U.S. patent application number 14/686008 was filed with the patent office on 2015-10-01 for analysis device.
This patent application is currently assigned to ARKRAY, INC.. The applicant listed for this patent is ARKRAY, Inc.. Invention is credited to Masashi Tsukada.
Application Number | 20150276652 14/686008 |
Document ID | / |
Family ID | 46298227 |
Filed Date | 2015-10-01 |
United States Patent
Application |
20150276652 |
Kind Code |
A1 |
Tsukada; Masashi |
October 1, 2015 |
Analysis Device
Abstract
An analysis device is disclosed which includes an electron
detection medium to obtain information needed for analyzing an
analyte in correlation with an electron transfer level, and a
reagent part which is disposed on the electron detection medium and
includes an electron transporting substance to transport electrons
between the analyte and the electron detection medium, the electron
transporting substance including a water-soluble aromatic
heterocycle compound, and being free of a metal complex. An
analysis method using the analysis device is also disclosed.
Inventors: |
Tsukada; Masashi; (Kyoto,
JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ARKRAY, Inc. |
Kyoto |
|
JP |
|
|
Assignee: |
ARKRAY, INC.
Kyoto
JP
|
Family ID: |
46298227 |
Appl. No.: |
14/686008 |
Filed: |
April 14, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13455555 |
Apr 25, 2012 |
9045790 |
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14686008 |
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Current U.S.
Class: |
205/777.5 ;
204/403.14 |
Current CPC
Class: |
G01N 27/3271 20130101;
G01N 2333/902 20130101; C12Q 1/004 20130101; G01N 2333/904
20130101; G01N 27/3273 20130101; C12Q 1/006 20130101 |
International
Class: |
G01N 27/327 20060101
G01N027/327 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 26, 2011 |
JP |
2011-098742 |
Apr 5, 2012 |
JP |
2012-086736 |
Claims
1. An analysis device comprising: an electron detection medium to
obtain information needed for analyzing an analyte in correlation
with an electron transfer level; and a reagent part which is
disposed on the electron detection medium and comprises an
oxidoreductase that transfers an electron to or from the analyte,
and an electron transporting substance to transport electrons
between the oxidoreductase and the electron detection medium
without involving any oxidation-reduction reaction, wherein the
electron transporting substance is a water-soluble aromatic
heterocycle compound that is free of a metal complex.
2. The analysis device according to claim 1, wherein the electron
transporting substance comprises at least one water-soluble
aromatic heterocycle compound selected from the group consisting of
pyridine compounds and imidazole compounds.
3. The analysis device according to claim 1, wherein the
water-soluble aromatic heterocycle compound has a molecular weight
of 1,000 or less.
4-5. (canceled)
6. The analysis device according to claim 1, wherein a
concentration of the electron transporting substance is a
concentration that allows transportation of electrons between the
electron detection medium and the oxidoreductase.
7. The analysis device according to claim 1, wherein a
concentration of the electron transporting substance is from 10
mass % to 60 mass % based on a total mass of the reagent part.
8. The analysis device according to claim 1, wherein the electron
transporting substance comprises at least one water-soluble
aromatic heterocycle compound selected from the group consisting of
pyridine and aminomethyl pyridine.
9. (canceled)
10. The analysis device according to claim 1, wherein the reagent
part comprises a crosslinked substance obtained by crosslinking
using at least one selected from the group consisting of
glutaraldehyde, carbodiimide compounds and succinimide esters.
11. The analysis device according to claim 1, wherein the electron
detection medium is an electric conductor.
12. The analysis device according to claim 1, wherein the analyte
is a saccharide.
13. An analysis method comprising obtaining information needed for
analyzing an analyte based on an electron transfer level of the
analyte using the analysis device according to claim 1.
14. The analysis device according to claim 1, wherein the electron
transporting substance comprises an electron cloud of pi-electrons
perpendicular to the surfaces of the molecules of the electron
transporting substance.
15. The analysis device according to claim 1, wherein the electron
transporting substance comprises 4-aminomethyl pyridine or
5-amino-4-imidazolecarboxamide.
16. The analysis device according to claim 1, wherein the electron
transporting substance has a molecular weight of 800 or less.
17. The analysis device according to claim 1, wherein the
oxidoreductase and the electrode have a gap in between, and the
electron transporting substance is configured to enter into the gap
in the presence of the analyte.
18. The analysis device according to claim 1, wherein the electron
transporting substance is configured to enter into a gap between
the oxidoreductase and the electrode, and the electron transporting
substance and the oxidoreductase overlap one another to construct
an electron transfer pathway with pi-electrons of the electron
transporting substance in the presence of the analyte.
19. The analysis device according to claim 1, wherein the electron
transporting substance is not a polymer.
20. An analysis method comprising transporting electrons in a
reagent part of an analysis device by electron transporting
substances between an analyte and an electron detection medium
without involving oxidation-reduction of the electron transporting
substances, wherein the reagent part is disposed on the electron
detection medium and comprises an oxidoreductase that transfers an
electron to or from the analyte, and the electron transporting
substance is a water-soluble aromatic heterocycle compound that is
free of a metal complex; and obtaining information about the
analyte based on an electron transfer level of the analyte by the
transporting electrons step.
Description
BACKGROUND
[0001] 1. Technical Field
[0002] The present invention relates to an analysis device.
[0003] 2. Related Art
[0004] As methods for measuring various substances contained in
biological samples such as blood, for example, methods with enzyme
sensors using enzymes are known. In an enzyme sensor utilizing an
electrochemical reaction system in which a general enzyme is used,
a signal is obtained by detecting, on an electrode surface, a
transfer of electrons generated based on an enzymatic catalytic
reaction. Even when a colorimetric reaction system, which depends
on changes in the optical properties of a pigment, is used as a
detection method, its basic reaction is derived from the enzymatic
catalytic reaction (oxidation-reduction) and involves electron
transfer. In these reaction systems, electron transport efficiency
in the reaction systems influences detection sensitivities. Various
techniques in which the electron transport efficiency is improved
in order to increase the sensitivity of enzyme sensors are
known.
[0005] For example, National phase publication (Translation of PCT
Application) No. 2002-514305 discloses a sensor in which a surface
of an electrode is modified with a substance having a helical
structure, such as nucleic acid, as a conductive polymer, to
promote electron transfer to or from an enzyme molecule to be
targeted.
[0006] Since electron transfer between an electrode and an enzyme
occurs through the active center of the enzyme, the manner of the
arrangement of a site, in which the active center is present, with
respect to the electrode is important. In order to decrease
influences on the orientation of the active center of an enzyme and
therefore to transport electrons efficiently, molecules which
function like an electron transport mediator which is
oxidized/reduced to transport electrons, for example, complexes
having, as an active center, a transition metal such as iron,
copper, osmium, or ruthenium, are known, and sensors using such a
complex are also known (for example, see Japanese National phase
publication (Translation of PCT Application) Nos. 2006-509837 and
2005-520172).
[0007] As an enzyme electrode, a system in which a polypyrrole is
used is known (for example, Biosensors & Bioelectronics, Vol.
7, (1992) pp. 461-471 and Sensors and Actuators B, Vol. 106, (2005)
pp. 289-295). Since pyrrole, which is a monomer, has poor
water-solubility, it is not possible to directly mix pyrrole with
an enzyme liquid to prepare an enzyme electrode on the surface of
an electrode material. Therefore, for example, in these documents
(i.e., Biosensors & Bioelectronics, Vol. 7, (1992) pp. 461-471
and Sensors and Actuators B, Vol. 106, (2005) pp. 289-295), a
polymerization reaction is carried out using ferric chloride and a
pyrrole solution in a track etched membrane and, thereafter, the
membrane is impregnated with an enzyme liquid to obtain an
electrode.
SUMMARY OF THE INVENTION
[0008] However, there is still room for improvement with respect to
the sensitivity of analysis devices such as sensors for detecting
analytes in samples. In a sensor, a higher detection sensitivity
not only allows detection of a small amount of analyte in a sample,
but also becomes advantageous when miniaturization of the sensor in
itself is required. In addition, a metal complex used as an
electron transport mediator is generally an expensive material and
may become unstable as a substance or interfere with a reaction
potential since the metal complex in itself is oxidized and
reduced.
[0009] Further, with respect to an enzyme electrode in which a
polypyrrole is used, a manufacturing method thereof is complicated.
Moreover, when voltage is continuously applied to polypyrrole in an
aqueous system environment, polypyrrole may be decomposed (see
Sensors and Actuators B, Vol. 106, (2005) pp. 289-295), and,
therefore, the long-time use reliability may be low when using an
enzyme electrode in which polypyrrole is used.
[0010] Thus, an analysis device that has higher sensitivity and
reliability than those conventionally used in the art by using an
electron transporting substance with stability has been
demanded.
[0011] Accordingly, it is an object of the present invention to
provide an analysis device that has higher sensitivity and
reliability than those conventionally used in the art; and also to
provide an analysis method using the analysis device.
[0012] Exemplary embodiments of the present invention include the
followings, but the present invention is not limited to the
following exemplary embodiments.
[0013] <1> An analysis device comprising:
[0014] an electron detection medium to obtain information needed
for analyzing an analyte in correlation with an electron transfer
level; and
[0015] a reagent part which is disposed on the electron detection
medium and comprises an electron transporting substance to
transport electrons between the analyte and the electron detection
medium, the electron transporting substance comprising a
water-soluble aromatic heterocycle compound, and being free of a
metal complex.
[0016] <2> The analysis device according to <1>,
wherein the electron transporting substance comprises at least one
water-soluble aromatic heterocycle compound selected from the group
consisting of pyridine compounds and imidazole compounds (the group
consisting of pyridine, imidazole and derivatives thereof).
[0017] <3> The analysis device according to <1> or
<2>, wherein the water-soluble aromatic heterocycle compound
has a molecular weight of 1,000 or less.
[0018] <4> The analysis device according to any one of
<1> to <3>, wherein the reagent part further comprises
an electron transfer compound that transfers an electron to or from
the analyte.
[0019] <5> The analysis device according to <4>,
wherein the electron transfer compound is an oxidoreductase.
[0020] <6> The analysis device according to <4> or
<5>, wherein a concentration of the electron transporting
substance is a concentration that allows transportation of
electrons between the electron detection medium and the electron
transfer compound.
[0021] <7> The analysis device according to any one of
<1> to <6>, wherein a concentration of the electron
transporting substance is from 10 mass % to 60 mass % based on a
total mass of the reagent part.
[0022] <8> The analysis device according to any one of
<1> to <7>, wherein the electron transporting substance
comprises at least one water-soluble aromatic heterocycle compound
selected from the group consisting of pyridine and aminomethyl
pyridine.
[0023] <9> The analysis device according to any one of
<1> to <8>, wherein the reagent part comprises a
crosslinked substance.
[0024] <10> The analysis device according to any one of
<1> to <9>, wherein the reagent part comprises a
crosslinked substance obtained by crosslinking using at least one
selected from the group consisting of glutaraldehyde, carbodiimide
compounds and succinimide esters.
[0025] <11> The analysis device according to any one of
<1> to <10>, wherein the electron detection medium is
an electric conductor.
[0026] <12> The analysis device according to any one of
<1> to <11>, wherein the analyte is a saccharide.
[0027] <13> An analysis method comprising obtaining
information needed for analyzing an analyte based on an electron
transfer level of the analyte using the analysis device according
to any one of <1> to <12>.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Exemplary embodiments of the present invention are described
in detail based on the following figures, wherein:
[0029] FIG. 1 is a conception diagram of an analysis device
according to an exemplary embodiment of the present invention;
[0030] FIG. 2 is a graph indicating glucose response currents from
enzyme electrodes in Example 1 of the present invention;
[0031] FIG. 3 is a voltammogram (oxidation wave) indicating glucose
response currents from enzyme electrodes in Example 2 of the
present invention;
[0032] FIG. 4 is a graph indicating glucose response currents from
enzyme electrodes in Example 3 of the present invention;
[0033] FIG. 5 is a graph in which stability is confirmed in
continuous measurement of enzyme electrodes in Example 4 of the
present invention;
[0034] FIG. 6 is a graph indicating glucose response currents from
enzyme electrodes in Example 5 of the present invention; and
[0035] FIG. 7 is a graph indicating glucose response currents from
enzyme electrodes in Comparative Example 1 of the present
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0036] An analysis device according to the present invention
includes: an electron detection medium to obtain information needed
for analyzing an analyte in correlation with an electron transfer
level; and a reagent part which is disposed on the electron
detection medium and includes an electron transporting substance to
transport electrons between the analyte and the electron detection
medium, the electron transporting substance including a
water-soluble aromatic heterocycle compound, and being free of a
metal complex.
[0037] An analysis method according to the present invention
includes obtaining information needed for analyzing an analyte
based on an electron transfer level of the analyte using the
analysis device.
[0038] According to the present invention, electrons are
efficiently transported between the analyte and the electron
detection medium since the electron transporting substance in the
reagent part includes a water-soluble aromatic heterocycle compound
(hereinafter, may also be simply referred to as an "aromatic
heterocycle compound"). Since the aromatic heterocycle compound may
simply function as a field for transporting electrons and it is not
necessary that the aromatic heterocycle compound in itself is
subjected to oxidation-reduction as in the case of a metal complex,
the aromatic heterocycle compound is considered to have no
dependency on a reaction potential in a system and to be a stable
compound. As a result, the analysis device according to the present
invention including the reagent part containing such a
water-soluble aromatic heterocycle compound can have higher
sensitivity and reliability than those conventionally used in the
art, for example, when being applied to a sensor and/or the
like.
[0039] In addition, by using such an analysis device, an analyte
can be analyzed with better sensitivity and higher reliability than
those conventionally used in the art.
[0040] More specifically, it is supposed that, when the
water-soluble aromatic heterocycle compound is present in the
reagent part, the molecules of the aromatic heterocycle compound
are close to each other, the electron cloud of pi-electrons present
to be perpendicular to the surfaces of the molecules of the
aromatic heterocycle compound becomes an electron-transporting
path, and, therefore, electrons are efficiently transported. In
addition, it is supposed that the reason for not involving any
oxidation-reduction reaction of the water-soluble aromatic
heterocyclic compounds in itself is that the electron cloud of
delocalized pi-electrons is utilized as the electron-transporting
path.
[0041] An analysis device according to an exemplary embodiment of
the present invention is described taking as an example the case of
using as an enzyme electrode, with reference to FIG. 1. However,
the present invention is not bound by the following theory.
[0042] An electron transfer layer 14 as the reagent part is
disposed on an electrode 12 as the electron detection medium in the
enzyme electrode 10 and molecules of electron transfer compound(s)
such as a oxidoreductase 16 are present to be independent of each
other in the electron transfer layer 14. The active centers 18 of
the molecules of the oxidoreductase 16 are not oriented in the same
direction with respect to the electrode 12 because the active
center 18 is often localized in a part of a molecule of the
oxidoreductase 16. That is, in one molecule of the oxidoreductase,
the active center 18 is placed in the vicinity of the electrode 12,
and in another molecule of the oxidoreductase 16, the active center
18 is placed in a place away from the electrode 12. Therefore, the
distances of electron transfer (directions of arrows in FIG. 1)
between the active centers 18 of the oxidoreductase 16 and the
electrode 12 are considered to be varied. When a distance from the
active center 18 of a molecule of the oxidoreductase 16 to the
electrode 12 is long, electron transfer between the oxidoreductase
16 and the electrode 12 by the oxidoreductase 16 alone becomes
difficult.
[0043] According to the exemplary embodiment of the present
invention, it is supposed that an aromatic heterocycle compound 20
(nitrogen-containing aromatic heterocycle compound in FIG. 1) is
present between an oxidoreductase 16 and the electrode 12 in the
electron transfer layer 14. Therefore, it is supposed that even
when a distance between the active center 18 of an oxidoreductase
16 and the electrode 12 is long, if an aromatic heterocycle
compound 20 is present therebetween, the aromatic heterocycle
compound 20 enters into a gap between the oxidoreductase 16 and the
electrode 12 and the aromatic heterocycle compound 20 and the
oxidoreductase 16 overlap one another to construct an electron
transfer pathway with the pi-electrons of the aromatic heterocycle
compound 20.
[0044] Construction of such an electron transfer pathway allows
electron transfer via the electron transfer pathway with an
aromatic heterocycle compound 20 not only between an oxidoreductase
16 present at a position near to the electrode 12 and the electrode
12 but also between an oxidoreductase 16 present at a position
farther away from the electrode 12 and the electrode 12.
[0045] As a result, electrons depending on the amount of an analyte
which is present are transferred between the oxidoreductase 16 and
the electrode 12 and are converted into the amount of the analyte
in a sample by a detection system which is not illustrated, so that
the presence or absence and amount of the analyte can be
detected.
[0046] In the analysis device, the concentration of the electron
transporting substance in the reagent part may be a concentration
that allows transportation of electrons between the electron
transfer compound and the electron detection medium and, for
example, the concentration of the electron transporting substance
may be from 10 mass % to 60 mass %, preferably from 10 mass % to 50
mass %, based on the total mass of the reagent part, when the
electron transfer compound is present. When the concentration of
the electron transporting substance is such a concentration, the
molecules of the aromatic heterocycle compound included in the
electron transporting substance around the analyte can become close
to each other to construct the electron transfer pathway with a
thickness enabling better electron transport. As a result, the
analysis device with better sensitivity can be made.
[0047] In the analysis device, the aromatic heterocycle compound
may be, for example, a nitrogen-containing aromatic heterocycle
compound and, for example, is preferably at least one selected from
the group consisting of pyridine, imidazole, and derivatives
thereof. The analysis device with higher sensitivity can be made
with, as the aromatic heterocycle compound, the nitrogen-containing
aromatic heterocycle compound, for example, at least one selected
from the group consisting of pyridine, imidazole, and derivatives
thereof.
[0048] In the analysis device, the reagent part preferably includes
an electron transfer compound which transfers electrons to or from
an analyte and the electron transfer compound is, for example, an
oxidoreductase. A naturally-occurring substance or a substance
derived from a living body can easily be detected with higher
sensitivity by incorporating the electron transfer compound such as
an oxidoreductase into the reagent part. In the analysis device,
the analyte may be, for example, a saccharide, and in this case, a
saccharide can be detected with good sensitivity.
[0049] The term "step" as used herein encompasses not only an
individual step but also a step in which an expected effect of this
step is attained even when the step cannot be clearly distinguished
from other step(s).
[0050] A numerical value range indicated by using "from A to B" as
used herein refers to a range including A and B as the minimum and
maximum values, respectively.
[0051] In the present invention, when a plurality of substances
corresponding to one component are present in a composition, the
amount of the component in the composition means the total amount
of the plurality of substances present in the composition unless
otherwise specified.
[0052] The present invention is described below.
[0053] The analysis device according to an exemplary embodiment of
the present invention includes: an electron detection medium for
obtaining information needed for analyzing an analyte in
correlation with an electron transfer level; and a reagent part
which is disposed on the electron detection medium and includes an
electron transporting substance for transporting electrons between
the analyte and the electron detection medium, wherein the electron
transporting substance includes a water-soluble aromatic
heterocycle compound, with the proviso that the electron
transporting substance is free of a metal complex.
[0054] The electron detection medium is for obtaining information
needed for analyzing an analyte in correlation with an electron
transfer level, and preferably includes a conductive layer which
can transfer electrons to or from the analyte and a supporting
member for defining or ensuring physical characteristics of the
electron detection medium, for example, defining a shape or
ensuring rigidity, which are disposed in this order.
[0055] The reagent part is disposed on the electron detection
medium and includes the electron transporting substance for
transporting electrons between the analyte and the electron
detection medium. In the reagent part, the electron transporting
substance may be included in a layer placed on the electron
detection medium. A layer that corresponds to the reagent part and
includes the electron transporting substance is referred to herein
as "electron transfer layer."
[0056] Electron Detection Medium
[0057] (1) Supporting Member
[0058] The material of the supporting member may be either a
material having insulating properties or a material having
conductivity. As the supporting member having insulating
properties, a commercially available engineering plastic such as
polyethylene terephthalate, polyimide, polystyrene, or Duracon
(registered trademark from Polyplastics Co., Ltd.) may be used.
[0059] As the supporting member having conductivity, for example,
conductive carbon paper, a carbon fiber web, or a metal in plate,
bar, or thin film form (e.g., gold or platinum) may be used. When
the supporting member having conductivity is used, the supporting
member may also function as a lead for taking an output from the
analysis device.
[0060] The supporting member may be a member having sufficient
rigidity in measurement. The supporting member may optionally have
flexibility. The form of the supporting member is not particularly
limited. The supporting member may have, for example, the form of a
film or a rod, the form may variously be changed depending on a
purpose.
[0061] The thickness of the supporting member may generally be from
0.1 mm to 1 mm, depending on the application of the analysis
device, but is not limited thereto.
[0062] (2) Conductive Layer
[0063] The conductive layer in the electron detection medium is
placed on the supporting member and includes a conductive substance
which can transfer electrons to or from the electron transfer layer
described below. By containing such a conductive substance, there
are such advantages that, for example, the electron detection
medium becomes an electric conductor and electrons can easily be
detected as electric signals.
[0064] As the conductive substance, which is not particularly
limited, a known substance which can transfer electrons may be
used. Examples of such substances may include carbon materials,
metals, metal-supported carbon, and the like, and one substance may
be used singly or two or more substances may be used in
combination.
[0065] A carbon material used as the conductive material may be
used in the form of carbon particles or a structure in which carbon
particles are high-density arranged or integrated. Examples of such
carbon particles may include activated carbon, graphite, carbon
black, and particles forming a higher-order structure, represented
by diamond-like carbon, a carbon nanotube, or fullerene. Such
structures in which carbon particles are high-density arranged or
integrated include glassy carbon, pyrolytic graphite carbon,
plastic-formed carbon, and/or the like. For example, such an
advantage that molding to a desirable shape is enabled can be
obtained by using such a carbon material. The carbon particles,
which have primary particle sizes, for example, ranging from 3 nm
to 150 nm, more preferably from 3 nm to 50 nm, are used. The
conductive substance in which the carbon particles have such
particle diameters has such an advantage that the specific surface
area of the electron detection medium is increased or
three-dimensional interaction of a fine structure may easily occur
in electron transfer.
[0066] The metal as the conductive material may be present as metal
particles in the conductive layer. When the metal particles are
used, the metal particles may be present independently of the
carbon particles or supported on the carbon particles.
[0067] Typical examples of such metal may include noble metals such
as platinum (Pt), rhodium (Rh), gold (Au), silver (Ag), palladium
(Pd), ruthenium (Ru), iridium (Ir), or osmium (Os), and one of
these noble metals may be used alone or two or more thereof may be
used in combination. Preferably, platinum is used alone or platinum
and any one or more other noble metal(s) are used in
combination.
[0068] When the metal is supported as the metal particles on the
carbon particles, the particle size of the metal such a size that
allows the metal particles to be suitably supported on the carbon
particles, for example, a size of a colloid level ranging from 1 nm
to 20 .mu.m, preferably from 1 nm to 4 nm. The amount of the metal
particles supported on the carbon particles may be, for example,
from 0.1 part by mass to 60 parts by mass based on 100 parts by
mass of the carbon particles. The amount of the metal particles
supported on the carbon particles of not less than 0.1 part by mass
can further improve sensitivity while the amount of not more than
60 parts by mass of the metal particles may provide, for example, a
better relationship between the amount of the metal used and the
sensitivity, and thus provide an economical advantage. Preferably,
the amount of the metal particles supported on the carbon particles
is from 0.5 part by mass to 40 parts by mass based on 100 parts by
mass of the carbon particles.
[0069] The metal contained in the conductive layer may be present
as a component in a different layer from that of the carbon
particles. The conductive layer in this case may be composed of the
layer having the carbon particles (carbon-containing layer) and the
layer having the metallic element (metal-containing layer). As the
kinds of metals which may be contained in the metal-containing
layer, the above-mentioned metals may be applied as is. Such a
metal-containing layer is preferably placed between the supporting
member and the carbon-containing layer.
[0070] The forms of the above-mentioned carbon material and metal
are not particularly limited and may be the forms of particles and
any of other forms such as plate, rod, and thin film forms. The
forms of the carbon and the metal may be identical to each other or
different.
[0071] The thickness of the conductive layer may generally be from
0.01 .mu.m to 10 .mu.m but is not limited thereto, although varying
according to the application of the analysis device.
[0072] Electron Transfer Layer
[0073] The electron transfer layer includes the electron
transporting substance and preferably further includes an electron
transfer compound which transfers electrons to or from an
analyte.
[0074] The electron transfer layer may preferably be placed at a
position allowing electron transfer to or from the electron
detection medium. The electron transfer layer may be disposed in
contact with the electron detection medium, but is not necessarily
in contact with the electron detection medium, and, for example,
another layer which does not interfere with electron transporting
may be disposed between the electron transfer layer and the
electron detection medium.
[0075] The thickness of the electron transfer layer may generally
be from 0.1 .mu.m to 5 .mu.m but is not limited thereto, although
varying according to the application of the analysis device.
[0076] (1) Electron Transfer Compound
[0077] The electron transfer compound in the analysis device may be
a compound which transfers electrons, depending on the amount of an
analyte which is present, to or from the analyte. Examples of such
electron transfer compounds may include a compound involved in an
enzyme reaction with an analyte. These electron transfer compounds
may appropriately be selected depending on the application of the
analysis device.
[0078] The electron transfer compound is preferably an enzyme,
further preferably an oxidoreductase. In the analysis device using
the enzyme as the electron transfer compound, electrons transferred
between an analyte and the electron transfer compound can simply
and easily be assessed as electrons depending on the amount of the
analyte which is present, based on an enzyme-substrate
relationship. Therefore, the analysis device is suitable for
quantitatively measuring the concentration of a certain analyte in
a sample, in which various substances are mixed, by a specific
enzymatic reaction.
[0079] The oxidoreductase is an enzyme that catalyzes an
oxidation-reduction reaction and a single enzyme or a combination
of a plurality of different enzymes may be used in the same
analysis device depending on the kind of an analyte and the details
of detection of interest. Examples of the oxidoreductase include
glucose oxidase (GOD), galactose oxidase, bilirubin oxidase,
pyruvate oxidase, D- or L-amino acid oxidase, amine oxidase,
cholesterol oxidase, choline oxidase, xanthine oxidase, sarcosine
oxidase, L-lactate oxidase, ascorbate oxidase, cytochrome oxidase,
alcohol dehydrogenase, glutamate dehydrogenase, cholesterol
dehydrogenase, aldehyde dehydrogenase, glucose dehydrogenase (GDH),
fructose dehydrogenase, sorbitol dehydrogenase, lactate
dehydrogenase, malate dehydrogenase, glycerol dehydrogenase,
17B-hydroxysteroid dehydrogenase, estradiol 17B-dehydrogenase,
amino acid dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase,
3-hydroxysteroid dehydrogenase, diaphorase, cytochrome
oxidoreductase, catalase, peroxidase, and glutathione
reductase.
[0080] Especially, an oxidoreductase for a saccharide is
preferable, and examples such oxideredutase include glucose oxidase
(GOD), galactose oxidase, glucose dehydrogenase (GDH), fructose
dehydrogenase, and sorbitol dehydrogenase.
[0081] The amount of the enzymes used is not particularly limited
and may appropriately be set.
[0082] (2) Aromatic Heterocycle Compound as Electron Transporting
Substance
[0083] The aromatic heterocycle compound included in the electron
transfer layer is a water-soluble aromatic heterocycle compound.
Incorporating the water-soluble aromatic heterocycle compounds in
the electron transfer layer allows transportation of electrons
between the electron transfer compound and the electron detection
medium. An analysis device with favorable stability under aqueous
environment, higher reliability and better sensitivity can be
provided by incorporating such an aromatic heterocycle compound
rather than a metal complex into the electron transfer layer. In
addition, there is also such an advantage that the aromatic
heterocycle compound does not depend on a reaction potential in a
system.
[0084] In the present invention, the term "water-soluble" means
dissolving in pure water of 20.degree. C. in a mass ratio of 6% or
higher under the environment at 20.degree. C.
[0085] Since the water-soluble aromatic heterocycle compound
includes an aromatic heterocycle, the electron clouds of
pi-electrons can be formed to transport electrons. The aromatic
heterocycle may be a five- or six-membered ring. The aromatic
heterocycle may be a condensed or non-condensed aromatic
heterocycle. Examples of the hetero atom include a nitrogen atom,
an oxygen atom, and a sulfur atom.
[0086] The aromatic heterocycle compound may have at least one
substituent on the aromatic heterocycle as long as the
water-solubility of the compound is not impaired.
[0087] Examples of the substituent on the aromatic heterocycle of
the aromatic heterocycle compound may include halogen atoms, amino
group, alkyl groups, alkenyl groups, and alkoxy groups.
[0088] These substituents may further have at least one
substituent, examples of which include ones mentioned as the
examples of the substituents on the aromatic heterocycle.
[0089] The aromatic heterocycle compound may be, from the viewpoint
of, for example, sensitivity, preferably a nitrogen-containing
aromatic heterocycle compound, and examples of the
nitrogen-containing aromatic heterocycle compound may include
imidazole, pyrazole, pyridine, pyrimidine, purine, and derivatives
thereof. The aromatic heterocycle compound is preferably, from the
viewpoint of, for example, sensitivity, pyridine, imidazole or a
derivative thereof, and is more preferably pyridine or a derivative
thereof. Examples of pyridine or a derivative thereof may include
pyridine and aminomethyl pyridine. One of such aromatic heterocycle
compounds may be used singly, or two or more thereof may be used in
combination. As the aromatic heterocycle compound, especially,
aminomethyl pyridine is further preferred from a viewpoint of, for
example, the sensitivity of the analysis device.
[0090] The aromatic heterocycle compound is preferably an aromatic
heterocycle compound having a molecular weight of 1,000 or less,
more preferably an aromatic heterocycle compound having a molecular
weight of 800 or less. Therefore, in an embodiment, it is
preferable that the aromatic heterocycle compound according to the
present invention does not encompass an aromatic heterocycle
compound having a molecular weight of more than 1,000, such as a
polymer. When the aromatic heterocycle compound having a molecular
weight of 1,000 or less is used, better transportation of electrons
between the electron transfer compound and the electron detection
medium may be attained.
[0091] The electron transfer layer may be a layer including the
aromatic heterocycle compound(s) in a concentration that allows
transportation of electrons between the electron transfer compound
and the electron detection medium. As used herein, "concentration
that allows transportation of electrons between an electron
transfer compound and an electron detection medium" means a
concentration at which at least between the electron transfer
compound and the electron detection medium in the electron transfer
layer the electron cloud of pi-electrons is present to be
delocalized. Thus, when only a part of the electron transfer layer
is in contact with the electron detection medium in the analysis
device, the concentration of the aromatic heterocycle compound(s)
may be a concentration that allows the transportation of electrons
between the electron transfer compound and the electron detection
medium in the region of the electron transfer layer in contact with
the electron detection medium, and is not necessarily a
concentration in the whole electron transfer layer.
[0092] The concentration that allows transportation of electrons
between the electron transfer compound and the electron detection
medium can specifically be varied according to the kind or
concentration of the electron transfer compound used and, in an
embodiment, the concentration of the aromatic heterocycle
compound(s) may be 10 mass % or more, from 10 mass % to 60 mass %,
preferably from 15 mass % to 50 mass %, based on the total mass of
the electron transfer layer (reagent part) (when the whole electron
transfer layer is in contact with the electron detection medium).
The concentration of the aromatic heterocycle compound(s) according
to the present invention means mass % based on the total mass that
is the mass of the electron transfer layer including the aromatic
heterocycle compound(s) together with an electron transfer compound
and another additive that has been developed on the electron
detection medium and has been dried.
[0093] The aromatic heterocycle compound may be subjected to
crosslinking treatment or contained in and coated with a polymer.
By being subjected to such treatment, the aromatic heterocycle
compound is crosslinked between the molecules thereof or the
aromatic heterocycle compound is crosslinked with an electron
transfer compound, and can be maintained in an electron transfer
layer for a long term. An analysis device having the electron
transfer layer (reagent part) which is such a crosslinked substance
allows the aromatic heterocycle compound to remain around a
conductive layer and an analyte, for example, even in continuous
measurement for a long term, so that a relative position to the
conductive layer or the analyte can be further favorably
maintained, for example, such an advantage as to be able to
maintain a stable output is obtained.
[0094] The crosslinking treatment may be a crosslinking treatment
that is commonly used in crosslinking of, for example, protein.
Examples of crosslinking agents used for such crosslinking
treatment may include glutaraldehyde and carbodiimide compounds,
and succinimide esters. One of these crosslinking agents may be
used alone or two or more thereof may be used in combination.
[0095] The amount of such an added crosslinking agent is not
particularly limited if being a commonly used amount and may
appropriately be set in a sufficient range such as, for example, 10
times or more that of a material to be crosslinked.
[0096] An electron transporting substance in the electron transfer
layer includes the aromatic heterocycle compound, the electron
transporting substance is free of a metal complex. Example of such
a metal complex which is not included in the electron transporting
substance according to the present invention include metal
complexes conventionally used as so-called electron transport
mediators. Specific examples of such a metal complex include osmium
complexes.
[0097] Other Layers
[0098] The analysis device according to the present invention may
include another layer(s) at any position as far as electron
transfer between the electron detection medium and the electron
transfer layer is possible. Examples of such other layers may
include a protective layer, a layer for restricting penetration of
substances, and a functional layer for modifying the surface of the
electron detection medium; and the analysis device may include one
or two or more in combination thereof.
[0099] The protective layer is not particularly limited as long as
being able to protect the surface of the electron transfer layer or
the analysis device. Examples of the protective layer may include
cellulose acetate polymer, polyurea, polyamide, polyester,
polyethylene oxide, polyvinyl alcohol, and lipid bilayer
membranes.
[0100] The thickness of the protective layer is not particularly
limited and may be, for example, from 0.5 .mu.m to 5 .mu.m.
[0101] Examples of a membrane for restricting penetration of
substances in the analysis device, which membranes are intended to
adjust the detection concentration range of an analyte, may include
polyurea, polyamide, polyester, polyethylene oxide, and polyvinyl
alcohol.
[0102] The thickness of the membrane for restricting penetration of
substances is not particularly limited and may be, for example,
from 0.5 .mu.m to 5 .mu.m.
[0103] Examples of the functional layers for modifying the surface
of the electron detection medium, which layers are intended to
improve the reactivity of a conductive member, may include thiol
compounds, silane coupling agents, and lipid bilayer membranes.
[0104] The thickness of the functional layer is not particularly
limited and may be, for example, from 0.001 .mu.m to 5 .mu.m.
[0105] Analyte
[0106] An analyte to be detected by the analysis device is not
particularly limited as long as being a substance which can
transfer electrons, depending on the amount of an electron transfer
compound which is present, to or from the electron transfer
compound and may be appropriately set according to the kind of the
electron transfer compound.
[0107] For example, when the analysis device is used in a clinical
application, various substrates contained in a clinical sample may
be analytes. Examples of such clinical sample may include blood,
serum, plasma, interstitial fluid, urine, sweat, tears, and saliva.
Typical examples of the substrate may include glucose, uric acid,
and glycosylated proteins.
[0108] For example, when the analysis device is utilized in a
non-clinical application such as monitoring of fermentation,
control of an industrial process, or environmental monitoring
(e.g., suppression of efflux of liquid and gas and contamination),
a food test, or veterinary medicine, various substrates contained
in a non-clinical sample such as fermentation liquid, effluent,
waste fluid, food, or milk may be an analyte.
[0109] Method for Producing Analysis Device
[0110] As a method for producing an analysis device, which is not
particularly limited, any method in which each layer or each member
as described above can be placed may be applied.
[0111] For example, by molding, shaping, or printing a mixture of
components for a conductive layer, the conductive layer may be
disposed on a supporting member to form an electrode, followed by
disposing a layer of a liquid reaction mixture for an electron
transfer layer containing an enzyme and an aromatic heterocycle
compound.
[0112] In a case where crosslinking treatment of an electron
transfer compound and an aromatic heterocycle compound in an
electron transfer layer is performed, the method of crosslinking
may vary depending on the kinds of the crosslinking agent and the
aromatic compound. In an embodiment, a crosslinking agent may be
incorporated into a reaction mixture, and the crosslinking may be
performed simultaneously with forming an electron transfer layer.
Alternatively, the crosslinking agent may be incorporated into a
treatment liquid which is different from the reaction mixture, and
after an analysis device is obtained without crosslinking as
described above, the treatment liquid containing the crosslinking
agent is applied to the electron transfer layer by adding,
spraying, or immersing the liquid to perform crosslinking
treatment.
[0113] As a print medium, for example, a film- or plate-like print
medium may be used. The analysis device may be used after being
removed from the print medium or may be used while being supported
on the print medium. In the latter case, the print medium may
function as a supporting member. The print medium may include a
recess which is formed in the printed part of the mixture. In this
case, a mask may be omitted.
[0114] It will be appreciated that drying during the production is
preferably performed at a lower temperature than a temperature at
which substantial deactivation of an enzyme occurs.
[0115] The analysis device according to the present invention can
basically be formed only in a simple step such as forming a
mixture, molding, and drying. That is, it can be expected to reduce
a production cost to such a degree that a high mass-production
technology can be utilized and a disposable analysis device can be
produced.
[0116] The analysis method according to the present invention
includes obtaining information needed for analyzing an analyte
based on the electron transfer level of the analyte using the
analysis device (referred to as an information obtaining step). In
the analysis method, information for analyzing the analyte can be
obtained with good sensitivity and high reliability because the
analysis device as described above is used.
[0117] In the information obtaining step in the analysis method,
the information needed for performing analysis is obtained based on
the electron transfer level of the analyte. As used herein,
"information needed" includes, for example, the amount, kind, and
oxidation-reduction state of the analyte, time-dependent change in
the analyte, and the like.
[0118] The analysis device as described above as an exemplary
embodiment includes the conductive layer in the electron detection
medium but the electron detection medium in the analysis toll of
the present invention is not limited thereto. For example, the
electron detection medium may include a color former.
[0119] When the electron detection medium includes a color former,
the electron detection medium preferably has a configuration in
which the color former is retained in a porous material insoluble
in a sample. Typical examples of such a porous material may include
a gelled material of, for example, polyacrylamide or polyvinyl
alcohol. Examples of a color former include MTT
(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide),
INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium
chloride), WST-4
(2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-te-
trazolium, monosodium salt), and 4AA (4-aminoantipyrine).
[0120] Application
[0121] The analysis device according to the present invention is
applicable to various applications because of having such an
advantage that electrons can efficiently be transported to an
electron detection medium through an electron transporting
substance. Examples of such applications may include uses in, for
example, an enzyme electrode; a sensor by which, as a platform,
measurement of a substance by a colorimetric reaction system
utilizing a color former and/or the like is performed; or the
reaction system of a bio-fuel cell.
EXAMPLES
[0122] The present invention is described in detail below with
reference to Examples. However, the present invention is not
limited thereto at all. Unless otherwise specified, "%" is based on
a mass.
Example 1
(1) Production of Enzyme Electrode
[0123] For an enzyme electrode, Pt (Au) was sputtered on a
polyimide (PI) film to obtain a substrate having a platinum layer.
As an electrode material, a printing ink in which 40 wt % Ketjen
black (manufactured by Lion Corporation) was mixed with 40 wt %
polyester resin as a binder and 20 wt % isophorone as a solvent was
used. The surface of the polyimide film was printed with the
printing ink so that the printing ink had a thickness of 10
.mu.m.
[0124] Then, an enzyme liquid containing 1,250 U/ml of a wild-type
GDH solution (0.1M MES buffer solution), 1% adonitol as a
stabilizer, and 1 wt % of 4-aminomethyl pyridine as a water-soluble
aromatic heterocycle compound was prepared.
[0125] The prepared enzyme liquid was dropwise added to the surface
of the electrode using a precise syringe, and thereafter, the
resulting electrode was left to stand for 4 hours under the
environment of 23.degree. C. and a relative humidity of <8% to
be dried, whereby an enzyme electrode was obtained.
[0126] As a comparative enzyme electrode, a comparative enzyme
electrode was produced in substantially the same manner as
described above except that 4-aminomethyl pyridine was not
added.
(2) Measurement by Enzyme Electrode
[0127] Electrode responses to glucose (100 mg/dL or 600 mg/dL) in
0.1 M phosphate buffer (pH 7.4) at 23.degree. C. and +0.6 V (vs.
Ag/AgCl) were detected by an amperometric method using the enzyme
electrodes obtained as described above. The results are shown in
FIG. 2. In FIG. 2, the black circles and the black triangles
indicate measurements using the enzyme electrode added with 17%
4-aminomethyl pyridine and the comparative enzyme electrode added
with no 4-aminomethyl pyridine, respectively.
[0128] As indicated in FIG. 2, about 13 times higher current
density was detected in the glucose response current from the
enzyme electrode containing 4-aminomethyl pyridine than that in the
response current from the comparative enzyme electrode added with
no 4-aminomethyl pyridine. This indicates that sensitivity is
increased by using 4-aminomethyl pyridine as the aromatic
heterocycle compound.
Example 2
(1) Production of Enzyme Electrode
[0129] For an enzyme electrode, Au was sputtered on a
polyetherimide (PEI) film to obtain a substrate having a gold
layer. As an electrode material, a printing ink in which 40 wt %
Ketjen black (manufactured by Lion Corporation) was mixed with 40
wt % polyester resin as a binder and 20 wt % isophorone as a
solvent was used. The surface of the polyetherimide film was
printed with the printing ink so that the printing ink had a
thickness of 10 .mu.m, whereby a a working electrode was
obtained.
[0130] Then, enzyme liquid containing 2,500 U/ml of a wild-type GDH
solution (0.1 M MES buffer solution), 2% sucrose as a stabilizer, 1
v/v % glutaraldehyde as a crosslinking agent, and 1% of
4-aminomethyl pyridine as the aromatic heterocycle compound was
prepared.
[0131] The prepared enzyme liquid was dropwise added to an
electrode surface using a precise syringe and the resulting
electrode was left to stand for 10 minutes at a normal temperature
(23.degree. C.) and a normal relative humidity (40% RH) to dry the
surface. Then, the electrode was heat-treated at 40.degree. C. for
15 minutes for drying, and then was left to stand for 2 hours under
the environment of 23.degree. C. and <2% RH for further drying,
whereby an enzyme electrode was obtained.
[0132] As a comparative enzyme electrode, a comparative enzyme
electrode was produced in substantially the same manner as
described above except that 4-aminomethyl pyridine was not
added.
(2) Measurement by Enzyme Electrode
[0133] Electrode responses to glucose in 0.1 M phosphate buffer (pH
7.4) were detected by voltammetry using the enzyme electrode
obtained as described above. The voltammetry was performed at a
sweep speed of 20 mV/s using the enzyme electrode produced in the
above described (1), Pt, and Ag/AgCl as working, counter, and
reference electrodes, respectively, at a measurement temperature of
37.degree. C. A glucose concentration was 100 mg/dL or no glucose
was added.
[0134] The results are shown in FIG. 3 (third scan is indicated).
In FIG. 3, the lozenges, the quadrangles, the circles, and the
triangles indicate the measurement results in the case of 100 mg/dL
glucose using the enzyme electrode added with 4-aminomethyl
pyridine, the measurement results in the case of 0 mg/dL glucose
using the enzyme electrode added with 4-aminomethyl pyridine, the
measurement results in the case of 100 mg/dL glucose using the
comparative enzyme electrode added with no 4-aminomethyl pyridine,
and the measurement results in the case of 0 mg/dL glucose using
the comparative enzyme electrode added with no 4-aminomethyl
pyridine, respectively.
[0135] As indicated in FIG. 3, since a potential at which
oxidization of glucose begins to occur is around -0.2 V with or
without the addition of 4-aminomethyl pyridine and any specific
peaks are not observed in the other potential regions, the
oxidation-reduction of 4-aminomethyl pyridine in itself is not
considered to occur.
[0136] Thus, it was exhibited that, in the enzyme electrodes added
with 4-aminomethyl pyridine, oxidation-reduction of a compound in
itself did not occur to transport electrons as in the case of a
metal complex, and glucose was able to be stably detected with good
sensitivity in the absence of an electron transfer mediator such as
a metal complex.
Example 3
[0137] Enzyme electrodes 3A to 3F were obtained in substantially
the same manner as in Example 2 (1) except that the concentrations
of 4-aminomethyl pyridine were in the range of from 1% to 6% in
preparation of enzyme solutions. In the final form of the enzyme
electrodes, the concentrations of from 1% to 6% correspond to dry
masses of from 23% to 64%, respectively.
[0138] Measurement for 100 mg/dL glucose in 0.1 M phosphate buffer
(pH 7.4) was performed using the enzyme electrodes 3A to 3F and the
comparative enzyme electrode containing no 4-aminomethyl pyridine
produced in Example 2 (1), at a measurement temperature of
37.degree. C. The enzyme electrodes 3A to 3F and the comparative
enzyme electrode; Pt; and Ag/AgCl were used as working, counter,
and reference electrodes, respectively. The results are shown in
FIG. 4.
[0139] As indicated in FIG. 4, it is found that glucose can be
detected by setting the concentrations of 4-aminomethyl pyridine
added in the enzyme solutions to the range of from 1% to 6% (from
23% to 64% on a dry mass basis in final form). It is also found
that respective sensitivities are improved by setting the
concentrations of 4-aminomethyl pyridine into the range of from 2%
to 5% (from 38 to 60% on a dry mass basis in final form),
especially to 4% (55% on a dry mass basis in final form).
Example 4
[0140] Crosslinked enzyme electrodes 4A and 4B were obtained in
substantially the same manner as in Example 2 (1) except that after
dropwise adding of the prepared enzyme liquid to the electrode
surface using a precise syringe and drying, glutaraldehyde was
added to perform crosslinking treatment.
[0141] The crosslinking treatment was performed as described below.
A dried electrode surface was gently rinsed with dH.sub.2O and the
electrode was immersed in 1 v/v % glutaraldehyde (GA) solution
(containing 1% adonitol). After the immersion for 45 minutes or 120
minutes, each electrode was taken out and incubation was performed
overnight in the environment of a room temperature and a low
humidity (23.degree. C. and <2% RH). As a result, each of the
enzyme electrodes 4A (treatment time of 45 minutes) and 4B
(treatment time of 120 minutes) was obtained.
[0142] Continuous measurement for glucose at a predetermined
concentration was performed using each of the obtained enzyme
electrodes 4A and 4B. For the continuous measurement, electrode
responses to glucose in a phosphate buffer were detected by an
amperometric method at 25.degree. C. and +0.6 V (vs. Ag/AgCl) for
100 mg/dL glucose. The evaluation for a change in relative value
over time, based on a current density just after the start of the
measurement as an initial value of 100%, was performed. The results
are shown in FIG. 5. In FIG. 5, the quadrangles, the triangles, and
the lozenges indicate the cases of using the enzyme electrode 4A,
the enzyme electrode 4B, and an uncrosslinked enzyme electrode,
respectively.
[0143] As indicated in FIG. 5, it is found that, by performing the
crosslinking treatment of 4-aminomethyl pyridine with
glutaraldehyde, the localization of 4-aminomethyl pyridine is
suppressed, the efflux from the electron transfer layer can also be
prevented, and more stable outputs than that from the untreated
enzyme electrode is maintained.
Example 5
[0144] Electrode responses to glucose were detected in
substantially the same manner as in Example 1 except that
5-amino-4-imidazolecarboxamide/HCl, instead of 4-aminomethyl
pyridine, was added to be 17%. The results are shown in FIG. 6. In
FIG. 6, the black quadrangles and the black lozenges indicate the
cases of using the enzyme electrode added with 17% of
5-amino-4-imidazolecarboxamide and the comparative enzyme electrode
added with no aromatic heterocycle compound, respectively.
[0145] As indicated in FIG. 6, it is found that the current
densities are further favorably detected compared with the
comparative enzyme electrode by using
5-amino-4-imidazolecarboxamide/HCl instead of 4-aminomethyl
pyridine, as in the case of using 4-aminomethyl pyridine, and it
was found that glucose can be detected with good sensitivity by
using an imidazole derivative.
Comparative Example 1
[0146] Enzyme electrodes in Comparative Example were obtained in
substantially the same manner as in Example 4 except that from
0.01% to 1.0% of poly(2-vinylpyridine) (weight molecular weight of
about 21,000, Fluka Corporation) was used instead of 4-aminomethyl
pyridine. Responses to 100 mg/dL of glucose were compared with
relative values for an enzyme electrode containing no water-soluble
aromatic heterocycle compound in substantially the same manner as
in Example 4 except that the comparative enzyme electrodes were
used. The results are shown in FIG. 7.
[0147] As indicated in FIG. 7, it was found that the responsiveness
in the case of using poly(2-vinylpyridine) was similar to that in
the enzyme electrode containing no water-soluble aromatic
heterocycle compound or, at a concentration of 0.1% or more, was
lower than that in the case of using the enzyme electrode
containing no water-soluble aromatic heterocycle compound.
[0148] As described above, it is found that glucose in a sample can
be detected with high sensitivity without treatment of adjusting
the orientation of enzymes and without using any metal complex
and/or the like, by using the enzyme electrodes of the present
examples containing 4-aminomethyl pyridine. In addition, it is
found that stable measurement is possible since 4-aminomethyl
pyridine in itself is not involved in oxidation-reduction.
Furthermore, the enzyme electrodes are hard to be affected by a
reaction potential, unlike a metal complex and/or the like, and are
applicable to a wide application such as a sensing device in a
clinical situation represented by SMBG (self-monitoring of blood
glucose), continuous blood glucose monitoring (CGM), or the like, a
reaction system such as a bio-fuel cell, or a sensing device for
industrial or environmental use in a non-clinical situation.
[0149] All references, patent applications, and technical standards
described in the present specification are herein incorporated in
their entirety by reference into the specification, to the same
extent as if each individual reference, patent application or
technical standard was specifically and individually indicated to
be incorporated herein by reference.
* * * * *