Method for correcting electrochemical sensor using electrochemical impedance measurement

Abstract

Provided is a method for managing the accuracy of an electrochemical sensor which includes a base material, an electrode pair that includes a counter electrode and a working electrode including an oxidoreductase electrode provided on the base material, and an outer layer film that covers the electrode pair and which quantitatively measures a target substance on the basis of an electrical signal generated by an oxidoreductase reaction, said method comprising periodically performing electrochemical impedance measurement between the working electrode and the counter electrode and monitoring a change in electrochemical impedance.

Description

Correction Method for Electrochemical Sensor Using Electrochemical Impedance Measurement

[0001] The present invention relates to a technique for detecting changes in electrochemical characteristics that may occur due to external shocks or the like in an electrochemical sensor used by being indwelling in the body, and correcting measured values by means of an electrochemical impedance measurement method.

[0002] In addition to the problems of initial stabilization and wetting of the electrochemical sensor, during its lifetime, the surface area of the electrode becomes narrower due to contaminating species such as peptides and microprotein molecules in the electrochemical sensor, or the diffusion path of the analyte and / or reaction by-products becomes narrower, and as a result, the accuracy of the sensor may decrease. There has been no explanation yet about how to determine that such contaminants have started to affect the sensor signal, and how to deal with such a state. Therefore, as a method and system for real-time maintenance, a technique has been proposed in which an electrochemical impedance spectroscopy (EIS) is used to verify that the sensor is functioning properly, and a voltage is applied for sensor recovery based on its impedance value (Patent Document 1).

[0003] Special Table 2010-537198

[0004] In the EIS and sensor recovery techniques proposed in Patent Document 1, it is difficult to determine whether the measured impedance value is derived from the components of the sensor or from an electrochemical reaction, correctly estimate the component state of the sensor, and perform correction suitable for the recovery of the sensor sensitivity. Also, the current value of the sensor components may change discontinuously due to external shocks or deterioration. Therefore, there is a need for a method of measuring and recognizing by some means whether the current value being measured currently can correctly reflect the concentration of the measurement target substance, and correcting it.

[0005] Therefore, an object of the present invention is to provide a technique capable of accurately grasping the state of an electrochemical sensor, correcting a measurement current value as necessary, and managing the accuracy of measurement using the electrochemical sensor in the quantification of a measurement target substance using the electrochemical sensor.

[0006] The inventors diligently conducted research to solve the above problems. As a result, they found that in quantitative measurement of a target substance using an electrochemical sensor which includes a substrate, an electrode pair including an oxidoreductase electrode provided on the substrate, and an outer layer film covering the electrode pair, and which quantitatively measures a target substance based on an electrical signal generated by an oxidoreductase reaction, it is possible to monitor changes in the state of the electrochemical sensor by periodically measuring the electrochemical impedance between the working electrode and the counter electrode and monitoring the changes in electrochemical impedance, thereby detecting changes in the state of the electrochemical sensor and controlling its accuracy. Specifically, they found that elements other than charge transfer resistance, namely the resistance and capacitance components of the outer layer film and reagent layer, are dominant in electrochemical impedance, and that by monitoring changes in electrochemical impedance, it is possible to detect changes in the state of the electrochemical sensor due to external shocks, etc.

[0007] Furthermore, we discovered that quantitative analysis can be performed with greater accuracy by measuring electrochemical impedance between the working electrode and the counter electrode, calculating the change in resistance from the measured electrochemical impedance, calculating the change in the electrical signal based on the change in resistance, and correcting the value of the electrical signal. Specifically, we found that the change in resistance calculated from the measured electrochemical impedance correlates with the change in current, and that quantitative analysis can be performed with greater accuracy by calculating the change in current affected by the change in resistance due to damage or deterioration of the outer film, and correcting the measured value with this change in current. Based on these discoveries, we have completed the present invention.

[0008] In one embodiment, the present invention relates to a method for quality control of 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 periodically measuring the electrochemical impedance between the working electrode and the counter electrode to monitor changes in the electrochemical impedance. Monitoring changes in the electrochemical impedance may also be done by monitoring changes in resistance values ​​calculated based on the intersection of a semicircle and the real axis of a Nyquist diagram.

[0009] In another embodiment, the present invention relates to a method for quality control of 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 the change in resistance from the measured electrochemical impedance; calculating the change in the electrical signal based on the change in resistance; and correcting the value of the electrical signal. The method for quality control of an electrochemical sensor may be a method comprising: measuring electrochemical impedance between the working electrode and the counter electrode; measuring resistance from the measured electrochemical impedance; calculating the resistance at a reference time or the change from the reference resistance; calculating the change in the electrical signal from the change in resistance; and correcting the measured value of the electrical signal using the change in the electrical signal.

[0010] In one embodiment, the electrochemical sensor may be bio-implantable. In one embodiment, the value of the electrical signal may be a current value. In one embodiment, the oxidoreductase may be a direct electron-transfer type oxidoreductase. In one embodiment, the outer layer film may contain hydrophilic polymers and hydrophobic polymers. In one embodiment, the potential in the electrochemical impedance measurement may be a potential at which the substance being measured reacts sufficiently, for example, a potential sufficiently higher than the redox potential of the active site of the 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 may be a frequency band between 100 kHz and 0.1 Hz.

[0011] According to the present invention, in quantitative analysis of a target substance using an electrochemical sensor, the state of the electrochemical sensor can be accurately grasped, and the measured current value can be corrected as necessary to perform more accurate measurements. More specifically, in an implantable sensor, when the reaction current during measurement changes discontinuously due to external shock or the like, this can be detected and corrected. A discontinuous change in the current value appears as a rapid change in the impedance value (for example, a significant decrease in the impedance value as shown in Figure 4). Furthermore, this is a method that can non-destructively and periodically diagnose the damage and deterioration status of sensor components (e.g., electrodes, enzyme layer, outer membrane, etc.).

[0012] Figure 1 is a graph showing the chronoamperometry results (change in current value over time) evaluated in Comparative Example 1. Figure 2 is a graph showing the chronoamperometry results (change in current value over time) evaluated in Example 1. Figure 3 is a graph showing the Nyquist plot measured in Comparative Example 1. Figure 4 is a graph showing the Nyquist plot measured in Example 1, showing the change in impedance value before and after external impact to the sensor. Figure 5 is a graph showing the results of electrochemical impedance measurements performed with varying glucose concentrations. Figure 6 is a graph plotting the change in resistance value obtained from the impedance value before and after damaging the sensor against the change in glucose reaction current value.

[0013] A method for controlling the accuracy of an electrochemical sensor according to one aspect of the present invention is characterized in that, in quantitative measurement using an electrochemical sensor that quantitatively measures a target substance based on an electrical signal generated by an oxidoreductase reaction, electrochemical impedance measurements are periodically performed between the working electrode and the counter electrode to monitor changes in electrochemical impedance. Here, "periodically" includes performing measurements at intervals ranging from 10 minutes to 24 hours. Electrochemical impedance measurements may also be performed when the current changes abruptly or when abnormal values ​​are observed.

[0014] Furthermore, another aspect of the present invention relates to a method for controlling the accuracy of an electrochemical sensor, which 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. The method involves quantitatively measuring a target substance based on an electrical signal generated by an oxidoreductase reaction using an electrochemical sensor, and includes measuring the 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 the electrical signal based on the change in resistance, and correcting the value of the electrical signal. Here, the "change" in resistance or current can be a change from a predetermined reference point (for example, at the time of the initial measurement or the immediately preceding measurement) or a change relative to a predetermined reference value.

[0015] (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.

[0016] (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.

[0017] (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.

[0018] (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.

[0019] 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.

[0020] 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).

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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).

[0025] (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.

[0026] (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.

[0027] 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.

[0028] (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.

[0029] 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.

[0030] (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).

[0031] 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.

[0032] 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.

[0033] 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.

[0034] (Accuracy control method based on electrochemical impedance) In an accuracy control method for an electrochemical sensor according to one aspect of the present invention, in parallel with electrical signal measurement (current measurement), electrochemical impedance measurements are periodically performed between the working electrode and the counter electrode to monitor changes in electrochemical impedance. As shown in Figure 4, if an abnormality occurs in the state of the electrochemical sensor due to deterioration or damage to the outer layer film of the electrochemical sensor, fluctuations will occur in the graph showing the change in impedance over time (such as a Nyquist plot). By detecting such fluctuations, abnormalities in the electrochemical sensor, such as deterioration or damage to the outer layer film, can be detected, and appropriate measures such as concentration correction or stopping the measurement can be taken as needed. Therefore, the accuracy control method based on electrochemical impedance may further include a step of flagging the measured value or stopping the measurement if the amount of change in the resistance value exceeds a predetermined threshold.

[0035] A method for monitoring changes in electrochemical impedance according to one aspect of the present invention can be used to detect abnormalities in an electrochemical sensor, such as deterioration or damage to the outer layer film, and can also be used as a prerequisite step for correcting electrical signal values. That is, if an abnormality is detected by monitoring changes in impedance, more accurate measurement results can be obtained by correcting the electrical signal based on the amount of change.

[0036] Furthermore, in another aspect of the present invention, in a method for controlling the accuracy of an electrochemical sensor, electrochemical impedance is measured between the working electrode and the counter electrode in parallel with electrical signal measurement (current measurement), the change in resistance is calculated from the measured value of the electrochemical impedance, the change in the electrical signal is calculated based on the change in the resistance, and the value of the electrical signal actually measured is corrected based on the calculated change in the electrical signal. Here, the "change" in resistance or current can be a change from a predetermined reference point (for example, at the time of the initial measurement or the immediately preceding measurement) or a change from a predetermined reference value. That is, the resistance is calculated from the measured value of the electrochemical impedance, and the change in the resistance from the resistance at the reference point or from a reference resistance value can be taken as the "change in resistance." In other words, in the present invention, as shown in Figure 6, it was found that a negative correlation is observed between the change in resistance obtained from the impedance value and the change in current. Therefore, by utilizing this correlation, it is possible to calculate the change in the sensor's current value (deviation in current value) that is deviated from external disturbance factors such as damage or deterioration of the outer layer film, and it is possible to correct the measured value of the actual electrical signal using this change in current value.

[0037] For example, as shown in Figure 6, a calibration curve plotting the relationship between the change in resistance and the change in current can be prepared in advance. Based on this calibration curve, the change in current from the reference current at the time of impedance measurement can be calculated from the change in resistance at the time of impedance measurement (change in resistance), and the current at the time of measurement can be corrected considering this change in current. Then, based on the corrected current, a more accurate amount of the target substance can be calculated.

[0038] For example, correction based on the correlation between the change in resistance and the change in current can be performed in the following steps: (1) Determine the reference resistance value (R0) from the impedance measurement at a reference point (e.g., when the sensor is first used). (2) Determine the resistance value at the time of measurement (R1) from the impedance measurement at the time of measurement. (3) Calculate the change in resistance value (ΔR = R1 - R0). (4) Determine the change in current value (ΔI) corresponding to the change in resistance value (ΔR) from a pre-created calibration curve or relationship. (5) Calculate the corrected current value (I_corrected = I_measured - ΔI) from the measured current value (I_measured). (6) Calculate the concentration of the substance being measured from the corrected current value (I_corrected).

[0039] (Example 1) <Electrode Manufacturing Example: Common to Examples and Comparative Examples> 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. 36 nL of the following reagents (dissolved in pure water to their respective final concentrations) were applied to the electrode (metal layer), and an enzyme layer (reagent layer) in which an electrochemical reaction occurred was prepared by drying and heating.

[0040] Final concentration reagent: 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%

[0041] <Preparation Example of Outer Layer Membrane: Common to Examples and Comparative Examples> For the purpose of restricting the glucose supply amount to GDH and for in-vivo retention, an outer layer membrane was formed on the above reagent layer of the sensor. The specific method for forming the outer layer membrane is as follows. (1) Cellulose acetate (CA) and polyvinyl pyrrolidone (PVP) were weighed so that their weight ratios were 82.5 wt% and 17.5 wt% respectively. (2) The powder in (1) was dissolved in a mixed solvent of tetrahydrofuran / ethanol (THF:EtOH = 7:3 volume ratio) with 24 vol% ethyl lactate 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 (API-40RSII, Mini-DLCS specification manufactured by Yamana Kahutech Co., Ltd.).

[0042] The conditions for spray coating were set as follows. Distance from the set nozzle to the sensor: 85 mm Moving speed of the nozzle: 200 mm / s Moving interval of the nozzle: 1 mm Nozzle pressure: 300 kPa Solution delivery rate: 2.5 mL / min

[0043] (4) The spray coating operation was repeated 6 times. Before each spray coating, the sensor with the outer layer membrane formed was left standing in the spray coating chamber for 5 minutes to remove the solvent in the outer layer membrane. The sensor with the outer layer membrane formed was stored under low humidity (humidity 10% or less) and reduced pressure to prevent surface contamination.

[0044] The procedures for chronoamperometry and electrochemical impedance measurement are as follows. (1) Measurement was performed for approximately 70 hours or more under an applied voltage of 150 mV (vsAg / AgCl) in a phosphate buffer solution containing 550 mg / dL glucose. Chronoamperometry was performed using a self-made potentiostat. (2) The sensor during measurement was removed, immersed in the phosphate buffer solution for about 10 minutes, and then electrochemical impedance measurement was performed in a buffer solution containing 100 mg / dL glucose. Electrochemical impedance measurement was performed using a potentiostat (VSP-300) manufactured by Biologic Co., Ltd.

[0045] The conditions for electrochemical impedance measurement are as follows. Applied voltage: +150 mV (vs Ag / AgCl), Measurement frequency: 100 kHz - 0.1 Hz decade, 20 steps, Amplitude: 10 mV

[0046] (3) The sensor that had been measured in (2) was intentionally damaged by pinching a part of the measurement section with tweezers, and then the electrochemical impedance was measured again under the measurement conditions of (2). (4) After the operation in (3) was carried out, the measurement in (1) was performed again.

[0047] (Comparative Example 1) Among the operations of (Example 1), an evaluation was conducted on a case where the operation in (3) was not carried out as a comparative example.

[0048] Figure 1 shows the evaluation results of chronoamperometry evaluated in (Comparative Example 1). The concentration of the glucose solution during the measurement was approximately 550 mg / dL. The sensor was removed during the measurement for electrochemical impedance measurement, but almost no change in the current value was observed before and after that.

[0049] On the other hand, as shown in Figure 2, in the evaluation of chronoamperometry evaluated in (Example 1), the current value clearly increased by 30% or more after the EIS measurement and damage. This is because damage to the outer layer film allowed more glucose, which is the reactant, to be supplied to the reaction part on the electrode. On the other hand, it was confirmed that the current noise did not change significantly before and after the impact.

[0050] Figure 3 shows the Nyquist plot measured in (Comparative Example 1). Continuous measurements were performed in the above-mentioned approximately 550 mg / dL glucose solution, and electrochemical impedance measurements were performed in a 100 mg / dL glucose solution on the 3rd and 7th days after the start of measurements. Nyquist plots obtained from impedance measurements generally include solution resistance, membrane resistance, diffusion resistance of reactants, and charge transfer resistance for the reaction of reactants. In the results for (Comparative Example 1) shown in (Figure 3), a portion of the arc and a linearly extended plot were observed, and these plots were observed in almost the same position regardless of the continuous measurement time. In this system, the impedance component of the arc observed on the high-frequency side is thought to be due to the sensor components and electrochemical reaction, and the resistance value was estimated by finding the intersection of the arc and the real axis. The impedance plot in this system was observed in almost the same position regardless of the continuous measurement time. This indicates that the impedance value does not change much due to disturbance factors introduced by the sensor components and reactants over the measurement elapsed time.

[0051] Figure 4 shows the Nyquist plot measured in (Example 1). Continuous measurements were performed in the approximately 550 mg / dL glucose solution, and electrochemical impedance measurements were taken in a 100 mg / dL glucose solution on the 3rd and 7th days after the start of measurements. As shown in (Figure 4), the impedance value decreased significantly when the sensor was damaged by an external impact. On the other hand, the value did not change much regardless of the measurement time, so it is clear that the impedance value changed only when an impact was applied.

[0052] Figure 5 shows electrochemical impedance measurements performed at varying glucose concentrations. Generally, changing glucose concentration alters charge transfer resistance, resulting in changes in impedance values, but in this system, there was almost no change. This result indicates that in the high-frequency range up to ~100 Hz, the impedance component is dominated by factors other than charge transfer resistance, specifically the resistance and capacitance components of the outer film and reagent layer, making it possible to non-destructively investigate the state of the sensor components.

[0053] Figure 6 shows a plot of the change in resistance value, calculated from the impedance value of the sensor before and after damage, and the change in glucose reaction current value. A clear negative correlation was observed. This correlation can be used to correct the sensor's current value that has deviated due to external disturbances.

Claims

1. A method for quality control of 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 periodically measuring the electrochemical impedance between the working electrode and the counter electrode to monitor changes in the electrochemical impedance.

2. A method for quality control of 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 the 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 the electrical signal based on the change in resistance; and correcting the value of the electrical signal.

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

4. The method according to claim 1 or 2, wherein the value of the electrical signal is a current value.

5. The method according to claim 1 or 2, wherein the oxidoreductase is a direct electron transfer type oxidoreductase.

6. The method according to claim 1 or 2, wherein the outer layer film comprises a hydrophilic polymer and a hydrophobic polymer.

7. The method according to claim 1 or 2, wherein the potential in the electrochemical impedance measurement is a potential at which the substance to be measured reacts sufficiently.

8. The method according to claim 1 or 2, wherein the measurement frequency band in the electrochemical impedance measurement is a frequency band between 0.1 Hz and 100 kHz.

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