JP2018042797A - Sensor element and measuring device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a sensor element which sufficiently fixes a mediator participating in electron transfer between an enzyme and a working electrode onto the working electrode and is hardly diffused in a sensor, and a measuring device comprising the sensor element.SOLUTION: A sensor element comprises: a base section (substrate); a working electrode provided on the base section; a detection layer provided on the working electrode; and a counter electrode where electrons move between the counter electrode and the working electrode. The detection layer includes: an enzyme acting on a target substance contained in liquid; a water-soluble mediator mediating electron transfer between the enzyme and the working electrode; and a hydrophilicity host material crosslinked to the mediator.SELECTED DRAWING: None

Description

  The present invention relates to a sensor element and a measuring device.

Diabetes treatment is based on daily blood glucose control, that is, the amount of glucose in the blood, so simple glucose sensors are used not only at medical institutions but also at home to understand blood glucose levels. Yes. The glucose sensor measures the glucose concentration in blood or plasma / interstitial fluid, and is a blood glucose self-measurement of blood glucose (SMBG) device that quantifies the glucose concentration in blood, and a subcutaneous stroma. Continuous glucose monitor (CGM; Continuous) that monitors fluctuations in blood glucose level by continuously measuring and analyzing the glucose concentration in the liquid
Glucose Monitoring) is known (for example, refer to Patent Documents 1 and 2). It should be noted that since the glucose concentration is slightly different between the subcutaneous interstitial fluid and the blood, in the case of monitoring with CGM, it is necessary to periodically measure the blood glucose level using SMBG or the like, and to correct by that value.

  SMBG can be further divided into an enzyme electrode method and an enzyme colorimetric (colorimetric determination) method depending on the measurement principle. In either case, a sample (blood / interstitial fluid, etc.) is collected and collected in the sample. By measuring the amount of glucose in the blood, the glucose concentration in the blood is quantified. For example, in the enzyme electrode method, an enzyme is used for quantification of glucose. Blood is collected using a puncture needle, and the blood (specimen, sample) collected is collected with an accessory called a test strip. This is performed by mixing and acting with a drug such as an enzyme held on the strip, and converting the amount of electricity (current amount) caused by the application of voltage into a glucose concentration.

  Examples of enzymes used in the glucose sensor of the above enzyme electrode method include glucose oxidase (GOD: glucose oxidase) and glucose dehydrogenase (GDH: glucose dehydrogenase). The GOD method using GOD as an enzyme has been used in the past because of its extremely high ability to recognize glucose, but it is measured lower than the actual value due to the influence of the dissolved oxygen partial pressure. Because of this tendency, the GDH method using GDH as an enzyme is more preferably used at present. In the GDH method, glucose in the blood is converted to gluconolactone by GDH held in the sensor, and simultaneously generated electrons reduce the mediator also held in the sensor to a reduced (hydrogenated) mediator. When a voltage is applied to the reduced mediator to oxidize it to the original oxidized mediator, an electromotive force is generated at the electrode, which is utilized in proportion to the glucose concentration in blood. Here, the mediator changes from the oxidized type to the reduced type, and further returns to the oxidized type, so that the cycle of the sensor is completed, so that the mediator can be reused. A mediator is a compound that is involved in electron transfer between an enzyme and an electrode and that is reversibly oxidized / reduced, and may be used because the voltage required for measurement can be lowered.

On the other hand, the CGM is a device that continuously measures blood sugar levels as the name suggests, and is a device that constantly measures blood sugar levels while wearing the CGM. In CGM, a sensor needle containing an enzyme is always embedded in the body and is always exposed to the specimen (sample), and the current flowing is measured by applying voltage continuously or periodically. Therefore, the fluctuation | variation of the glucose level in subcutaneous interstitial fluid is monitored. The enzyme electrode method is also used in CGM, and glucose in the interstitial fluid permeates the sensor, and the permeated glucose and enzyme (such as GDH may require a coenzyme during an enzymatic reaction). And a mediator produces a reduced mediator. By electrically detecting the amount of this reduced mediator, the glucose concentration in the blood can be observed. The enzyme and mediator used here are held by a host material made of a polymer resin on the electrode surface of the sensor. This host material is a material that is prepared by a solution using water as a solvent because CGM is embedded in the human body and the enzyme is easily deactivated in the presence of an organic solvent. It has a water-soluble property that can be dissolved, and has a hydrophilic property (water absorption, water content) that can sufficiently contain water after film formation. It is required to be a material.

  In addition, in SMBG by the enzyme electrode method, it is necessary to measure one measurement accurately and with good reproducibility. Therefore, in order to measure accurately, the “amount” of the collected sample is accurate and a voltage is applied. Two important points are that the measured current value is accurate. Therefore, uniform and quick mixing of the sample (specimen) with the mediator and other drugs on the test strip is required.

  On the other hand, CGM is a human-embedded type and is measured by time differentiation, and there is no concept of the amount of sample liquid. For this reason, the accuracy of the “amount” of the collected sample is not required, but it is necessary to accurately measure the amount of reduced mediator produced, that is, the measurement current. The amount of reduced mediator is proportional to the glucose concentration in the blood, but since the mediator is a relatively small molecule, it is difficult to diffusely hold it in the host material. Furthermore, if the mediator diffuses into the liquid containing the target substance to be measured and is too far away from the vicinity of the sensor electrode, electronic transfer with the enzyme cannot be performed, or the reduced mediator is too far from the electrode. If it is no longer oxidized, a baseline fluctuation (drift) of the current obtained from the measurement will occur, and even if the blood glucose level has not changed, data will appear as if the blood glucose level has changed. There is a case. This variation in baseline is a problem specific to CGM that occurs because CGM is a device that performs continuous measurement, and CGM continuously monitors blood glucose levels, thereby allowing diabetic patients to monitor their daily blood glucose levels. This is one of the reasons why it cannot be used as a determination device at the time of insulin administration, while it is used for grasping the change and determining a treatment policy by a doctor.

  Thus, the measurement by CGM is not currently “measurement of blood glucose level as an index for calculating insulin dose”. This is because, in the measurement by CGM, if correction of the value by SMBG is required several times / day, the calculation index of the insulin dose is sufficient for the measurement by SMBG which is more accurate and highly reliable (for example, Non-Patent Document 1).

  Therefore, the future direction of CGM development is “to understand changes in blood glucose levels in diabetic patients and determine the treatment policy by the doctor.” And “blood glucose as a calculation index for insulin dosage It is to achieve the two purposes of “to be value measurement”. In order to achieve these two objectives, first of all, it is not necessary to correct the measurement value by SMBG, that is, to suppress the drift of the baseline in the measurement. It is necessary to suppress this. In order to suppress the diffusion of the mediator in the sensor, it is necessary to fix the mediator to the host material. That is, it is desired that the mediator does not diffuse into the sensor within a range where the potential of the mediator can be transferred to and from the enzyme.

  Thus, for example, there is a technique for fixing a mediator composed of a derivative of a quinone molecule to an anode electrode using a crosslinking agent (for example, Patent Document 3).

International Publication No. 2011/024487 Special table 2013-500793 gazette JP 2005-79001 A

Nippon Medtronic Co., Ltd. Glucose Monitor System “Medtronic Mini Med CGMS-Gold” Instruction Manual

  However, the crosslinking agent used in the above technique does not progress in the crosslinking reaction unless the enzyme activity is significantly reduced, such as it is difficult to dissolve in water or the crosslinking reaction does not proceed unless it is in a basic or high temperature environment. Absent. In addition, these crosslinking agents are insufficient even if they are crosslinked with the hydrophilic host material used in CGM, resulting in non-uniform film quality. Therefore, even if the mediator is to be crosslinked with the hydrophilic host material using the disclosed crosslinking agent, it cannot be sufficiently fixed to the host material. Therefore, in the above technique, it is conceivable to use an organic solvent in the crosslinking reaction. However, the enzyme exhibits a catalytic function in an aqueous solution, and is easily denatured and deactivated in the presence of the organic solvent. It is not preferable to use it for the crosslinking reaction.

  Accordingly, some aspects of the present invention provide a sensor in which a mediator involved in electron transfer between an enzyme and a working electrode is sufficiently fixed on the working electrode by solving at least a part of the problems described above. It is an object of the present invention to provide a sensor element that is difficult to diffuse inside and a measuring device including the sensor element.

  SUMMARY An advantage of some aspects of the invention is to solve at least a part of the problems described above, and the invention can be implemented as the following aspects or application examples.

[Application Example 1]
One aspect of the sensor element according to the present invention is:
The base,
A working electrode provided at the base;
A sensing layer provided on the working electrode;
A counter electrode from which electrons move between the working electrode and
The sensing layer is
An enzyme that acts on the target substance contained in the liquid;
A water-soluble mediator that mediates electron transfer between the enzyme and the working electrode;
And a hydrophilic host material cross-linked to the mediator.

  According to the aspect of Application Example 1, the water-soluble mediator is cross-linked to the hydrophilic host material, so that the mediator is sufficiently fixed on the working electrode. Therefore, it is possible to provide a sensor element in which the mediator is less likely to diffuse in the sensor and the baseline drift is less likely to occur.

[Application Example 2]
In the above application example,
The mediator and the host material may have crosslinkable functional groups that crosslink each other.

The sensor element according to the present invention has a crosslinkable functional group in which each of the mediator and the host material is cross-linked, whereby the mediator and the host material are cross-linked.
The mediator is fully immobilized on the working electrode. For this reason, it becomes difficult for the mediator to diffuse in the sensor, and the drift of the baseline does not easily occur.

[Application Example 3]
In the above application example,
The mediator and the host material can be crosslinked by a water-soluble crosslinking agent.

  In the sensor element according to the present invention, the mediator and the host material are cross-linked by a water-soluble cross-linking agent that dissolves in water used as a solvent, and the mediator is sufficiently fixed on the working electrode. For this reason, it becomes difficult for the mediator to diffuse in the sensor, and the drift of the baseline does not easily occur.

[Application Example 4]
In the above application example,
The crosslinking agent may be one or more compounds selected from the group consisting of bis (vinylsulfonyl) compounds, bisacrylamide compounds, and bisstyrene compounds.

  The sensor element according to the present invention uses one or more compounds selected from the group consisting of bis (vinylsulfonyl) -based compounds, bisacrylamide-based compounds, and bisstyrene-based compounds as a cross-linking agent. Without lowering, the mediator and the host material are cross-linked, and the mediator is sufficiently fixed on the working electrode.

[Application Example 5]
In the above application example,
The mediator may be one or more quinone compounds selected from the group consisting of benzoquinone compounds, naphthoquinone compounds, phenanthrenequinone compounds, and acenaphthenequinone compounds.

  The sensor element according to the present invention is such that the mediator is one or more quinone compounds selected from the group consisting of benzoquinone compounds, naphthoquinone compounds, phenanthrenequinone compounds, and acenaphthenequinone compounds. Thus, it is possible to provide a sensor element that detects a target substance efficiently and is less likely to cause a baseline drift.

[Application Example 6]
In the above application example,
The crosslinkable functional group may be one or more functional groups selected from the group consisting of a double bond, a hydroxyl group, an imino group, a thiol group, and an amino group.

  In the sensor element according to the present invention, the crosslinkable functional group is one or more functional groups selected from the group consisting of a double bond, a hydroxyl group, an imino group, a thiol group, and an amino group. Are cross-linked so that the mediator is fully immobilized on the working electrode.

[Application Example 7]
In the above application example,
The host material may be one or more compounds selected from the group consisting of polyvinyl alcohol compounds, glycol compounds, and cellulose compounds.

  In the sensor element according to the present invention, the host material is one or more compounds selected from the group consisting of a polyvinyl alcohol compound, a glycol compound, and a cellulose compound. And has a sufficiently hydrophilic (water-absorbing) property after film formation. By using a host material formed of these compounds, not only is the effect of retaining the enzyme in the host material without deactivating the enzyme, but also the cross-linking with the mediator allows the mediator to be placed on the working electrode. It can be fixed sufficiently.

[Application Example 8]
In the above application example,
The mediator may have a redox potential in a range of −0.25V to 0.2V.

  The sensor element according to the present invention is less susceptible to interference components during measurement when the redox potential of the mediator is in the range of −0.25 V to 0.2 V.

[Application Example 9]
In the above application example,
The target substance may be glucose.

  The sensor element according to the present invention is suitable when the target substance is glucose.

[Application Example 10]
In the above application example,
The enzyme can be glucose dehydrogenase.

The sensor element according to the present invention is also suitable when the enzyme is a glucose dehydrogenase target substance.
[Application Example 11]
In the above application example,
A reference electrode can be further included.

  The sensor element according to the present invention is also suitable when it further includes a reference electrode.

[Application Example 12]
One aspect of the measuring apparatus according to the present invention is:
The sensor element according to any one of the application examples 1 to 11 is provided.

  According to the application example 12, by including the sensor element according to the present invention, the water-soluble mediator involved in the electron transfer between the enzyme and the working electrode is cross-linked to the hydrophilic host material, and the mediator is It is well fixed on the working electrode. Therefore, it is possible to provide a measurement device in which the mediator is less likely to diffuse in the sensor and the baseline drift is less likely to occur.

[Application Example 13]
In the above application example,
The measurement device may be a continuous blood glucose measurement type.

  The measurement apparatus according to the present invention is particularly suitable as a continuous blood glucose measurement type measurement apparatus by including the sensor element according to the present invention.

The perspective view which shows typically the state which connected the sensor element and measuring device which concern on embodiment of this invention. The perspective view which shows typically the state which pulled out the needle part from the sensor element. The side view which shows the state which mounted | wore the skin with the sensor element. Sectional drawing which expands and shows the cannula with which a sensor element is provided. The top view which shows the detection part with which a sensor element is provided. Sectional drawing which shows the detection part with which a sensor element is provided. Sectional drawing which shows the other structural example of the detection part with which a sensor element is provided. The longitudinal cross-sectional view which shows the other structural example of the detection part with which a sensor element is provided. The figure which shows typically the structure of the circuit which drives the detection part with which a sensor element is provided. The perspective view which shows typically the state which mounted | wore the insulin pump with the sensor element.

  DESCRIPTION OF EMBODIMENTS Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings. The embodiments described below do not unduly limit the contents of the present invention described in the claims. In addition, not all of the configurations described below are essential constituent requirements of the present invention.

1. Sensor Element and Measuring Device First, a measuring device including a sensor element and a sensor element according to the present embodiment will be described with reference to the drawings in the order of the device configuration, the detection unit configuration, and the glucose level measurement. In this embodiment, as a sensor element and a measurement device, a continuous blood glucose measurement type glucose sensor used for measuring the glucose concentration in subcutaneous interstitial fluid, in which the target substance is glucose in subcutaneous interstitial fluid. Although an example is given and demonstrated, this invention is not limited to this aspect.

1.1. Apparatus Configuration A measuring apparatus according to an embodiment of the present invention includes a sensor element according to an embodiment of the present invention.

  FIG. 1 is a perspective view schematically showing a state in which the sensor element 100 and the measuring device 101 according to the embodiment of the present invention are connected, and FIG. 2 is a schematic view in which the needle part 121 is pulled out from the sensor element 100. FIG. FIG. 3 is a side view showing a state where the sensor element 100 is attached to the skin, and FIG. 4 is an enlarged cross-sectional view showing a cannula 111 provided in the sensor element 100.

  The measuring apparatus 101 shown in FIG. 1 is used by connecting the sensor element 100 to the measuring apparatus 101 shown in FIG. 1, and processing for analyzing the sensor element 100 and the current value obtained by the sensor element 100. An arithmetic device 210 including the circuit 200, a display unit 155 including a monitor 151 that displays a measurement value obtained by the arithmetic device 210, and a connector 131 that attaches (connects) the sensor element 100 to the display unit 155. And a wiring 132 for connecting the processing circuit 200 and the connector 131.

  As shown in FIG. 1-3, the sensor element 100 has a main body part 110 having a dome shape as a whole, and a removable part 120 that is detachable from the main body part 110 and includes a needle part 121 on the distal end side. Have

  As shown in FIG. 2, the detachable portion 120 includes a grip portion 122 located on the proximal end side and a needle portion 121 located on the distal end side. The needle part 121 is formed in a semi-cylindrical shape as a whole, and is sharply formed on the tip side. The needle part 121 surrounds a part of the side surface of the cannula 111 by penetrating the through-hole 112 and projecting from the lower surface of the main body part 110 when the detachable part 120 is attached to the main body part 110. It is configured to mate with the cannula 111 (see FIG. 4).

  As shown in FIG. 3, the main body 110 has an adhesive layer (not shown) on the lower surface 110 a and is inserted into the subcutaneous tissue 502 from the epidermis 501 and collected by sucking subcutaneous interstitial fluid. Projectingly formed. The cannula 111 is connected to a through hole 112 formed through the main body 110 from the upper surface 110b toward the lower surface 110a.

  In the sensor element 100 having such a configuration, when the detachable portion 120 is attached to the main body portion 110, the needle portion 121 is inserted into the through hole 112 of the main body portion 110 while the grip portion 122 is gripped. (See FIG. 1). On the other hand, when the detachable part 120 is removed from the main body part 110, it is performed by pulling out the needle part 121 while holding the grip part 122 (see FIG. 2). As described above, the detachable portion 120 of the sensor element 100 according to the present embodiment is configured to be detachable from the main body portion 110.

  When attaching the sensor element 100 to the epidermis 501, as shown in FIG. 3, the lower surface 110a of the main body 110 is brought into contact with the epidermis 501 and the needle part 121 with the cannula 111 is pierced into the epidermis 501. This causes the needle 121 and cannula 111 to be inserted into the subcutaneous tissue 502. Thereby, the main body 110 is fixed to the epidermis 501, and the cannula 111 is placed in the subcutaneous tissue 502 by the guidance of the needle 121 of the detachable part 120.

  Next, by grasping the grasping part 122 and detaching the attaching / detaching part 120 from the main body part 110, with the cannula 111 inserted (remaining) in the subcutaneous tissue 502, the needle part 121 is pulled out from the subcutaneous tissue 502. remove. In this way, only the main body part 110 of the sensor element 100 can be attached to the epidermis 501, and the needle part 121 of the detachable part 120 allows the cannula 111 to be percutaneously attached when the main body part 110 is attached to the epidermis 501. In particular, it is used as a guide member for placement into the subcutaneous tissue 502.

  As shown in FIG. 4, the cannula 111 is formed in a cylindrical shape as a whole, and has a hollow portion 114 formed of a communication hole (through hole) communicating from the base end to the tip connected to the lower surface 110 a of the main body 110. , And a window portion 113 that opens the hollow portion 114 to the outside of the cannula 111.

  The hollow portion 114 is provided with a detection unit 300 included in the main body 110. Interstitial fluid contained in the detection unit 300 by moving from the blood vessel 503 to the subcutaneous tissue 502 comes into contact with the detection unit 300 through the window 113, and the detection unit 300 can detect glucose in the interstitial fluid. . By attaching the main body 110 to the epidermis 501, the cannula 111 can be placed in the subcutaneous tissue 502 over a long period of time, so that glucose in the interstitial fluid can be detected continuously. As described above, the sensor element 100 according to the embodiment of the present invention is used as a CGMS (continuous glucose monitoring system) for continuously observing the glucose value in the interstitial fluid.

1.2. Configuration of Detection Unit Next, the detection unit 300 included in the main body 110 will be described. FIG. 5 is a plan view showing the detection unit 300 included in the main body 110 of the sensor element 100 according to the present embodiment, and FIG. 6 is a cross-sectional view showing the detection unit 300.

As shown in FIGS. 5 and 6, the detection unit 300 includes a substrate (base) 301 and an electrode layer 315.
And a detection layer 321.

1.2.1. Substrate The substrate 301 supports each part (in this embodiment, the electrode layer 315 and the detection layer 321) constituting the detection unit 300.

  As a constituent material of the substrate 301, various materials can be used without particular limitation as long as they are stable without chemically reacting with air, water, body fluid, blood, and interstitial fluid. Specifically, for example, inorganic materials such as glass and SUS, amorphous polyarylate (PAR), polysulfone (PSF: Polysulfone), polyethersulfone (PES), polyphenylene sulfide (PPS: Polyphenylene sulfide). , Polyether ether ketone (PEEK: Polyether ether / Also known as: aromatic polyetherketone), Polyimide (PI: Polyimide), Polyetherimide (PEI: Polyetherimide), Fluorocarbon (Fluorocarbon polymer), Polyamide containing nylon and amide (PA), such as polyethylene terephthalate (PET) Resin material and the like, such as ether, it may be used singly or in combination of two or more of them.

1.2.2. Electrode layer The electrode layer 315 detects electrons generated in the detection layer 321 and measures the detected electrons as a current amount. The electrode layer 315 is formed on the substrate 301 and has a working electrode 311, a counter electrode 312 and a reference electrode. 313 and wiring 314 are provided.

  Each of the electrodes 311, 312, and 313 is electrically connected to the processing circuit 200 through the wirings 314 and 132 and the connector 131 independently. As a result, the current value measured at each of the electrodes 311 and 312 (electrode layer 315) is transmitted to the processing circuit 200 via the circuit 400 and the wiring 314 included in the main body 110, and the arithmetic device 210 including the processing circuit 200 is transmitted. By the analysis, the glucose value in the interstitial fluid is calculated as the measured value. And this measured value (glucose value) is displayed on the monitor 151, and a glucose value is continuously notified to a wearer.

  The constituent material of each electrode 311, 312, 313 is not particularly limited as long as it can be used as an enzyme electrode, and for example, gold, silver, platinum, an alloy containing these or an alloy mainly containing these, Metal oxide-based materials such as ITO, carbon-based materials such as carbon (graphite), conductive carbon tape, and the like can be used as electrodes.

  The electrodes 311, 312, and 313 can be formed by sputtering, plating, or vacuum heating vapor deposition when the electrodes 311, 312, and 313 are made of platinum, gold, or an alloy thereof. Further, when each of the electrodes 311, 312, and 313 is made of carbon graphite, it can be realized by mixing carbon graphite in a binder dissolved in an appropriate solvent and applying it. In the case of a conductive carbon tape, it is configured by being directly pasted on the substrate 301.

  The counter electrode 312 is made of the same material as that of the working electrode 311 so that the counter electrode 312 is stable without chemically reacting with water, body fluid, blood, and interstitial fluid, like the working electrode 311 described later. It is preferable to be a film. In addition, if high accuracy and stabilization of measurement are desired, it is preferable that the area is large. Even if the area is about half that of the working electrode 311, there is no problem in operation. It is preferably about −2 times.

  The reference electrode 313 is preferably formed using silver, and more preferably, the silver surface is converted to AgCl. Further, a platinum wire (wire) containing platinum as a main component may be used, and a working electrode 311 described later is used so as to be a stable material without chemically reacting with water, body fluid, blood, or interstitial fluid. The surface may be formed of the same material as in FIG.

1.2.3. Detection Layer The detection layer 321 is a layer containing an enzyme and a mediator, and is formed by being laminated on the electrode layer 315 as shown in FIG. That is, the detection layer 321 is formed so as to cover the working electrode 311, the counter electrode 312, and the reference electrode 313, and by the action of the enzyme in contact with glucose that has permeated from the interstitial fluid in contact with the upper surface of the detection layer 321, for example, Glucose is oxidized and converted to gluconolactone. Then, the electrons generated at the same time as the oxidation of glucose reduce the mediator to a reduced (hydrogenated) mediator, and when the voltage is applied to the reduced mediator to oxidize it to the original oxidized mediator, Supply to the working electrode 311 of the electrode layer 315.

  The detection layer 321 having such an action includes an enzyme that acts on the target substance, a water-soluble mediator that mediates electron transfer between the enzyme and the working electrode 311, and a hydrophilic host material that is cross-linked to the mediator. If necessary, it has a binder or a curing agent that bonds the constituent materials.

<Enzyme>
As the enzyme contained in the detection layer 321, glucose oxidase (GOD) can be used for the purpose of detecting glucose in the specimen, but it is not affected by the partial pressure of dissolved oxygen in the blood, and maltose. It is preferable to use glucose dehydrogenase (glucose dehydrogenase; GDH) because it has substrate specificity that does not act on other sugars such as. In this case, it is preferable to use flavin adenine dinucleotide (FAD) or nicotinamide adenine dinucleotide (NAD) as a coenzyme. When a coenzyme is used, it can be used whether it is chemically bound to the enzyme or not, but it is preferably chemically bound to the enzyme.

<Mediator>
The mediator included in the detection layer 321 has a function of mediating electron transfer between the enzyme and the working electrode 311 and can be cross-linked with a hydrophilic host material directly or via a cross-linking agent. It is a water-soluble compound having a functional group, and particularly preferably a quinone compound. Here, in this specification, “water-soluble” refers to the property of being dissolved in water used as a solvent. Specifically, at a temperature of 60 ° C. or lower, the reaction solution concentration is 20% by mass or less, preferably 10% by mass. It refers to the property that can be dissolved at a concentration of less than%. By using such a water-soluble compound as a mediator, the detection layer 321 can be produced without reducing the enzyme activity when the detection layer 321 is produced. Further, in the manufactured detection layer 321, the mediator and the host material are cross-linked, so that the mediator is sufficiently fixed on the working electrode 311.

As such a quinone-based mediator, specifically, as a functional group, a double bond, a hydroxyl group (—OH), an imino group (—NH—), a thiol group (—SH), an amino group (—NH ₂ ). ), Benzoquinone compounds, naphthoquinone compounds, phenanthrenequinone compounds, and acenaphthenequinone compounds.

More specifically, vitamin-MK3, vitamin-MK4, vitamin-K1,2-hydroxy-1,4-naphthoquinone, 5-hydroxy-1,4-naphthoquinone, trihydroxynaphthoquinone, 3- Hydroxy-1,4-naphthoquinone-2
-7- (4-styryl) -9,10 which is Na-sulfonic acid, 7-hydroxy-1,2-naphthoquinone-4-sulfonic acid Na, 1,2-naphthoquinone 4-sulfonic acid Na, phenanthrenequinone compound -Na phenanthrenequinone-2-sulfonic acid, 7- (propane-1-ene) -9,10-phenanthrenequinone, 7- (1-hydroxypropane) -9,10-phenanthrenequinone-2-sulfonic acid Na, 2 -Hydroxy-9,10-phenanthrenequinone, 1,4,8-trihydroxy-2-methyl-3- (4-methylvaleryl) -9,10-phenanthrenequinone, 2- (1-aminopropyl) -9,10 -Phenanthrenequinone, an acenaphthenequinone compound, 3- (2,7-dimethyloctane-2,6-dien-8-yl -7,8 acenaphthenequinone, 3- (1-hydroxypropyl) -7,8 acenaphthenequinone, 3- (1-aminopropyl) -7,8-acenaphthenequinone, and the like.

  These mediators preferably have a redox potential in the range of −0.25 V or more and 0.2 V or less. In this embodiment, when the oxidation-reduction potential of the mediator is in the range of −0.25 V or more and 0.2 V or less, it is less likely to be affected by blood interference components when measuring the glucose concentration.

  The quinone compound that is considered to be used as a mediator is a voltage-current graph obtained by measurement using cyclic voltammetry and a silver-silver chloride electrode (Ag | Agcl) as a reference electrode. As a result of the swept sweep measurement at −0.5 V, a peak potential where the compound is reduced to become a reductant and a return oxidation peak potential when the reductant is oxidized and returned to the original state are observed. In the sensor element 100 according to the present embodiment, the reduction of the mediator is generated by the reaction between the enzyme-coenzyme acting on glucose and the enzyme-coenzyme reductant that has been changed. Peak potential.

  On the other hand, examples of disturbing components in blood include, for example, ascorbic acid, uric acid, and acetaminophen whose concentration is increased by medication. When these redox potentials are measured by cyclic voltammetry, they are considered to be used as mediators. A compound may overlap with an electrochemically reacting potential. Therefore, in the measurement of the oxidation-reduction potential by cyclic voltammetry, it is necessary to select the mediator to be used so that the potential of the blood interfering component and the mediator do not overlap.

  In other words, when the redox potential of the mediator is in the range of −0.25 V or more and 0.2 V or less, the blood interference component signal is not picked up. Can be removed.

<Host material>
The hydrophilic host material that constitutes the detection layer 321 and retains the enzyme and mediator in the detection layer 321 has hydrophilicity and can be crosslinked directly with the mediator or via a crosslinking agent. Any material can be used as long as it is a material formed of a compound having the following. Furthermore, it has water-soluble properties before film formation by self-crosslinking or crosslinking reaction with a crosslinking agent or curing by ultraviolet irradiation, etc., and maintains affinity with water after film formation while maintaining blood and subcutaneous properties. Any material that is insoluble in water that is not dissolved by moisture in the body, such as interstitial fluid, can be used without particular limitation. In this embodiment, by using such a hydrophilic host material, the detection layer 321 can be manufactured without reducing the enzyme activity when the detection layer 321 is manufactured. In the manufactured detection layer 321, the mediator and the host material are cross-linked and the mediator is sufficiently fixed on the working electrode 311.

Here, in the present specification, “hydrophilic” means having a strong affinity for water. Specifically, it means a property capable of containing (holding water, containing water) 10% by mass or more, preferably 20% by mass, and more preferably 50% by mass or more with respect to its own weight. Therefore, “affinity with water” after the formation of the host material means that the host material after the film formation has a property of sufficiently containing water.

  Examples of such a crosslinkable functional group include a double bond, a hydroxyl group, an imino group, a thiol group, and an amino group. Examples of the hydrophilic host material include methyl cellulose (MC), acetyl cellulose (cellulose acetate), polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), and polyvinyl alcohol-polyvinyl acetate copolymer (PVA-PVAc). Etc. are preferably used, and one or more of these can be used in combination. By using these materials, not only can the enzyme be held in the host material, but also it can be immobilized on the membrane that is the host material by crosslinking with the mediator. In addition, a decrease in enzyme activity can be suppressed during film formation and after film formation.

  Such a host material is a material having the largest content in the detection layer 321 containing an enzyme. The sensor element 100 according to the present embodiment is a sensor that is placed in the body and constantly measures the concentration of glucose contained in the interstitial fluid and plasma. Therefore, the required function is to detect interstitial fluid and plasma containing glucose. It is necessary to be able to penetrate constantly. For this purpose, it is desirable that water, which is the main component of interstitial fluid and plasma, has high hydrophilicity and permeability. In addition, the sensor element 100 according to the present embodiment is a sensor that is placed in the body for a period as long as several days to several weeks, and the detection layer 321 needs to be continuously present for the sensor to function during the period. For this purpose, the physical property that the detection layer 321 is melted in the body is not useful. Therefore, the host material that is the main component of the detection layer 321 is required to be a material that does not dissolve due to internal components including water, and in order to realize this, after applying the host material material to the substrate 301, There is a need for a method of insolubilization by using a functional group possessed therein or by separately using a crosslinking material or the like.

  Thus, in the present embodiment, “crosslinking” is an essential step for making the host material insoluble in water since the sensor element 100 is premised on insertion into the body. In the present embodiment, the cross-linking between the mediator and the host material includes two cases, that is, a case where the mediator is cross-linked by a cross-linking agent and a case where it is cross-linked without using a cross-linking agent. In this embodiment, the water-soluble compound serving as the host material is formed into a film by forming a cross-linking bond by cross-linking using a cross-linking agent and / or self-cross-linking not using a cross-linking agent. It becomes an insoluble host material.

  In this “crosslinking”, when the water-soluble compound as a host material has a reactive functional group capable of crosslinking by self-crosslinking or a mediator, crosslinking can be performed without using a crosslinking agent. is there. On the other hand, when the water-soluble compound serving as the host material does not have such a reactive functional group, it undergoes a crosslinking reaction by using a crosslinking agent and crosslinks with the film-forming and mediator.

  Examples of such reactive functional groups capable of self-crosslinking include a double bond and an azide group. When the compound serving as the host material has these reactive functional groups, the reactive functional groups can react with each other to be crosslinked without using a crosslinking agent. On the other hand, when such a reactive functional group is not present, the use of a crosslinking agent is necessary, and the host material requires a functional group capable of reacting with the crosslinking agent. Examples of such a functional group capable of reacting with a crosslinking agent include a hydroxyl group, an imino group, an amino group, and a thiol group. In addition, in the case of forming a film by crosslinking a compound having a reactive functional group, such as a double bond or an azide group, a crosslinking agent can also be used.

  Even if the mediator and the cross-linking agent are cross-linked, if the mediator does not cross-link with the host material, the mediator is not fixed to the host material and is easily eluted from the detection layer and diffuses into the sensor. Therefore, in this embodiment, it is essential that the mediator and the host material are cross-linked.

  In addition, since the host material is required to hold an enzyme, it is desirable to use a carrier binding method by a covalent bond or a lattice-type inclusion method. In this case, if the covalent bond method is used, it is necessary to select a means for binding without reducing the enzyme activity. In order to crosslink, the functional group introduced into the host material and the functional group such as amino group possessed by the enzyme are used, or the host material and the enzyme are combined with a bifunctional or higher functional crosslinking agent. It may be combined. When the lattice type is used, a means for controlling the degree of crosslinking when the host material is insolubilized is generally used.

  Further, since the host material is required to immobilize the mediator, it is desirable to use a carrier binding method using a covalent bond. In general, most mediator molecules used in enzyme sensors have a small molecular weight and a molecular weight of 1000 or less. On the other hand, since the enzyme has a molecular weight exceeding tens of thousands, even if the enzyme can be immobilized in a lattice form, it is almost impossible to immobilize the mediator. Therefore, if the enzyme and the mediator are coexisted in the same layer in the detection layer 321 and coating and crosslinking are performed, the activity of the enzyme can be deactivated by a functional group (reactive group) used for fixing the mediator. It is necessary to consider that hydrophilic materials should be used.

  As will be described later, since the detection layer 321 is formed by applying, drying, and insolubilizing a solution in which the host material, the mediator, and the enzyme are simultaneously dissolved to the substrate 301, they may be dissolved in the same solvent. desirable. Since it is known that the activity of an enzyme decreases due to a change in the three-dimensional structure even with an organic solvent, the solvent used is preferably an aqueous solvent containing water as a main component.

<Binder, curing agent>
In the detection layer 321, the binder and curing agent that binds between the constituent materials are compounds that do not reduce the enzyme activity, and the functional groups include acrylamide groups, styryl groups or double bonds, vinyl sulfone groups, and the like. A compound having two or more functional groups in one molecule can be used.

Such a compound is not particularly limited as long as it significantly reduces enzyme activity. For example, 1,2-bis (vinylsulfonylacetamido) ethane (CAS registration number 66710-66-7), 1,3- Bis (vinylsulfonylacetamido) propane (CAS registration number 93629-90-4), 1,3-bis (vinylsulfonyl) propan-2-ol (CAS registration number 67006-32-0), N, N′-methylenebis Acrylamide (CAS registration number 110-26-9), 1,4-bis (4-vinylphenoxy) -butane (CAS registration number 112309-98-5) and the like. These compounds may be used as a mixture. When such a binder or curing agent is included in the detection layer 321, the holding power of the enzyme and the mediator is improved in the detection layer 321.

<Stabilizer>
In the present embodiment, the detection layer 321 may further include a stabilizer for protecting and stabilizing the enzyme held in the detection layer 321. Examples of such a stabilizer include albumin. Among albumins, human-derived albumin such as human serum albumin, bovine-derived albumin such as bovine serum albumin, and the like can be given.

<Buffer agent>
Furthermore, in this embodiment, the detection layer 321 may include a buffering agent (buffer solution) for suppressing pH fluctuations caused by enzymes. The buffering agent preferably has a buffer region in the same region as the optimum pH of the enzyme used and is water-soluble (aqueous solution), and examples thereof include a phosphate buffer.

<Layer structure of detection layer>
The detection layer 321 may be composed of two or more layers in addition to the one layer as described above.

  For example, when the detection layer 321 is composed of two or more layers, for example, the above-described detection layer 321 is provided as a glucose detection layer, and the glucose permeation adjustment layer, noise, or the like laminated on the upper side or the lower side of the glucose detection layer. The structure provided with at least 1 sort (s) of a removal layer and an enzyme protective layer is mentioned.

  The glucose permeation control layer is laminated on the upper side of the glucose detection layer. The glucose permeation control layer suppresses or prevents the glucose detection layer from coming into contact with the measurement target (interstitial fluid, blood), while It is a layer that exhibits the function of permeation and has the function of controlling the transmittance of oxygen and glucose. It is preferable that the glucose permeation regulating layer having such a function can transmit more oxygen than glucose. As a result, when GOD is used as an enzyme, it is possible to suppress or prevent an apparent decrease in the measurement value due to lack of oxygen in the detection of glucose, and to improve the detection accuracy of the glucose measurement value. Can be planned.

  Although it does not specifically limit as this glucose permeation | regulation control layer, For example, what mixed the crosslinking agent, such as an isocyanate compound, and polyethyleneglycol (PEG) which is a polymer provided with a terminal hydroxyl group, 4-hydroxybutyl acrylate, etc. single or mixed And those in which a urethane bond is generated to form a crosslinked structure.

  In addition, those composed of aminopropyl polysiloxane or the like in which a urea resin is formed by using an isocyanate and an amino group are preferably used, and those composed of a siloxane resin are more preferably used. Those composed of siloxane are particularly preferably used.

  The noise removal layer is laminated on the lower side of the glucose detection layer, and is a layer aimed at improving the adhesion between the electrode and the glucose detection layer. The electrical noise can be reduced by improving the adhesion between the electrode and the glucose detection layer. Moreover, the drift of a measured value can be suppressed.

The constituent material of the noise removal layer is not particularly limited as long as it can exhibit the adhesive function as described above. For example, polyethylene glycol (PEG), polypropylene glycol (PPG), methyl cellulose (MC), acetyl cellulose (Cellulose acetate), hydroxyethylcellulose, hydroxypropylcellulose, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), polyvinyl alcohol-polyvinyl acetate copolymer (PVA-PVAc), poly (vinyl alcohol) -styrylpyridinium compound (PVA) -SbQ), hydroxyethyl methacrylate, poly (2-hydroxyethyl methacrylate) (HEMA) and the like, and one or more of these can be used in combination. Among these materials, PVA-SbQ is a material utilizing UV curability. In order to insolubilize these noise removal layers, an isocyanate compound may be added to the noise removal layer so that isocyanate can be used as a functional group.

  Further, a surface modifying agent such as a silane coupling agent or a titanium coupling agent may be used as the noise removing layer, or the surface modifying layer may be provided separately from the noise removing layer. Moreover, you may contain albumin in a noise removal layer in order to protect a glucose detection layer and a boundary surface.

  The enzyme protective layer is a layer that is laminated on the glucose detection layer and has a function of protecting the interface of the glucose detection layer. When the detection layer 321 includes a glucose permeation control layer, the protective layer is located between the glucose detection layer and the glucose permeation control layer.

  The material constituting the enzyme protective layer is not particularly limited as long as it can function to protect the interface of the glucose detection layer. For example, polyethylene glycol (PEG), polypropylene glycol (PPG), methyl cellulose (MC) , Acetyl cellulose (cellulose acetate), hydroxyethyl cellulose, hydroxypropyl cellulose, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), polyvinyl alcohol-polyvinyl acetate copolymer (PVA-PVAc) poly (vinyl alcohol) -styrylpyridinium compound (PVA-SbQ) hydroxyethyl methacrylate, poly (2-hydroxyethyl methacrylate) (HEMA) and the like can be used, and one or more of these can be used in combination. Yes.

  For the purpose of protecting the glucose detection layer, albumin similar to the above may be contained in the enzyme protection layer. Further, in order to make the enzyme protective layer insoluble, the binder or curing agent is a material using a material having two or more functional groups such as aldehyde and isocyanate in the molecule, and those functional groups. You may mix and use the polymer material which has the functional group which can couple | bond, specifically a hydroxyl group, an amino group, an epoxy group, etc. at the terminal, and an enzyme. In this case, as the binder and the curing agent, for example, glutaraldehyde, toluene diisocyanate, isophorone diisocyanate and the like can be used.

<Method for producing detection layer>
In the present embodiment, the detection layer 321 includes, for example, an enzyme, a mediator, and a host material (a binder, a curing agent, a stabilizer, and a buffer as necessary) that dissolve the detection layer 321 in water. Alternatively, after being dispersed, it is applied to the substrate 301 on which the electrode layer 315 is formed, dried, and insolubilized. In this case, when a material using UV curable properties such as PVA-SbQ is used as the host material, it is performed by appropriately irradiating ultraviolet rays.

  Thus, a film is formed by reacting each material dissolved or dispersed in water to form a host material which is a polymer film (polymer) insoluble in water. Then, the mediator is cross-linked and fixed to the host material which is a polymer having a large molecular weight. For this reason, when the sensor element including the detection layer formed in this way is embedded in the body, small molecule mediators are prevented from eluting from the detection layer and diffusing.

1.2.4. Other Configurations of Detection Unit In addition, the detection unit 300 has been described as having a three-layer configuration including a substrate 301, an electrode layer 315, and a detection layer 321 as shown in FIG. For example, as shown in FIG. 7, a four-layer structure having a partition wall layer 331, and a five-layer structure having a partition wall layer 331 and an opening layer 341 as shown in FIG. It may be what constitutes. These will be described in detail below.

  7 includes a partition layer 331 in addition to the substrate 301, the electrode layer 315, and the detection layer 321. The detection unit 300 having a four-layer configuration illustrated in FIG.

  The partition layer 331 is stacked on the electrode layer 315, that is, is interposed between the electrode layer 315 and the detection layer 321, and corresponds to the working electrode 311, the counter electrode 312 and the reference electrode 313 included in the electrode layer 315. A partition wall having an opening is formed at the position. By the partition layer 331, regions corresponding to the electrodes 311, 312, and 313 are partitioned in the thickness direction of the detection unit 300. That is, for each of the electrodes 311, 312, and 313, a region where electrons generated by an enzyme acting on glucose are supplied from the detection layer 321 is strictly partitioned. As a result, signal noise can be reduced and influence between adjacent electrodes 311, 312, and 313 can be reduced.

  In addition to the function as a partition wall, the partition layer 331 can also function as an edge cover and / or a bank.

  By causing the partition layer 331 to function as an edge cover, erosion from the ends of the electrodes 311, 312, and 313 can be suppressed or prevented.

  Further, by causing the partition layer 331 to function as a bank, when the detection layer 321 is formed using a liquid phase film formation method, the detection layer 321 is formed in the opening of the partition layer 331. When supplying the liquid material, the size of the liquid pool of the liquid material can be easily defined.

  The height, thickness and constituent materials of such a partition layer 331 are appropriately selected according to the purpose.

  In addition, a method for forming the partition layer 331 is not particularly limited. For example, a photolithography method using a negative or positive photosensitive material including a polyimide resin, an acrylic resin, or the like, or a photoresist using a dry resist sheet. Law.

  8 includes a partition layer 331 and an opening layer 341 in addition to the substrate 301, the electrode layer 315, and the detection layer 321.

  This partition layer 331 has the same configuration as the partition layer 331 described in the detection unit 300 having the four-layer configuration shown in FIG. 7 and exhibits the same function.

  Further, the opening layer 341 is stacked on the detection layer 321 and forms a partition including an opening at a position corresponding to the working electrode 311, the counter electrode 312, and the reference electrode 313 included in the electrode layer 315. Similar to the partition wall layer 331, regions corresponding to the electrodes 311, 312, and 313 are strictly partitioned by the opening layer 341 in the thickness direction of the detection unit 300. Thereby, glucose that permeates into the detection layer 321 from the interstitial fluid is limited to only the glucose that is located in the opening of the opening layer 341, that is, supplied from the thickness direction (upward direction) of the detection layer 321. It is possible to accurately suppress or prevent glucose from permeating from an oblique direction. Therefore, the amount of glucose can be detected more accurately.

  A method for forming such an opening layer 341 is not particularly limited. For example, when the opening layer 341 is made of titanium, a method of forming a titanium film using a mask sputtering method can be given. In the case where the opening layer 341 is formed of a resin material such as naphthoquinone azide polymer (polysulfonic acid ester), a photolithography method using a negative photosensitive material containing the resin material can be given.

  In addition to the configuration shown in FIGS. 5 to 8, the detection unit 300 further includes an electrode layer 315 including a working electrode 311, a counter electrode 312, and a reference electrode 313, and each of these electrodes 311, 312, 313. In addition, a wiring layer including a wiring 314 that is electrically connected independently can be provided between the substrate 301 and the electrode layer 315.

  Further, in the present embodiment, in the detection unit 300, the case where the working electrode 311, the counter electrode 312, the reference electrode 313, and the wiring 314 included in the electrode layer 315 are entirely covered with the detection layer 321 has been described. Alternatively, the detection layer 321 may be selectively formed on the working electrode 311 and the counter electrode 312.

1.2.5. Configuration of Circuit The main body 110 has a circuit that is disposed therein and drives the detection unit 300. FIG. 9 is a diagram schematically illustrating a configuration of a circuit 400 that drives the detection unit 300 included in the sensor element 100.

  The circuit 400 includes an amplifier 401 for applying an amplified constant voltage between the reference electrode 313 and the working electrode 311, and an amplifier 402 for amplifying the current value flowing between the working electrode 311 and the counter electrode 312. Are electrically connected to each other through the wiring 314. The amplifier 401 is electrically connected to the reference electrode 313 via the wiring 314, and applies a constant voltage, for example, 0.1 V amplified between the reference electrode 313 and the amplifier 401. The amplifier 402 amplifies the current value that flows between the working electrode 311 and the counter electrode 312 via the wiring 314, and transmits the amplified current value to the processing circuit 200 via the wiring 132.

  The formation of the reference electrode 313 can be omitted. In this case, the amplifier 401 is electrically connected so that a constant voltage can be applied between the working electrode 311 and the counter electrode 312. You can do it.

1.3. Measurement of Glucose Value Next, an example of a method for measuring the glucose value in the interstitial fluid using the measuring device 101 including the sensor element 100 according to the present embodiment will be described.

  First, the cannula 111 is inserted into the subcutaneous tissue 502, and the detection layer 321 of the detection unit 300 is brought into contact with the interstitial fluid to be stabilized.

  Next, a constant voltage is applied between the working electrode 311 and the reference electrode 313 for a predetermined time. The generated electron is measured as a current value flowing between the working electrode 311 and the counter electrode 312 to obtain a glucose value in the interstitial fluid.

1.4. As described above, in the sensor element 100 and the measuring apparatus 101 according to the present embodiment, the mediator is sufficiently fixed on the working electrode 311 because the water-soluble mediator is cross-linked to the hydrophilic host material. The For this reason, it becomes possible to provide the sensor element 100 and the measuring apparatus 101 in which the mediator is less likely to be eluted and diffused in the sensor, and the baseline of the measured value is less likely to drift. In addition, since the detection layer 321 is formed of a water-soluble or hydrophilic material as described above, the mediator is sufficiently fixed to the film formed of the host material without significantly reducing the enzyme activity. Can do.

Thereby, in the sensor element 100 and the measuring apparatus 101 which concern on this embodiment, SMB
The measurement value is not corrected by G, and measurement of the glucose value using the measurement apparatus 101 according to the present embodiment makes it possible for the doctor to determine the diabetic patient's daily blood glucose level and determine the treatment policy. It is possible to satisfy both of the two objectives of “being blood glucose level measurement as a calculation index of insulin dose”.

1.5. Others The sensor element 100 according to the present embodiment may be attached to, for example, an insulin pump in addition to being attached to the measurement apparatus 101 as described above.

  FIG. 10 is a perspective view schematically showing a state in which the sensor element 100 according to the present embodiment is attached to the insulin pump 171.

  An insulin pump 171 shown in FIG. 10 is used by connecting the sensor element 100, and includes an arithmetic unit 210 including the sensor element 100 and a processing circuit 200 that analyzes a current value obtained by the sensor element 100, and A supply unit 175 including a needle unit 172 that supplies (administers) insulin to the subcutaneous tissue 502 based on a measurement value obtained by calculation by the calculation device 210 and a sensor element 100 are attached (connected) to the supply unit 175. The connector 131 includes wiring 132 that connects the processing circuit 200 and the connector 131. In such an insulin pump 171, the current value flowing between the working electrode 311 and the counter electrode 312 is transmitted to the processing circuit 200 via the circuit 400 and the wiring 132 provided in the main body 110, and the processing circuit 200 is The glucose value (glucose concentration) in the interstitial fluid is calculated as a measured value by analysis of the arithmetic device 210 provided. Based on this measured value (glucose value), that is, when the measured value is higher than the set concentration, the insulin pump 171 is activated, and insulin is automatically administered to the wearer via the needle portion 172. Is done.

  In the measuring apparatus 101 and the insulin pump 171, the current value measured by the sensor element 100 may be transmitted to the processing circuit without passing through the wiring. For example, the current value is measured wirelessly through the communication means. The value may be transmitted to the processing circuit.

  In the measuring apparatus 101, the sensor element 100 and the display unit 155 are not limited to those connected via the wiring 132, but may be integrally formed, or the insulin pump 171. In this case, the sensor element 100 and the supply unit 175 are not limited to those connected via the wiring 132 but may be integrally formed.

2. EXAMPLES Hereinafter, the present invention will be described more specifically with reference to experimental examples and comparative examples, but the present invention is not limited to these examples.

2.1. Preparation of sensor element <Example 1>
First, a transparent glass substrate having an average thickness of 0.5 mm was prepared as a substrate. Next, a patterned conductive ITO film having an average thickness of 100 nm was formed as a wiring layer on the substrate by mask sputtering.

  Next, a patterned platinum film having an average thickness of 200 nm was formed as an electrode layer including a working electrode, a counter electrode, and a reference electrode on the obtained ITO film by a mask sputtering method.

On the obtained platinum film, a bank having an average height of 8 μm is formed by a photoresist using an acrylic material, and an opening that is in contact with a liquid to be measured (blood, interstitial liquid) is opened to thereby form a partition layer. Formed.

  And the board | substrate with which the electroconductive ITO film | membrane, the platinum film | membrane, and the partition layer were laminated | stacked was immersed in order of acetone and 2-propanol, and after ultrasonically cleaning, the oxygen plasma process and the argon plasma process were performed. Each of these plasma treatments was performed at a plasma power of 100 W, a gas flow rate of 20 sccm, and a treatment time of 5 seconds with the substrate heated to 70-90 ° C.

  An ink containing silver-silver chloride particles is filled on the platinum electrode of the reference electrode by an ink jet method so that the film thickness becomes 3 μm, and dried in an oven at 120 ° C., so that the silver-silver chloride film is formed. Formed.

  Next, the liquid composition prepared to have the following composition was applied to the entire surface of the reference electrode, the working electrode, and the counter electrode using a spin coating method, and a film thickness of 2 μm was obtained after drying. After drying, an insolubilized film was obtained by irradiating ultraviolet rays having a wavelength of 365 nm at 1000 mJ, and used as a detection layer for detecting glucose.

Liquid composition for detecting layer formation-1 part by weight of FAD-GDH (FAD glucose dehydrogenase)-600 parts by weight of PVA-SbQ-2.8 parts by weight of vitamin-K1-29000 parts by weight of water (solvent)

<Example 2-4>
A composition was prepared under the same conditions as in Example 1 except that the mediators listed in Table 4 were selected.

<Example 5>
PVA-SbQ and 5 (polypropylene glycol) listed in Table 1 are used as host materials, and D (2-hydroxy-1,4-naphthoquinone) listed in Table 2 is used instead of vitamin-K1 as a mediator. Further, under the same conditions as in Example 1, except that a liquid composition for forming a detection layer was prepared using α (1,3-bis (vinylsulfonylacetamido) propane) described in Table 3 as a crosslinking agent. A sensor element was produced.

Liquid composition for detecting layer formation-FAD-GDH (FAD glucose dehydrogenase) 1 part by weight-PVA-SbQ 500 parts by weight-PPG-300 (polypropylene glycol) 100 parts by weight-2-hydroxy-1,4-naphthoquinone 1 .2 parts by weight-1,3-bis (vinylsulfonylacetamido) propane 50 parts by weight-Water (solvent) 29000 parts by weight

<Example 6-20>
A composition was prepared under the same conditions as in Example 5 except that the host material and mediator listed in Table 4 were selected.

<Examples 21-28>
Same as Example 5 except that the host materials and mediators listed in Table 4 were selected, and β (1,3-bis (vinylsulfonyl) propan-2-ol) listed in Table 3 was used as a crosslinking agent. A composition was prepared under the following conditions.

<Examples 29-33>
The composition was prepared under the same conditions as in Example 5 except that the host material and mediator listed in Table 4 were selected and that γ (N, N′-methylenebisacrylamide) listed in Table 3 was used as the crosslinking agent. Prepared.

<Examples 34-38>
The host material and mediator described in Table 4 were selected, and δ (1,4-bis (4-vinylphenoxy) -butane) described in Table 3 was used as a crosslinking agent. A composition was prepared under conditions.

<Examples 39-41>
A composition was prepared under the same conditions as in Example 21 except that the three types of host materials described in Table 4 were used and the mediators described in Table 4 were selected.

<Comparative Examples 1 and 2>
A composition was prepared under the same conditions as in Example 1 except that the mediators listed in Table 4 were selected.

<Comparative Examples 3 and 4>
A mediator described in Table 4 was selected, and a composition was prepared under the same conditions as in Example 1 except that γ was used as a crosslinking agent.

<Comparative Example 5-7>
A composition was prepared under the same conditions as in Example 1 except that the host material, mediator and crosslinking agent shown in Table 4 were selected.

<Comparative Example 8>
As shown in Table 4, a composition was prepared under the same conditions as in Example 1 except that no mediator and crosslinking agent were used and only the host material was used.

2.2. Evaluation test <Measurement of detected current density (initial characteristics)>
The glucose concentration was adjusted to 100 mg / dL in phosphate physiological saline and prepared as a standard solution, and this preparation solution was heated at 35 ° C. so as to be constant. The sensor element obtained above was immersed in this solution, and the reference electrode part, the counter electrode part, and the working electrode part were completely immersed in the solution. Next, a voltage was applied so that the gap between the reference electrode and the working electrode was 100 mV, and this state was maintained for 5 minutes to stabilize the state. The obtained current value was taken as the detected current density. As an apparatus, an electrochemical analyzer ALS608B manufactured by BAS Co., Ltd. was used.

<Measurement of change in current density after 24 hours (drift test)>
In the above evaluation test, the current value was measured every 0.5 hours, the current ground for 24 hours from the voltage application was measured, and the amount of change from the value obtained in the evaluation test was examined.

2.3. Evaluation Results In all of the examples, the initial detected current density observed was about 3 μA / mm ² , and the change in current density after 24 hours was 3% or less, and the amount of change was small, and almost no drift occurred. . On the other hand, in all of the comparative examples, the initial detected current density was 0.020 μA / mm ² or less, and only the noise level value was observed, and the change after 24 hours could not be discussed. In Comparative Examples 1 and 2, since Compound 1 used as the host material does not react with the hydroxyl group of the mediator, the mediator cannot be immobilized, and electronically exchanged with FAD-GDH used as the enzyme. This is thought to be due to the fact that it was not possible. Thus, the compound 1 used as the host material is fixed to the film formed of the host material when a mediator having a functional group other than a hydroxyl group is used as in Example 1-4. However, in the case of a functional group that does not crosslink with the host material even if it has a functional group, the mediator was not fixed. Even when the mediators (M, R) used in Comparative Examples 1 and 2 are used, as in Examples 14, 19, 25, and 27, the host material can be crosslinked with a crosslinking agent. Although it is fixed to the film formed of the material, as in Comparative Example 3-5, when the crosslinking agent is not used to crosslink the host material, the mediator may be fixed to the film formed of the host material. could not. Furthermore, since the mediators used in Comparative Examples 6 and 7 were not cross-linked with the host material even when a cross-linking agent was used, the mediator could not be fixed to the film formed of the host material. In this way, even if the mediator and the cross-linking agent react to form a cross-linking bond, if the host material is not involved in this reaction, the molecular weight of the mediator does not increase so much, it is not fixed and is eluted from the sensor, The sensor behavior became unstable. Comparative Example 8 is an example in which a film was formed without using a mediator or a cross-linking agent. Even when the same amount of enzyme was used as in the other examples, only a detection current density of noise level was observed.

  From the above examples, when the mediator is cross-linked to the host material, the mediator involved in electron transfer between the enzyme and the working electrode is fixed to the host material, so that it is sufficiently fixed on the working electrode. A sensor element that did not diffuse and hardly drifted was obtained.

  The present invention is not limited to the embodiments described above, and various modifications are possible. For example, the present invention includes substantially the same configuration (for example, a configuration having the same function, method, and result, or a configuration having the same purpose and effect) as the configuration described in the embodiment. In addition, the invention includes a configuration in which a non-essential part of the configuration described in the embodiment is replaced. In addition, the present invention includes a configuration that achieves the same effect as the configuration described in the embodiment or a configuration that can achieve the same object. In addition, the invention includes a configuration in which a known technique is added to the configuration described in the embodiment.

  DESCRIPTION OF SYMBOLS 100 ... Sensor element 101 ... Measuring apparatus 110 ... Main body part 111 ... Cannula 112 ... Through-hole 113 ... Window part 114 ... Hollow part 120 ... Detachable part 121 ... Needle part 122 ... Gripping part 131 ... Connector, 132 ... Wiring, 151 ... Monitor, 155 ... Display part, 171 ... Insulin pump, 172 ... Needle part, 175 ... Supply part, 200 ... Processing circuit, 210 ... Calculation device, 300 ... Detection part, 301 ... Board ( (Base), 311 ... working electrode, 312 ... counter electrode, 313 ... reference electrode, 314 ... wiring, 315 ... electrode layer, 321 ... sensing layer, 331 ... partition layer, 341 ... opening layer, 400 ... circuit, 401, 402 ... Amplifier, 501 ... epidermis, 502 ... subcutaneous tissue, 503 ... blood vessel

Claims (13)

The base,
A working electrode provided at the base;
A sensing layer provided on the working electrode;
A counter electrode from which electrons move between the working electrode and
The sensing layer is
An enzyme that acts on the target substance contained in the liquid;
A water-soluble mediator that mediates electron transfer between the enzyme and the working electrode;
And a hydrophilic host material cross-linked to the mediator.   The sensor element according to claim 1, wherein the mediator and the host material have crosslinkable functional groups that crosslink each other.   The sensor element according to claim 1 or 2, wherein the mediator and the host material are crosslinked by a water-soluble crosslinking agent.   The sensor element according to claim 3, wherein the cross-linking agent is at least one compound selected from the group consisting of bis (vinylsulfonyl) -based compounds, bisacrylamide-based compounds, and bisstyrene-based compounds.   5. The mediator according to claim 1, wherein the mediator is one or more quinone compounds selected from the group consisting of benzoquinone compounds, naphthoquinone compounds, phenanthrenequinone compounds, and acenaphthenequinone compounds. The described sensor element.   The said crosslinkable functional group is 1 or more types of functional groups selected from the group which consists of a double bond, a hydroxyl group, an imino group, a thiol group, and an amino group as described in any one of Claims 2 thru | or 5. Sensor element.   The sensor element according to any one of claims 1 to 6, wherein the host material is at least one compound selected from the group consisting of a polyvinyl alcohol compound, a glycol compound, and a cellulose compound.   The sensor element according to claim 1, wherein the mediator has an oxidation-reduction potential in a range of −0.25 V or more and 0.2 V or less.   The sensor element according to any one of claims 1 to 8, wherein the target substance is glucose.   The sensor element according to any one of claims 1 to 9, wherein the enzyme is glucose dehydrogenase.   The sensor element according to claim 1, further comprising a reference electrode.   A measuring device comprising the sensor element according to any one of claims 1 to 11.   The measurement device according to claim 12, wherein the measurement device is a continuous blood glucose measurement type.