U.S. patent application number 09/748229 was filed with the patent office on 2001-07-05 for biosensor.
This patent application is currently assigned to Matsushita Electric Industrial Co., Ltd.. Invention is credited to Ikeda, Shin, Nankai, Shiro, Taniike, Yuko.
Application Number | 20010006150 09/748229 |
Document ID | / |
Family ID | 18495434 |
Filed Date | 2001-07-05 |
United States Patent
Application |
20010006150 |
Kind Code |
A1 |
Taniike, Yuko ; et
al. |
July 5, 2001 |
Biosensor
Abstract
The present invention provides a biosensor having a high current
response sensitivity, a low blank response and a high storage
stability. This sensor comprises an electrode system including a
working electrode and a counter electrode, for forming an
electrochemical measurement system by coming in contact with a
supplied solution; an insulating supporting member for supporting
the electrode system; a first reagent layer formed on the working
electrode; and a second reagent layer formed on the counter
electrode, wherein the first reagent layer does not contain an
enzyme, but it contains at least an electron mediator, and the
second reagent layer does not contain an electron mediator, but it
contains at least an enzyme.
Inventors: |
Taniike, Yuko; (Osaka,
JP) ; Ikeda, Shin; (Osaka, JP) ; Nankai,
Shiro; (Osaka, JP) |
Correspondence
Address: |
Brian F. Ferguson
MCDERMOTT, WILL & EMERY
600 13th Street, N.W.
WASHINGTON
DC
20005-3096
US
|
Assignee: |
Matsushita Electric Industrial Co.,
Ltd.
|
Family ID: |
18495434 |
Appl. No.: |
09/748229 |
Filed: |
December 27, 2000 |
Current U.S.
Class: |
204/403.14 ;
204/403.08; 204/403.11; 205/777.5 |
Current CPC
Class: |
C12Q 1/004 20130101;
C12Q 1/001 20130101 |
Class at
Publication: |
204/403 ;
205/777.5 |
International
Class: |
G01N 027/327 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 27, 1999 |
JP |
HEI 11-369835 |
Claims
1. A biosensor comprising: an electrode system including a working
electrode and a counter electrode, for forming an electrochemical
measurement system by coming in contact with a supplied sample
solution; an electrically insulating supporting member for
supporting said electrode system; a first reagent layer formed on
said working electrode; and a second reagent layer formed on said
counter electrode, wherein said first reagent layer does not
contain an enzyme, but it contains at least an electron mediator,
and said second reagent layer does not contain an electron
mediator, but it contains at least an enzyme.
2. The biosensor in accordance with claim 1, wherein said
supporting member comprises an electrically insulating base plate
on which said working electrode and said counter electrode are
formed.
3. The biosensor in accordance with claim 1, wherein said
supporting member comprises an electrically insulating base plate
and an electrically insulating cover member for forming a sample
solution supply pathway or a sample solution storage section
between said cover member and said base plate, said working
electrode is formed on said base plate, and said counter electrode
is formed on an inner surface of said cover member so as to face
said working electrode.
4. The biosensor in accordance with claim 3, wherein said cover
member comprises a sheet member having an outwardly expanded curved
section, for forming a sample solution supply pathway or a sample
solution storage section between said cover member and said base
plate.
5. The biosensor in accordance with claim 3, wherein said cover
member comprises a spacer having a slit for forming said sample
solution supply pathway and a cover for covering said spacer.
6. The biosensor in accordance with claim 1, wherein said first
reagent layer contains a hydrophilic polymer.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates to a biosensor for rapid
quantification of a substrate contained in a sample with high
accuracy.
[0002] Conventionally, methods using polarimetry, colorimetry,
reductimetry and a variety of chromatography have been developed as
the measure for quantitative analysis of sugars such as sucrose and
glucose. However, those conventional methods are all poorly
specific to sugars and hence have poor accuracy. Among them, the
polarimetry is simple in manipulation, but it is largely affected
by the temperature during the manipulation. Therefore, this method
is not suitable for simple quantification of sugars at home by
ordinary people.
[0003] In recent years, a variety of biosensors have been developed
which best utilize a specific catalytic action of enzymes.
[0004] In the following, a method of quantitative analysis of
glucose will be explained as an example of the method for
quantifying a substrate contained in a sample. Conventionally known
electrochemical quantification of glucose includes a method using a
combination of glucose oxidase (EC 1.1.3.4: hereinafter abbreviated
to "GOD") as an enzyme with an oxygen electrode or a hydrogen
peroxide electrode (see "Biosensor" ed. by Shuichi Suzuki,
Kodansha, for example).
[0005] GOD selectively oxidizes .beta.-D-glucose as a substrate to
D-glucono-.delta.-lactone using oxygen as an electron mediator.
Oxygen is reduced to hydrogen peroxide during the oxidation
reaction by GOD in the presence of oxygen. A decreased volume of
oxygen is measured by the oxygen electrode, or an increased volume
of hydrogen peroxide is measured by the hydrogen peroxide
electrode. The decreased volume of oxygen or, otherwise, the
increased volume of hydrogen peroxide is proportional to the
content of glucose in the sample. It is therefore possible to
quantify glucose based on the decreased volume of oxygen or the
increased volume of hydrogen peroxide.
[0006] In the above method, it is possible to quantify glucose in
the sample accurately by using the specificity of the enzyme
reaction. However, as speculated from the reaction, this prior art
method has a drawback that the measurement result is greatly
affected by the oxygen concentration in the sample. Hence, in the
event where oxygen is absent in the sample, measurement is
infeasible.
[0007] Under such a circumstance, a glucose sensor of new type has
been developed which uses as the electron mediator an organic
compound or a metal complex such as potassium ferricyanide, a
ferrocene derivative and a quinone derivative, in place of oxygen
in the sample. The sensor of this type oxidizes the reduced
electron mediator resulting from the enzyme reaction on a working
electrode so as to determine the glucose concentration in the
sample based on an oxidation current produced by the oxidation
reaction. At this time, on a counter electrode, the oxidized
electron mediator is reduced, and a reaction for generating the
reduced electron mediator proceeds. With the use of such an organic
compound or metal complex as the electron mediator in place of
oxygen, it is possible to form a reagent layer by precisely placing
a known amount of GOD together with the electron mediator in their
stable state on the electrode, thereby enabling accurate
quantification of glucose without being affected by the oxygen
concentration in the sample. In this case, it is also possible to
integrate the reagent layer containing the enzyme and electron
mediator with an electrode system while keeping the reagent layer
in an almost dry state, and therefore a disposable glucose sensor
based on this technology has recently been noted considerably. A
typical example of such a glucose sensor is a biosensor disclosed
in Japanese Laid-Open Patent Publication Hei 3-202764. With such a
disposable glucose sensor, it is possible to measure the glucose
concentration easily with a measurement device by simply
introducing a sample into the sensor connected detachably to the
measurement device. The application of such a technique is not
limited to quantification of glucose and may be extended to
quantification of any other substrate contained in the sample.
[0008] In recent years, there has been demand for a biosensor
having a higher current response sensitivity and a biosensor
exhibiting a low response when the substrate concentration is zero
and excellent storage stability. The response when the substrate
concentration is zero is hereinafter referred to as "blank
response".
BRIEF SUMMARY OF THE INVENTION
[0009] The present invention provides a biosensor comprising: an
electrode system including a working electrode and a counter
electrode, for forming an electrochemical measurement system by
coming in contact with a supplied sample solution; an electrically
insulating supporting member for supporting the electrode system; a
first reagent layer formed on the working electrode; and a second
reagent layer formed on the counter electrode, wherein the first
reagent layer does not contain an enzyme, but it contains at least
an electron mediator, and the second reagent layer does not contain
an electron mediator, but it contains at least an enzyme.
[0010] In a preferred mode of the present invention, the supporting
member comprises an electrically insulating base plate on which the
working electrode and the counter electrode are formed.
[0011] In another preferred mode of the present invention, the
supporting member comprises an electrically insulating base plate
and an electrically insulating cover member for forming a sample
solution supply pathway or a sample solution storage section
between the cover member and the base plate, the working electrode
is formed on the base plate, and the counter electrode is formed on
an inner surface of the cover member so as to face the working
electrode.
[0012] It is preferred that the cover member comprises a sheet
member having an outwardly expanded curved section, for forming a
sample solution supply pathway or a sample solution storage section
between the cover member and the base plate.
[0013] A more preferred cover member comprises a spacer having a
slit for forming the sample solution supply pathway and a cover for
covering the spacer.
[0014] It is preferred that the first reagent layer contains a
hydrophilic polymer.
[0015] While the novel features of the invention are set forth
particularly in the appended claims, the invention, both as to
organization and content, will be better understood and
appreciated, along with other objects and features thereof, from
the following detailed description taken in conjunction with the
drawings.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING
[0016] FIG. 1 is a vertical cross-sectional view of a glucose
sensor according to one example of the present invention.
[0017] FIG. 2 is an exploded perspective view of the glucose
sensor, omitting the reagent layers and surface active agent layer
therefrom.
[0018] FIG. 3 is a vertical cross-sectional view of a glucose
sensor according to another example of the present invention.
[0019] FIG. 4 is an exploded perspective view of the glucose
sensor, omitting the reagent layers and surface active agent layer
therefrom.
[0020] FIG. 5 is a vertical cross-sectional view of a glucose
sensor according to still another example of the present
invention.
[0021] FIG. 6 is an exploded perspective view of the glucose
sensor, omitting the reagent layers and surface active agent layer
therefrom.
[0022] FIG. 7 is a vertical cross-sectional view of a glucose
sensor of a comparative example.
DETAILED DESCRIPTION OF THE INVENTION
[0023] A biosensor according to a preferred embodiment of the
present invention comprises an electrically insulating base plate;
a working electrode and a counter electrode formed on the base
plate; a first reagent layer formed on the working electrode; and a
second reagent layer formed on the counter electrode, wherein the
first reagent layer does not contain an enzyme, but it contains at
least an electron mediator, and the second reagent layer does not
contain an electron mediator, but it contains at least an
enzyme.
[0024] In this biosensor, since an enzyme is not present on the
working electrode, the electrode reaction on the working electrode
can never be hindered by adsorption of the enzyme to the working
electrode and the oxidation reaction of the reduced electron
mediator on the working electrode proceeds smoothly, thereby
improving the current response sensitivity. Moreover, since the
enzyme and the electron mediator are separated from each other, it
is possible to prevent contact and interaction between the enzyme
and the electron mediator, thereby suppressing an increase in the
blank response and a degradation in the enzyme activity during
long-time storage.
[0025] A biosensor according to another preferred embodiment of the
present invention comprises an electrically insulating base plate;
an electrically insulating cover member for forming a sample
solution supply pathway or a sample solution storage section
between the cover member and the base plate; a working electrode
formed on the base plate; a counter electrode formed on an inner
surface of the cover member so as to face the working electrode; a
first reagent layer formed on the working electrode; and a second
reagent layer formed on the counter electrode, wherein the first
reagent layer does not contain an enzyme, but it contains at least
an electron mediator, and the second reagent layer does not contain
an electron mediator, but it contains at least an enzyme.
[0026] The cover member comprises a sheet member having an
outwardly expanded curved section, for forming a sample solution
supply pathway or a sample solution storage section between the
cover member and the base plate.
[0027] A more preferred cover member comprises a spacer with a slit
for forming the sample solution supply pathway and a cover for
covering the spacer.
[0028] In such a biosensor, since the first reagent layer and
second reagent layer are formed on separate members, respectively,
the first reagent layer and second reagent layer having different
compositions can be readily separated from each other. Moreover,
since the working electrode and counter electrode are formed at
opposite positions, the ion transfer between the electrodes is
facilitated, thereby further increasing the current response.
[0029] In a biosensor whose cover member comprises the spacer and
cover, since the physical strength of the cover is enhanced, the
first reagent layer and second reagent layer are not brought into
contact with each other by an external physical pressure, thereby
preventing degradation in the enzyme activity due to the contact
between the enzyme and the electron mediator.
[0030] In either of the biosensors of the above-described
embodiments, it is preferred that at least the first reagent layer
contains a hydrophilic polymer. Since the hydrophilic polymer
prevents adsorption of proteins, etc. to the working electrode, the
current response sensitivity is further improved. Besides, during
the measurement, since the viscosity of a sample solution is
increased by the hydrophilic polymer dissolved in the sample
solution, the effects of physical impact, etc. on the current
response are reduced, thereby improving the stability of the
current response.
[0031] In the present invention, for the base plate, spacer and
cover, it is possible to use any material having an insulating
property and sufficient rigidity during storage and measurement.
Examples of such a material include thermoplastic resins such as
polyethylene, polystyrene, polyvinyl chloride, polyamide and
saturated polyester resin, or thermosetting resins such as a urea
resin, melamine resin, phenol resin, epoxy resin and unsaturated
polyester resin. Among these resins, polyethylene terephthalate is
preferred in view of the adhesiveness to the electrode.
[0032] For the working electrode, it is possible to use any
conductive material if it is not oxidized itself in oxidizing the
electron mediator. For the counter electrode, it is possible to use
a generally used conductive material such as palladium, silver,
platinum, and carbon.
[0033] As the enzyme, it is possible to use the one suitable for
the type of a substrate in the sample, which is the subject of
measurement. Examples of the enzyme include fructose dehydrogenase,
glucose oxidase, alcohol oxidase, lactate oxidase, cholesterol
oxidase, xanthine oxidase, and amino acid oxidase.
[0034] Examples of the electron mediator include potassium
ferricyanide, p-benzoquinone, phenazine methosulfate, methylene
blue, and ferrocene derivatives. Besides, even when oxygen is used
as the electron mediator, a current response is obtained. These
electron mediators are used singly or in combinations of two or
more.
[0035] A variety of hydrophilic polymers are applicable. Examples
of the hydrophilic polymer include hydroxyethyl cellulose,
hydroxypropyl cellulose, methyl cellulose, ethyl cellulose,
ethylhydroxyethyl cellulose, carboxymethyl cellulose, polyvinyl
pyrrolidone, polyvinyl alcohol, polyamino acid such as polylysine,
polystyrene sulfonate, gelatin and its derivatives, polyacrylic
acid and its salts, plolymethacrylic acid and its salts, starch and
its derivatives, and a polymer of maleic anhydride or a maleate.
Among them, carboxymethyl cellulose, hydroxyethyl cellulose and
hydroxypropyl cellulose are particularly preferred.
[0036] The following description will explain the present invention
in further detail by illustrating examples thereof.
EXAMPLE 1
[0037] A glucose sensor will be explained as an example of a
biosensor.
[0038] FIG. 1 is a vertical cross-sectional view of a glucose
sensor of this example, and FIG. 2 is an exploded perspective view
of the glucose sensor, omitting the reagent layers and surface
active agent layer therefrom.
[0039] First, a silver paste was printed on an electrically
insulating base plate 1 made of polyethylene terephthalate by
screen printing to form leads 2 and 3 and the base of
later-described electrodes. Then, a conductive carbon paste
containing a resin binder was printed on the base plate 1 to form a
working electrode 4. This working electrode 4 was in contact with
the lead 2. Further, an insulating paste was printed on the base
plate 1 to form an insulating layer 6. The insulating layer 6
covered the peripheral portion of the working electrode 4 so that a
fixed area of the working electrode 4 was exposed. Next, a counter
electrode 5 was formed by printing a conductive carbon paste
containing a resin binder so as to be in contact with the lead
3.
[0040] A first aqueous solution containing potassium ferricyanide
as an electron mediator and no enzyme was dropped on the working
electrode 4 of the base plate 1 and then dried to form a first
reagent layer 7. Besides, a second aqueous solution containing GOD
as an enzyme and no electron mediator was dropped on the counter
electrode 5 of the base plate 1 and then dried to form a second
reagent layer 8. Further, in order to achieve smooth supply of a
sample, a layer 9 containing lecithin as a surface active agent was
formed so as to cover the first reagent layer 7 and second reagent
layer 8.
[0041] Finally, the base plate 1, a cover 12 and a spacer 10 were
adhered to each other in a positional relationship as shown by the
dashed lines in FIG. 2 to fabricate the glucose sensor.
[0042] The spacer 10 to be inserted between the base plate 1 and
the cover 12 has a slit 11 for forming a sample solution supply
pathway between the base plate 1 and the cover 12.
[0043] Since an air vent 14 of the cover 12 communicates with this
sample solution supply pathway, when the sample is brought into
contact with a sample supply port 13 formed at an open end of the
slit 11, the sample readily reaches the first reagent layer 7 and
second reagent layer 8 in the sample solution supply pathway
because of capillary phenomenon.
[0044] As a comparative example, a glucose sensor was fabricated in
the same manner as in Example 1 with the exception of the process
of forming the reagent layers. FIG. 7 is a vertical cross-sectional
view of the glucose sensor of the comparative example. A reagent
layer 30 was formed by dropping an aqueous solution containing GOD
and potassium ferricyanide on the working electrode 4 and counter
electrode 5 and then drying the aqueous solution. Moreover, a layer
9 containing lecithin as a surface active agent was formed on the
reagent layer 30.
[0045] Next, with the glucose sensors of Example 1 and the
comparative example, the concentration of glucose was measured
using a solution containing a certain amount of glucose as a
sample. The sample was supplied to the sample solution supply
pathway from the sample supply port 13 and, after elapse of a
certain time, a voltage of 500 mV was applied to the working
electrode 4 using the counter electrode 5 as reference. Since the
spacer 10 is interposed between the cover 12 and the base plate 1,
the strength of the sensor against an external physical pressure is
increased. Consequently, the volume of the sample solution supply
pathway is readily kept constant, and the effects of physical
pressure, etc. on the current response are reduced.
[0046] The value of a current which flowed across the working
electrode 4 and the counter electrode 5 upon the application of
this voltage was measured. As a result, in both of Example 1 and
the comparative example, a current response proportional to the
glucose concentration in the sample was observed. When the sample
comes into contact with the first reagent layer 7, potassium
ferricyanide as the oxidized form of the electron mediator
dissociates into ferricyanide ion and potassium ion. The glucose in
the sample, the ferricyanide ion dissolved in the sample from the
first reagent layer 7 and the GOD dissolved in the sample from the
second reagent layer 8 react. As a result, the glucose is oxidized
into glucono lactone, and the oxidized form ferricyanide ion is
reduced to the reduced form ferrocyanide ion. A reaction of
oxidizing ferrocyanide ion into ferricyanide ion proceeds on the
working electrode 4, while a reaction of reducing ferricyanide ion
into ferrocyanide ion proceeds on the counter electrode 5. Since
the concentration of ferrocyanide ion is proportional to the
concentration of glucose, it is possible to measure the
concentration of glucose based on the oxidation current of the
ferrocyanide ion.
[0047] In comparison with the glucose sensor of the comparative
example that contains GOD as an enzyme in the reagent layer 30 on
the working electrode 4, the current response was increased in the
glucose sensor of Example 1 for the following reason. Since the
first reagent layer 7 did not contain GOD, it was possible to
prevent a lowering of the current response due to the adsorption of
GOD onto the working electrode 4.
[0048] Moreover, in comparison with the comparative example, the
blank response was lowered and the current response was not changed
so much even after a long-time storage for the following reason.
Since the GOD and potassium ferricyanide were separated from each
other, it was possible to prevent contact and interaction between
the GOD and potassium ferricyanide. Hence, it was possible to
suppress an increase in the blank response and degradation in the
enzyme activity during a long-time storage.
EXAMPLE 2
[0049] FIG. 3 is a vertical cross-sectional view of a glucose
sensor of this example, and FIG. 4 is an exploded perspective view
of the glucose sensor, omitting the reagent layers and surface
active agent layer therefrom.
[0050] A working electrode 4 and a lead 2 were formed by sputtering
palladium on an electrically insulating base plate 21. Next, by
pasting an insulating sheet 23 on the base plate 21, the working
electrode 4 and a terminal section to be inserted into a
measurement device were defined.
[0051] Meanwhile, a counter electrode 5 was formed by sputtering
palladium onto the inner wall surface of an outwardly expanded
curved section 24 of an electrically insulating cover member 22. An
end portion of the curved section 24 was provided with an air vent
14.
[0052] A first aqueous solution containing potassium ferricyanide
as an electron mediator and no enzyme was dropped on the working
electrode 4 of the base plate 21 and then dried to form a first
reagent layer 7. Besides, a second aqueous solution containing GOD
as an enzyme and no electron mediator was dropped on the counter
electrode 5 of the cover member 22 to form a second reagent layer
8. Further, a layer 9 containing lecithin as a surface active agent
was formed on the first reagent layer 7.
[0053] Finally, the base plate 21 and cover 22 were adhered to each
other to fabricate the glucose sensor. Accordingly, the working
electrode 4 and the counter electrode 5 are positioned to face each
other with a space formed between the base plate 21 and the curved
section 24 of the cover member 22 therebetween. This space serves
as a sample storage section and, when a sample is brought into
contact with an open end of the space, the sample readily moves
toward the air vent 14 due to capillary phenomenon and comes into
contact with the first reagent layer 7 and second reagent layer
8.
[0054] Next, the concentration of glucose was measured according to
the same procedure as in Example 1. As a result, a current response
proportional to the concentration of glucose in the sample was
observed. The counter electrode 5 was electrically connected by
holding an end portion of the curved section 24 with a clip
connected to a lead wire.
[0055] In comparison with the glucose sensor of Example 1, a
further increase in the response value was observed in the glucose
sensor of Example 2 for the following reason. Since the first
reagent layer 7 did not contain GOD like Example 1 and the working
electrode 4 and counter electrode 5 were formed at opposite
positions, ion transfer between the working electrode 4 and counter
electrode 5 was facilitated.
[0056] Moreover, since the GOD and potassium ferricyanide were
separated from each other, like Example 1, the blank response was
lowered and the current response was not changed so much even after
a long-time storage in comparison with the comparative example.
EXAMPLE 3
[0057] FIG. 5 is a vertical cross-sectional view of a glucose
sensor of this example, and FIG. 6 is an exploded perspective view
of the glucose sensor, omitting the reagent layers and surface
active agent layer therefrom.
[0058] First, a silver paste was printed on an electrically
insulating base plate 31 made of polyethylene terephthalate by
screen printing to form a lead 2. Then, a conductive carbon paste
containing a resin-binder was printed on the base plate 31 to form
a working electrode 4. This working electrode 4 was in contact with
the lead 2. Further, an insulating paste was printed on the base
plate 31 to form an insulating layer 6. The insulating layer 6
covered the peripheral portion of the working electrode 4 so that a
fixed area of the working electrode 4 was exposed.
[0059] Next, a silver paste was printed on the inner surface of an
electrically insulating cover 32 to form a lead 3, and then a
conductive carbon paste was printed to form a counter electrode 5.
Further, an insulating paste was printed to form an insulating
layer 6. The cover 32 was provided with an air vent 14.
[0060] A first aqueous solution containing potassium ferricyanide
as an electron mediator and no enzyme was dropped on the working
electrode 4 of the base plate 31 and then dried to form a first
reagent layer 7, while a second aqueous solution containing GOD as
an enzyme and no electron mediator was dropped on the counter
electrode 5 of the cover 32 and then dried to form a second reagent
layer 8. Further, a layer 9 containing lecithin as a surface active
agent was formed on the first reagent layer 7.
[0061] Finally, the base plate 31, the cover 32 and a spacer 10
were adhered to each other in a positional relationship as shown by
the dashed lines of FIG. 6 to fabricate the glucose sensor.
[0062] The spacer 10 interposed between the base plate 31 and the
cover 32 has a slit 11 for forming a sample solution supply pathway
between the base plate 31 and the cover 32. The working electrode 4
and counter electrode 5 are positioned to face each other in the
sample solution supply pathway formed in the slit 11 of the spacer
10.
[0063] Since the air vent 14 of the cover 32 communicates with this
sample solution supply pathway, when a sample is brought into
contact with a sample supply port 13 formed at an open end of the
slit 11, the sample readily reaches the first reagent layer 7 and
second reagent layer 8 in the sample solution supply pathway
because of capillary phenomenon.
[0064] Next, the concentration of glucose was measured according to
the same procedure as in Example 1. As a result of the measurement,
a current response proportional to the concentration of glucose in
the sample was observed.
[0065] In the glucose sensor of Example 3, the first reagent layer
7 did not contain GOD, and the working electrode 4 and counter
electrode 5 were formed at opposite positions. Therefore, like
Example 2, the response value was increased in comparison with the
glucose sensor of Example 1.
[0066] Moreover, since the GOD and potassium ferricyanide were
separated from each other, like Example 1, the blank response was
lowered and the current response was not changed so much even after
a long-time storage in comparison with the comparative example.
[0067] Furthermore, since the spacer 10 was interposed between the
base plate 31 and cover 32, the strength of the sensor against an
external physical pressure was enhanced. As a result, the first
reagent layer 7 and the second reagent layer 8 were never brought
into contact with each other by the physical pressure, thereby
preventing the current response from being varied by the
degradation of the enzyme activity caused by the contact between
GOD and potassium ferricyanide. In addition, since the volume of
the sample solution supply pathway was readily kept constant, the
stability of the current response was improved in comparison with
Example 2.
EXAMPLE 4
[0068] In this embodiment, a glucose sensor was fabricated in the
same manner as in Example 3 with the exception of the process of
forming the first reagent layer 7 and second reagent layer 8.
[0069] A first aqueous solution containing potassium ferricyanide
as an electron mediator, carboxymethyl cellulose as a hydrophilic
polymer and no enzyme was dropped on the working electrode 4 of the
base plate 31 and then dried to form the first reagent layer 7,
while a second aqueous solution containing GOD as an enzyme,
carboxymethyl cellulose and no electron mediator was dropped on the
counter electrode 5 of the cover 32 and then dried to form the
second reagent layer 8. Moreover, the layer 9 containing lecithin
as a surface active agent was formed on the first reagent layer
7.
[0070] Next, the concentration of glucose was measured according to
the same procedure as in Example 1. As a result of the measurement,
a current response proportional to the concentration of glucose in
the sample was observed.
[0071] In the glucose sensor of Example 4, the first reagent layer
7 did not contain GOD, and the working electrode 4 and counter
electrode 5 were formed at opposite positions. Therefore, like
Example 2, the current response was increased in comparison with
the glucose sensor of Example 1.
[0072] Moreover, since the GOD and potassium ferricyanide were
separated from each other, like Example 1, the blank response was
lowered and the current response was not changed so much even after
a long-time storage in comparison with the comparative example.
[0073] Furthermore, since the spacer 10 was interposed between the
base plate 31 and cover 32, like Example 3, it was possible to
prevent the current response from being varied by the degradation
in the enzyme activity caused by the contact between GOD and
potassium ferricyanide. In addition, since the volume of the sample
solution supply pathway was readily kept constant, like Example 3,
the stability of the current response was improved in comparison
with Example 2.
[0074] Besides, in comparison with Examples 2 and 3, the current
response was further increased for the following reason. The
presence of carboxymethyl cellulose in the first reagent layer 7
prevented adsorption of proteins to the surface of the working
electrode 4, and hence the electrode reaction on the working
electrode 4 proceeded smoothly. Furthermore, since the viscosity of
the sample was increased during the measurement, the effects of
physical impact, etc. on the sensor were reduced and variations in
the sensor response were decreased.
[0075] In the above-described examples, while a voltage of 500 mV
was applied to the working electrode 4 using the counter electrode
5 as reference, the voltage is not necessarily limited to 500 mV.
Any voltage that enables oxidation of the electron mediator reduced
with the enzyme reaction may be applied.
[0076] In the above-described examples, while the first reagent
layer 7 contained one kind of electron mediator, it may contain two
or more kinds of electron mediators.
[0077] The first reagent layer 7 and second reagent layer 8 may be
immobilized on the working electrode 4 or the counter electrode 5
so as to insolubilize the enzyme or the electron mediator. In the
case where the first reagent layer 7 and second reagent layer 8 are
immobilized, it is preferable to use a crosslinking immobilization
method or an adsorption method. Alternatively, the electron
mediator and the enzyme may be mixed into the working electrode and
the counter electrode, respectively.
[0078] As the surface active agent, it is possible to use a
material other than lecithin. Besides, in the above-described
examples, although the surface active agent layer 9 was formed only
on the first reagent layer 7, or on the first reagent layer 7 and
second reagent layer 8, the formation of the surface active agent
layer 9 is not necessarily limited to these examples, and the
surface active agent layer 9 may be formed at a position facing the
sample solution supply pathway, such as a side face of the slit 11
of the spacer 10.
[0079] In the above-described examples, a two-electrode system
consisting only of the working electrode and counter electrode is
described. However, if a three-electrode system including an
additional reference electrode is adopted, it is possible to
perform a more accurate measurement.
[0080] It is preferred that the first reagent layer and second
reagent layer are not in contact with each other and are separated
from each other with a space therebetween. Accordingly, it is
possible to further enhance the effect of suppressing an increase
in the blank response and the effect of improving the storage
stability.
[0081] As described above, according to the present invention, it
is possible to obtain a biosensor having a high current response, a
low blank response and a high storage stability.
[0082] Although the present invention has been described in terms
of the presently preferred embodiments, it is to be understood that
such disclosure is not to be interpreted as limiting. Various
alterations and modifications will no doubt become apparent to
those skilled in the art to which the present invention pertains,
after having read the above disclosure. Accordingly, it is intended
that the appended claims be interpreted as covering all alterations
and modifications as fall within the true spirit and scope of the
invention.
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