC bias voltage and applied to the working electrode. It is preferable that this applied AC voltage is as low as possible while still allowing the impedance between the working electrode and the counter electrode to be measured. If the value of the AC voltage applied to the working electrode is too high, an electrochemical reaction will occur on the electrode surface, causing problems such as a change in the surface condition of the electrode. Conversely, if the value of the AC voltage is too low, the signal-to-noise ratio will decrease, and the impedance measurement will become inaccurate. For this reason, the voltage applied between the working electrode and the counter electrode is preferably an amplitude of 5 to 20 mV.

[0030] Then, using a frequency response analyzer, the impedance between the working electrode and the counter electrode is measured by changing the frequency of the applied AC voltage. The variable range of this frequency can be any range in which impedance measurement is possible, but for example, the frequency can be changed between a first frequency (lower limit) of 0.1 mHz to 100 mHz and a second frequency (upper limit) of 10 kHz to 1 MHz. More specifically, it is appropriate to change the frequency within the range of 0.1 mHz to 100 kHz or 0.1 Hz to 100 kHz.

[0031] Next, the resistance value is calculated from the impedance value obtained by electrochemical impedance measurement. The resistance value can be determined, for example, by drawing a Nyquist plot based on data obtained from a frequency response analyzer and finding the intersection of the semicircles with the real axis. Specifically, the resistance value can be calculated as the distance from the intersection of the high-frequency arc and the real axis to the intersection of the low-frequency arc and the real axis.

[0032] (Concentration correction method based on electrochemical impedance measurement) The concentration correction method of the present invention includes measuring electrochemical impedance between the working electrode and the counter electrode, calculating the change in resistance from the measured value of the electrochemical impedance, calculating the change in electrical signal from the value of the electrical signal during the electrochemical impedance measurement, and correcting the measured concentration of the target substance based on a correction calculation formula using the change in resistance and the change in electrical signal.

[0033] In this invention, it was found that the change in resistance value calculated by electrochemical impedance measurement and the change in measured current value are inversely proportional. The inverse relationship between current and resistance corresponds to the relationship I=E / R (Ohm's law), and in the embodiment, a linear relationship between 1 / R1 and the current value has been confirmed (Figure 4). By utilizing this relationship, changes in the permeability characteristics of the outer layer film can be compensated for. Therefore, the measured concentration of the target substance can be corrected by multiplying the measured value of the target substance concentration (Gt) calculated by chronoamperometry, etc., by the product of the current change value (It / Iinitial), that is, the ratio of the current value at measurement to the current value at the reference time (e.g., at the start of measurement), and the resistance change value (Rt / Rinitial), that is, the ratio of the resistance value at measurement to the resistance value at the reference time (e.g., at the start of measurement).

[0034] The formula for converting glucose concentration using the resistance value obtained by EIS is as follows: Glu(corrected value) = (It / Iinitial) × (Rt / Rinitial) × Glu(measured value) where It is the current value at the time of measurement (t), Iinitial is the current value at the reference time (e.g., at the start of measurement), and Glu(measured value) is the glucose concentration obtained by chronoamperometry, etc., Rt is the resistance value at the time of measurement (t) (calculated from the impedance value at t), and Rinitial is the resistance value at the reference time (e.g., at the start of measurement) (calculated from the impedance value at the reference time). Note that if current measurement and electrochemical impedance measurement cannot be measured simultaneously, Rt and Rinitial may be values ​​calculated from electrochemical impedance measurements at a time close to the measurement of It and Iinitial, respectively. In addition, although the above formula was explained using glucose concentration, the same correction is possible for other substances being measured.

[0035] The reference point (initial) is preferably the time when the first EIS is performed after the target substance concentration has been adjusted to a predetermined value and the electrochemical reaction has stabilized. Performing EIS immediately after starting continuous measurement reduces the acquisition error of Rinitial and improves the accuracy of correction. In continuous measurement, even if the target substance concentration remains constant, the current may increase over time due to changes in the hydration structure of the outer layer film, etc. Therefore, it is desirable to perform EIS periodically and update Rt to perform correction.

[0036] For example, a concentration correction method based on electrochemical impedance measurement may include the following steps: Step (1): Obtain Rinitial by initial EIS and simultaneously obtain the initial current Iinitial. Step (2): Obtain It by CA at measurement time t, and obtain Rt by performing EIS at a nearby time (if simultaneous measurement is not possible, it can be done at a nearby time). Step (3): Calculate each ratio: ΔI ratio = It / Iinitial, ΔR ratio = Rt / Rinitial. Step (4): Concentration correction: Glu (corrected value) = (It / Iinitial) × (Rt / Rinitial) × Glu (measured value). Applicable to other analytes as well.

[0037] The present invention will be specifically described below with reference to examples. However, the present invention is not limited to the following embodiments.

[0038] (Example 1) <Procedure for fabricating an electrochemical sensor - common to both the example and comparative example> A metal layer was formed on a polyether ether ketone substrate by sputtering metal (gold). Furthermore, a portion of the metal layer was removed by etching to form an electrode pattern on the metal layer. The following reagents (dissolved in pure water to their respective final concentrations) were applied to the electrodes. By drying and heating, an enzyme layer (reagent layer) in which an electrochemical reaction occurs was fabricated.

[0039] Final reagent concentration: Phosphate buffer 24 mM, Trehalose 1.03 wt%, Cytochrome c-modified glucose dehydrogenase (GDH) 20.4 mg / mL, Crosslinking agent 12%, Carbon black dispersion 0.96 wt%

[0040] <Outer film deposition procedure common to both examples and comparative examples> An outer film was deposited on the reagent layer described above in order to limit the amount of glucose supplied to GDH and for the purpose of biological implantation. The specific method for depositing the outer film is as follows: (1) Cellulose acetate (CA) and polyvinylpyrrolidone (PVP) were weighed out in weight ratios of 84 wt% and 16 wt%, respectively. (2) The powders from (1) were dissolved in a tetrahydrofuran / ethanol mixed solvent (THF:EtOH = 7:3 volume ratio) to which 17.5 vol% ethyl lactate was added to make a composition of 2 wt%. (3) The solution prepared in (2) was spray-coated onto the sensor using a spray-coating device (rCoater Model No.: ETA09710 Serial No.: FS2670, manufactured by Asahi Sanac Co., Ltd.).

[0041] The spray coating conditions were set as follows: Distance from nozzle to sensor: 55 mm; Nozzle movement speed: 200 mm / s; Nozzle movement interval: 1 mm; Nozzle pressure: 150 kPa; Solution discharge rate: 2.5 mL / min

[0042] (4) The spray coating process was repeated six times. Before each spray coating, the sensor with the outer film was left to stand in the spray coating chamber for 5 minutes to remove the solvent from the outer film. The sensor with the outer film was stored at low humidity (humidity 10% or less).

[0043] (Electrochemical Measurement Procedure) In Example 1, the following procedure was followed: (1) Continuous measurement in a 550 mg / dL glucose aqueous solution (using a homemade potentiostat) Chronoamperometry (CA measurement) was started in a phosphate buffer containing 100 mg / dL glucose using a vial cell. Then, the concentration was adjusted by adding phosphate buffer and 2 mol / L glucose solution to achieve glucose concentrations of approximately 70 → 180 → 500 → 550 mg / dL. Liquid paraffin was then added to the measurement solution. The applied voltage for continuous measurement was set to +150 mV (vs Ag / AgCl).

[0044] (2) Performing Electrochemical Impedance Spectroscopy (using a Biologic potentiostat VSP-300) (2-1) The sensor on which CA measurement was being performed in (1) was disconnected from the self-made potentiostat after a certain period of time had elapsed after preparing the glucose concentration to 550 mg / dL, and then connected to the Biologic potentiostat VSP-300 to perform EIS. EIS was performed 1 h, 3 h, and 5 h after preparing an aqueous glucose solution at 550 mg / dL, and the change in impedance value during the period when the measured current change amount was large was examined. Thereafter, EIS was performed periodically during the measurement period.

[0045] (2-2) Measurement Conditions for EIS EIS was performed according to the following procedure. (1) Before performing the alternating current impedance measurement, a potential holding operation of 150 mV (vs Ag / AgCl) for 1 minute was performed to stabilize the electrochemical reaction on the enzyme. (2) The measurement conditions were as follows.

[0046] Applied voltage; +150 mV (vsAg / AgCl) Amplitude; +10 mV Measurement frequency; 100 kHz to 0.1 Hz decade; 20

[0047] EIS was performed in a combination so as to be a normal three-electrode measurement. Working electrode: Reaction part of the reagent-coated sensor with an outer layer film Counter electrode: Gold exposed part of the reagent-coated sensor with an outer layer film Reference electrode: Ag / AgCl (coated on the sensor) Measurement solution: Phosphate buffer solution containing approximately 550 mg / dL glucose

[0048] The sensor resistance value (R1) was calculated by obtaining the intersection of the semi-circle (thought to include film resistance and charge transfer resistance) seen on the high-frequency side and the real axis in the obtained Nyquist plot.

[0049] (Comparative Example 1) As a control and comparative condition for Example 1, CA measurement was continuously performed without performing (2) of (the procedure for electrochemical measurement). The influence of the presence or absence of EIS on the CA measurement values was evaluated based on the chronoamperogram of this Comparative Example 1.

[0050] 1. Relationship between glucose current value and impedance resistance value. Figure 1 shows the results under conditions (Example 1, N1-7) in which CA measurements were performed while EIS was periodically conducted.

[0051] Figure 2 shows the CA measurement results in Comparative Example 1 (N=8-18), where EIS was not performed. Similar to Example 1, an upward trend in the measured current value was observed. The current value 14 days after the start of measurement was approximately 1.8 times higher than the initial current value of 550 mg / dL. Despite the glucose concentration of the reactant remaining constant, this continuous increase in current is thought to be due to the permeation limiting performance of the outer film changing in the aqueous solution. It is presumed that when the outer film is in the aqueous solution, the distribution of each component of the polymer constituting the outer film changes over time due to changes in the hydration structure, etc.

[0052] Figure 3 shows the Nyquist plot for Example 1. A high-frequency semicircular component originating from charge transfer resistance or membrane resistance and Warburg impedance originating from mass diffusion were observed. The decrease in the diameter of the semicircle over time indicates that glucose permeation into the membrane became easier. This suggests that structural changes occurred in the CA / PVP membrane while it was immersed in the measurement solution. The diameter of the semicircle was determined and its value (R1) was calculated.

[0053] In general, Ohm's law holds true for electronic circuits, as shown in the following equation, and the current is proportional to the reciprocal of the resistance: I = E / R ... (1) Here, I is the current, E is the electric potential, and R is the resistance.

[0054] Figure 4 shows the results of plotting the reciprocal value of the calculated R1 value (1 / R1) against the corresponding glucose oxidation current value. Except for N7, the plots for the six sensor conditions lie on a straight line. This indicates that Ohm's law holds true in this measurement system. Therefore, if the current and resistance values ​​at a known glucose concentration can be measured, it can be predicted that the sensor's characteristics will change along the above straight line (master curve). Furthermore, if the measured value deviates from this master curve, it can be determined that the sensor is faulty, or a different master curve can be used for case analysis and prediction. The reason why the N7 plot differs from the other sensors is unknown, but it is thought to be due to something specific to that sensor.

[0055] 2. Glucose Concentration Conversion Using Impedance Measurements In this study, we attempted to convert and correct the glucose concentration under a constant value of 550 mg / dL. First, we will explain the method for calculating glucose concentration in normal measurements. The normal method for calculating glucose concentration was calculated according to the following formula.

[0056] (Cur. Glu) = (It / Iinitial) × Glu(real) ... (2) Here, Cur. Glu: glucose concentration conversion value, It: measurement current value at time point (t) 1 hour after adjusting to a glucose concentration of 550 mg / dL, Iinitial: measurement current value at time point 1 hour after adjusting to a glucose concentration of 550 mg / dL (start of measurement), and Glu(real): glucose concentration obtained using GA-1153 (Arkray).

[0057] Next, the glucose concentration conversion formula using the resistance value obtained by EIS is as follows: (Cal. Glu) = (It / Iinitial) × (Rt / Rinitial) × Glu(real) ... (3) Here, Rinitial is the resistance value obtained 1 hour after adjusting to a glucose concentration of 550 mg / dL (at the start of measurement) (calculated from the impedance value at the start of measurement), and Rt is the resistance value obtained at a point in time (t) after 1 hour has elapsed since adjusting to a glucose concentration of 550 mg / dL (calculated from the impedance value at time t).

[0058] Figure 5 shows the results calculated based on each conversion formula. Cur. Glu is the graph obtained using formula (2), and Cal. Glu is the graph of the glucose concentration calculation result obtained using formula (3). In the conversion using glucose oxidation current, the calculated glucose concentration increased along with the increase in the current value, and after 16 days, the calculated glucose concentration was approximately twice the initial value. On the other hand, in the conversion formula using the resistance value obtained by EIS, the increase was significantly smaller and the correction was well performed, with the calculated glucose concentration after 16 days being approximately 1.3 times the initial value. In the correction for the N4 sensor, despite the correction, the value remained somewhat high (Cal. Glu = 700 mg / dL) compared to the other conditions. This is because the initial EIS was performed on the N4 sensor one day after the start of measurement, so the Rinitial measurement value was 0.85 times that of the other conditions. Therefore, in the correction method using formula (3) which utilizes the resistance value obtained from EIS, it is important to perform EIS at the beginning of continuous measurement for more accurate correction.

Claims

1. A method for correcting a measured concentration of a target substance in an electrochemical sensor that quantitatively measures a target substance based on an electrical signal generated by an oxidoreductase reaction, comprising a substrate, an electrode pair including a working electrode and a counter electrode provided on the substrate, and an outer layer film covering the electrode pair, the method comprising: measuring electrochemical impedance between the working electrode and the counter electrode; calculating a change in resistance from the measured value of the electrochemical impedance; calculating a change in electrical signal from the value of the electrical signal during the electrochemical impedance measurement; and correcting the measured concentration of the target substance based on a correction formula using the change in resistance and the change in electrical signal.

2. The method according to claim 1, wherein the electrochemical sensor is implantable in a biomedical environment.

3. The method according to claim 1, wherein the value of the electrical signal is a current value.

4. The method according to claim 1, wherein the oxidoreductase is a direct electron transfer type oxidoreductase.

5. The method according to claim 1, wherein the outer layer film comprises a hydrophilic polymer and a hydrophobic polymer.

6. The method according to claim 1, wherein the potential in the electrochemical impedance measurement is a potential at which the substance to be measured reacts sufficiently.

7. The method according to claim 1, wherein the measurement frequency band in the electrochemical impedance measurement is a frequency band between 0.1 Hz and 100 kHz.

8. The method according to any one of claims 1 to 7, wherein the resistance change is the ratio of the resistance value Rt at time t of electrochemical impedance measurement to the resistance value Rinitial at reference time, Rt / Rinitial, the electrical signal change is the ratio of the electrical signal value It at time t of electrochemical impedance measurement to the electrical signal value Iinitial at reference time, It / Iinitial, and the correction calculation formula is the product of It / Iinitial and Rt / Rinitial.

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