JP5088148B2 - Method for measuring metabolite concentration and electrochemical sensor used therefor - Google Patents

Description

  The present invention relates to a method for measuring a metabolite concentration in an exudate and an electrochemical sensor used therefor.

  As diabetes progresses, it causes various complications and can develop into serious symptoms such as blindness and gangrene. Diabetes tends to cause arteriosclerosis, which is a cause of cerebral infarction and myocardial infarction, which are causes of high death together with cancer. Preventing these problems not only contributes to the improvement of human health, but is also important in avoiding the surge in medical expenses that has become a social problem in recent years. Measurement of fasting blood glucose level is important for diagnosis of diabetes and discovery of disease, and management such as insulin injection according to blood glucose level is important for treatment. However, the examinee must suffer from blood sampling pain. In particular, diabetic patients need to measure blood glucose level with invasion by blood sampling several times a day, which is a great burden for many patients to receive continuous treatment.

  Under such a background, a noninvasive glucose sensor that can measure a blood glucose level (glucose concentration) in a noninvasive manner in medical examination and treatment is desired. The GlucoWatch (trademark), which is a noninvasive glucose sensor that measures the glucose concentration in the exudate from the skin whose blood and glucose concentrations substantially correspond, has already been put into practical use by Cygnus Corporation for the first time. This is to extract the extracellular fluid of the skin as an exudate by passing an electric current through the skin, and the principle of iontophoresis that accelerates the penetration of drugs into the skin by passing an electric current through the skin. This is the reverse of the above. Iontophoresis is a method in which a charged drug is actively absorbed into the body by an electrochemical potential by applying an external voltage to the skin or mucous membrane, and is known as a non-invasive medication method. . If this is used in reverse, a neutral substance can be extracted from the body by electroosmosis, and a diagnosis can be performed using the substance. In Patent Document 1, this is called ion permeation.

  In the biosensor disclosed in Patent Document 1, a low background current is ensured by increasing the S / N ratio using an electrode formed using a platinum and graphite paste.

On the other hand, it is Ben-Aryeh et. al. (Non-Patent Document 1). Furthermore, although it is still in the research stage, the glucose concentration in tears can also be targeted (Non-patent Document 2). If the blood glucose level can be diagnosed based on the glucose concentration in saliva or tears, a noninvasive glucose sensor can be configured.
Japanese Patent No. 3155523 Diabetical Complications, 1988, Volume 2, p. 96 Viadimir et al., Clinical. Chemstry, 2004, 50, p. 2353

  However, the conventional non-invasive method has problems such as polarization occurring on the skin surface, skin irritation and burning due to concentration of current density, and further improvement has been demanded in this respect.

  According to the method of measuring the glucose concentration of saliva, the problem of irritation to the skin is avoided, but the glucose concentration in saliva is extremely low, about 1-2% compared to that in blood, so an inexpensive device is required. It is difficult to easily measure the glucose concentration with high accuracy, and it has not spread to clinical practice.

  Therefore, an object of the present invention is to provide a method that enables highly accurate measurement of metabolite concentration of a non-invasive extracorporeal fluid while suppressing irritation to the skin.

The present invention provides a working electrode of an electrochemical sensor having a conductive DLC film or a conductive diamond film in contact with the skin while a pulse current or a direct current of 0.1 to 10 μAcm ⁻² is applied thereto. Extracting extracorporeal exudate by applying an electric current, bringing the extracted extracorporeal exudate into contact with the working electrode, and determining the metabolite concentration of the extracorporeal exudate in contact with the working electrode. Measuring the metabolite concentration of the exudate with a chemical sensor.

In the method according to the present invention, since the exudate is efficiently extracted by applying a pulse current or a direct current in the specific range, irritation to the skin is sufficiently suppressed. The amount of extracorporeal exudate extracted is very small because pulse current or relatively weak direct current is used, but by using a highly sensitive electrochemical sensor equipped with a working electrode having a DLC film or diamond film, Even in such a small amount of exudate, the metabolite concentration can be measured with high accuracy. In order to further suppress irritation to the skin, it is preferable that the applied pulse current has a frequency of 1 to 100 kHz, a duty ratio of 5 to 30% or 65 to 85%, and a pulse current density of 0.0001 to 10 mAcm ⁻² .

  An electrochemical sensor according to the present invention includes a working electrode having a conductive DLC film or a conductive diamond film, and is used for measuring the concentration of a metabolite in an extracorporeal exudate by the method according to the present invention. is there. The electrochemical sensor system according to the present invention includes the electrochemical sensor according to the present invention and a control unit that detects an oxidation-reduction current generated in the electrochemical sensor.

  According to the electrochemical sensor of the present invention, it is possible to measure the metabolite concentration of the extracorporeal exudate noninvasively with high accuracy while suppressing irritation to the skin.

  ADVANTAGE OF THE INVENTION According to this invention, it is possible to measure the metabolite density | concentration of the extracorporeal exudate by noninvasiveness with high precision, suppressing the irritation | stimulation to skin.

  Further, according to the present invention, a high SN ratio can be obtained by using a DLC film or a diamond film with a small residual current. Therefore, a pulse current can be used in order to significantly suppress the applied current for electroosmosis and further reduce the burden on the skin. Therefore, skin irritation and burning during non-invasive measurement are sufficiently suppressed.

  Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the following embodiments.

  The method according to the present embodiment is a non-invasive measurement method using an electrochemical sensor having a working electrode and a counter electrode. Specifically, in the method according to the present embodiment, the extracellular fluid extracted from the extracellular fluid of the skin by applying a direct current or a pulsed current to the working electrode of the electrochemical sensor in contact with the skin. And contacting the working electrode with the electrochemical sensor to measure the metabolite concentration of the exudate in contact with the working electrode.

  A direct current or a pulsed current is applied to the skin where the working electrode of the electrochemical sensor is in contact. The application of the current is performed, for example, by a method of applying a pair of electrodes to the skin near the working electrode.

In the case of a direct current, a current of 0.1 to 10 μAcm ⁻² is applied. In the case of a pulse current, it is preferable that the frequency is 1 to 100 kHz, the duty ratio is 5 to 30% or 65 to 85%, and the pulse current density is 0.0001 to 10 mAcm ⁻² . By applying an electric current in such a range, extracorporeal exudate can be extracted from the skin while suppressing irritation to the skin particularly remarkably. From the viewpoint of balance between skin irritation and measurement accuracy, the frequency of the pulse current is preferably 1 to 50 kHz, more preferably 10 to 50 kHz. The duty ratio of the pulse current is preferably 5 to 20% or 70 to 85%, more preferably 5 to 15% or 70 to 80%. The pulse current density is preferably 0.001 to 5 mAcm ⁻² , more preferably 0.01 to ² mAcm ⁻² .

  The extracorporeal exudate extracted from the skin is brought into contact with the working electrode, and then the oxidation-reduction current value generated between the working electrode and the counter electrode is measured. Based on the relationship between the metabolite concentration at a predetermined potential and the oxidation current value, the metabolite concentration of the extracorporeal exudate is calculated.

  A metabolite that is a measurement target substance of the method according to the present embodiment is a substance related to metabolism in a living body, and the entire metabolite may be generally referred to as a metabolome. Specific examples of metabolites include sugars (such as glucose), amino acids, and proteins. In addition to basic metabolites, these include oxidative stress markers, substances that can be treated as mental stress markers, and vitamins such as ascorbic acid produced outside the body. It is clinically significant to detect the concentration of metabolites.

  An oxidative stress marker is a substance that serves as an indicator of cell aging, DNA damage, arteriosclerosis, and the like. Oxygen stress markers are, for example, due to oxidative reactions caused by superoxide generated from mitochondria, nitric oxide, peroxynitride, hypochlorous acid or their free radicals, metal ions, lipoxygenase, and myeloperoxidinase. Generate. Representative oxidative stress markers, 8-hydroxy-2'-deoxysideguanosine (8-OHdG), 8-hydroxyguanine (8-OH-Gua), 8-oxo-7,8-dihydro-2'-deoxy DNA damage markers such as guanosine (8-oxodG) and 8-nitroguanosine are caused by oxidative stress damage of DNA. Other oxidative stress markers include 8-isoplastane, a free radical oxidation product of arachidonic acid, and hydroxyleucine, hydroxyvaline, nitrotyrosine, carboxymethyllysine, pentosidine, nitrotyrosine, which are amino acid / protein oxidation disorder markers. And thymine glycol, α-tocophenol which is an in-vivo oxidizing component, biopyrine, oleic acid, thyrodoxine, oxidized coenzyme Q10, carboxymethyllysine, peroxynitrite, nitrogen oxides such as nitric oxide, nitrosothiol reactant , Lipid peroxide (LPO), malondialdehyde, oxidized LDL and oxidized LP (a), creatol MDA-LDL, and the like.

  Examples of mental stress markers include cortisol, norepinephrine, chromogranin A, IgA, and β-endorphin.

  Other metabolites are not particularly limited as long as they are chemical substances that cause a redox reaction. For example, cholesterol, lactic acid, creatinine, protein, hydrogen peroxide, alcohol, glutamic acid, alcohol amino acids, fructosamine, glycerol, acyl-coenzyme A , Tyramine, amino acid, glycolate, pyridoxal-4-, sorbose, guronolactose, galactose, pyranose, uric acid, pyruvic acid, ascorbic acid, ammonia, trimethylamine, acetone, ethane, pentane, hydrogen, oxygen, methane, propane, butane , Isoprene and mercaptans.

  An embodiment of an electrochemical sensor and an electrochemical sensor system suitably used in the above method will be described below.

  FIG. 1 is a block diagram showing an outline of an embodiment of an electrochemical sensor system. An electrochemical sensor system 100 shown in FIG. 1 has an electrochemical sensor 1 having a working electrode 2, a counter electrode 3, and a reference electrode 5, and the electrochemical sensor 1 detects an oxidation-reduction current of a chemical substance to be analyzed in a specimen. And a control unit 10 for controlling. The working electrode 2, the counter electrode 3, and the reference electrode 5 are each electrically connected to the control unit 10. The working electrode 2 is controlled by the control unit 10 so as to be maintained at a predetermined potential with respect to the reference electrode 5. Then, a current between the working electrode 2 and the counter electrode 3 is detected by the control unit 10.

  The electrochemical sensor system according to the present invention may not include the reference electrode as in the embodiment shown in FIG. In this case, the potential of the working electrode 2 is controlled with reference to the counter electrode 3.

  FIG. 3 is a plan view showing an embodiment of an electrochemical sensor. The electrochemical sensor 1 shown in FIG. 3 includes a first substrate 21, a working electrode 2, a counter electrode 3, a reference electrode 5, and a working electrode 2 provided near one end on one surface of the first substrate 21. Lead wires 2a, 3a and 5a formed on the first substrate 21 so as to be connected to the counter electrode 3 and the reference electrode 5 and to extend to the other end of the first substrate 21, respectively. And a second substrate 22 formed with a window 25 that is exposed to the working electrode 2, the counter electrode 3, and the reference electrode 5. The second substrate 22 covers portions of the lead wires 2a, 3a, and 5a other than the vicinity of the other end of the first substrate 21.

The working electrode 2 is a conductive DLC film or a conductive diamond film formed on the first substrate 21, and is particularly preferably a DLC film. The DLC film is generally called a diamond-like carbon film, a diamond-like carbon film, a diamond-like carbon film, or an i-carbon film. The DLC film is an amorphous carbon film mainly composed of carbon and hydrogen and having a mixture of sp ³ bonds and sp ² bonds. On the other hand, the diamond film is a film containing a crystal having a diamond structure, and its structure is different from that of the DLC film. It is known that a DLC film and a diamond film can be clearly distinguished by Raman spectroscopy. In the Raman spectrum, a clear peak is observed at 1333 cm ⁻¹ for the diamond film, whereas a broad peak is observed in the Disordered band near 1350 cm ⁻¹ and the graphic band near 1550 cm ⁻¹ for the DLC film. The

The DLC film preferably contains 10-40 atomic% hydrogen and 30-85 atomic% carbon. When the DLC film has a composition in a specific range, the sensitivity as an electrochemical sensor is improved while maintaining an appropriate resistivity. When hydrogen is contained in this range, the proportion of carbon atoms in the film that form sp ³ bonds including dangling bonds terminated with hydrogen increases, and the film becomes amorphous. An amorphous DLC film is advantageous for industrial production because the film formation temperature is low, film formation with uniform quality is easy, and the film formation rate is high.

In addition, a DLC film mainly composed of sp ³ -bonded carbon has a very small process of adsorbing chemical substances that are oxidized or reduced by an electrochemical reaction. For example, it is caused by hydrogen, hydroxide, or ions caused by water. The inner sphere redox reaction through adsorption to the electrode is extremely difficult to occur. As a result, a noise current called a residual current becomes extremely small, so that an electrochemical reaction of a chemical substance to be detected can be detected with a high SN ratio.

  The DLC film may contain at least one element selected from the group consisting of nitrogen, phosphorus, arsenic, antimony, and bismuth. By containing 5 to 30 atomic% of these elements, the DLC film becomes an n-type semiconductor film. Alternatively, the DLC film may further contain an element selected from the group consisting of boron, gallium and indium. By containing 5 to 30 atomic% of these elements, the DLC film becomes a p-type semiconductor film.

  The DLC film preferably further contains 0.01 to 20 atomic% oxygen. The DLC film preferably further contains 0.002 to 1 atom% of chlorine. If the proportion of chlorine is 0.002 atomic% or less, insulation tends to be difficult to occur, and if it exceeds 1 atomic%, the conductivity tends to decrease.

In order to obtain the sensitivity improvement effect particularly remarkably, the resistivity of the DLC film is preferably 10 ⁻³ to 10 ⁶ Ωcm, more preferably 10 ⁻³ to 10 ³ Ωcm, and further preferably 10 ⁻³ to 10 ² Ωcm. is there.

  The film thickness of the DLC film or diamond film is desirably 0.01 to 20 μm. If the thickness of the DLC film or diamond film is less than 0.01 μm, the resistivity of the working electrode tends to increase or pinholes tend to occur. If many pinholes occur, the sensor electrode may not function normally. On the other hand, when the thickness of the DLC film or diamond film exceeds 20 μm, the first substrate 21 as a base tends to be warped or cracked. Further, when the DLC film or the diamond film is thick, a voltage drop generated in proportion to the product of the current and the resistance between the first substrate 21 and the solution becomes large, and the sensitivity tends to decrease. . The thickness of the DLC film or diamond film is preferably as small as possible from the viewpoint of miniaturization of the sensor. From the same viewpoint, the thickness of the DLC film or diamond film is more preferably 0.05 to 10 μm, and further preferably 0.1 to 5 μm.

  The conductive DLC film can be formed by, for example, a vapor phase method using a source gas containing a hydrocarbon gas while heating the first substrate 21. As the vapor phase method, an ionized vapor deposition method and a high-frequency plasma CVD method are suitable. By adjusting the composition of the source gas, the heating temperature of the first substrate 21 during film formation, and the plasma conditions, a DLC film having the above-described specific composition can be formed. Specifically, for example, a DLC film containing 10 to 40 atomic% hydrogen and 30 to 85 atomic% carbon by forming the film while heating the temperature of the first substrate 21 to 150 to 450 ° C. Is often formed.

  Nitrogen gas, ammonia gas, nitrogen-containing organic substances such as pyridine, boron oxide and alcohols or keto, or a gas such as boron alkoxide, together with hydrocarbon gas as a carbon source during film formation, nitrogen is used. Then, a DLC film doped with boron or the like is formed. For example, a mixed gas containing hydrocarbon gas, nitrogen gas, and optionally at least one additive gas selected from the group consisting of hydrogen, neon, helium, argon and oxygen is introduced as a source gas into the vacuum film forming apparatus. However, the conductive DLC film can be formed by applying a voltage of 13.56 MHz to the raw material gas to form plasma and depositing hydrocarbons generated from the plasmaized raw material gas.

  The working electrode 2 may have a functional film formed on the DLC film or the diamond film. This functional membrane contains an enzyme that selectively causes an electrochemical reaction of a metabolite in a specimen. Specific examples of enzymes include glucose oxidase, glucose dehydrogenase, cholesterol oxidase, cholesterol dehydrogenase, alcohol oxidase, alcohol dehydrogenase, xanthine oxidase, lactate oxidase, lactate dehydrogenase, fructose dehydrogenase, ascorbate oxidase, creatine phosphokinase, glycerol oxidase, acyl- Coenzyme A oxidase, tyramine oxidase, amino acid oxidase, glycolate oxidase, pyridoxal-4-oxidase, sorbose oxidase, glonolactooxidase, galactose oxidase, pyranose oxidase, uricase, pyruvate dehydrogenase, pyruvate oxidase Fine bilirubin oxidase and the like.

  By the action of glucose oxidase, when a necessary potential is applied to the electrode, glucose in the sample solution is oxidized to gluconic acid to generate hydrogen peroxide. At this time, ferrocene derivatives such as potassium ferricyanide, ferrocene, 1,1′-dimethylferrocene, ferrocenecarboxylic acid and ferrocenecarboxaldehyde, quinones such as hydroquinone, chloranil and bromanyl, or ferricyanide, octacyanotungstate and octane. When a mediator such as a metal complex ion such as cyanomolybdate ion is used, a current proportional to the glucose concentration can be detected selectively and with high sensitivity by a DLC film or a diamond film.

  The functional film containing an enzyme can be formed, for example, by forming a film of a solution containing an enzyme and, if necessary, other components such as a mediator on a DLC film or a diamond film and drying the film.

  The first substrate 21 is a support that supports the working electrode 2, the counter electrode 3, and the reference electrode 5. The first substrate 21 only needs to have a physical strength that can withstand use as an electrochemical sensor.

  When the DLC film is an n-type semiconductor film, the first substrate 21 is preferably an n-type crystalline silicon substrate. This makes it difficult for interface resistance such as a Schottky barrier to occur between the first substrate 21 and the DLC film. Since the detection current of the electrochemical sensor is usually a minute current of about 1 mA or less, the occurrence of IR drop is sufficiently prevented. From the same viewpoint, when the DLC film is a p-type semiconductor film, the first substrate 21 is preferably a p-type crystalline silicon substrate.

  The counter electrode 3 is comprised from the electroconductive material for electrodes normally used in an electrochemical reaction. Preferably, the counter electrode 3 is composed of a noble metal such as platinum and gold, or a conductive DLC film similar to the working electrode 2.

  The reference electrode 5 is typically composed of silver-silver chloride or mercury-mercury chloride. The reference electrode 5 can be formed by a method using a paste.

  The lead wires 2a, 3a, 5a are made of a conductive material such as copper and can be formed by a normal method using a metal mask.

  As the second substrate 22, a substrate similar to the first substrate 21 is used. The first substrate 21 and the second substrate 22 are bonded by an adhesive with the working electrode 2 and the like interposed therebetween.

  FIG. 4 is a plan view illustrating an embodiment of an electrochemical sensor that does not include a reference electrode. The electrochemical sensor 1 shown in FIG. 4 has the same configuration as the electrochemical sensor 1 of FIG. 3 except that the reference electrode 5 and the lead wire 5a connected thereto are not formed.

  5 and 6 are plan views showing an embodiment of an electrochemical sensor provided with an application electrode for applying an electric current to the skin. The electrochemical sensor 1 shown in FIG. 5 is connected to the application electrode 51 and a pair of application electrodes 51, 52 arranged to face each other with the working electrode 2, the counter electrode 3, and the reference electrode 5 interposed therebetween. A lead wire 51 a formed on the first substrate 21 so as to extend to the other end portion of the substrate 21 and an application electrode 52 and extend to the other end portion of the first substrate 21. In this way, a lead wire 52a formed on the first substrate 21 is provided. Other configurations are the same as those of the electrochemical sensor of FIGS. The application electrodes 51 and 52 apply current to the skin where the working electrode 2 is in contact. Similarly, the electrochemical sensor 1 shown in FIG. 6 includes a pair of application electrodes 51 and 52 arranged to face each other with the working electrode 2 and the counter electrode 3 interposed therebetween, and a first substrate connected to the application electrode 51. The lead wire 51 a formed on the first substrate 21 so as to extend to the other end of the first substrate 21 and the application electrode 52 and extend to the other end of the first substrate 21. And a lead wire 52 a formed on the first substrate 21.

  Extraction of the extracorporeal exudate and measurement of its metabolite concentration are performed by applying a predetermined direct current or pulse current between the application electrodes 51 and 52 while pressing the portion in the window 25 of the electrochemical sensor 1 against the skin. Done.

  The controller 10 constituting the electrochemical sensor system 100 includes, for example, a potentiostat that controls the potential of the working electrode 2 with respect to the reference electrode 5, and an ammeter that measures a current flowing between the working electrode 2 and the counter electrode 3. , A processor for controlling the potentiostat and the ammeter, and software for controlling the potentiostat and the ammeter. The processor (CPU = central processing unit) controls the potentiostat and the ammeter by executing the software. The potentiostat is controlled so that the potential of the working electrode 2 is maintained at a predetermined potential such that the redox current inherent to the chemical substance to be analyzed in the sample is appropriately detected. Further, the processor reads a calibration curve relating to the relationship between the concentration of the chemical substance to be analyzed in the sample at a predetermined potential and the oxidation current value, and calculates the concentration of the chemical substance to be analyzed in the sample based on this calibration curve. The electrochemical sensor system 100 preferably includes a monitor that displays the calculated results.

FIG. 7 is a flow diagram illustrating one embodiment of a method for measuring the metabolite concentration of an extracted extracorporeal exudate. In step S1, calibration curve data created in advance is read into the processor. The working electrode in step S2 is held at a predetermined potential E _n, the working electrode in the step S3 - measuring a current value I _n at a given time t _n seconds after the start of the voltage application between the counter electrode. It reads the current value I _n in step S4, the concentration C of metabolite in vitro leaching solution is sample based on interpolation from the calibration curve in step S5 is calculated. In step S6, the validity of the calculation result is determined. If it is valid, the result is displayed in step S7. If it is determined that the calculation result is not valid, the process returns to step S2.

  According to such an electrochemical sensor system, a plurality of types of metabolites can be separated and detected by setting a potential using a redox potential unique to the metabolite in the exudate. In this respect, the system according to this embodiment is superior to a semiconductor sensor that cannot distinguish and detect a plurality of types of metabolites.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the following examples.

Example 1
An n-type (100) plane single crystal silicon wafer was placed in a vacuum film forming apparatus provided with electrodes arranged to face each other. Then, while heating the silicon wafer to 200 ° C., ethylene and nitrogen are introduced as source gases at 5: 5 sccm, and the inside of the vacuum apparatus is turned into plasma with a power of 500 W at a frequency of 13.56 MHz. A conductive DLC film was formed. The resistivity of the formed DLC film was 0.3 Ωcm, and its composition was 10 atomic% hydrogen, 5 atomic% nitrogen, and 85 atomic% carbon. The silicon wafer on which the DLC film was formed was cut into 5 mm square to obtain a chip in which the DLC film was formed on the n-type single crystal silicon substrate.

  Next, 2 U glucose dehydrogenase as an enzyme, 0.025 mg flavin adenine dinucleotide as a coenzyme, and 0.025 mg potassium ferricyanide as a mediator were dissolved in 5 μL of a 0.05 M phosphate buffer solution at pH 7.2. Appropriately apply the obtained solution onto the conductive DLC membrane, and then apply 1 μL of 1 wt% bovine albumin solution and 1 μL of 20 wt% glutaraldehyde solution prepared using 0.05 M phosphate buffer solution of pH 7.2 in order. did. Thereafter, the film was dried in an environment of 30 ° C. and a humidity of 10% for 30 minutes to form a functional film on the DLC film.

  A silver-silver chloride electrode as a reference electrode is formed by printing a silver / silver chloride paste using a DLC film as a working electrode, and a DLC film as a counter electrode is formed in the same manner as above, as shown in FIG. An electrochemical sensor having a simple circuit was constructed.

  The produced electrochemical sensor was connected to a self-made potentiostat controlled by a processor loaded with driving software, and an electrochemical sensor system was configured to measure the current value flowing between the working electrode and the counter electrode. The driving software was set so that the detection potential was 1.2 V with respect to the reference electrode.

  A glucose solution in which glucose was dissolved at a concentration of 3 mM in a buffer solution of pH 7.2 with 0.05 M disodium hydrogen phosphate-potassium dihydrogen phosphate was prepared, and an electrochemical sensor was immersed therein to measure the current value. Moreover, the electric current value of the buffer solution which did not melt | dissolve glucose as a blank was also measured. Table 1 shows the glucose concentration calculated from the measurement results together with the SN ratio.

Examples 2-5
By changing the introduction ratio of ethylene and nitrogen gas in the same manner as in Example 1, conductive DLC films having different compositions were formed on the silicon substrate. Except this, it carried out similarly to Example 7, and prepared the electrochemical sensor and the electrochemical sensor system. The glucose concentration was measured in the same manner as in Example 1 using the obtained electrochemical sensor. The results are summarized in Table 1.

Comparative Example 1
An electrochemical sensor system was prepared in the same manner as in Example 1 except that a platinum plate was used as the working electrode, and the glucose concentration was measured. The calculated glucose concentration was 2.8 mM and the SN ratio was 6.7.

Comparative Example 2
An electrochemical sensor system was prepared in the same manner as in Example 1 except that glassy carbon was used as the working electrode, and the glucose concentration was measured. The calculated glucose concentration was 2.7 mM, and the SN ratio was 7.7.

Example 6
An electrochemical sensor having the same configuration as that of the embodiment shown in FIG. 5 was prepared in the same manner as in Example 1 except that the application electrode was further formed.

Using the obtained electrochemical sensor, the glucose concentration in plasma, tears, saliva and exudate from the skin collected from blood was measured. The measurement of the glucose concentration in the exudate was carried out by pressing the counter electrode and the application electrode against the skin of the subject, and changing the frequency, duty ratio, and pulse current density parameters between the application electrodes in Examples 6-1 to Table 6-1. The test was performed on the exudate that was captured by applying the pulse current set to the value shown in 6-5 or the direct current shown in Examples 6-6 to 6-8. Regarding the feeling of irritation, the irritation feeling when applying a current of DC 3 mA / cm ² was set as 100, and the magnitude of the irritation feeling when measuring the exuding fluid was relatively evaluated based on the judgment of the subject.

Comparative Example 3
An electrochemical sensor having the same configuration as that of Example 6 was prepared as Comparative Example 3 except that a platinum plate was used as the working electrode, and the same glucose concentration measurement was performed.

Comparative Example 4
With the same electrochemical sensor as in Example 6, the glucose concentration in plasma, tears, saliva and exudates from the skin collected from blood was measured. The measurement of the glucose concentration in the exudate was carried out by pressing the counter electrode and the applied electrode against the subject's skin while changing the frequency, duty ratio, and pulse current density parameters between the applied electrodes in Comparative Example 4-1 in Table 3. The test was performed on the exudate that was captured by applying the pulse current set to the value shown in 4-2 or the direct current shown in Comparative Examples 4-3 to 4-5.

  Further, glucose concentrations in plasma, tears and saliva collected from blood were measured by the hexonase method, and the values are shown in Table 3 for comparison.

As shown in Table 3, Example 6 well reproduced the results of the hexonase method, but Comparative Example 3 had a large measurement error. In Comparative Examples 4-1, 4-2, 4-4, and 4-5, although the measurement accuracy was good, the feeling of irritation to the skin was great. In Comparative Example 4-3 to which a direct current less than 0.1 μA / cm ⁻² was applied, although the skin irritation was small, the glucose concentration could not be measured because the amount of extracorporeal exudate was small.

Example 7
A device capable of transcutaneous extraction using the electrochemical sensor used in Example 6 (details are in accordance with Example 1 of Patent 23328290, 23 columns, 20 rows to 24 columns, 4 rows, FIGS. 1A to C). And the sensor was applied to the skin, the exudate was captured through a direct current of ² μA / cm ^{2 at} the applied electrode, and then the glucose concentration of the exudate was measured by the sensor.

Example 8
The glucose concentration of the exudate was measured in the same manner as in Example 7 except that a pulse current having a frequency of 10 kHz, a duty ratio of 25%, and a pulse current density of ² mAcm ⁻² was applied.

Comparative Example 5
The glucose concentration of the extracorporeal exudate was measured in the same manner as in Example 7 except that a direct current of 0.32 mA / cm ² was applied.

Comparative Example 6
The glucose concentration of the extracorporeal exudate was measured in the same manner as in Example 7 except that the electrochemical sensor of Comparative Example 3 having a platinum electrode as a working electrode was used.

Comparative Example 7
The glucose concentration of the extracorporeal exudate was measured in the same manner as in Comparative Example 6 except that a direct current of 0.32 mA / cm ² was applied.

In the above Examples and Comparative Examples, the stimulation feeling when measuring the glucose concentration of the extracorporeal exudate was set to 100 in Comparative Example 5 (0.32 mA / cm ² ), and the magnitude of the stimulation feeling was determined by the subject. Based on relative evaluation. We also measured flux, which indicates the rate at which extracorporeal exudate was extracted from the skin. Furthermore, using each electrochemical sensor, blood was collected separately and the glucose concentration in the plasma was also measured.

As shown in Table 4, the feeling of irritation to the patient's skin could be reduced by direct current ² μA / cm ² or pulse current. Even when a small amount of extracorporeal exudate is collected with a weak direct current such as ² μA / cm ² , if a conductive DLC film is used as in Example 7, Comparative Example 6 using platinum is used. In comparison, the glucose concentration can be detected with high sensitivity. In addition, it was found that the pulse current is excellent in the flux representing the speed of extracting the exudate and can be measured with high sensitivity while suppressing the patient's irritation. The present invention can be applied not only to glucose but also to all metabolites that can be detected by an electrochemical reaction.

It is a block diagram showing one embodiment of an electrochemical sensor system. It is a block diagram showing one embodiment of an electrochemical sensor system. It is a top view which shows one Embodiment of an electrochemical sensor. It is a top view which shows one Embodiment of an electrochemical sensor. It is a top view which shows one Embodiment of an electrochemical sensor. It is a top view which shows one Embodiment of an electrochemical sensor. It is a flowchart which shows one Embodiment of the method of measuring the metabolite density | concentration of extracorporeal exudate.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 1 ... Electrochemical sensor, 2 ... Working electrode, 3 ... Counter electrode, 5 ... Reference electrode, 10 ... Control part, 21 ... 1st board | substrate (support body), 22 ... 2nd board | substrate, 51, 52 ... Applied electrode, 100: Electrochemical sensor system.

Claims (6)

While the working electrode having the DLC film was a conductive working electrode of an electrochemical sensor in contact with the skin, external exuding liquid by applying a pulse current to the skin of the portion in contact with the working electrode Extracting the extracorporeal exudate into contact with the working electrode;
Measuring the metabolite concentration of the exudate in contact with the working electrode with the electrochemical sensor;
Equipped with a,
The pulse current has a frequency of 1 to 100 kHz, a duty ratio of 5 to 30% or 65 to 85%, and a pulse current density of 0.0001 to 10 mAcm ⁻² .
A method for measuring the concentration of metabolites in exudates. The method of claim 1, wherein the DLC film comprises 10-40 atomic percent hydrogen and 30-85 atomic percent carbon. The method of claim 2, wherein the DLC film further comprises 0.01-20 atomic percent oxygen. The DLC film is an n-type semiconductor film containing 5 to 30 atomic% of at least one element selected from the group consisting of nitrogen, phosphorus, arsenic, antimony and bismuth, or from the group consisting of boron, gallium and indium. The method as described in any one of Claims 1-3 which is a p-type semiconductor film containing 5-30 atomic% of the selected elements. An electrochemical sensor, comprising a working electrode having a conductive DLC film, for measuring a metabolite concentration of an exudate by means of the method according to any one of claims 1 to 4 . An electrochemical sensor system comprising: the electrochemical sensor according to claim 4; and a control unit that detects an oxidation-reduction current generated in the electrochemical sensor.

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