U.S. patent application number 12/456199 was filed with the patent office on 2010-01-14 for biosensor.
Invention is credited to Masaki Fujiwara, Shoji Miyazaki, Takahiro Nakaminami, Junko Nakayama, Hiroyuki Tokunaga, Eriko Yamanishi.
Application Number | 20100006432 12/456199 |
Document ID | / |
Family ID | 26588660 |
Filed Date | 2010-01-14 |
United States Patent
Application |
20100006432 |
Kind Code |
A1 |
Miyazaki; Shoji ; et
al. |
January 14, 2010 |
Biosensor
Abstract
In a biosensor for measuring a specific substance in a liquid
sample, one or a combination of sugar alcohol, metallic salt,
organic acid or organic acid salt which has at least one carboxyl
group in a molecule, and organic acid or organic acid salt which
has at least one carboxyl group and one amino group in a molecule,
is included in a reagent layer provided on electrodes, thereby
providing a highly-accurate biosensor which is excellent in
stability and has high response (sensitivity, linearity) of the
sensor to the substrate concentration.
Inventors: |
Miyazaki; Shoji; (Ehime,
JP) ; Tokunaga; Hiroyuki; (Ehime, JP) ;
Fujiwara; Masaki; (Ehime, JP) ; Nakaminami;
Takahiro; (Kyoto, JP) ; Nakayama; Junko;
(Ehime, JP) ; Yamanishi; Eriko; (Ehime,
JP) |
Correspondence
Address: |
AMSTER, ROTHSTEIN & EBENSTEIN LLP
90 PARK AVENUE
NEW YORK
NY
10016
US
|
Family ID: |
26588660 |
Appl. No.: |
12/456199 |
Filed: |
June 12, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11106078 |
Apr 14, 2005 |
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12456199 |
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09979842 |
May 1, 2002 |
6911131 |
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PCT/JP01/02558 |
Mar 28, 2001 |
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11106078 |
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Current U.S.
Class: |
204/403.14 ;
204/403.01 |
Current CPC
Class: |
C12Q 1/003 20130101 |
Class at
Publication: |
204/403.14 ;
204/403.01 |
International
Class: |
G01N 27/30 20060101
G01N027/30; G01N 27/26 20060101 G01N027/26 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 29, 2000 |
JP |
2000-90362 |
Nov 9, 2000 |
JP |
2000-342537 |
Claims
1. A biosensor for measuring the concentration of a specific
substance in a sample solution, wherein sugar alcohol is included
in a previously-provided reagent layer so that it is dissolved in
the sample solution and specifically reacts to the specific
substance in the sample solution.
2. The biosensor as defined in claim 1, wherein the concentration
of the specific substance is measured employing electrodes
comprising at least a working electrode and a counter electrode
provided on an insulating support.
3. The biosensor as defined in claim 2, wherein the reagent layer
is formed on the electrodes or so that the electrodes are arranged
in a diffusion area where a reagent in the reagent layer is
dissolved and diffused in the sample solution, and the reagent
layer includes at least an enzyme and an electron transfer
agent.
4. The biosensor as defined in claim 1, wherein the sugar alcohol
is chain polyhydric alcohol or cyclic sugar alcohol, or a
substitution product or derivative of the sugar alcohol.
5. The biosensor as defined in claim 1, wherein the sugar alcohol
is either or both of multiol and lactitol.
6. A biosensor for measuring the concentration of a specific
substance in a sample solution, wherein metallic salt is included
in a previously-provided reagent layer so that it is dissolved in
the sample solution and specifically reacts to the specific
substance in the sample solution.
7. The biosensor as defined in claim 6, wherein the concentration
of the specific substance is measured employing electrodes
comprising at least a working electrode and a counter electrode
provided on an insulating support.
8. The biosensor as defined in claim 7, wherein the reagent layer
is formed on the electrodes or so that the electrodes are arranged
in a diffusion area where a reagent in the reagent layer is
dissolved and diffused in the sample solution, and the reagent
layer includes at least an enzyme and an electron transfer
agent.
9. The biosensor as defined in claim 6, wherein the metallic salt
is metallic salt sulfate, metallic salt hydrogensulfate, metallic
salt sulfite, metallic salt hydrogensulfite, or metallic salt
hyposulfite.
10. The biosensor as defined in claim 6, wherein the metallic salt
is either or both of magnesium sulfate and calcium sulfate.
11. The biosensor as defined in claim 6, wherein the metallic salt
is metallic salt nitrate, metallic salt hydrogennitrate, metallic
salt nitrite, metallic salt hydrogennitrite, or metallic salt
hyponitrite.
12. The biosensor as defined in claim 6, wherein the metallic salt
is either or both of magnesium nitrate and calcium nitrate.
13. A biosensor for measuring the concentration of a specific
substance in a sample solution, wherein organic acid or organic
acid salt which has at least one carboxyl group in its molecule is
included in a previously-provided reagent layer so that it is
dissolved in the sample solution and specifically reacts to the
specific substance in the sample solution.
14. The biosensor as defined in claim 13, wherein the concentration
of the specific substance is measured employing electrodes
comprising at least a working electrode and a counter electrode
provided on an insulating support.
15. The biosensor as defined in claim 14, wherein the reagent layer
is formed on the electrodes or so that the electrodes are arranged
in a diffusion area where a reagent in the reagent layer is
dissolved and diffused in the sample solution, and the reagent
layer includes at least an enzyme and an electron transfer
agent.
16. The biosensor as defined in claim 13, wherein the organic acid
is aliphatic carboxylic acid, carbocyclic carboxylic acid, or
heterocyclic carboxylic acid, or a substitution product or
derivative of the organic acid.
17. The biosensor as defined in claim 13, wherein the carboxylic
acid is one of glutaric acid, adipic acid, phthalic acid, and
benzoic acid, or a combination of these acids.
18. A biosensor for measuring the concentration of a specific
substance in a sample solution, wherein organic acid or organic
acid salt which has at least one carboxyl group and one amino group
in its molecule is included in a previously-provided reagent layer
so that it is dissolved in the sample solution and specifically
reacts to the specific substance in the sample solution.
19. The biosensor as defined in claim 18, wherein the concentration
of the specific substance is measured employing electrodes
comprising at least a working electrode and a counter electrode
provided on an insulating support.
20. The biosensor as defined in claim 19, wherein the reagent layer
is formed on the electrodes or so that the electrodes are arranged
in a diffusion area where a reagent in the reagent layer is
dissolved and diffused in the sample solution, and the reagent
layer includes at least an enzyme and an electron transfer
agent.
21. The biosensor as defined in claim 18, wherein the organic acid
is amino acid, or a substitution product or derivative of amino
acid.
22. The biosensor as defined in claim 18, wherein the amino acid is
one of glycine, serine, proline, threonine, lysine, and taurine, or
a combination of these acids.
23. A biosensor for measuring the concentration of a specific
substance in a sample solution, wherein a combination of at least
two of sugar alcohol, metallic salt, organic acid or organic acid
salt which has at least one carboxyl group in its molecule, and
organic acid or organic acid salt which has at least one carboxyl
group and one amino group in its molecule, is included in a
previously-provided reagent layer so that it is dissolved in the
sample solution and specifically reacts to the specific substance
in the sample solution.
24. The biosensor as defined in claim 1, wherein the reagent layer
further includes a hydrophilic polymer.
25. The biosensor as defined in claim 6, wherein the reagent layer
further includes a hydrophilic polymer.
26. The biosensor as defined in claim 13, wherein the reagent layer
further includes a hydrophilic polymer.
27. The biosensor as defined in claim 18, wherein the reagent layer
further includes a hydrophilic polymer.
28. The biosensor as defined in claim 23, wherein the reagent layer
further includes a hydrophilic polymer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/106,078, filed Apr. 14, 2005, which is a
continuation of U.S. patent application Ser. No. 09/979,842, filed
May 1, 2002, now U.S. Pat. No. 6,911,131, which is a 35 U.S.C.
.sctn.371 National Stage of PCT International Patent Application
No. PCT/JP01/02558, filed Mar. 28, 2001, which claims priority of
Japanese Patent Application No. 2000-90362, filed Mar. 29, 2000,
and of Japanese Patent Application No. 2000-342537, filed Nov. 9,
2000, the contents of all of which are hereby incorporated by
reference into the subject application.
TECHNICAL FIELD
[0002] The present invention relates to a biosensor for analyzing a
specific component in a liquid sample and, more particularly, to a
reagent formulation for composing a reagent layer of the
biosensor.
BACKGROUND ART
[0003] A biosensor is a sensor which utilizes the molecule
identifying abilities of organic materials such as microorganisms,
enzymes, and antibodies, and applies the organic materials as
molecular recognition elements. That is, the biosensor utilizes a
reaction which occurs when an immobilized organic material
recognizes a target specific substance, such as oxygen consumption
by respiration of a microorganism, an enzyme reaction, and
luminescence.
[0004] Among biosensors, enzyme sensors have been put to practical
use. For example, enzyme sensors for glucose, lactic acid,
cholesterol, lactose, urea, and amino acid are utilized in medical
measurement or food industry. An enzyme sensor reduces an electron
acceptor by an electron generated by a reaction between a substrate
included in a sample solution as a specimen and an enzyme, and a
measuring device electrochemically measures the reduction quantity
of the electron acceptor, thereby performing quantitative analysis
of the specimen. As an example of such biosensor, a sensor proposed
in Japanese Published Patent Application No. Hei. 11-1324511 has
been known.
[0005] FIGS. 11 and 12 are exploded perspective views illustrating
conventional biosensors for measuring a blood sugar level. A
measuring electrode 2 or 102 (also referred to as a working
electrode), a counter electrode 3 or 103, and a detecting electrode
4, which electrodes comprise a conductive material, are formed on
an insulating support 1 or 101 comprising polyethylene
terephthalate or the like, and a reagent layer 5 or 105 including
an enzyme which specifically reacts to a specific component in a
sample solution, an electron transfer agent, and a hydrophilic
polymer is formed on these electrodes.
[0006] In order to form a cavity for detecting an electric current
value generated by a reaction between the specific component in the
sample solution and a reagent in the reagent layer 5 or 105 with
the above-mentioned electrodes 2, 3, 4, 102, 103, a spacer 6 or 106
having a spindly cutout pail 7 or 107 in a position on the
electrodes and the reagent layer, and a cover 8 or 108 having an
air vent 9 or 109 are attached onto the insulating support.
[0007] In the biosensor constructed as described above, the sample
solution is supplied from the inlet of the cavity (sample suction
inlet) to the inside of the cavity by capillary phenomenon and is
let to the position of the electrodes and the reagent layer. When a
specific component in the sample solution reacts to the reagent of
the reagent layer, an electric current is generated, and the
generated electric current is read by an external measuring device
through leads of the biosensor, whereby quantitative analysis of
the specimen is carried out.
[0008] However, in the biosensor with the above-described reagent
composition, under the environment where heat and moisture exist,
particularly under the environment of high temperature and humidity
where the temperature is over 30.degree. C. and the humidity is
over 80%, a reduction reaction occurs between the electron transfer
agent and a portion of enzyme protein or hydrophilic polymer which
is included in the reagent layer 5 or 105, thereby generating a
background electric current (noise electric current). As the value
of the background electric current increases with time, the sensor
performance is deteriorated.
[0009] Furthermore, as a means to solve the problem, it is possible
to eliminate moisture and prevent the deterioration of the sensor
performance by enclosing a desiccant such as silica gel or
activated alumina into a biosensor preservation container which
employs a molded container of resin or aluminium seal. However, it
is impossible to completely eliminate water of molecular level
remaining in the reagent included in the biosensor, with the
desiccant alone.
[0010] Further, it is extremely hard to keep the preservation
container free of moisture penetration over long term, and the
reduction reaction between a portion of enzyme protein or
hydrophilic polymer and the electron transfer agent is promoted
when only a slight amount of moisture exists. Therefore, it is
extremely difficult to effectively suppress the increase in the
background electric current with time.
[0011] Further, when an inorganic salt such as potassium
ferricyanide is included in the mixed reagent layer composed of
various reagents such as an enzyme and an electron transfer agent,
the reagent layer is extremely easily crystallized in the process
of drying the reagent solution, whereby the surface of the reagent
layer becomes rough and uneven, resulting in deterioration in the
response (linearity, sensitivity) of the sensor to the substrate
concentration and the measurement accuracy.
[0012] The present invention is made to solve the above-mentioned
problems and has for its object to provide a highly-accurate
biosensor which efficiently prevents deterioration of the
performance of the biosensor due to contact with moisture, and has
high response (linearity, sensitivity) of the sensor to the
substrate concentration.
DISCLOSURE OF THE INVENTION
[0013] According to Claim 1 of the present invention, there is
provided a biosensor for measuring the concentration of a specific
substance in a sample solution, wherein sugar alcohol is included
in a previously-provided reagent layer so that it is dissolved in
the sample solution and specifically reacts to the specific
substance in the sample solution. Therefore, it is possible to
suppress an increase in a background electric current with time,
and suppress a needles reaction with various contaminants existing
in the blood, without preventing an enzyme reaction or the like,
thereby providing a high-performance biosensor which is excellent
in linearity (having a big slope and a small intercept of a
regression expression) and has small variations among individual
sensors.
[0014] According to Claim 2 of the present invention, in the
biosensor as defined in Claim 1, the concentration of the specific
substance is measured employing electrodes comprising at least a
working electrode and a counter electrode provided on an insulating
support. Therefore, it is possible to provide a biosensor which is
suitable for an examination employing electrodes.
[0015] According to Claim 3 of the present invention, in the
biosensor as defined in Claim 2, the reagent layer is formed on the
electrodes or so that the electrodes are arranged in a diffusion
area where a reagent in the reagent layer is dissolved and diffused
in the sample solution, and the reagent layer includes at least an
enzyme and an electron transfer agent. Therefore, it is possible to
provide a biosensor which is suitable for an examination employing
a reaction between an enzyme and an electron transfer agent.
[0016] According to Claim 4 of the present invention, in the
biosensor as defined in any of Claims 1 to 3, the sugar alcohol is
chain polyhydric alcohol or cyclic sugar alcohol, or a substitution
product or derivative of the sugar alcohol. Claim 4 embodies the
sugar alcohol defined in Claim 1 and, therefore, the same effect as
in Claim 1 can be achieved.
[0017] According to Claim 5 of the present invention, in the
biosensor as defined in any of Claims 1 to 3, the sugar alcohol is
either or both of multiol and lactitol. Claim 5 embodies the sugar
alcohol in Claim 1 and therefore, the same effects as in Claim 1
are achieved.
[0018] According to Claim 6 of the present invention, there is
provided a biosensor for measuring the concentration of a specific
substance in a sample solution, wherein metallic salt is included
in a previously-provided reagent layer so that it is dissolved in
the sample solution and specifically reacts to the specific
substance in the sample solution. Therefore, it is possible to
provide a biosensor which suppresses an increase in a background
electric current with time, without preventing an enzyme reaction
or the like.
[0019] According to Claim 7 of the present invention, in the
biosensor as defined in Claim 6, the concentration of the specific
substance is measured employing electrodes comprising at least a
working electrode and a counter electrode provided on an insulating
support. Therefore, it is possible to provide a biosensor which is
suitable for an examination employing electrodes.
[0020] According to Claim 8 of the present invention, in the
biosensor as defined in Claim 7, the reagent layer is formed on the
electrodes or so that the electrodes are arranged in a diffusion
area where a reagent in the reagent layer is dissolved and diffused
in the sample solution, and the reagent layer includes at least an
enzyme and an electron transfer agent. Therefore, it is possible to
provide a biosensor which is suitable for an examination employing
a reaction between an enzyme and an electron transfer agent.
[0021] According to Claim 9 of the present invention, in the
biosensor as defined in any of Claims 6 to 8, the metallic salt is
metallic salt sulfate, metallic salt hydrogensulfate, metallic salt
sulfite, metallic salt hydrogensulfite, or metallic salt
hyposulfite. Claim 9 embodies the metallic salt in Claim 6 and,
therefore, the same effect as in Claim 6 is achieved.
[0022] According to Claim 10 of the present invention, in the
biosensor as defined in any of Claims 6 to 8, the metallic salt is
either or both of magnesium sulfate and calcium sulfate. Claim 10
embodies the metallic salt in Claim 6 and, therefore, the same
effect as in Claim 6 is achieved.
[0023] According to Claim 11 of the present invention, in the
biosensor as defined in any of Claims 6 to 8, the metallic salt is
metallic salt nitrate, metallic salt hydrogennitrate, metallic salt
nitrite, metallic salt hydrogennitrite, or metallic salt
hyponitrite. Claim 11 embodies the metallic salt in Claim 6 and,
therefore, the same effect as in Claim 6 is achieved.
[0024] According to Claim 12 of the present invention, in the
biosensor as defined in any of Claims 6 to 8, the metallic salt is
either or both of magnesium nitrate and calcium nitrate. Claim 12
embodies the metallic salt in Claim 6 and, therefore, the same
effect as in Claim 6 is achieved.
[0025] According to Claim 13 of the present invention, there is
provided a biosensor for measuring the concentration of a specific
substance in a sample solution, wherein organic acid or organic
acid salt which has at least one carboxyl group in its molecule is
included in a previously-provided reagent layer so that it is
dissolved in the sample solution and specifically reacts to the
specific substance in the sample solution. Therefore, it is
possible to suppress an increase in a background electric current
with time, and suppress a needles reaction with various
contaminants existing in the blood, without preventing an enzyme
reaction or the like, thereby providing a high performance
biosensor which is excellent in linearity (having a big slope and a
small intercept of a regression expression) and has small
variations among individual sensors.
[0026] According to Claim 14 of the present invention, in the
biosensor as defined in Claim 13, the concentration of the specific
substance is measured employing electrodes comprising at least a
working electrode and a counter electrode provided on an insulating
support. Therefore, it is possible to provide a biosensor which is
suitable for an examination employing electrodes.
[0027] According to Claim 15 of the present invention, in the
biosensor as defined in Claim 14, the reagent layer is formed on
the electrodes or so that the electrodes are arranged in a
diffusion area where a reagent in the reagent layer is dissolved
and diffused in the sample solution, and the reagent layer includes
at least an enzyme and an electron transfer agent. Therefore, it is
possible to provide a biosensor which is suitable for an
examination employing a reaction between an enzyme and an electron
transfer agent.
[0028] According to Claim 16 of the present invention, in the
biosensor as defined in any of Claims 13 to 15, the organic acid is
aliphatic carboxylic acid, carbocyclic carboxylic acid, or
heterocyclic carboxylic acid, or a substitution product or
derivative of the organic acid. Claim 19 embodies the organic acid
in Claim 13 and, therefore, the same effect as in Claim 13 is
achieved.
[0029] According to Claim 17 of the present invention, in the
biosensor as defined in any of Claims 13 to 15, the carboxylic acid
is one of glutaric acid, adipic acid, phthalic acid, and benzoic
acid, or a combination of these acids. Claim 17 embodies the
carboxylic acid in Claim 13 and, therefore, the same effect as in
Claim 13 is achieved.
[0030] According to Claim 18 of the present invention, there is
provided a biosensor for measuring the concentration of a specific
substance in a sample solution, wherein organic acid or organic
acid salt which has at least one carboxyl group and one amino group
in its molecule is included in a previously-provided reagent layer
so that it is dissolved in the sample solution and specifically
reacts to the specific substance in the sample solution. Therefore,
it is possible to form the reagent layer closely and homogeneously
packed, thereby providing a biosensor which can dramatically
enhance a response (sensitivity, linearity) of the sensor to the
substrate concentration.
[0031] According to Claim 19 of the present invention, in the
biosensor as defined in Claim 18, the concentration of the specific
substance is measured employing electrodes comprising at least a
working electrode and a counter electrode provided on an insulating
support. Therefore, it is possible to provide a biosensor which is
suitable for an examination employing electrodes.
[0032] According to Claim 20 of the present invention, in the
biosensor as defined in Claim 19, the reagent layer is formed on
the electrodes or so that the electrodes are arranged in a
diffusion area where a reagent in the reagent layer is dissolved
and diffused in the sample solution, and the reagent layer includes
at least an enzyme and an electron transfer agent. Therefore, it is
possible to provide a biosensor which is suitable for an
examination employing a reaction between enzyme and an electron
transfer agent.
[0033] According to Claim 21 of the present invention, in the
biosensor as defined in any of Claims 18 to 20, the organic acid is
amino acid, or a substitution product or derivative of the amino
acid. Claim 21 embodies the organic acid in Claim 18 and,
therefore, the same effect as in Claim 18 is achieved.
[0034] According to Claim 22 of the present invention, in the
biosensor as defined in any of Claims 18 to 20, the amino acid is
one of glycine, serine, proline, threonine, lysine, and taurine, or
a combination of these acids. Claim 22 embodies the amino acid in
Claim 18 and, therefore, the same effect as in Claim 18 is
achieved.
[0035] According to Claim 23 of the present invention, there is
provided a biosensor for measuring the concentration of a specific
substance in a sample solution, wherein a combination of at least
two of sugar alcohol, metallic salt, organic acid or organic acid
salt which has at least one carboxyl group in its molecule, and
organic acid or organic acid salt which has at least one carboxyl
group and one amino group in its molecule, is included in a
previously-provided reagent layer so that it is dissolved in the
sample solution and specifically reacts to the specific substance
in the sample solution. Therefore, it is possible to provide a
highly-accurate biosensor which is excellent in stability and has
high response (sensitivity, linearity) of the sensor to the
substrate concentration.
[0036] According to Claim 24 of the present invention, in the
biosensor as defined in any of Claims 1 to 23, the reagent layer
further includes a hydrophilic polymer. Since the reagent layer
includes the hydrophilic polymer, formation of homogeneous reagent
on the surface of the electrodes is facilitated, and adhesion
between the electrodes and the reagent is enhanced. Further, since
the respective substances are homogeneously dispersed in the
reagent layer, homogeneous reagent formation can be realized,
thereby providing a high performance biosensor which has small
variations among individual sensors.
BRIEF DESCRIPTION OF DRAWINGS
[0037] FIG. 1 is a diagram illustrating sensor response
characteristics when lactitol is added as sugar alcohol into a
reagent solution, in a first embodiment.
[0038] FIG. 2 is a diagram illustrating sensor response
characteristics when maltitol is added as sugar alcohol into the
reagent solution, in the first embodiment.
[0039] FIG. 3 is a diagram illustrating an increase in the
background electric current under a harsh environment when purified
water is employed as a sample solution in the first embodiment.
[0040] FIG. 4 is a diagram illustrating an increase in the whole
blood response value under a harsh environment when the whole blood
is employed as the sample solution in the first embodiment.
[0041] FIG. 5 is a diagram illustrating an increase in the
background electric current under a harsh environment when purified
water is employed as a sample solution in a second embodiment.
[0042] FIG. 6 is a diagram illustrating an increase in the whole
blood response value under a harsh environment when the whole blood
is employed as the sample solution in the second embodiment.
[0043] FIG. 7 is a diagram illustrating an increase in the
background electric current under a harsh environment when purified
water is employed as a sample solution in a third embodiment.
[0044] FIG. 8 is a diagram illustrating an increase in the
background electric current under a harsh environment when purified
water is employed as a sample solution in a fourth embodiment.
[0045] FIG. 9 is a diagram illustrating the whole blood response
value when the whole blood is employed as a sample solution in a
fifth embodiment.
[0046] FIG. 10 is a diagram illustrating the whole blood response
value when the whole blood is employed as the sample solution in
the fifth embodiment.
[0047] FIG. 11 exemplifies an exploded perspective view of a
three-electrode-system biosensor.
[0048] FIG. 12 exemplifies an exploded perspective view of a
two-electrode-system biosensor.
BEST MODE TO EXECUTE THE INVENTION
Embodiment 1
[0049] Hereinafter, a biosensor according to a first embodiment of
the present invention will be described. In the respective
embodiments of the invention described below, an enzyme sensor,
which employs an enzyme as a molecular recognition element that
specifically reacts to a specific substance in a sample solution,
is exemplified.
[0050] FIG. 11 exemplifies an exploded perspective view of a
three-electrode-system biosensor, and FIG. 12 exemplifies an
exploded perspective view of a two-electrode-system biosensor. In
FIGS. 11 and 12, numeral 1 or 101 denotes an insulating support, on
which a working electrode 2 or 102 and a counter electrode 3 or 103
are formed at prescribed positions in prescribed shapes. Further,
in the three-electrode-system biosensor shown in FIG. 11, a
detecting electrode 4 is formed on the insulating support 1 at a
prescribed position in a prescribed shape. The detecting electrode
4 here serves as an electrode for detecting shortage of a specimen
quantity and, further, it can be employed as a part of a reference
electrode or the counter electrode.
[0051] Preferably, a material of the insulating support 1 or 101 is
polyethylene terephthalate, polycarbonate, polyimide, or the
like.
[0052] Further, a conductive substance constituting each electrode
is a simple substance such as a carbon or a noble metal such as
gold, platinum, palladium, or the like, or a compound material such
as a carbon paste or a noble-metal paste.
[0053] The simple substance such as carbon or noble metal such as
gold, platinum, palladium, or the like can be easily formed into a
conductive layer on the insulating support 1 or 101 employing
spattering deposition or the like, and the compound material such
as a carbon paste or a noble-metal paste can be easily formed into
a conductive layer on the insulating support 1 or 101 employing
screen printing or the like.
[0054] In the formation of the respective electrodes, the
conductive layer is formed over the entire or partial insulating
support 1 or 101 by the above-mentioned spattering deposition or
screen printing and, thereafter, slits are formed 11 using a laser
or the like to divide the conductive layer into the respective
electrodes. Further, the electrodes can be similarly formed by
screen printing or spattering deposition employing a printing plate
or a mask plate on which an electrode pattern is previously
formed.
[0055] A reagent layer 5 or 105, which includes an enzyme, an
electron transfer agent, a hydrophilic polymer, and sugar alcohol,
is formed on the electrodes formed as described above.
[0056] The first embodiment of the invention is characterized by
that the sugar alcohol is included in the reagent layer 5 or 105,
and the sugar alcohol prevents that the oxidized-form electron
transfer agent gets contact with a part of a reactive functional
group existing in enzyme protein or hydrophilic polymer included in
the reagent, so that the electron transfer agent is denatured
(reduced) from the oxidized-form to reduced-form in the reagent
layer 5 or 105 formed on the electrodes.
[0057] Accordingly, in the biosensor with the above-described
reagent formulation, under the environment where heat and moisture
exist, particularly under the environment of high temperature and
humidity where the temperature is over 30.degree. C. and the
humidity is over 80%, it is possible to suppress a background
electric current (noise electric current) which occurs due to a
reduction reaction between the electron transfer agent and a part
of enzyme protein or hydrophilic polymer included in the reagent
layer 5 or 105, and is increased with time, thereby preventing the
performance of the biosensor from being deteriorated.
[0058] Further, since the sugar alcohol is included in the reagent
layer, a needles reaction with various contaminants existing in the
blood, especially in blood corpuscles, can be also suppressed,
thereby providing high performance biosensors which are excellent
in linearity (having a big slope and a small segment of a
regression expression) and have less variations among the
individual sensors.
[0059] The sugar alcohol included in the reagent layer 5 or 105 may
be chain polyhydric alcohol or cyclic sugar alcohol such as
sorbitol, maltitol, xylitol, mannitol, lactitol, reduced
paratinose, arabinitol, glycerol, ribitol, galactitol,
sedoheptitol, perseitol, boremitol, styratitol, polygalitol,
iditol, talitol, allitol, ishylitol, reduced starch saccharified
material, and ishylitol.
[0060] The same effect as above can also be achieved by using the
stereoisomer, substitution product, or derivative of any of the
sugar alcohols.
[0061] Among these sugar alcohols, maltitol and lactitol are the
most suitable material since they are relatively low in the unit
price, are easily available, and are highly effective in
suppressing the background electric current.
[0062] The addition quantity of the sugar alcohol is preferably
0.1-500 mM as the reagent solution concentration, and more
preferably, 1-100 mM.
[0063] Thereafter, in the biosensor as shown in FIG. 11 or 12, the
spacer 6 or 106 having the cutout part 7 or 107 and the cover 8 or
108 are attached onto the reagent layer 5 or 105 and the electrodes
2, 3, 4, 102, 103, whereby a cavity through which the sample
solution is supplied is formed.
[0064] A material suitable for the spacer 6 or 106 and the cover 8
or 108 may be polyethylene terephthalate, polycarbonate, polyimide,
polybutylene terephthalate, polyamide, polyvinyl chloride,
polyvinylidene chloride, nylon, and the like.
[0065] The supply of the sample solution to the biosensor having
such cavity is realized by capillary phenomenon, and an air vent 9
or 109 for letting air out of the biosensor is required in the
cavity for smooth supply of the sample solution.
[0066] The air vent 9 or 109 can be arranged at any position in the
cavity unless it prevents the supply of the sample solution.
[0067] In the so-formed biosensor, the electric current value
obtained by the reaction between a specific component in the sample
solution and the reagent layer 5 or 105 including an enzyme or the
like is read by an external measuring device connected through lead
parts 10, 11, 12, 110, 111 of the working electrodes 2 or 102, the
counter electrode 3 or 103, and the detecting electrode 4.
Embodiment 2
[0068] Hereinafter, a biosensor according to a second embodiment of
the present invention will be described.
[0069] The biosensor according to the second embodiment of the
invention is the biosensor shown in FIG. 11 or 12 whose reagent
layer 5 or 105 is formed of an enzyme, an electron transfer agent,
a hydrophilic polymer, and a metallic salt. Other constituents are
the same as those of the above-described biosensor according to the
first embodiment and, therefore, descriptions thereof will be
omitted.
[0070] The second embodiment of the invention is characterized by
that a metallic salt is included in the reagent layer 5 or 105, and
the metallic salt prevents that the oxidized-form electron transfer
agent gets contact with a part of a reactive functional group
existing in enzyme protein and hydrophilic polymer included in the
reagent, so that the electron transfer agent is denatured (reduced)
from the oxidized form to reduced form in the reagent layer 5 or
105 formed on the electrodes.
[0071] Accordingly, in the biosensor with the above-described
reagent constitution, under the environment where heat and moisture
exist, particularly under the environment of high temperature and
humidity where the temperature is over 30.degree. C. and the
humidity is over 80%, it is possible to suppress a background
electric current (noise electric current) which occurs due to the
reduction reaction between the electron transfer agent and a part
of enzyme protein or hydrophilic polymer and is increased with
time, thereby preventing the performance of the biosensor from
being deteriorated.
[0072] As the metallic salt included in the reagent layer 5 or 105,
metallic salt sulfate or metallic salt nitrate is particularly
effective. As metallic salt sulfate, metallic salt hydrogensulfate,
metallic salt sulfite, metallic salt hydrogensulfite, or metallic
salt hyposulfite, for example, there are aluminium sulfate,
magnesium sulfate, zinc sulfate, antimony sulfate, indium sulfate,
uranyl sulfate, uranium sulfate, cadmium sulfate, potassium
sulfate, gallium sulfate, calcium sulfate, silver sulfate, chromium
sulfate, cobalt sulfate, potassium bisulfate, zirconium sulfate,
mercury sulfate, tin sulfate, strontium sulfate, caesium sulfate,
cerium sulfate, thallium sulfate, titanium sulfate, iron sulfate,
copper sulfate, sodium sulfate, lead sulfate, nickel sulfate,
neodymium sulfate, vanadium sulfate, palladium sulfate, barium
sulfate, bismuth sulfate, praseodymium sulfate, beryllium sulfate,
manganese sulfate, lanthanum sulfate, lithium sulfate, rubidium
sulfate, aluminium potassium sulfate, aluminium sodium sulfate,
uranyl potassium sulfate, potassium chromium sulfate, disodium
magnesium sulfate, magnesium dipotassium sulfate, manganese caesium
sulfate, rubidium aluminium sulfate, potassium hydrogensulfate,
sodium hydrogensulfate, potassium sulfite, calcium sulfite, sodium
sulfite, barium sulfite, bismuth sulfite, sodium hyposulfite,
potassium bisulfite, sodium bisulfite, and the like, while, as
metallic salt nitrate, metallic salt hydrogennitrate, metallic salt
nitrite, metallic salt hydrogennitrite, or metallic salt
hyponitrite, there are aluminium nitrate, magnesium nitrate, zinc
nitrate, antimony sulfate, ytterbium nitrate, yttrium nitrate,
indium nitrate, uranyl nitrate, erbium nitrate, cadmium nitrate,
gadolinium nitrate, potassium nitrate, calcium nitrate, silver
nitrate, chromium nitrate, cobalt nitrate, samarium nitrate,
zirconium nitrate, dysprosium nitrate, mercury nitrate, tin
nitrate, strontium nitrate, caesium nitrate, cerium nitrate,
thallium nitrate, iron nitrate, terbium nitrate, copper nitrate,
thorium nitrate, sodium nitrate, lead nitrate, nickel nitrate,
neodymium nitrate, palladium nitrate, barium nitrate, bismuth
nitrate, praseodymium nitrate, beryllium nitrate, holmium nitrate,
manganese nitrate, europium nitrate, lanthanum nitrate, lithium
nitrate, ruthenium nitrate, rubidium nitrate, rhodium nitrate,
thallium mercury nitrate, potassium nitrite, silver nitrite,
calcium nitrite, sodium nitrite, potassium cobaltinitrite, sodium
cobaltinitrite, sodium hyponitrite, and the like.
[0073] Among these metallic salts, magnesium sulfate, calcium
sulfate, magnesium nitrate, and calcium nitrate are particularly
suitable because they are highly effective in preventing moisture
absorption.
[0074] The addition quantity of the metallic salt is suitably
0.1-500 mM as a reagent solution concentration, and more suitably,
1-50 mM.
Embodiment 3
[0075] Hereinafter, a biosensor according to a third embodiment of
the present invention will be described.
[0076] The biosensor according to the third embodiment of the
invention has a reagent layer 5 or 105 shown in FIG. 11 or 12,
which is formed of an enzyme, an electron transfer agent, a
hydrophilic polymer, and an organic acid or organic acid salt which
has at least one carboxyl group in a molecule. Other constituents
are the same as those of the above-described biosensor according to
the first embodiment and, therefore, descriptions thereof will be
omitted.
[0077] The third embodiment of the invention is characterized by
that an organic acid or organic acid salt having at least one
carboxyl group in a molecule is included in the reagent layer 5 or
105, and the organic acid or organic acid salt prevents that the
oxidized-form electron transfer agent gets contact with a part of a
reactive functional group existing in enzyme protein and
hydrophilic polymer included in the reagent, so that the electron
transfer agent gets denatured (reduced) from the oxidized form to
reduced form in the reagent layer 5 or 105 formed on the
electrodes.
[0078] Accordingly, in the biosensor with the above-described
reagent constitution, under the environment where heat and moisture
exist, particularly under the environment of high temperature and
humidity where the temperature is over 30.degree. C. and the
humidity is over 80%, it is possible to suppress a background
electric current (noise electric current) which occurs due to the
reduction reaction between a part of enzyme protein or hydrophilic
polymer and the electron transfer agent and is increased with time,
thereby preventing the performance of the biosensor from being
deteriorated.
[0079] Further, since the organic acid or organic acid salt having
at least one carboxyl group in a molecule is included in the
reagent layer, a needles reaction with various contaminants
existing in the blood, particularly in blood corpuscles, can be
also suppressed, thereby providing high performance biosensors
which are excellent in linearity (having a big slope and a small
intersept of a regression expression) and have less variations
among individual sensors.
[0080] The organic acid or organic acid salt which has at least one
carboxyl group in a molecule and is included in the reagent layer 5
or 105 may be aliphatic carboxylic acid, carbocyclic carboxylic
acid, heterocyclic carboxylic acid, or their salt.
[0081] For example, the aliphatic carboxylic acid may be malonic
acid, succinic acid, glutaric acid, adipic acid, maleic acid,
fumaric acid, and their salt.
[0082] The degree of the effect becomes larger as the straight
chain is longer and the molecular weight is larger, and
particularly, an organic acid or organic acid salt having three or
more hydrocarbon chains is desirable. Further, as for a reagent
employed for the biosensor, a reagent having more hydrophilic
functional groups in the molecular structure is preferable since
the reagent is required to be highly soluble in water.
[0083] Further, the carbocyclic carboxylic acid may be benzoic
acid, phthalic acid, isophthalic acid, terephthalic acid, and their
salt, and the same effect as the above can be obtained by employing
these.
[0084] The heterocyclic carboxylic acid may be 2-phthalic-acid,
nicotinic acid, isonicotinic acid, and their salt, and the same
effect as the above can be obtained by employing these.
[0085] Further, in addition to the above-described aliphatic and
carbocyclic carboxylic acid, as well as heterocyclic carboxylic
acid or carboxylate salt, malic acid, oxaloacetic acid, citric
acid, ketoglutaric acid, and their salt, for example, in which
functional groups of the carboxylic acid or carboxylate salt are
partially replaced by other functional groups, can also achieve the
same effect as described above.
[0086] Among such organic acids or organic acid salts, glutaric
acid, adipic acid, phthalic acid, and benzoic acid are most
suitable.
[0087] The addition quantity of the organic acid or organic acid
salt is suitably 0.01-100 mM as a reagent solution concentration
and, more suitably, 0.1-10 mM.
Embodiment 4
[0088] Hereinafter, a biosensor according to a fourth embodiment of
the present invention will be described.
[0089] The biosensor according to the fourth embodiment of the
invention has a reagent layer 5 or 105 shown in FIG. 11 or 12,
which is formed of an enzyme, an electron transfer agent, a
hydrophilic polymer, and an organic acid or organic acid salt which
has at least one carboxyl group and one amino group in a molecule.
Other constituents are the same as those of the above-described
biosensor according to the first embodiment and, therefore,
descriptions thereof will be omitted.
[0090] The fourth embodiment of the invention is characterized by
the an organic acid or organic acid salt having at least one
carboxyl group and one amino group in a molecule is included in the
reagent layer 5 or 105, and addition of the organic acid or organic
acid salt into the reagent layer 5 or 105 provides the effect that
the surface state of the reagent layer 5 or 105 can be made
extremely smooth and homogeneous. Although the reagent layer 5 or
105 is easily crystallized in the process of drying the reagent
solution when inorganic salt such as potassium ferricyanide
employed as an electron transfer agent is included in the reagent
layer, the crystallization of the inorganic salt can be prevented
by the organic acid or organic acid salt which has at least one
carboxyl group and one amino group in a molecule and is included in
the reagent.
[0091] Since the inorganic salt prevented from being crystallized
exists in the reagent layer as a particulate, it can get close and
uniform contact with an enzyme molecule, resulting in a reagent
layer condition which is excellent in efficiency of electron
transfer with the enzyme molecule.
[0092] Further, since the solubility of the reagent layer can be
enhanced, the sensitivity and linearity of the sensor can be
dramatically improved.
[0093] The organic acid or organic acid salt, which has at least
one carboxyl group and one amino group in a molecule and is
included in the reagent layer 5 or 105, may be an organic acid or
organic acid salt such as glycine, alanine, valine, leucine,
isoleucine, serine, threonine, methionine, asparagine, glutamine,
arginine, lysine, histidine, phenylalanine, tryptophan, proline, or
their salt, or sarcosine, betaine, taurine, or the like.
[0094] The same effect as above can be achieved by employing the
substitution product or derivative of any of these organic acids or
organic acid salts.
[0095] Among the organic acids or organic acid salts, glycine,
serine, proline, threonine, lysine, and taurine are particularly
suitable since they are effective in preventing
crystallization.
[0096] The addition quantity of the organic acid or organic acid
salt is suitably 0.1-100 mM as a reagent solution concentration
and, more suitably, 10-500 mM.
[0097] In the first to fourth embodiments of the invention,
descriptions are given of the cases where sugar alcohol, metallic
salt, organic acid or organic acid salt which has at least one
carboxyl group in a molecule, and organic acid or organic acid salt
which has at least one carboxyl group and one amino group in a
molecule are respectively added to the reagent layer 5 or 105,
these additives may be appropriately combined.
[0098] The enzyme, which is included in the reagent in the first to
fourth embodiments of the invention, may be glucose oxidase,
lactate oxidase, cholesterol oxidase, cholesterol esterase,
uricase, ascorbia acid oxidase, bilirubin oxidase, glucose
dehydrogenase, lactate dehydrogenase, or the like, and the electron
transfer agent may be potassium ferricyanide, p-benzoquinone or its
derivative, phenazine methosulfate, methylene blue, ferrocene or
its derivative, or the like.
[0099] In the first to fourth embodiments of the invention, the
reagent layer 5 or 105 includes a hydrophilic polymer, whereby the
sample solution has a viscosity, and the formation of the reagent
on the electrodes is facilitated and homogenized and, further, the
adhesion between the electrodes and the reagent is enhanced.
Furthermore, the hydrophilic polymer included in the reagent layer
makes the condition of the reagent crystal after the reagent is
dried even and homogeneous, resulting in a high-accurate
biosensor.
[0100] Hydrophilic polymers used for the above-described purpose
may be carboxymethyl cellulose, hydroxyethyl cellulose,
hydroxypropylcellulose, methyl cellulose, ethyl cellulose, ethyl
hydroxyethyl cellulose, carboxymethyl ethyl cellulose, polyvinyl
alcohol, polyvinyl pyrrolidone, polyamino acid such as polylysine,
polystyrene sulfonate, or gelatine and its derivative, acrylic acid
and its salt, methacrylic acid and its salt, starch and its
derivative, maleic anhydride and its salt, and agarose gel and its
derivative.
[0101] While in the first to fourth embodiments of the invention
the above-described reagent layer 5 or 105 is provided on the
electrodes, specifically the reagent layer 5 or 105 can be arranged
over the entire surface or part of the electrodes. Besides, the
reagent layer 5 or 105 may be arranged within a range where the
performance of the biosensor is not deteriorated, i.e., a range
where the electrodes are provided within an area in which the
reagent in the reagent layer is dissolved and diffused into the
sample solution.
Example 1
[0102] A palladium thin film of approximately 10 nm thick is formed
over the entire surface of an insulating support comprising
polyethylene terephthalate by spattering deposition and,
thereafter, slits are formed in part of the thin film using a YAG
laser so as to divide the thin film into a working electrode, a
counter electrode, and a detecting electrode. On the electrodes, an
aqueous solution including an enzyme (glucose oxidase), an electron
transfer agent (potassium ferricyanide), a hydrophilic polymer
(carboxymethyl cellulose), and sugar alcohol is circularly dropped
centering around the working electrode so as to cover parts of the
counter electrode and the detecting electrode, and then it is
dried, thereby forming a reagent layer. On the reagent layer, a
spacer having a cutout part and comprising polyethylene
terephthalate and a cover having an air vent and comprising
polyethylene terephthalate are attached, thereby manufacturing a
three-electrode-system sensor for measuring a blood sugar level, in
which a cavity as a capillary for leading blood is formed.
[0103] FIG. 1 illustrates sensor response characteristics when
lactitol is added into the reagent solution as sugar alcohol, in
the case where the sample solution is whole blood and the lactitol
concentration is varied in four levels. Likewise, FIG. 2
illustrates sensor response characteristics in the case where
maltitol is added as sugar alcohol and the maltitol concentration
is varied in four levels. Here, a sensor in which the sugar alcohol
addition concentration (the concentration as a sample aqueous
solution) is 0 is handled as a conventional sensor, and sensors in
which the sugar alcohol addition concentrations are 5, 10, 25, and
50 mM are employed as invention sensors.
[0104] FIG. 3 illustrates change of a background electric current
value with time under a harsh environment (exposure in temperature
of 30.degree. C. and humidity of 80%), which is measured using the
sensor manufactured as described above. Purified water including no
glucose is employed as a sample solution. FIG. 4 illustrates change
of a sensor response value with time in the case where whole blood
adjusted to have the glucose concentration of 80 mg/dL is employed
as a sample solution. In either case, there are four points of
measurement in total: just after the sensor is manufactured
(O-hour), 6-hour, 12-hour, and 24-hour after the manufacture.
[0105] The current measurement condition is as follows. After
confirming that the cavity is filled with the sample solution,
enzyme reaction is promoted for twenty-five seconds. Thereafter, a
voltage of 0.2V is applied among the working electrode, the counter
electrode, and the detecting electrode, and an electric current
value obtained five seconds after the application is measured.
Here, the detecting electrode is also employed as a part of the
counter electrode.
[0106] The number of measurement n is n=10 for each concentration
and measurement time, and the average thereof is plotted in the
figures.
[0107] As is evident from FIG. 1, the response characteristics of
the sensor in which lactitol is added as sugar alcohol tend to be
high particularly in a range where the glucose concentration is
high, i.e., over 400 mg/dL, as compared with the conventional
sensor including no sugar alcohol, and this result indicates that
excellent response characteristics with a favorable regression
expression (having a small intercept and a big slope) are
obtained.
[0108] As is evident from FIG. 2, also when maltitol is employed as
sugar alcohol, excellent response characteristics are obtained as
in the above-mentioned case of employing lactitol.
[0109] Furthermore, as is evident from FIGS. 3 and 4, in the sensor
to which the sugar alcohol is added, increase in the background
electric current in the exposure environment under the high
temperature and humidity is efficiently suppressed, resulting in
excellent preservation stability with small variations of the
sensor response value with time.
Example 2
[0110] A sensor for measuring a blood sugar level is manufactured
by the same procedure as in the first example. In this second
example, magnesium sulfate as metallic salt sulfate is added
instead of sugar alcohol as an addition agent for suppressing
increase of a background electric current with time.
[0111] FIG. 5 illustrates change of a background electric current
value with time under a harsh environment (exposure in temperature
of 30.degree. C. and humidity of 80%), which is measured employing
the sensor manufactured as described above. Purified water
including no glucose is employed as a sample solution. FIG. 6
illustrates change of a sensor response value with time in the case
where whole blood adjusted to have the glucose concentration of 80
mg/dL is employed as a sample solution.
[0112] In either case, there are four points of measurement in
total: just after the sensor is manufactured (0-hour), 6-hour,
12-hour, and 24-hour after the manufacture.
[0113] The number of measurement n is the same as that in the first
example.
[0114] As is evident from FIGS. 5 and 6, also in the sensor in
which metallic salt sulfate is added, as well as sugar alcohol in
the first example, increase in the background electric current in
the exposure environment under the high temperature and humidity is
efficiently suppressed, resulting in excellent preservation
stability with small variations of the sensor response value with
time.
Example 3
[0115] An electrode layer comprising a working electrode and a
counter electrode is formed on an insulating support comprising
polyethylene terephthalate by screen printing, and a reagent layer
including an enzyme (glucose oxidase), an electron transfer agent
(potassium ferricyanide), a hydrophilic polymer (carboxymethyl
cellulose), and aliphatic carboxylic acid (concentration of which
is 5 mM as a sample solution) is formed on the electrode layer.
Thereafter, a spacer comprising polyethylene terephthalate and a
cover also comprising polyethylene terephthalate are attached,
thereby manufacturing a two-electrode-system sensor for measuring a
blood sugar level, in which a cavity as a capillary for leading
blood is formed.
[0116] Here, four kinds of two-electrode-system sensors in total
are manufactured: three sensors respectively including, as organic
acids, malonic acid (HOOC-CH.sub.2-COOH) as aliphatic carboxylic
acid, glutaric acid (HOOC-CH.sub.2-CH.sub.2-CH.sub.2-COOH), and
adipic acid (HOOC-CH.sub.2-CH.sub.2-CH.sub.2-CH.sub.2-COOH), and a
conventional sensor including no aliphatic carboxylic acid.
[0117] FIG. 7 illustrates background electric currents under a
harsh environment (temperature of 40.degree. C. and humidity of
80%), which are measured employing the four sensors manufactured as
mentioned above. Purified water including no glucose is employed as
a sample solution. There are four points of measurement in total:
just after the sensor is manufactured (0-hour), 7-day, 14-day, and
30-day after the manufacture. The measurement condition is as
follows. The cavity is filled with the sample solution (purified
water), and the reaction is promoted for twenty-five seconds.
Thereafter, a voltage of 0.5V is applied between the working
electrode and the counter electrode, and an electric current value
obtained five seconds after the application is measured.
[0118] The number of measurement n is n=10 for each measurement
point, and the average thereof is plotted in FIG. 7.
[0119] As is evident from FIG. 7, increase in the background
electric current is reliably suppressed in the sensors added with
the aliphatic carboxylic acids, and the pace of the increase is
reduced in the order of malonic acid, glutaric acid, and adipic
acid, thereby suggesting that the sensor with more complicated
molecular structure, longer straight chain, and larger molecular
weight is more effective in suppressing the increase in the
background electric current. The electric current value thus
obtained corresponds to the quantity of potassium ferrocyanide
which is generated by a reaction between glucose oxidase and
potassium ferricyanide as well as carboxymethyl cellulose and
potassium ferricyanide.
Example 4
[0120] A biosensor is manufactured by the same procedure as in the
third example and the same evaluation as in the third example is
made. This fourth example employs three kinds of organic acids as
follows: benzoic acid and phthalic acid as carbocyclic carboxylic
acids, and malic acid (derivative of succinic acid) with the
structure in which a part of hydrocarbon chain of succinic acid is
replaced by a hydroxyl group.
[0121] As is evident from FIG. 8, the effect of suppressing
increase in the background electric current is confirmed whichever
organic acid of benzoic acid, phthalic acid, or malic acid is
employed as in the third example.
Example 5
[0122] An electrode layer comprising a working electrode and a
counter electrode is formed on an insulating support comprising
polyethylene terephthalate by screen printing, and a reagent layer
including an enzyme (glucose dehydrogenase with pyrrolo-quinoline
quinone as coenzyme), an electron transfer agent (potassium
ferricyanide), a hydrophilic polymer (carboxymethyl cellulose),
aliphatic carboxylic acid (phthalic acid), and amino acid is formed
on the electrodes. Thereafter, a spacer comprising polyethylene
terephthalate and a cover comprising polyethylene terephthalate as
well are attached, thereby manufacturing a two-electrode-system
sensor for measuring a blood sugar level, in which a cavity as a
capillary for leading blood is formed.
[0123] In this fifth example, eight kinds of sensors in total are
manufactured as follows: sensors respectively including, as organic
acids, glycine (Gly), serine (Ser), proline (Pro), threonine (Thr),
lysine (Lys), sarcosine (derivative of glycine), and taurine, which
are amino acids having at least one carboxyl group and one amino
group in a molecule, and a conventional sensor including no amino
acid.
[0124] FIGS. 9 and 10 illustrate sensor response characteristics
when glucose in human whole blood is measured employing the eight
sensors manufactured as described above. This fifth example employs
the glucose concentrations in whole blood, 40, 80, 350, 600, and
700 mg/dl.
[0125] The measurement condition is as follows. After the cavity is
filled with the sample solution (human whole blood), a reaction is
promoted for twenty-five seconds. Thereafter, a voltage of 0.5V is
applied between the working electrode and the counter electrode,
and an electric current value obtained five seconds after the
application is measured.
[0126] The number of measurement n is n=20 for each concentration,
and the average thereof is plotted in the figure.
[0127] As is evident from FIGS. 9 and 10, dramatic enhancement of
the response value and linearity is confirmed particularly in a
range where the glucose concentration is higher than 480 mg/dL, as
compared with the conventional sensor including no amino acid,
although there are slight differences in the response values
according to the kinds of amino acids.
[0128] Table 1 shows variations of the sensor response value at the
measurement of n=20 by CV values. As is evident from table 1,
considerable improvement of the CV value is recognized in the
sensors of the invention which are added with amino acids. The
reason is as follows. Since the amino acid added into the reagent
layer prevents potassium ferricyanide from being crystallized, the
reagent layer is formed smoothly and homogeneously, whereby
solubility and diffusion of the reagent becomes homogeneous,
resulting in a reduction of response variations.
TABLE-US-00001 TABLE 1 Concentration (mg/dl) 40 80 350 600 700
Conventional 3.25% 2.48% 2.05% 2.11% 4.15% sensor Gly-133 mN 2.15%
1.75% 1.55% 1.28% 1.05% Ser-95 mM 2.54% 2.11% 1.75% 1.45% 1.22%
Pro-87 mN 2.38% 1.98% 1.69% 1.40% 1.18% Thr-84 mN 2.18% 2.00% 1.65%
1.39% 1.24% Lys-HCl-55 mM 2.48% 1.89% 1.58% 1.41% 1.14% Sarcosine-
2.65% 2.08% 1.68% 1.35% 1.25% 122 mM Taurine-80 mM 2.18% 1.78%
1.43% 1.22% 1.10%
[0129] While the first to fifth examples describe the biosensors
for measuring the glucose concentration in blood, the sample
solution and substance as targets of measurement and the type of
biosensor are not restricted thereto. For example, a liquid vital
specimen such as saliva, intercellular substance liquid, urine, or
sweat, as well as food or drinking water, can also be employed as a
target sample solution besides blood. Further, lactic acid,
cholesterol, uric acid, ascorbic acid, or bilirubin, can also be
also employed as a target substance besides glucose. Furthermore,
As a current measurement method, there are the three-electrode
system constituted by the working electrode, the counter electrode,
and the detecting electrode, which is employed in the first and
second examples, or the two-electrode system constituted by the
working electrode and the counter electrode, which is employed in
the third and fifth examples, and the same effects as described
above can be obtained whichever system is employed. The
three-electrode system enables more accurate measurement as
compared with the two-electrode system.
[0130] While the examples are described exemplifying the enzyme
sensor as a biosensor, the present invention is similarly
applicable to a biosensor utilizing antibodies, microorganisms,
DNA, RNA, or the like, besides enzyme, as a molecular recognition
element which specifically reacts to a specific substance in a
sample solution.
APPLICABILITY IN INDUSTRY
[0131] A biosensor according to the present invention includes
either one or a combination of sugar alcohol, metallic salt,
organic acid or organic acid salt which has at least one carboxyl
group in a molecule, or organic acid or organic acid salt which has
at least one carboxyl group and amino group in a molecule in its
reagent, so that increase of a background electric current with
time can be suppressed, a needles reaction with various
contaminants existing in the blood can be suppressed, or the
reagent layer can be closely and homogeneously formed, without
preventing an enzyme reaction or the like, thereby providing a
highly stable and efficient biosensor.
* * * * *