Patent Description:
A glucose sensor such as a blood sugar level sensor using electrochemistry is known. Generally, such a glucose sensor includes two or more electrodes at least including a working electrode and a counter electrode, and after a spacer for forming a cavity is bonded on the electrodes, a reagent layer including an enzyme, a mediator and the like is formed at a portion of the cavity, and a cover is attached to structure the glucose sensor.

When a specimen (blood, interstitial fluid, sweat, etc.) is introduced into the cavity of the glucose sensor (a space formed by a groove formed in the spacer), the glucose (or a substrate) contained in the specimen reduces the mediator (or an electrode active material) via the enzyme. Here, when a predetermined voltage is applied to the electrode, the reduced mediator is oxidized by an electrochemical reaction. When the reduced mediator is oxidized, an oxidation current is generated, and the amount of the glucose can be detected by measuring this oxidation current.

Furthermore, using electrically conductive fine particles such as carbon nanotubes (hereinafter also abbreviated as "CNTs") to measure a current generated by direct electron transfer between a glucose-bound enzyme and an electrode through the CNTs to measure a glucose level, is also considered.

For example, Patent Literature <NUM> (<CIT>) discloses a reagent layer for a glucose sensor including an oxidoreductase, a water-soluble electrically conductive polymer and electrically conductive fine particles. Patent Literature <NUM> discloses using flavin adenine dinucleotide-dependent glucose dehydrogenase (hereinafter also abbreviated as "FAD-GDH") as the oxidoreductase.

Furthermore, Patent Literature <NUM> (<CIT>) discloses a reagent layer for a glucose sensor including an enzyme, electrically conductive fine particles and a non-electrically conductive polymer. Patent Literature <NUM> discloses using CNTs as the electrically conductive fine particles.

Non-Patent Literature <NUM> (<NPL>) specifically discloses using CNT as a mediator in a reagent layer of a glucose sensor (a reagent for a glucose sensor).

However, the present inventors have found through an investigation that when FAD-GDH is used as the enzyme, direct electron transfer may not occur in some case depending on the type of CNT.

The present invention has been made in view of the above problem, and it is an object of the present invention to provide a glucose sensor which is highly sensitive and accurate when CNT is used and FAD-GDH is used as an enzyme.

The present invention is directed to a reagent used for a glucose sensor for electrochemical, quantitative determination of glucose, comprising
a flavin adenine dinucleotide glucose dehydrogenase (FAD-GDH), single-walled carbon nanotubes (CNTs), and a dispersant, the dispersant including at least one type of compound selected from an anionic compound, a cationic compound, and a nonionic compound.

In one aspect, the flavin adenine dinucleotide glucose dehydrogenase is glycosylated.

In another aspect, the flavin adenine dinucleotide glucose dehydrogenase has a molecular weight of <NUM> KDa or more.

In a further aspect, the flavin adenine dinucleotide glucose dehydrogenase has a molecular weight of <NUM> KDa or more.

In a particular aspect, the anionic compound is at least any one of sodium dodecyl sulfate, sodium cholate and sodium dodecylbenzene sulfonate.

In another particular aspect, the cationic compound is cetyltrimethylammonium bromide.

In a further particular aspect, the nonionic compound is at least any one of octylphenol ethoxylate and polysorbates.

In one aspect, the flavin adenine dinucleotide glucose dehydrogenase is derived from Aspergillus filamentous fungi, Thermoascus filamentous fungi or Talaromyces filamentous fungi.

The present invention provides a sensor for electrochemical, quantitative determination of glucose, comprising an electrode,
the electrode having a surface at least partially covered with a reagent layer comprising said reagent.

The present invention provides a method for manufacturing said glucose sensor comprising
applying on the electrode a carbon nanotube liquid containing the single-walled carbon nanotubes and the dispersant, followed by an enzyme liquid containing the flavin adenine dinucleotide glucose dehydrogenase, and drying them to form the reagent layer.

The present invention provides a method for manufacturing said glucose sensor comprising
applying on the electrode an enzyme liquid containing the flavin adenine dinucleotide glucose dehydrogenase, followed by a carbon nanotube liquid containing the single-walled carbon nanotubes and the dispersant, and drying them to form the reagent layer.

The present invention provides a method for manufacturing said glucose sensor comprising
applying on the electrode a liquid mixture of a carbon nanotube liquid containing the single-walled carbon nanotubes and the dispersant and an enzyme liquid containing the flavin adenine dinucleotide glucose dehydrogenase, and drying it to form the reagent layer.

The present invention provides a glucose measuring device using said glucose sensor.

The present invention can thus provide a glucose sensor which is highly sensitive and accurate when CNT is used and FAD-GDH is used as an enzyme.

In the drawings, identical reference characters denote identical or equivalent components. Dimensional relationships such as length, width, thickness, depth, and the like have been appropriately changed for clarification and simplification of the drawings, and do not represent actual dimensional relationships. It is needless to say that each embodiment is an example and partial substitution or combination of configurations shown in different embodiments is possible.

With reference to <FIG>, a glucose sensor of the present embodiment is a glucose sensor (a sensor chip) for measuring glucose (a substrate) contained in a sample liquid, and includes an insulating substrate <NUM>, an electrode, a reagent layer <NUM>, a spacer <NUM>, and a cover <NUM>.

The electrode includes a working electrode <NUM> and a counter electrode <NUM> provided on one surface of insulating substrate <NUM>. Reagent layer <NUM> is formed on a portion of a surface of the electrode facing away from insulating substrate <NUM>.

Spacer <NUM> has a notch <NUM> for forming a cavity <NUM> for guiding a sample liquid to reagent layer <NUM>, and spacer <NUM> is disposed on the electrode so that reagent layer <NUM> is located inside notch <NUM> (or cavity <NUM>). Preferably, of the surface of the electrode, at least a portion exposed in cavity <NUM> is covered with the reagent layer.

Cover <NUM> is provided on a surface of spacer <NUM> facing away from insulating substrate <NUM> so as to cover at least notch <NUM>. Cover <NUM> has an air hole 5a in communication with cavity <NUM>.

In the present embodiment, reagent layer <NUM> is composed of a reagent (a reagent for a glucose sensor) including flavin adenine dinucleotide glucose dehydrogenase (FAD-GDH), single-walled carbon nanotubes (single-walled CNTs), and a dispersant.

The present inventors have conducted an inspection to find that in a glucose sensor using FAD-GDH as an enzyme, when a multi-walled CNT or bundled single-walled CNTs is/are used as CNT, direct electron transfer between the enzyme and the electrode via the CNT does not easily occur. On the other hand, it has been found that direct electron transfer occurs when debundled single-walled CNTs are used.

It is inferred that these result because FAD-GDH (or an enzyme <NUM>) has an active center having a size of around <NUM> and when a multi-walled CNT or bundled single-walled CNTs having a particle size larger than that is/are used, CNT <NUM> cannot enter the active center (see <FIG>), and CNT <NUM> cannot pass/receive electrons to/from enzyme <NUM>. On the other hand, it is inferred that when debundled single-walled CNTs are used, (i.e., when single-walled CNTs and a dispersant are blended in the reagent liquid), CNT <NUM> can enter the active center of enzyme <NUM> (see <FIG>), and CNT <NUM> can pass/receive electrons to/from enzyme <NUM>.

Therefore, when FAD-GDH is used as an enzyme, using debundled single-walled CNTs (a reagent liquid containing single-walled CNTs and a dispersant) allows a glucose sensor to be provided which is highly sensitive and accurate using CNT as a mediator.

In a relation in size with the active center of FAD-GDH, the single-walled CNT preferably has an outer diameter (or the cylinder has a diameter) of <NUM> to <NUM>, more preferably <NUM> to <NUM>. The particle diameter of the single-walled CNT can be measured with a transmission electron microscope (TEM) or an atomic force microscope (AFM).

Note, for example, that the electrode may have a surface with a hydrophilic polymer membrane formed thereon. This is because when the electrode (or an electrode membrane) has a surface covered with the reagent layer, the hydrophilic polymer membrane allows the electrode to have a surface enhanced in wettability by the reagent liquid and thus helps to form the reagent layer. The hydrophilic polymer membrane for example includes an acetonitrile plasma-polymerized membrane, a membrane composed of a hydrophilic polymer such as carboxymethyl cellulose and methyl cellulose or an amphiphilic polymer such as polyvinyl pyrrolidone or the like.

Preferably, FAD-GDH is glycosylated (i.e., has a carbohydrate attached). The glycosylated FAD-GDH suppresses detachment of the reagent layer and suppresses deactivation of the enzyme (or FAD-GDH).

In the process for forming the reagent layer, when introducing fine particles such as CNTs, a reagent layer such as an enzyme may peel off due to the fine-particle layer's membrane stress. When the reagent peels off, direct electron transfer between the enzyme and the electrode via the CNT is not performed effectively. In addition, produced sensors have large variation in quality, characteristics, and the like.

In addition, when the CNT liquid is brought into contact with the enzyme, the enzyme may be inactivated due to an effect of the dispersant contained in the CNT liquid.

In contrast, when FAD-GDH is glycosylateed, the enzyme's three-dimensional structure is rigidly held. This can suppress detachment of the reagent layer when with the enzyme as a base the CNT liquid is dropped to form the reagent layer. As a result, direct electron transfer between the enzyme and the electrode via the CNT can be performed effectively. This reduces variation between produced sensors in quality, characteristics, and the like.

In addition, when the CNT liquid is brought into contact with the enzyme, and the dispersant or the CNT acts on the enzyme, the enzyme having its three-dimensional structure rigidly held by a carbohydrate is not inactivated and its original activity can be ensured.

As the FAD-GDH a FAD-GDH derived for example from Aspergillus filamentous fungi, Thermoascus filamentous fungi or Talaromyces filamentous fungi can suitably be used (see, for example, <CIT>, <CIT>, <CIT>, and <CIT>).

The dispersant is a compound which can prevent bundling of single-walled CNTs. The dispersant can be at least one type of compound selected from an anionic compound, a cationic compound and a nonionic compound.

The anionic compound is sodium dodecyl sulfate, sodium cholate or sodium dodecylbenzene sulfonate, for example. The cationic compound is cetyltrimethylammonium bromide for example. The nonionic compound is octylphenol ethoxylate (Triton-X-<NUM>, Triton-X-<NUM>, Triton-X-<NUM>, Triton-X- <NUM> and the like produced by Dow Chemical Company), or polysorbates (polysorbate <NUM> (Tween <NUM>), polysorbate <NUM> (Tween <NUM>), polysorbate <NUM> (Tween <NUM>), polysorbate <NUM> (Tween <NUM>), etc.), for example.

Reagent layer <NUM> may include a hydrophilic polymer (carboxymethylcellulose or the like). Such a hydrophilic polymer effectively helps to immobilize reagent layer <NUM> to a surface of the electrode or filters impurity (such as blood cells in the blood) in a sample liquid.

While insulating substrate <NUM> is not particularly limited in what material is used to form it, it is formed of plastic materials such as PET (polyethylene terephthalate) film, photosensitive materials, paper, glass, ceramics, biodegradable materials, or the like. These materials are also used as materials for spacer <NUM> and cover <NUM>.

An electrode provided on insulating substrate <NUM> includes at least working electrode <NUM> and counter electrode <NUM>. In addition to working electrode <NUM> and counter electrode <NUM>, the electrode may include a reference electrode serving as a reference for potential in measuring the electrode's potential and a detection electrode for detecting that a sample is supplied to cavity <NUM>.

These electrodes (the working, counter, reference, and detection electrodes, etc.) are formed of materials such as platinum, gold, palladium or a similar noble metal, carbon, copper, aluminum, nickel, titanium, ITO (indium tin oxide), ZnO (zinc oxide), and the like.

Cover <NUM> is preferably formed of an insulating material, and it can for example be plastics such as PET film, photosensitive materials, paper, glass, ceramics, and biodegradable materials. It is preferable that cover <NUM> have air hole 5a in communication with cavity <NUM> formed by spacer <NUM>. This is because capillarity allows a sample to be attracted toward air hole 5a and thus helps introducing the sample into cavity <NUM>.

An example of a method for manufacturing the glucose sensor of the present embodiment will be described with reference to <FIG>. <FIG> is an exploded perspective view showing a configuration of a glucose sensor according to the present embodiment. <FIG> are diagrams for illustrating an example of a process for manufacturing the glucose sensor of the present embodiment, and each shows a different step. In this embodiment, a plurality of glucose sensors can be fabricated at the same time.

Initially, referring to <FIG>, an electrode (working electrode <NUM> and counter electrode <NUM> for quantitatively determining a substrate) is formed on each of a plurality of insulating substrates <NUM>. Specifically, an electrically conductive layer is formed on one surface of insulating substrate <NUM> by sputtering or the like, and the formed electrically conductive layer is subjected to laser-processing, photolithography, or the like to form a pattern to thus form the electrode (or electrode membrane). Other than working electrode <NUM> and counter electrode <NUM>, the reference electrode, detection electrode, and the like mentioned above may be formed. A plasma treatment may be applied to the surface of the electrode and that of insulating substrate <NUM>.

The electrode membrane can be formed, for example, by a sputtering method, a vacuum deposition method, an ion plating method, a CVD (chemical vapor deposition) method, an MBE (molecular beam epitaxy) method, a melt transporting method, a melt temperature lowering method, a sol-gel method, a plating method, a coating method, screen-printing, or the like. While the electrode membrane is not particularly limited in thickness, it is for example <NUM> to <NUM>.

Subsequently, referring to <FIG>, spacer <NUM> having notch <NUM> is bonded to a portion of the electrode (working electrode <NUM> and counter electrode <NUM>) on a side thereof facing away from insulating substrate <NUM>, and a portion of a surface of insulating substrate <NUM> on a side facing the electrode in a region in which the electrode is not formed.

Referring to <FIG>, a reagent liquid including an enzyme (FAD-GDH), single-walled CNTs and a dispersant can be dropped on a side of the electrode (working electrode <NUM> and counter electrode <NUM>) facing away from insulating substrate <NUM> (in notch <NUM>) and dried to form reagent layer <NUM>.

More specifically, referring to <FIG>, for example, reagent layer <NUM> can be formed through a process in which a carbon nanotube (CNT) liquid (CNT <NUM> and a dispersant <NUM>) and subsequently, an enzyme liquid (enzyme <NUM>) are applied on an electrode <NUM> (a hydrophilic polymer membrane <NUM>) (process A).

Referring to <FIG>, reagent layer <NUM> may be formed through a process in which the enzyme liquid (enzyme <NUM>) is applied prior to the CNT liquid (CNT <NUM> and dispersant <NUM>) (process B).

Referring to <FIG>, reagent layer <NUM> may be formed through a process in which initially the CNT liquid (CNT <NUM> and dispersant <NUM>) and the enzyme liquid (enzyme <NUM>) are mixed together to prepare a liquid mixture which is in turn applied to electrode <NUM> (hydrophilic polymer membrane <NUM>) (Process C).

Subsequently, referring to <FIG>, cover <NUM> having air hole 5a is disposed on spacer <NUM> so as to cover at least notch <NUM> to thus form cavity <NUM> for guiding a sample liquid to reagent layer <NUM>. Air hole 5a is provided on a side opposite to an opening of cavity <NUM> in communication with an interior of cavity <NUM>.

Subsequently, a set of substrates of glucose sensors formed through the above process are divided to provide glucose sensors having cavity <NUM> (see <FIG> and <FIG>).

The glucose sensor (or sensor chip) of the present invention is attached to a measuring instrument in use. That is, when a sample (blood or the like) is supplied to cavity <NUM> of the glucose sensor attached to the measuring instrument, a substance in the sample to be measured (i.e., glucose) is bound to the enzyme (FAD-GDH) and electrons are transferred by the tunnel effect to a CNT significantly close to the FAD serving as an active center, and an electric current is thus generated. The measuring instrument electrically connected to working electrode <NUM> and counter electrode <NUM> of the glucose sensor measures the current to thus quantitatively determine the substance contained in the sample to be measured.

Hereinafter, an example of usage of the glucose sensor of the present invention will be described. Initially, blood is brought into contact with an end portion (an inlet 41c) of cavity <NUM>, and introduced into cavity <NUM> through capillarity. Then, a voltage is applied between working electrode <NUM> and counter electrode <NUM>, and a value of a current is measured, as timed as determined. The applied voltage is, for example, <NUM> V. When blood is introduced into cavity <NUM>, an analyte in the blood allows the enzyme and the CNT to directly transfer electrons. The current flowing when a voltage is applied between working electrode <NUM> and counter electrode <NUM> has a correlation with the analyte's concentration.

Subsequently, a value of the current is measured after the voltage is applied when a predetermined period of time has elapsed. For example, a value of the current after a period of <NUM> to <NUM> seconds has elapsed is measured. This value of the current can be used to determine the analyte's concentration from a previously obtained calibration curve.

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

Basically, a glucose sensor having the structure as shown in <FIG> described in the first embodiment was fabricated. The electrode was formed of gold. A metal film made of gold was formed by sputtering and patterned to form the electrode (the working electrode and the counter electrode). In addition, an acetonitrile plasma-polymerized membrane was applied on a surface of the electrode (that is, a portion where the reagent layer was formed). No reference or detection electrode was fabricated.

The enzyme liquid was an aqueous dispersion of FAD-GDH. The FAD-GDH was an FAD-GDH having an enzyme No. <NUM> shown in Table <NUM>.

The CNT liquid was a dispersion liquid containing <NUM>% by mass of single-walled CNTs (debundled and having an outer diameter of <NUM> to <NUM>) and <NUM>% by mass of a dispersant (sodium cholate) with water used as a dispersion medium.

With reference to <FIG>, in the case of such a CNT liquid, even when bundled single-walled CNTs <NUM> are added, single-walled CNTs <NUM> are debundled due to the presence of dispersant <NUM>. For obtaining such a single-walled CNT, and preparing such a CNT liquid, etc., for example, reference can be made to Non-Patent Literature <NUM>.

Then, as shown in <FIG>, the CNT liquid (including single-walled CNTs <NUM> and dispersant <NUM>) and subsequently, the enzyme liquid (including enzyme <NUM>) are dropped on electrode <NUM> (hydrophilic polymer membrane <NUM>) and dried to form reagent layer <NUM> (Process A).

A glucose sensor of Comparative Example <NUM> was fabricated in a manner similar to that for Example <NUM> except that the CNT liquid was a dispersion liquid containing <NUM>% by mass of multi-walled CNTs (debundled and having an outer diameter of <NUM> to <NUM> and <NUM> to <NUM> layers) and <NUM>% by mass of a dispersant (sodium cholate) with water used as a dispersion medium. Reagent layer <NUM> was formed through the same process as in Example <NUM> shown in <FIG> (process A).

A glucose sensor of Comparative Example <NUM> was fabricated in a manner similar to that for Example <NUM> except that no dispersant was added to the CNT liquid. That is, in Comparative Example <NUM>, the CNT liquid was a dispersion liquid containing <NUM>% by mass of single-walled CNTs (bundled) and no dispersant with a liquid mixture of water and ethanol (water:ethanol = <NUM>:<NUM>) used as a dispersion medium. Reagent layer <NUM> was formed through the same process as in Example <NUM> shown in <FIG> (process A).

A liquid in which glucose was dissolved in a <NUM> phosphate buffer liquid (pH: <NUM>) was prepared as a sample liquid (a glucose liquid) to be measured. The glucose's concentration was <NUM>, <NUM>, <NUM> or <NUM>.

This sample liquid was supplied into the cavity of each glucose sensor fabricated in Example <NUM>, Comparative Example <NUM> and Comparative Example <NUM>, and cyclic voltammetry was employed (scanning speed: <NUM> V/s) to measure a value of a current passing between the working electrode and the counter electrode.

A result of the measurement of the value of the current is shown in <FIG> correspond to Example <NUM>, Comparative Example <NUM> and Comparative Example <NUM>, respectively.

As shown in <FIG> (see a value for a voltage of <NUM> V in particular), when debundled multi-walled CNT (Comparative Example <NUM>) and bundled CNTs (Comparative Example <NUM>) are used, an electrochemical reaction (a current) depending on glucose concentration could not be detected. In contrast, when debundled single-walled CNTs (a reagent liquid containing single-walled CNTs and a dispersant) was used (Example <NUM>), a current was detected.

It is inferred that this is because, with reference to <FIG>, the FAD-GDH (or enzyme <NUM>) has an active center (located between two solid triangles) having a size of around <NUM> and when multi-walled CNT <NUM> (<FIG>) or bundled single-walled CNTs (<FIG>) having a larger particle diameter is/are used (Comparative Examples <NUM> and <NUM>), CNT <NUM> cannot pass/receive electrons to/from enzyme <NUM>. On the other hand, with reference to <FIG>, it is inferred that when debundled single-walled CNTs <NUM> are used (Example <NUM>), CNT <NUM> can enter the active center of enzyme <NUM>, and CNT <NUM> can pass/receive electrons to/from enzyme <NUM>.

For Example <NUM>, glucose sensors (of seven types) were fabricated through the same process as in Example <NUM> except that seven types of FAD-GDHs (enzyme Nos. <NUM> to <NUM>) shown in Table <NUM> were used. Note that a case with enzyme No. <NUM> used overlaps with Example <NUM>. For preparation of the FAD-GDHs, reference can be made for example to <CIT>, <CIT> <CIT>, and <CIT>.

For Example <NUM>, glucose sensors (of seven types) were fabricated in the same manner as in Example <NUM> except that instead of the reagent layer formation process (process A) in Example <NUM>, a reagent layer was formed through process B (i.e., applying the enzyme liquid before the CNT liquid (see <FIG>).

For Example <NUM>, glucose sensors (of seven types) were fabricated in the same manner as in Example <NUM> except that instead of the reagent layer formation process (process A) in Example <NUM>, a reagent layer was formed through process C (i.e., initially, mixing the CNT liquid and the enzyme liquid together to prepare a liquid mixture and applying it on electrode <NUM> (hydrophilic polymer membrane <NUM>) (see <FIG>).

A liquid in which glucose was dissolved in a <NUM> phosphate buffer liquid (pH: <NUM>) was prepared as a sample liquid (a glucose liquid) to be measured. The glucose's concentration was <NUM>, <NUM> or <NUM>.

This sample liquid was supplied into the cavity of each of the glucose sensors (of <NUM> types in total) fabricated in Examples <NUM> to <NUM>, and cyclic voltammetry was employed to measure a value of a current, similarly as done for Test Example <NUM>. A result of the measurement is shown in <FIG> and <FIG>.

For the measurement results in <FIG> and <FIG>, a result of evaluation of a sensing operation (i.e., whether a current is sensed or not) is shown in Table <NUM>. In Table <NUM> at the "sensing operation (sensing of current)" column, "+" means that a current is sensed and "-" means that no current is sensed.

From the results shown in Table <NUM> and <FIG> and <FIG>, it can be seen that a glucose sensor of Example <NUM> in which the single-walled CNT dispersion liquid was dropped after the enzyme liquid was dropped (Process B) and a glucose sensor of Example <NUM> in which the enzyme liquid and the single-walled CNT dispersion liquid were mixed together and then dropped (Process C) can steadily sense a current for FAD-GDHs having generally larger apparent molecular weights (that is, having larger amounts of glycosylation).

In particular, it is believed that a FAD-GDH having a molecular weight of <NUM>,<NUM> or more tends to ensure direct electron transfer between the enzyme and the electrode via the CNT for any of Processes A and B (Examples <NUM> and <NUM>).

Furthermore, it is believed that a FAD-GDH having a molecular weight of <NUM>,<NUM>,<NUM> or more tends to ensure direct electron transfer between the enzyme and the electrode via the CNT for any of Processes A, B and C (Examples <NUM>, <NUM> and <NUM>).

It is believed that Example <NUM> manufactured through Process A tends to be most reliably sense a current, followed by Example <NUM> manufactured through Process B followed by Example <NUM> manufactured through Process C. By thus reliably sensing a current, a highly sensitive and accurate glucose sensor can be provided.

Claim 1:
A reagent used for a glucose sensor for electrochemical, quantitative determination of glucose, comprising
a flavin adenine dinucleotide glucose dehydrogenase, single-walled carbon nanotubes, and a dispersant, which can prevent bundling of the single-walled carbon nanotubes, the dispersant including at least one type of compound selected from an anionic compound, a cationic compound, and a nonionic compound.