U.S. patent number RE36,991 [Application Number 09/375,705] was granted by the patent office on 2000-12-19 for biosensor and method for producing the same.
This patent grant is currently assigned to Matsushita Electric Industrial Co., Ltd.. Invention is credited to Mariko Miyashita, Shiro Nankai, Satoko Tsuji (nee Fujisawa), Tomohiro Yamamoto, Toshihiko Yoshioka.
United States Patent |
RE36,991 |
Yamamoto , et al. |
December 19, 2000 |
Biosensor and method for producing the same
Abstract
A biosensor for rapid quantification of a specific component
contained in various biological samples with high accuracy has an
electrically insulating base, an electrode system including a
working electrode and a counter electrode formed on one face of the
insulating base, and a reaction layer formed on the insulating base
in close contact with the electrode system. The reaction layer
contains at least a hydrophilic polymer, a buffer and an enzyme
which is separated from the buffer.
Inventors: |
Yamamoto; Tomohiro (Neyagawa,
JP), Miyashita; Mariko (Nishinomiya, JP),
Yoshioka; Toshihiko (Hirakata, JP), Tsuji (nee
Fujisawa); Satoko (Katano, JP), Nankai; Shiro
(Hirakata, JP) |
Assignee: |
Matsushita Electric Industrial Co.,
Ltd. (Osaka-fu, JP)
|
Family
ID: |
16120827 |
Appl.
No.: |
09/375,705 |
Filed: |
August 13, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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Reissue of: |
277556 |
Jul 19, 1994 |
05658443 |
Aug 19, 1997 |
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Foreign Application Priority Data
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Jul 23, 1993 [JP] |
|
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5-182583 |
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Current U.S.
Class: |
204/403.14;
204/415; 205/777.5; 435/287.9; 435/817; 205/778; 204/418;
204/403.1 |
Current CPC
Class: |
C12Q
1/001 (20130101); C12Q 1/002 (20130101); Y10S
435/817 (20130101) |
Current International
Class: |
C12Q
1/00 (20060101); G01N 027/26 (); C12M 001/34 () |
Field of
Search: |
;204/403,415,418
;435/817,287.9 ;205/777.5,778 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0 251 915 |
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Jan 1988 |
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EP |
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0 502 504 |
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Sep 1992 |
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EP |
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3537915 |
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Apr 1987 |
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DE |
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1-114747 |
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May 1989 |
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JP |
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2-062952 |
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Mar 1990 |
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JP |
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WO 90/05910 |
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May 1990 |
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WO |
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Primary Examiner: Bell; Bruce F.
Attorney, Agent or Firm: Akin, Gump, Strauss, Hauer &
Feld, L.L.P.
Claims
What is claimed is:
1. A method for producing a biosensor comprising the steps of:
forming a first layer containing an enzyme and a hydrophilic
polymer by using water as the medium on a face of an insulating
base in close contact with an electrode system including a working
electrode and a counter electrode which are provided on said
insulating base; and
forming a second layer containing a buffer on said first layer by
using an organic solvent solution of a lipid which does not
dissolve said hydrophilic polymer.
2. The method for producing a biosensor in accordance with claim 1,
wherein said step of forming the first layer comprises spreading an
aqueous solution which dissolves the enzyme and the hydrophilic
polymer on the insulating base and drying the spread solution, and
wherein said step of forming the second layer comprises spreading a
solution obtained by dispersing the buffer in an organic solvent
solution and drying the spread solution.
3. The method for producing a biosensor in accordance with claim 2,
wherein said aqueous solution which dissolves the enzyme and the
hydrophilic polymer further dissolves an electron acceptor.
4. A method for producing a biosensor comprising the steps of:
forming a first layer containing a buffer and a hydrophilic polymer
by using water as the medium on a face of an insulating base in
close contact with an electrode system including a working
electrode and a counter electrode provided on said insulating base;
and
forming a second layer containing a hydrophilic polymer and an
enzyme on said first layer by using an organic solvent as the
medium that does not dissolve said hydrophilic polymer contained in
the first layer.
5. The method for producing a biosensor in accordance with claim 4,
wherein said step of forming the first layer comprises spreading an
aqueous solution which dissolves the buffer and the hydrophilic
polymer on the insulating base and drying the spread solution, and
wherein said step of forming the second layer comprises spreading
an organic solvent solution of the hydrophilic polymer on the first
layer and drying the spread solution, and further dropping an
aqueous solution of the enzyme on the second layer and drying the
dropped solution.
6. The method for producing a biosensor in accordance with claim 5,
wherein said aqueous solution of the enzyme further dissolves an
electron acceptor.
7. The method for producing a biosensor in accordance with claim 5,
further comprising a step of forming a third layer by spreading an
organic solvent solution of a lipid over the second layer and
drying the spread solution.
8. A method for producing a biosensor comprising:
a first step of spreading an aqueous solution containing a
hydrophilic polymer and a buffer on an insulating base in close
contact with an electrode system including a working electrode and
a counter electrode provided on a face of said insulating base and
drying the spread solution, and
a second step of spreading an organic solvent solution containing
at least an enzyme and a hydrophilic polymer over the layer and
drying the spread solution.
9. The method for producing a biosensor in accordance with claim 8,
wherein either of said aqueous solution employed in said first step
on the organic solvent solution employed in said second step
further contains an electron acceptor.
10. The method for producing a biosensor in accordance with claim
9, wherein the organic solvent solution employed in said second
step further contains a hydrophilic polymer.
11. A method for producing a biosensor comprising the steps of:
forming a first layer containing an enzyme and a hydrophilic
polymer by using water as the medium on a face of an insulating
base in close contact with an electrode system including a working
electrode and a counter electrode which are provided on said
insulating base; and
forming a second layer containing a buffer on said first layer by
using an organic solvent solution of a hydrophilic polymer, which
solvent does not dissolve said hydrophilic polymer contained in the
first layer.
12. The method for producing a biosensor in accordance with claim
11, further comprising a step of forming a third layer by spreading
an organic solvent solution of a lipid over the second layer and
drying the spread solution.
13. The method for producing a biosensor in accordance with claim
11, wherein said step of forming the first layer comprises
spreading an aqueous solution which dissolves the enzyme and the
hydrophilic polymer on the insulating base and drying the spread
solution, and wherein said step of forming the second layer
comprises spreading a solution obtained by dispersing the buffer in
the organic solvent solution and drying the spread solution.
14. The method for producing a biosensor in accordance with claim
13, wherein said aqueous solution which dissolves the enzyme and
the hydrophilic polymer further dissolves an electron acceptor.
.[.15. The method for producing a biosensor in accordance with
claim 13, wherein said aqueous solution which dissolves the enzyme
and the hydrophilic polymer
further dissolves an electron acceptor..].16. A biosensor
comprising:
an electrical insulating base,
an electrode system including a working electrode and a counter
electrode which are provided on a face of said insulating base,
and
a reaction layer formed on said insulating base in close contact
with said electrode system; wherein
said reaction layer is a laminate of at least two layers, and
wherein a first reaction layer is in contact with said electrode
system and contains an enzyme, an electron acceptor and a
hydrophilic polymer, and a second reaction layer is on top of said
first reaction layer and contains a buffer and a lipid,
said enzyme being separated from said buffer. 17. A biosensor
comprising,
an electrical insulating base,
an electrode system including a working electrode and a counter
electrode which are provided on a face of said insulating base,
and
a reaction layer formed on said insulating base in close contact
with said electrode system; wherein
said reaction layer is a laminate of at least two layers, and
wherein a first reaction layer is in contact with said electrode
system and contains an enzyme and an electron acceptor and a second
reaction layer is on top of said first reaction layer and contains
a buffer and a hydrophilic
polymer, said enzyme being separated from said buffer. 18. A
biosensor comprising,
an electrical insulating base,
an electrode system including a working electrode and a counter
electrode which are provided on a face of said insulating base
and
a reaction layer formed on said insulating base in close contact
with said electrode system; wherein
said reaction layer is a laminate of at least three layers, and
wherein a first reaction layer is in contact with said electrode
system and contains an enzyme and an electron acceptor, a second
reaction layer is on top of said first reaction layer and contains
a buffer and a hydrophilic polymer and a third reaction layer is on
top of said second reaction layer and contains a lipid,
said enzyme being separated from said buffer. .Iadd.19. A method
for producing a biosensor comprising the steps of:
forming a first layer containing a buffer and a hydrophilic polymer
by using water as the medium on a face of an insulating base in
close contact with an electrode system including a working
electrode and counter electrode provided on said insulating base;
and forming a second layer containing a lipid-modified enzyme on
said first layer by using an organic solvent as the medium that
does not dissolve said hydrophilic polymer contained in the first
layer..Iaddend..Iadd.20. The method for producing a biosensor in
accordance with claim 19, wherein said step of forming the first
layer comprises spreading an aqueous solution which dissolves the
buffer and the hydrophilic polymer on the insulating base and
drying the spread solution, and wherein said step of forming the
second layer comprises spreading an organic solvent solution of the
lipid-modified enzyme on the first layer and drying the spread
solution..Iaddend..Iadd.21. The method for producing a biosensor in
accordance with claim 20, wherein said aqueous solution further
dissolves an electron acceptor..Iaddend..Iadd.22. The method for
producing a biosensor in accordance with claim 20, wherein said
organic solvent solution of the lipid-modified enzyme is dispersed
with an electron acceptor..Iaddend..Iadd.23. The method for
producing a biosensor in accordance with claim 19, wherein said
step of forming the first layer comprises spreading an aqueous
solution which dissolves the buffer and the hydrophilic polymer on
the insulating base and drying the spread solution, and wherein
said step of forming the second layer comprises spreading an
organic solvent dispersed with the lipid-modified enzyme on the
first layer and drying the spread solution..Iaddend..Iadd.24. The
method for producing a biosensor in accordance with claim 23,
wherein said aqueous
solution further dissolves an electron acceptor..Iaddend..Iadd.25.
The method for producing a biosensor in accordance with claim 23,
wherein said organic solvent solution is further dispersed with an
electron acceptor..Iaddend..Iadd.26. A biosensor comprising:
an electrical insulating base,
an electrode system including a working electrode and a counter
electrode which are provided on a face of said insulating base,
and
a reaction layer formed on said insulating base in close contact
with said electrode system;
wherein said reaction layer is a laminate of at least two layers,
and
wherein a first layer is in contact with said electrode system and
contains a buffer and a hydrophilic polymer, and a second layer is
on top of said first layer and contains a lipid-modified enzyme,
said enzyme being separated from said buffer..Iaddend..Iadd.27. The
biosensor in accordance with claim 26, wherein said first layer
contains an electron acceptor..Iaddend..Iadd.28. The biosensor in
accordance with claim 26, wherein said second layer contains an
electron acceptor..Iaddend.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a biosensor capable of rapidly
quantifying a specific component in a sample solution with high
accuracy in a simplified manner, and to a method for producing the
same.
2. Description of the Related Art
Various types of biosensor have heretofore been proposed as a
system for quantifying the specific component in the sample
solution without requiring diluting or stirring of the sample
solution.
As an example of such biosensors, a glucose Sensor will be
described in the following paragraphs. In general, a system
combining glucose oxidase with an enzyme electrode or a hydrogen
peroxide electrode is already known as a method of quantifying
glucose utilizing the enzyme electrode. The glucose oxidase
selectively oxidizes a substrate, i.e., .beta.-D-glucose into
D-glucono-.delta.-lactone by using oxygen as an electron acceptor.
During this reaction, oxygen is reduced into hydrogen peroxide. By
measuring the amount of the oxygen consumed in this reaction by an
oxygen electrode, or by measuring the amount of the hydrogen
peroxide produced in this reaction by a hydrogen peroxide electrode
which utilizes a platinum electrode or the like, the glucose in the
sample solution can be quantified.
By the above-mentioned method, the measurement is however adversely
influenced with a concentration of the dissolved oxygen depending
on the subject of the measurement. Further, the measurement is made
completely impossible under a condition lacking oxygen. A type of
the glucose sensor that does not use oxygen as the electron
acceptor but uses a metal complex or an organic compound such as
potassium ferricyanide, a derivative of ferrocene or a derivative
of quinone as the electron acceptor has therefore been developed.
With this type of biosensor, by oxidizing a reductant of the
electron acceptor produced as the result of the enzyme reaction by
the electrode, the concentration of the glucose can be determined
based on the current consumed for this oxidation reaction. This
manner of measurement is not limited to glucose but has been widely
applied for the quantification of substrates other than
glucose.
As an example of this type of biosensor, a glucose sensor is known
(Japanese Laid-Open Patent Publication No. Hei 1-114,747) which
will be described below.
The disclosed biosensor has a configuration comprising an
electrical insulating base provided with an electrode system
including a working electrode and a counter electrode, a filter
layer composed of polycarbonate porous film, an electron acceptor
carrying layer, an enzyme carrying layer, a buffer carrying layer,
and a developing layer composed of woven cellulose, which are
sequentially laminated on the insulating base by placing some space
from the electrode system. In this configuration, the
above-mentioned carrying layers are prepared by impregnating
cellulosic porous films with aqueous solutions of the electron
acceptor, the enzyme, and the buffer, and then drying the
impregnated bodies.
The operation of this glucose sensor is as follows.
The sample solution titrated on the developing layer is first
passed to the buffer carrying layer, whereby the pH value of the
sample solution is adjusted to a pH value that can give the highest
activity to the enzyme by the buffering action of the buffer. Next,
the glucose in the sample solution reacts specifically with the
glucose oxidase in the enzyme carrying layer. At the same time, the
electron acceptor, such as potassium ferricyanide in the electron
acceptor carrying layer, is reduced by the electron produced by the
above-mentioned reaction to produce potassium ferrocyanide. The
amount of the produced potassium ferrocyanide is directly
proportional to the concentration of glucose contained in the
sample solution. After the substances having a large molecular
weight such as protein which disturb the electrode reaction
contained in the sample solution are filtered off by the filter
layer, the sample solution reaches the electrode system provided on
the insulating base. In order to prevent erroneous measurement,
part of the electrode system is covered with the insulating layer.
By measuring the value of the current for oxidizing the potassium
ferrocyanide produced in the sample solution by the electrode
system it is possible to determine the glucose concentration of the
sample solution.
the configuration of such prior art sensors, however, there is an
inconvenience that an adverse influence is given to the responsive
current, because wetting of the surface of the insulating base
including the electrode system with the sample solution is not
necessarily uniform and thus bubbles are retained between the
porous body of the filter layer and the insulating base. Further,
if the sample solution contains substances liable to be absorbed in
the electrode or substances having an electrode activity, there
would be a case wherein the response of the sensor is adversely
influenced.
As a method for overcoming the above-mentioned inconveniences, the
following biosensor is proposed and disclosed in Japanese Laid-Open
Patent Publication No. Hei 2-062,952.
In the disclosed configuration, the sensor comprises an
electrically insulating base, an electrode system composed of a
working electrode, a counter electrode and a reference electrode
formed on the insulating base by means of screen printing or the
like, and a reaction layer including a hydrophilic polymer, an
oxido-reductase, an electron acceptor, and a buffer as well if
required, formed on the electrode system in a manner that the
reaction layer is in close contact with the electrode system.
When the sample solution containing the substrate is titrated on
the reaction layer, the reaction layer dissolves in the sample
solution which is thereby adjusted to a pH value at which the
highest enzyme activity is achieved by the buffering action of the
buffer, the enzyme reacts with the substrate, and the electron
acceptor is reduced. After the completion of the enzyme reaction,
the reduced electron acceptor is electrochemically oxidized, and
the concentration of the substrate contained in the sample solution
is derived from the value Of the current consumed for oxidizing the
electron acceptor.
In the above-mentioned configuration of the prior art sensor, if
the biosensor is moistened, the buffer would be partly mixed with
the enzyme to induce a chemical interaction, thereby lowering the
enzyme activity and deteriorating the storing property of the
biosensor.
SUMMARY OF THE INVENTION
It is therefore a primary object of the present invention to
provide a biosensor that can be applied to quantification of a
specific component contained in various biological samples in a
rapid and simple manner with high accuracy.
It is another object of the present invention to provide a
biosensor that can be stored for a long period of time, and can be
utilized in quality control of foodstuffs as well as in clinical
tests.
It is still another object of the present invention to provide a
method for producing such biosensors while avoiding a possible
mixing of an enzyme with a buffer during its manufacturing
process.
The present invention provides a biosensor comprising,
an electrical insulating base,
an electrode system including at least a working electrode and a
counter electrode which are provided on a face of the insulating
base, and
a reaction layer formed on the insulating base in close contact
with the electrode system; wherein
the reaction layer contains at least a hydrophilic polymer, an
enzyme and a buffer, and
the enzyme being separated from the buffer.
In a preferred embodiment of the present invention, the reaction
layer preferably comprises at least two layers, wherein a first
layer is in contact with the electrode system and contains the
enzyme and the hydrophilic polymer, and a second layer contains the
buffer. It is also preferable for the second layer to comprise a
lipid of amphipathic (lipophilic and hydrophilic) property.
In another preferred embodiment of the present invention, the
reaction layer preferably comprises at least two layers, wherein a
first layer is in contact with the electrode system and contains
the buffer and the hydrophilic polymer, and a second layer contains
the enzyme. It is also preferable for the second layer to comprise
a hydrophilic polymer being soluble in an organic solvent that does
not dissolve the hydrophilic polymer contained in the first
layer.
It is preferable for the reaction layer of the biosensor in
accordance with the present invention to contain an electron
acceptor.
The present invention also provides a biosensor, wherein the
reaction layer preferably comprises at least three layers, and a
first layer contains the buffer and the hydrophilic polymer, and a
second layer contains the enzyme and the hydrophilic polymer. It is
also preferable for the second layer to further comprise an
electron acceptor.
Another preferred embodiment of the present invention further
comprises a layer containing a lipid, especially an amphipathic
lipid, placed to the outermost part of the reaction layer.
In a further preferred embodiment of the present invention, the
biosensor comprises a layer consisting essentially of a hydrophilic
polymer placed in close contact with the electrode system.
In still another preferred embodiment of the present invention, the
layer containing the buffer and the hydrophilic polymer is in close
contact with a layer containing the enzyme and the hydrophilic
polymer, wherein the hydrophilic polymers are different from each
other, and wherein the hydrophilic polymer contained in the upper
layer is soluble in an organic solvent that does not dissolve the
hydrophilic polymer contained in the underlying layer.
The present invention also provides a method for producing a
biosensor which comprises the steps of:
forming a first layer containing an enzyme and a hydrophilic
polymer by using water as the medium on a face of an insulating
base in close contact with an electrode system including at least a
working electrode and a counter electrode which are provided on the
insulating base; and
forming a second layer containing a buffer on the first layer by
using an organic solvent as the medium that does not dissolve the
hydrophilic polymer.
In a preferred embodiment of the above-mentioned method, the step
of forming the first layer comprises spreading an aqueous solution
which dissolves the enzyme and the hydrophilic polymer on the
insulating base and drying the spread solution, wherein the step of
forming the second layer comprises spreading a solution obtained by
dispersing the buffer in an organic solvent solution of a lipid and
drying the spread solution.
In another preferred embodiment of the present invention, the step
of forming the first layer comprises spreading an aqueous solution
which dissolves the enzyme and the hydrophilic polymer on the
insulating base and drying the spread solution, wherein the step of
forming the second layer comprises spreading a solution obtained by
dispersing the buffer in an organic solvent solution of the
hydrophilic polymer on the first layer and drying the spread
solution.
The present invention also provides a method for producing a
biosensor which comprises the steps of:
forming a first layer containing a buffer and a hydrophilic polymer
by using water as the medium on a face of an insulating base in
close contact with an electrode system including at least a working
electrode and a counter electrode provided on the insulating base;
and
forming a second layer containing a hydrophilic polymer and an
enzyme on the first layer by using an organic solvent as the medium
that does not dissolve the hydrophilic polymer contained in the
first layer.
In a preferred embodiment of the present invention, the step of
forming the first layer comprises spreading an aqueous solution
which dissolves the buffer and the hydrophilic polymer on the
insulating base and drying the spread solution, wherein the step of
forming the second layer comprises spreading an organic solvent
solution of the hydrophilic polymer on the first layer and drying
the spread solution, and further dropping an aqueous solution of
the enzyme on the second layer and drying the dropped solution.
It is preferable that the above-mentioned aqueous solution which
dissolves the enzyme and the hydrophilic polymer further dissolves
an electron acceptor.
In the same manner, it is also preferable that the above-mentioned
aqueous solution of the enzyme further dissolves an electron
acceptor.
Further, it is preferable that the method further comprises a step
of forming a third layer by spreading an organic solvent solution
of a lipid over the second layer and drying the spread
solution.
While novel features of the invention are set forth in the
preceding, the invention, both as to organization and content, can
be further understood and appreciated, along with other objects and
features thereof, from the following detailed description and
example when taken in conjunction with the attached drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a cross-sectional side view showing an essential part of
a biosensor prepared in accordance with Example 1 of the present
invention.
FIG. 2 is an exploded perspective view of the biosensor shown in
FIG. 1 removed of its reaction layer.
FIG. 3 is a cross-sectional side view showing an essential part of
a biosensor prepared in accordance with Example 2 of the present
invention.
FIG. 4 is a cross-sectional side view showing an essential part of
a biosensor prepared in accordance with Example 3 of the present
invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
In the following paragraphs, embodiments of the biosensor and
method for producing the same in accordance with the present
invention will be described in detail with reference to the
attached drawings.
As described in the above, the biosensor of the present invention
has a configuration that the reaction layer formed on the electrode
system on the insulating base is in close contact with the
electrode system, and contains at least the hydrophilic polymer,
the enzyme and the buffer, wherein the enzyme is separated from the
buffer. Since the reaction layer contains the buffer, even in the
case wherein the pH value of the sample solution does not coincide
with the pH value which gives the highest enzyme activity, the pH
value of the sample solution is automatically adjusted to the pH
value which gives the highest activity to the enzyme when the
sample solution reaches the buffer contained in the reaction layer.
Therefore, there is no need for previously adjusting the pH value
of the sample solution with a buffer or the like and it is possible
to measure the concentration of the specific component in the
sample solution by a simple operation.
Further, by separating the enzyme from the buffer in the reaction
layer, it is possible to prevent a partial mixing of the buffer
with the enzyme attributable to a possible wetting or moistening of
the biosensor and a lowering of the activity of the enzyme
attributable to the chemical interaction induced by the mixing, and
thus to maintain the enzyme at a condition that stabilizes the
enzyme during the storing period of the biosensor.
In the biosensor prepared in accordance with the present invention,
the layer containing the buffer is in close contact with the layer
containing the enzyme, but the hydrophilic polymers contained in
both layers are different from each other. By selecting the
hydrophilic polymer contained in the upper layer as the one that is
soluble in an organic solvent which does not dissolve the
hydrophilic polymer contained in the underlying layer, a direct
contact of the buffer with the enzyme can effectively be avoided
during the manufacturing process of the biosensor.
The biosensor having the above-mentioned configuration can be
obtained by the following manufacturing processes.
One of the processes comprises the steps of forming a first layer
composed of the enzyme and the hydrophilic polymer on the
insulating base, which is in close contact with the electrode
system, by using water as a medium, and forming a second layer
containing the buffer on the first layer by using an organic
solvent as the medium that does not dissolve the hydrophilic
polymer contained in the first layer.
The other process comprises steps of forming a first layer composed
of the buffer and the hydrophilic polymer on the insulating base,
being in close contact with the electrode system, by using water as
a medium, and forming a second layer composed of the enzyme and the
hydrophilic polymer on the first layer by using an organic solvent
as the medium that does not dissolve the first mentioned
hydrophilic polymer.
It is preferable that the biosensor of the present invention has a
layer containing a lipid that facilitates an infusion of the sample
solution into the reaction layer. In addition to lecithin
(phosphatidyl cholin) used in the following examples, an
amphipathic (lipophilic and hydrophilic) lipid such as
phospholipids, exemplified as phosphatidyl serine, phosphatidyl
ethanolamine and the like, are preferable as the lipid.
As the hydrophilic polymer for forming the reaction layer, in
addition to carboxymethyl cellulose and polyvinyl pyrrolidone which
are used in the following examples, there are exemplified polyvinyl
alcohol, water soluble cellulose derivatives such as ethyl
cellulose and hydroxypropyl cellulose; gelatin, polyacrylic acid
and its salts, starch and its derivatives, maleic anhydride and its
salts, polyacrylamide, methacrylate resin, poly-2-hydroxyethyl
methacrylate.
Although the description on the following examples is limited to
the two-electrode system composed only of the working electrode and
the counter electrode, a more accurate measurement can be performed
by employing a three-electrode system also including a reference
electrode.
In addition to potassium ferricyanide used in the following
examples, p-benzoquinone, phenadine methosulfate and ferrocene can
be used as the electron acceptor.
As the buffer, any buffer that can demonstrate a pH value which
gives the highest activity to the employed enzyme such as any salts
of citric acid can freely be used in addition to the phosphate
buffer used in the examples.
The present invention can widely be applied to any reaction system
where an enzyme participates, such as alcohol sensor, sucrose
sensor, and cholesterol sensor, in addition to the exemplified
glucose sensor, lactic acid sensor and glucose sensor. In these
cases, alcohol oxidase, lactic acid dehydrogenase, cholesterol
oxidase, cholesterol dehydrogenase, xanthine oxidase, and an amino
acid oxidase can be used in compliance with the specific substance
to be quantified, in addition to the fructose dehydrogenase, lactic
acid oxidase and glucose oxidase.
As described in the above, the biosensor of the present invention
can be applied to the quantification of the specific component
contained in the various biological samples in a rapid and simple
manner with a high accuracy. Further, since the biosensor can be
stored for a long period of time, its value of utilization is great
in quality control of foodstuffs as well as in clinical tests.
EXAMPLE 1
(Fructose Sensor I)
FIG. 1 is a cross-sectional side view showing a fructose sensor
prepared in accordance with an embodiment of the present invention
with its cover and a spacer omitted, and FIG. 2 is an exploded
perspective view of the fructose sensor with its reaction layer
omitted.
An insulating base i is made of polyethylene terephthalate. On the
insulating base 1, there are provided lead wires 2 and 3 of silver
by means of screen printing. An electrode system including a
working electrode 4 and a counter electrode 5 is also formed on the
insulating base 1 by printing an electrically-conductive carbon
paste containing a resin binder. Further, an insulating layer 6 is
formed on the insulating base 1 by printing an insulating paste.
The insulating layer 6 maintains areas of the exposed regions of
the working electrode 4 and the counter electrode 5 constant, and
partly covers the lead wires 2 and 3.
After the electrode region was prepared in this manner, a mixed
aqueous solution composed of an aqueous solution (0.5 wt %) of a
hydrophilic polymer, sodium salt of carboxymethyl cellulose
(hereinafter referred to CMC) which dissolved fructose
dehydrogenase (EC1. 1. 99. 11.; hereinafter referred to FDH) as an
enzyme and potassium ferricyanide as an electron acceptor, was
dropped on the electrode system. By being dried in a hot air dryer
at 40.degree. C. for 10 minutes, an FDH-potassium ferricyanide-CMC
layer 7 was formed.
On the FDH-potassium ferricyanide-CMC layer 7, there was dropped a
dispersion prepared by dispersing microcrystals of potassium
dihydrogenphosphate and dipotassium hydrogenphosphate as a buffer
in a toluene solution (0.5 wt %) of lecithin as a dispersing
medium, which was then dried to form a buffer-lecithin layer 8.
Since toluene used as the solvent for forming the layer 8 did not
dissolve CMC in the underlying layer, a direct contact of the
buffer in the layer 8 with the enzyme in the layer 7 was
effectively avoided. Further, by the provision of the layer
containing an amphipathic lipid such as lecithin on the surface of
the reaction layer, an infusion of the sample solution from the
surface into the reaction layer can be made with ease. As described
in the above, the reaction layer of the fructose sensor was
formed.
The manufacturing process of the biosensor can be simplified by
dropping the mixed solutions containing the hydrophilic polymer,
the enzyme and the electron acceptor, each in a stroke, and by the
subsequent drying. The temperature range during the drying step is
preferably from 20.degree. C. to 80.degree. C. which does not lead
to a deactivation of the enzyme but is sufficient for completing
the drying in a short period of time.
After forming the reaction layer in the above-mentioned manner, the
fructose sensor was completed by adhering a cover 14 and a spacer
13 to the insulating base in a positional relationship shown by
single dot-dash-lines in FIG. 2. By a simple operation of bringing
the sample solution to a contact with a sample supplying inlet 15
provided on a tip of the sensor, the sample solution can easily be
introduced into the reaction layer region. Since the supplying
amount of the sample solution is dependent on the volume of a space
formed by the cover 14 and the spacer 13, there is no need of
measuring the supplying amount beforehand.
Further, evaporation of the sample solution can be minimized during
the measurement thereby enabling a measurement of high accuracy. In
FIG. 2, a reference numeral 16 designates an air inlet opening
provided on the cover 14. When a transparent resin is used as the
material for constituting the cover 14 and the spacer 13, it is
possible to easily observe the condition of the reaction layer and
the state of introducing the sample solution from the outside.
Two minutes after supplying 3 .mu.l of a fructose standard solution
as the sample solution to the fructose sensor thus prepared through
the sample supplying inlet 15, a pulse voltage of +0.5 V on the
basis of the voltage at the counter electrode was applied to the
working electrode. Then the anodic current value 5 seconds after
the application was measured.
When the sample solution reached the reaction layer, the sample
solution dissolved the buffer-lecithin layer 8 to have a desirable
pH value, and subsequently dissolved the FDH-potassium
ferricyanide-CMC layer 7. During this process, the fructose
contained in the sample solution was oxidized so by the FDH, and
then the potassium ferricyanide was reduced to a potassium
ferrocyanide by shifting of electrons by the oxidation. Next, by
the application of the above-mentioned pulse voltage, a current was
generated for oxidizing the produced potassium ferrocyanide, and
this current value corresponded to the concentration of fructose
contained in the sample solution.
The activity of the enzyme employed in the fructose sensor
demonstrates its maximum value at pH 4.5 at 37.degree. C. Since the
fructose standard solution is substantially neutral, when the
standard solution reaches the buffer-lecithin layer 8, its pH value
is adjusted to 4.5, thereby making the enzyme activity highest.
Further, by separating the buffer from the enzyme, it is possible
to improve the storing property of the sensor.
The response obtained with the thus prepared fructose sensor to the
fructose standard solution demonstrates a linear relationship for
the fructose concentration, and the linear relationship can be
maintained in storage for a long period of time.
In the above-mentioned example, in place of the buffer-lecithin
layer 8, another buffer-hydrophilic polymer layer may be formed by
spreading a solution prepared by dispersing the buffer in a
solution of a hydrophilic polymer dissolved in an organic solvent
which does not dissolve the CMC contained in the underlying layer,
such as an ethanol solution of polyvinyl pyrrolidone, followed by
drying.
EXAMPLE 2
(Fructose Sensor II)
In a manner similar to that in Example 1, an electrode system
composed of the working electrode 4 and the counter electrode 5 was
formed on the insulating base 1 made of polyethylene terephthalate
by means of screen printing, as shown by FIG. 3. By dropping an
aqueous solution (0.5 wt %) of CMC on the electrode system and then
drying, a CMC layer was formed. Next, an aqueous solution of the
enzyme FDH and the electron acceptor potassium ferricyanide was
spread over the CMC layer and then dried to form an FDH-potassium
ferricyanide-CMC layer 7. In this case however, the CMC, the FDH as
well as the potassium ferricyanide were partially mixed together
and formed in a thin film of a thickness of several microns. That
is, when the above-mentioned aqueous solution was dropped on the
CMC layer, the previously formed CMC layer was once dissolved and
then formed a layer 7 in a state partly mixed with the enzyme and
the like during the subsequent drying process.
In this case however, since no stirring or the like operation was
performed, a completely mixed state was not brought about but a
state wherein the surface of the electrode system was covered only
with the CMC was brought about by this process. Since the enzyme,
the electron acceptor and the like are prevented from a direct
contact with the surface of the electrode system in this manner, it
is considered that
(i) there is a low possibility of an absorption of protein on the
surface of the electrode system and a change in the characteristics
of the electrode system by a chemical action of a substance having
an oxidizing ability such as potassium ferricyanide, and
(ii) as a result, it is possible to obtain a sensor having a sensor
response with high accuracy.
On this FDH-potassium ferricyanide-CMC layer 7, a dispersion
prepared by dispersing microcrystals of potassium
dihydrogenphosphate and dipotassium hydrogenphosphate, as the
buffer, in an ethanol solution of polyvinyl pyrrolidone
(hereinafter referred to PVP) as the hydrophilic polymer in 0.5 wt
% was dropped to cover the layer 7 completely, and then dried to
form a buffer-PVP layer 10. Since the ethanol employed in forming
the layer 10 does not dissolve the CMC contained in the underlying
layer, a direct contact of the enzyme in the layer 7 with the
buffer contained in the layer 10 can effectively be avoided.
By dropping a toluene solution of lecithin in 0.5 wt % on the
buffer-PVP layer 10 and then drying the dropped solution, a
lecithin layer 9 was formed on the layer 10. In the above-mentioned
manner, a reaction layer of the fructose sensor shown in FIG. 3 was
formed.
By combining the insulating base formed with the reaction layer
with a spacer 13 and a cover 14 shown by FIG. 2 in a similar manner
to that in Example 1, the fructose sensor of this example was
completed.
By the provision of the buffer-PVP layer 10, even in a case of
selecting a fruit Juice and the like containing solid components
such as fruit flesh or pulp as the sample solution, a possible
absorption of the above-mentioned flesh or pulp on the surface of
the electrode system and its adverse influence on the response of
the sensor can effectively be prevented by this buffer-PVP layer,
and at the same time, the pH value of the sample solution can be
made to a pH value that gives the maximum activity to the
enzyme.
The fructose sensor thus prepared demonstrates a rapid and a highly
accurate response and has an excellent storing property because the
buffer is separated from the enzyme as in Example 1.
EXAMPLE 3
(Lactic Acid Sensor)
In a manner similar to that in Example 1, an electrode system was
formed on the insulating base 1 made of polyethylene terephthalate
by means of screen printing, as shown by FIG. 4. By dropping an
aqueous solution (0.5 wt %) of CMC, which also dissolved the
buffer, potassium dihydrogenphosphate and dipotassium
hydrogenphosphate, on the electrode system and then drying, a
buffer-CMC layer 11 was formed. Next, an ethanol solution (0.5 wt
%) of PVP was spread over the buffer-CMC layer 11 so that it
covered the layer, and then dried to form a PVP layer. An aqueous
solution of lactic acid oxidase (available from TOYOBO Co., Ltd.,
hereinafter referred to LOD) as an enzyme and potassium
ferricyanide as an electron acceptor was spread over the PVP layer
and then dried. In this case, however, since the PVP layer was
partly dissolved in the above-mentioned aqueous solution, an
LOD-potassium ferricyanide-PVP layer 12 was formed. Further, since
the ethanol employed for forming the PVP layer did not dissolve the
CMC contained in the underlying layer, the PVP layer was not mixed
with the buffer, and the buffer was completely separated from the
enzyme.
By dropping a toluene solution of lecithin in 0.5 wt % on the
LOD-potassium ferricyanide-PVP layer 12 and by subsequent drying, a
lecithin layer 9 was formed. In the above-mentioned manner, a
reaction layer of a lactic acid sensor was formed. FIG. 4 is a
configuration of the reaction layer of the lactic acid sensor.
After forming the reaction layer in the above-mentioned manner, the
lactic acid sensor of this example was completed by combining the
insulating base formed with the reaction layer with a spacer 13 and
a cover 14 shown by FIG. 2 in a unitary body in a manner similar to
that in Example 1.
Three (3) .mu.l of a sample solution prepared by diluting lactic
acid with pure water to have a predetermined concentration was
supplied to the lactic acid sensor thus prepared through a sample
supplying inlet 15 thereof. The sample solution rapidly reached a
region of air outlet 16 to dissolve the reaction layer on the
electrode system.
When supplied with a sample solution, the reaction layer was
immediately dissolved in the sample solution, and the buffer
contained in the buffer-CMC layer 11 was dissolved in the sample
solution to give a desired pH value to the sample solution.
One minute after the supply of the sample solution, a pulse voltage
of +0.5 V on the basis of the voltage at the counter electrode 5
was applied to the working electrode 4 and the anodic current value
5 seconds after the application was measured. As a result of the
measurement, a response current value proportional to the
concentration of lactic acid in the sample solution was
obtained.
Since the optimum pH of the enzyme employed in the lactic acid
sensor is in a range from 6 to 7 but the standard solution of
lactic acid is more acidic than the value in the range, it is
possible to derive the maximum activity of the enzyme by causing
the sample solution to reach the buffer-CMC layer and thus
adjusting pH value of the sample solution from 6 to 7. Further,
since the buffer is separated from the enzyme, the lacti acid
sensor has an excellent storing property.
EXAMPLE 4
(Glucose Sensor I)
In a manner similar to that in Example 1, an electrode system
identical with the electrode region shown in FIG. 1 was formed on
the insulating base 1 made of polyethylene terephthalate by means
of screen printing. By dropping an aqueous solution (0.5 wt %) of
CMC, which also dissolved the buffer, potassium dihydrogenphosphate
and dipotassium hydrogenphosphate, on the electrode system and then
drying, a buffer-CMC layer was formed. Next, an ethanol solution
prepared by dispersing lipid-modified glucose oxidase (hereinafter
referred to as lipid-modified GOD) as an enzyme and potassium
ferricyanide as an electron acceptor was spread over the buffer-CMC
layer to cover the layer and then dried to form a lipid-modified
GOD-potassium ferricyanide layer. After a reaction layer was formed
in the above-mentioned manner, it was combined with a spacer 13 and
a cover 14 shown by FIG. 2 in a unitary body, whereby the glucose
sensor of this example was completed.
The above-mentioned lipid-modified GOD can be obtained by adding
glucose oxidase (available from TOYOBO Co., Ltd.) to a solution
prepared by dispersing an amphipathic lipid, DC-3-12L in water,
standing still at 4.degree. C. for 1.5 days, and freeze-drying the
stood product. The lipid-modified GOD is easily dispersible in an
organic solvent without being agglomerated, and is also soluble in
water.
EXAMPLE 5
(Glucose Sensor II)
In a manner similar to that in Example 1, an electrode system
identical with the electrode system shown in FIG. 1 was formed on
the insulating base 1 made of polyethylene terephthalate by means
of screen printing.
After producing the electrode system in the above-mentioned manner,
a buffer-potassium ferricyanide-CMC layer was formed by dropping an
aqueous solution of CMC in 0.5 wt %, which also dissolved potassium
dihydrogenphosphate and dipotassium hydrogenphosphate as a buffer,
and potassium ferricyanide as an electron acceptor, on the
electrode system, followed by drying. Next, a benzene solution of
lipid-modified GOD as the enzyme was spread over to cover the
buffer-potassium ferricyanide-CMC layer, and then dried to form a
lipid-modified GOD layer. After forming a reaction layer on the
insulating base in the above-mentioned manner, the insulating base
was combined with a spacer 13 and a cover 14 shown by FIG. 2 in a
unitary body in a manner similar to that in Example 1, whereby the
glucose sensor of this example was completed.
In the foregoing embodiments, although the electrode system was
formed by means of screen printing with an electrically-conductive
paint, it may alternately be formed by sputtering of platinum. In
this case, the potassium ferricyanide employed as the electron
acceptor can be dispensed with; in this enzyme reaction, hydrogen
peroxide generated by reducing the oxygen in the substrate solution
in proportion to the concentration of lactic acid (or glucose) can
be detected by the platinum electrodes, thereby quantifying the
concentration of the lactic acid (or glucose).
It is understood that various other modifications will be apparent
to and can be readily made by those skilled in the art to which
this invention pertains without departing from the scope and spirit
of this invention. Accordingly, it is not intended that the scope
of the claims appended hereto be limited to the description as set
forth herein, but rather that the claims be construed as
encompassing all the features of patentable novelty that reside in
the present invention, including all features that would be treated
as equivalents thereof, by those skilled in the art to which this
invention pertains.
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