U.S. patent application number 09/770946 was filed with the patent office on 2001-09-13 for biosensor and method of producing the same.
This patent application is currently assigned to Matsushita Electric Industrial Co., Ltd.. Invention is credited to Hasegawa, Miwa, Ikeda, Shin, Nankai, Shiro, Watanabe, Motokazu, Yamamoto, Tomohiro.
Application Number | 20010020591 09/770946 |
Document ID | / |
Family ID | 18545624 |
Filed Date | 2001-09-13 |
United States Patent
Application |
20010020591 |
Kind Code |
A1 |
Hasegawa, Miwa ; et
al. |
September 13, 2001 |
Biosensor and method of producing the same
Abstract
The present invention provides a biosensor that has a reaction
reagent system easily dissolved even in a sample solution
containing a high concentration of a substrate and ensures rapid
and highly accurate measurement. The method of forming a target
reaction layer according to the present invention dissolves a
material constituting the target reaction layer in a solvent of a
sublimable substance to prepare a solution, applies the solution in
a desired area to form the target reaction layer, freezes the
applied solution, and sublimates the solvent included in a solid
matter of the frozen solution under reduced pressure for removal.
The resultant reaction layer has a large surface area and is easily
dissolved in the sample solution to enable rapid measurement.
Inventors: |
Hasegawa, Miwa; (Nara-shi,
JP) ; Yamamoto, Tomohiro; (Osaka, JP) ;
Watanabe, Motokazu; (Osaka, JP) ; Ikeda, Shin;
(Osaka, JP) ; Nankai, Shiro; (Osaka, JP) |
Correspondence
Address: |
AKIN, GUMP, STRAUSS, HAUER & FELD, L.L.P.
ONE COMMERCE SQUARE
2005 MARKET STREET, SUITE 2200
PHILADELPHIA
PA
19103
US
|
Assignee: |
Matsushita Electric Industrial Co.,
Ltd.
|
Family ID: |
18545624 |
Appl. No.: |
09/770946 |
Filed: |
January 26, 2001 |
Current U.S.
Class: |
205/777.5 ;
204/403.11; 427/2.13; 435/25; 435/287.9 |
Current CPC
Class: |
C12Q 1/002 20130101 |
Class at
Publication: |
205/777.5 ;
204/403; 435/25; 435/287.9 |
International
Class: |
G01N 027/327; C12Q
001/26 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 27, 2000 |
JP |
2000-018834 |
Claims
1. A method of manufacturing a biosensor which comprises an
electrically insulating base plate, an electrode system including a
measuring electrode and a counter electrode formed on the base
plate, and a reaction reagent system including at least an
oxidoreductase and an electron mediator as reagents, wherein the
reagents of the reaction reagent system are present as a reaction
layer structure that comprises at least one reaction layer and is
formed on or in the vicinity of the electrode system, said method
comprising the step of forming a specific reaction layer of the
reaction layer structure that contains at least one specific
reagent of the reaction reagent system, said step comprising: (1)
dissolving the at least one specific reagent in a solvent of a
sublimable substance to prepare a solution; (2) applying the
solution in a desired area to form the specific reaction layer; (3)
freezing the applied solution; and (4) sublimating the solvent
included in a solid matter of the frozen solution under reduced
pressure for removal.
2. The method in accordance with claim 1, wherein the specific
reaction layer contains all the reagents of the reaction reagent
system.
3. The method in accordance with claim 1, wherein the reaction
layer structure comprises a plurality of reaction layers and only
the specific reaction layer contains the at least one specific
reagent of the reaction reagent system.
4. The method in accordance with claim 1, said method comprising
the step of forming a stack of plural reaction layers as the
reaction layer structure, wherein the at least one specific reagent
of the reaction reagent system is contained only in an upper-most
reaction layer of the stack, said step comprising: pre-forming the
stack of plural reaction layers except the upper-most reaction
layer; dissolving the at least one specific reagent in a solvent of
a sublimable substance to prepare a solution and applying the
solution on the pre-formed stack of plural reaction layers without
the upper-most reaction layer; freezing the applied solution; and
sublimating the solvent included in a solid matter of the frozen
solution under reduced pressure for removal.
5. The method in accordance with claim 1, wherein said biosensor
further comprises a cover member, which is joined with the base
plate to define a sample solution supply pathway, through which a
sample solution flows to the electrode system, the reaction layer
structure is exposed to the sample solution supply pathway, and at
least one reaction layer of the reaction layer structure is formed
on the cover member.
6. The method in accordance with claim 1, wherein at least a
lower-most reaction layer of the reaction layer structure includes
a hydrophilic polymer.
7. The method in accordance with claim 3, wherein said
oxidoreductase is an enzyme functioning as a catalyst of the
oxidation reaction of cholesterol, and said electron mediator is
contained in another reaction layer different from a reaction layer
containing said enzyme.
8. The method in accordance with claim 3, wherein said
oxidoreductase is an enzyme functioning as a catalyst of the
oxidation reaction of cholesterol, and the layer containing said
enzyme further contains a surface active agent.
9. The method in accordance with claim 1, wherein said electron
mediator is potassium ferricyanide.
10. The method in accordance with claims 3, wherein said at least
one specific reagent of the reaction reagent system is an
oxidoreductase or potassium ferricyanide which is an electron
mediator.
11. A method of manufacturing a biosensor which comprises an
electrically insulating base plate, an electrode system including a
measuring electrode and a counter electrode formed on said base
plate, and a reaction reagent system including at least an
oxidoreductase and an electron mediator, wherein reagents of said
reaction reagent system are included in one or more reaction layers
formed on or in the vicinity of the electrode system, said method
comprising the step of forming said one or more reaction layers,
said step comprising: dissolving said reagents in a solvent of a
sublimable substance to prepare a solution; applying the solution
in a desired area to form said one or more reaction layers;
freezing the applied solution; and sublimating the solvent included
in a solid matter of the frozen solution under reduced pressure for
removal.
12. The method in accordance with claim 11, wherein said biosensor
further comprises a cover member, which is joined with the base
plate to define a sample solution supply pathway, through which a
sample solution flows to the electrode system, the reaction layer
structure is exposed to the sample solution supply pathway, and at
least one reaction layer of the reaction layer structure is formed
on the cover member.
13. A method of manufacturing a biosensor which comprises an
electrically insulating base plate, an electrode system including a
measuring electrode and a counter electrode formed on said base
plate, and a reaction reagent system including at least an
oxidoreductase and an electron mediator, wherein reagents of said
reaction reagent system are included in a plurality of reaction
layers formed on or in the vicinity of the electrode system, and
only a specific reaction layer of the plurality of reaction layers
contains at least one specific reagent of said reaction reagent
system, said method comprising the step of forming said specific
reaction layer, said step comprising: dissolving the at least one
specific reagent in a solvent of a sublimable substance to prepare
a solution; applying the solution in a desired area to form the
reaction layer; freezing the applied solution; and sublimating the
solvent included in a solid matter of the frozen solution under
reduced pressure for removal.
14. The method in accordance with claim 13, wherein said biosensor
further comprises a cover member, which is joined with the base
plate to define a sample solution supply pathway, through which a
sample solution flows to the electrode system, the reaction layer
structure is exposed to the sample solution supply pathway, and at
least one reaction layer of the reaction layer structure is formed
on the cover member.
15. A method of manufacturing a biosensor which comprises an
electrically insulating base plate, an electrode system including a
measuring electrode and a counter electrode formed on said base
plate, and a reaction reagent system including at least an
oxidoreductase and an electron mediator, wherein reagents of said
reaction reagent system are included in one or more reaction layers
formed on or in the vicinity of the electrode system, and at least
one specific reagent of said reaction reagent system is contained
only in an upper-most layer of a specific reaction layer comprising
a stack of layers, said method comprising the step of forming said
specific reaction layer, said step comprising: pre-forming the
stack of layers except the upper-most layer; dissolving the at
least one specific reagent in a solvent of a sublimable substance
to prepare a solution; applying the solution on the pre-formed
stack of layers without the upper-most layer; freezing the applied
solution; and sublimating the solvent included in a solid matter of
the frozen solution under reduced pressure for removal.
16. The method in accordance with claim 15, wherein said biosensor
further comprises a cover member, which is joined with the base
plate to define a sample solution supply pathway, through which a
sample solution flows to the electrode system, the reaction layer
structure is exposed to the sample solution supply pathway, and at
least one reaction layer of the reaction layer structure is formed
on the cover member.
17. A biosensor manufactured by the method of claim 1.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates to a biosensor that ensures
rapid and highly accurate quantification of a specific component
contained in a sample by a simplified procedure, and more
specifically to a method of forming a reaction layer of the
biosensor.
[0002] One proposed biosensor adopts the simple technique that
quantifies a specific component in a sample without diluting or
stirring a sample solution (Japanese Laid-Open Patent Publication
No. Hei 2-062952).
[0003] This prior art biosensor is manufactured by forming an
electrode system, which includes a measuring electrode, a counter
electrode, and a reference electrode, on an electrically insulating
base plate by a known method like screen printing and further
forming an enzyme reaction layer, which includes a hydrophilic
polymer, an oxidoreductase, and an electron mediator, on the
electrode system. A buffer may be added to the enzyme reaction
layer according to the requirements.
[0004] When a sample solution including a substrate is dropped on
the enzyme reaction layer of the biosensor manufactured in the
above manner, the enzyme reaction layer is dissolved to make the
enzyme react with the substrate and reduce the electron mediator.
After completion of the enzyme reaction, the concentration of the
substrate included in the sample solution is determined, based on
the observed oxidation current flowing in the process of
electrochemically oxidizing the reduced electron mediator.
[0005] The following describes a glucose sensor as one example of
the biosensor.
[0006] A generally known method of quantifying glucose combines
glucose oxidase with either an oxygen electrode or a hydrogen
peroxide electrode.
[0007] Glucose oxidase selectively oxidizes a substrate
.beta.-D-glucose to D-glucono-.delta.-lactone with oxygen as the
electron mediator. In the course of this reaction, oxygen is
reduced to hydrogen peroxide. Glucose is quantified by measuring
the quantity of oxygen consumption due to the reduction with the
oxygen electrode or by measuring the quantity of hydrogen peroxide
production with the hydrogen peroxide electrode, such as a platinum
electrode.
[0008] The quantification of some substrates of interest according
to this prior art method is, however, significantly affected by the
concentration of dissolved oxygen. The measurement is unavailable
in the absence of oxygen. Another type of the glucose sensor has
accordingly be developed, which does not use oxygen as the electron
mediator but utilizes a metal complex or an organic compound, such
as potassium ferricyanide, a ferrocene derivative, or a quinone
derivative, for the electron mediator.
[0009] The glucose sensor of this type oxidizes the reduced form
electron mediator, which results from the enzyme reaction, on an
electrode and determines the concentration of glucose from the
observed oxidation current.
[0010] The biosensor according to this technique is, in principle,
applicable to measurement of various substances by using an enzyme
that acts upon each substance of interest as the substrate.
[0011] For example, application of cholesterol oxidase or
cholesterol dehydrogenase and cholesterol esterase for the
oxidoreductase enables measurement of serum cholesterol, which is
used as a diagnostic indication in a diversity of medical
institutes.
[0012] The progress of the enzyme reaction of cholesterol esterase
is remarkably slow. Addition of an appropriate surface active agent
enhances the activity of cholesterol esterase and shortens the time
required for the whole reaction.
[0013] In this prior art biosensor, for example, a reaction layer
is obtained by dissolving potassium ferricyanide, which is one of
the electron mediators discussed above, alone or with other
components in a solvent, dropping the solution in a desired area
for the reaction layer on a base plate, and drying the dropped
solution with warm blast. In this reaction layer, potassium
ferricyanide deposits in the form of needles having the
longitudinal dimension of even greater than 1 mm. The reaction
layer accordingly has the heterogeneous configuration, which
worsens the measurement accuracy of a resultant sensor.
[0014] Compared with that in the glucose sensor, the reaction layer
in the cholesterol sensor contains a higher concentration of the
corresponding enzyme. The prior art method that forms such a
reaction layer by drying the dropped solution with warm blast
causes the resultant reaction layer to be slowly dissolved in a
sample solution and have poor response.
BRIEF SUMMARY OF THE INVENTION
[0015] The object of the present invention is thus to provide a
biosensor that has high accuracy and excellent response to a
substrate even in a high concentration range of the substrate.
[0016] In order to form a reaction layer containing a reagent such
as potassium ferricyanide, which tends to deposit from an aqueous
solution in the form of crystals, or a high concentration of an
enzyme, the inventors have found the suitable method that dissolves
a material constituting a target reaction layer in a solvent of a
sublimable substance to prepare a solution, applies the solution in
a desired area to form the target reaction layer, freezes the
applied solution, and sublimates the solvent included in a solid
matter of the frozen solution under reduced pressure for removal.
This method gives the reaction layer that has a large surface area
and is easily dissolved in a sample solution. The technique of the
present invention is based on these findings.
[0017] At least part of the above and the other related objects of
the present invention is attained by a method of manufacturing a
biosensor, which includes an electrically insulating base plate, an
electrode system that is provided on the electrically insulating
base plate and includes a measuring electrode and a counter
electrode, and a reaction reagent system that includes at least an
oxidoreductase and an electron mediator as reagents. The reagents
of the reaction reagent system are present as a reaction layer
structure that includes at least one reaction layer and is formed
on or in the vicinity of the electrode system. The method includes
the step of forming a specific reaction layer of the reaction layer
structure that contains at least one specific reagent of the
reaction reagent system. The step comprises:
[0018] (1) dissolving the at least one specific reagent in a
solvent of a sublimable substance to prepare a solution;
[0019] (2) applying the solution in a desired area to form the
specific reaction layer;
[0020] (3) freezing the applied solution; and
[0021] (4) sublimating the solvent included in a solid matter of
the frozen solution under reduced pressure for removal.
[0022] In accordance with one preferable application of the present
invention, the specific reaction layer contains all the reagents of
the reaction reagent system.
[0023] In accordance with another preferable application of the
present invention, the reaction layer structure has a plurality of
reaction layers and only the specific reaction layer contains the
at least one specific reagent of the reaction reagent system.
[0024] In accordance with still another preferable application of
the present invention, the method includes the step of forming a
stack of plural reaction layers as the reaction layer structure,
wherein the at least one specific reagent of the reaction reagent
system is contained only in an upper-most reaction layer of the
stack. The step includes the sub-steps of: pre-forming the stack of
plural reaction layers except the upper-most reaction layer;
dissolving the at least one specific reagent in a solvent of a
sublimable substance to prepare a solution and applying the
solution on the pre-formed stack of plural reaction layers without
the upper-most reaction layer; freezing the applied solution; and
sublimating the solvent included in a solid matter of the frozen
solution under reduced pressure for removal.
[0025] It is preferable that the method further includes the steps
of: forming a cover member on the electrically insulating base
plate, which is joined with the base plate to define a sample
solution supply pathway, through which a sample solution flows to
the electrode system; causing the reaction layer structure to be
exposed to the sample solution supply pathway; and forming at least
one reaction layer of the reaction layer structure on the cover
member.
[0026] In the case where the oxidoreductase is an enzyme
functioning as a catalyst of the oxidation reaction of cholesterol,
it is preferable that the electron mediator is included in another
reaction layer different from a reaction layer containing the
enzyme.
[0027] In accordance with another preferable application of the
present invention, the oxidoreductase is an enzyme functioning as a
catalyst of the oxidation reaction of cholesterol, and the electron
mediator is contained in another reaction layer different from a
reaction layer containing the enzyme.
[0028] In accordance with still another preferable application of
the present invention, the oxidoreductase is an enzyme functioning
as a catalyst of the oxidation reaction of cholesterol, and the
layer containing the enzyme further contains a surface active
agent.
[0029] In accordance with one preferable application of the present
invention, the above-mentioned at least one specific reagent of the
reaction reagent system is an oxidoreductase or potassium
ferricyanide which is an electron mediator.
[0030] It is also preferable that at least lower-most reaction
layer of the reaction layer structure includes a hydrophilic
polymer.
[0031] While the novel features of the invention are set forth
particularly in the appended claims, the invention, both as to
organization and content, will be better understood and
appreciated, along with other objects and features thereof, from
the following detailed description taken in conjunction with the
drawings.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING
[0032] FIG. 1 is a disassembled perspective view illustrating the
structure of a biosensor without a reaction layer structure in one
embodiment of the present invention.
[0033] FIG. 2 is a vertical sectional view illustrating a main part
of the biosensor shown in FIG. 1.
[0034] FIG. 3 is a vertical sectional view illustrating a main part
of another biosensor in another embodiment of the present
invention.
[0035] FIG. 4 is a graph showing responses of cholesterol sensors
prepared as examples of the present invention and comparative
examples.
DETAILED DESCRIPTION OF THE INVENTION
[0036] One aspect of the present invention is a method of
manufacturing a biosensor which comprises an electrically
insulating base plate, an electrode system including a measuring
electrode and a counter electrode formed on the base plate, and a
reaction reagent system including at least an oxidoreductase and an
electron mediator, wherein reagents of the reaction reagent system
are included in one or more reaction layers formed on or in the
vicinity of the electrode system. This method includes the step of
forming the one or more reaction layers and the step comprises:
dissolving the reagents in a solvent of a sublimable substance to
prepare a solution; applying the solution in a desired area to form
the one or more reaction layers; freezing the applied solution; and
sublimating the solvent included in a solid matter of the frozen
solution under reduced pressure for removal.
[0037] Another aspect of the present invention is a method of
manufacturing a biosensor which comprises an electrically
insulating base plate, an electrode system including a measuring
electrode and a counter electrode formed on the base plate, and a
reaction reagent system including at least an oxidoreductase and an
electron mediator, wherein reagents of the reaction reagent system
are included in a plurality of reaction layers formed on or in the
vicinity of the electrode system, and only a specific reaction
layer of the plurality of reaction layers contains at least one
specific reagent of the reaction reagent system. The method
includes the step of forming the specific reaction layer, and the
step comprises: dissolving the at least one specific reagent in a
solvent of a sublimable substance to prepare a solution; applying
the solution in a desired area to form the reaction layer; freezing
the applied solution; and sublimating the solvent included in a
solid matter of the frozen solution under reduced pressure for
removal.
[0038] A further aspect of the present invention is a method of
manufacturing a biosensor which comprises an electrically
insulating base plate, an electrode system including a measuring
electrode and a counter electrode formed on the base plate, and a
reaction reagent system including at least an oxidoreductase and an
electron mediator, wherein reagents of the reaction reagent system
are included in one or more reaction layers formed on or in the
vicinity of the electrode system, and at least one specific reagent
of the reaction reagent system is contained only in an upper-most
layer of a specific reaction layer comprising a stack of layers.
The method includes the step of forming the specific reaction layer
and the step comprises: pre-forming the stack of layers except the
upper-most layer; dissolving the at least one specific reagent in a
solvent of a sublimable substance to prepare a solution; applying
the solution on the pre-formed stack of layers without the
upper-most layer; freezing the applied solution; and sublimating
the solvent included in a solid matter of the frozen solution under
reduced pressure for removal.
[0039] As described above, the method of the present invention
forms a specific reaction layer or an upper-most reaction layer in
a stack of plural reaction layers, which contains at least one
specific reagent, by dissolving a material constituting the
reaction layer including the specific reagent in a solvent of a
sublimable substance to prepare a solution, applying the solution
in a desired area to form the reaction layer, freezing the applied
solution, and sublimating the solvent included in a solid matter of
the frozen solution under reduced pressure for removal. This method
gives the reaction layer having a porous structure that is composed
of substantially uniform fine particles and has a large surface
area. This arrangement enhances the solubility of the reaction
layer in a sample solution and shortens the time required for
measurement. This technique is especially effective in the case
where the sample solution has a high concentration of a substrate.
The high concentration of the substrate in the sample solution
results in a high viscosity of the sample solution, so that the
prior art reaction layer is not easily dissolved. The arrangement
of the present invention enhances the solubility of the reaction
layer even in the sample solution having the high concentration of
the substrate.
[0040] This method is especially effective for the electron
mediator among the various reagents of the reaction reagent system.
The electron mediator may be potassium ferricyanide or a suitable
redox compound selected among a variety of redox compounds having
the electron transferring ability to and from an oxidoreductase
like cholesterol oxidase. It is preferable that the electron
mediator alone is carried on the biosensor, in order to increase
the surface area of the electron mediator and enhance the
solubility thereof.
[0041] The solvent of the sublimable substance preferably used in
the present invention is either electrochemically inactive or
oxidant and accordingly does not reduce nor modify the electron
mediator or the enzyme. Water is the best solvent for this purpose.
Dimethylformamide, dimethyl sulfoxide, and quinones like
para-benzoquinone are also available for the solvent of the
electron mediator. Water or a buffer is preferable for the solvent
of the enzyme. Other applicable solvents include about 40% aqueous
solution of dimethylformamide and about 20% aqueous solution of
ethanol.
[0042] The oxidoreductase used in the present invention is an
enzyme for a measuring subject as a substrate. Thus, glucose
oxidase is used in the glucose sensor. In order to measure serum
cholesterol concentrations as the diagnostic standard, cholesterol
oxidase or cholesterol dehydrogenase as the enzyme functioning as a
catalyst of the oxidation reaction of cholesterol, and cholesterol
esterase as the enzyme functioning as a catalyst of the process of
converting cholesterol ester into cholesterol are used. Since the
enzyme reaction of cholesterol esterase proceeds very slowly, an
addition of an appropriate surface active agent improves the
activity of cholesterol esterase, enabling reduction of the time
which is necessary for the whole reaction.
[0043] A cholesterol sensor according to the present invention has
a reaction layer structure that may include a plurality of reaction
layers, that is, a layer containing a hydrophilic polymer, a layer
containing an enzyme that functions as a catalyst of the oxidation
reaction of cholesterol, a layer containing a surface active agent,
a layer containing cholesterol esterase, and a layer containing the
electron mediator. The surface active agent, the enzyme functioning
as the catalyst of the oxidation reaction of cholesterol, and
cholesterol esterase may be included in one mixed reaction layer.
The reaction layers arranged at a plurality of different positions
may be formed as divisions of an identical composition or may
alternatively have different compositions. These reaction layers
are arranged on or in the vicinity of the electrode system in the
biosensor.
[0044] The biosensor may have a cover member that is joined with
the base plate with the electrode system formed thereon to define a
sample solution supply pathway, through which a sample solution
flows to the electrode system. In this case, the reaction layer may
be formed at a specific position exposed to the sample solution
supply pathway or at an opening of the sample solution supply
pathway. The reaction layer may be formed at any suitable positions
as long as the reaction layer is easily dissolved in a supply of
the sample solution and reaches the electrode system. A hydrophilic
polymer layer is preferably formed on the electrode system to
protect the electrode system and prevent the reaction layer from
being peeled off. It is also preferable that a hydrophilic polymer
layer is formed as the base of the reaction layer or that a
hydrophilic polymer is included in a lower-most reaction layer.
[0045] It is preferable that the reaction layer containing the
electron mediator is separate from the surface active agent for the
enhanced solubility, and is also separate from the enzyme
functioning as the catalyst of the oxidation reaction of
cholesterol, e.g. cholesterol esterase for the enhanced storage
stability.
[0046] In some biosensors for measuring the blood sugar (see, for
example, Japanese Laid-Open Patent Publication No. Hei 2-062952), a
lipid-containing layer is formed to cover the layer formed on the
electrode system, in order to facilitate introduction of the sample
solution into the reaction layer. The biosensor of the present
invention for measuring cholesterol includes the surface active
agent, which has the similar functions to those of the lipid, and
accordingly does not require the lipid layer.
[0047] Examples of the hydrophilic polymer include water-soluble
cellulose derivatives, especially ethyl cellulose, hydroxypropyl
cellulose, carboxymethyl cellulose, polyvinyl pyrrolidone,
polyvinyl alcohol, gelatin, polyacrylic acid and salts thereof,
starch and derivatives thereof, polymer of maleic anhydride and
salts thereof, polyacrylamide, methacrylate resin,
poly-2-hydroxyethyl methacrylate and the like.
[0048] As the surface active agent, it is possible to use an
arbitrary choice of n-octyl-.beta.-D-thioglucoside, polyethylene
glycol monododecyl ether, sodium cholate, dodecyl-.beta.-maltoside,
sucrose monolaurate, sodium deoxycholate, sodium taurodeoxycholate,
N,N-bis(3-D-gluconeamidopr- opyl)deoxycholeamide and
polyoxyethylene(10) octyl phenyl ether.
[0049] When a lipid is used, an amphipathic phospholipid such as
lecithin, phosphatidyl choline and phosphatidyl ethanolamine is
preferably used.
[0050] As the measuring method of the oxidation current, a
two-electrode system composed only of a measuring electrode and a
counter electrode and a three-electrode system further comprising a
reference electrode are applicable, and the three-electrode system
can give more accurate measurement results.
[0051] The present invention is described in detail by referring to
preferred embodiments.
[0052] FIG. 1 is a disassembled perspective view illustrating a
biosensor without a reaction layer in a first embodiment of the
present invention.
[0053] Silver paste is printed on an electrically insulating base
plate 1 of polyethylene terephthalate by the technique of screen
printing to form leads 2 and 3. Electrically conductive carbon
paste including a resin binder is further printed on the base plate
1 to form an electrode system including a measuring electrode 4 and
a counter electrode 5. Electrically insulating paste is also
printed on the base plate 1 to form an electrically insulating
layer 6. The electrically insulating layer 6 partly covers the
leads 2 and 3 and keeps an exposed area of the measuring electrode
4 and the counter electrode 5 fixed.
[0054] A cover 12 with an air vent 14 and a spacer 11 are bonded to
the electrically insulating base plate 1 according to the
positional relationship shown by the dashed line in FIG. 1. This
completed a cholesterol sensor. The spacer 11 has a slit 15, which
is joined with the base plate 1 and the cover 12 to define a sample
solution supply pathway. The sample solution supply pathway has an
opening 13.
[0055] FIG. 2 is a vertical sectional view illustrating the
biosensor of the first embodiment. A hydrophilic polymer layer 7 is
formed on the electrode system upon the electrically insulating
base plate 1. The hydrophilic polymer layer 7 is obtained by
dropping an aqueous solution of a hydrophilic polymer and drying
the dropped solution with warm blast. A reaction layer 8 including
reaction reagents is formed on the hydrophilic polymer layer 7. In
the case of a cholesterol sensor, the reaction layer 8 includes
cholesterol oxidase, cholesterol esterase, the surface active
agent, and the electron mediator. The reaction layer 8 is prepared
by dropping an aqueous solution of such reagents on the hydrophilic
polymer layer 7, freezing the dropped solution, and drying the
frozen solution under reduced pressure for sublimation of the water
content.
[0056] FIG. 3 is a vertical sectional view illustrating another
biosensor in a second embodiment of the present invention. Like the
first embodiment, the hydrophilic polymer layer 7 is formed on the
electrode system upon the electrically insulating base plate 1. In
the second embodiment, reaction layers 8a and 8b are formed
respectively on the hydrophilic polymer layer 7 and on the surface
of the cover 12 that is exposed to the sample solution supply
pathway. Each of these reaction layers 8a and 8b is prepared by
dropping an aqueous solution including the constituents of each
layer at a specified position, freezing the dropped solution, and
drying the frozen solution under reduced pressure for sublimation
of the water content.
[0057] In accordance with one embodiment of the cholesterol sensor,
the reaction layer 8a includes cholesterol oxidase, cholesterol
esterase, and the surface active agent, whereas the reaction layer
8b includes the electron mediator. In accordance with another
embodiment, the reaction layer 8a includes cholesterol oxidase and
cholesterol esterase, whereas the reaction layer 8b includes the
surface active agent and the electron mediator.
[0058] In accordance with still another embodiment, any one of the
reaction layers 8, 8a, and 8b is constructed as a stack of plural
reaction layers. At least an upper-most layer of the stack is
prepared through the steps of freezing an aqueous solution of at
least one reagent to be contained in the upper-most layer and
removing the water content by sublimation. The at least one reagent
included in the upper-most layer is, for example, potassium
ferricyanide, which forms coarse crystals when the aqueous solution
is dried with warm blast. It is preferable that the reaction layer
8b includes a hydrophilic polymer or is formed on a hydrophilic
polymer layer. This arrangement effectively prevents the reaction
layer 8b from being peeled off.
EXAMPLE 1
[0059] Example 1 is a cholesterol sensor having the structure of
FIG. 3, wherein the reaction layer 8a includes the electron
mediator and the reaction layer 8b includes cholesterol oxidase,
cholesterol esterase, and the surface active agent. This
cholesterol sensor was prepared in the following manner.
[0060] The procedure of Example 1 first prepared a 0.5% by weight
of aqueous solution containing sodium carboxymethyl cellulose
(hereinafter referred to as CMC), dropped 5 .mu.l of the aqueous
solution on the electrode system upon the base plate 1, and dried
the dropped solution in a drying apparatus with warm blast of
50.degree. C. for 10 minutes. This gave the CMC layer 7. The
procedure then dissolved potassium ferricyanide in water to prepare
a 1 M aqueous solution, dropped 1 .mu.l of the aqueous solution on
the CMC layer 7, and dried the dropped solution in the drying
apparatus with warm blast of 50.degree. C. for 10 minutes. This
gave the reaction layer 8a including potassium ferricyanide.
[0061] The procedure, on the other hand, prepared a mixed aqueous
solution by dissolving cholesterol oxidase coming from Nocardia
(EC1.1.3.6, hereinafter referred to as ChOD) and cholesterol
esterase coming from Pseudomonas (EC.3.1.1.13, hereinafter referred
to as ChE) in water and adding polyoxyethylene(10)octyl phenyl
ether (TritonX-100) as a surface active agent. The procedure then
dropped 2.5 .mu.l of the mixed aqueous solution in the recess
defined by the slit 15 of the cover member including the cover 12
and the spacer 11, froze the dropped solution with liquid nitrogen
of -196.degree. C., and dried the frozen solution in a Kjeldahl
flask set in a freeze-drying apparatus for 3 hours. This gave the
reaction layer 8b including 4 units (U) of cholesterol oxidase, 10
U of cholesterol esterase, and 3% by weight of the surface active
agent. The reaction layer 8b held the contour formed immediately
after dropping 2.5 .mu.l of the mixed aqueous solution and had the
porous structure having an extremely large surface area.
[0062] The cholesterol sensor was completed by bonding the cover
member to the base plate.
[0063] The procedure supplied 1 .mu.l of a cholesterol standard
solution containing cholesterol and cholesterol linoleate, which is
one of the cholesterol esters, through the opening 13 of the sample
solution supply pathway to the cholesterol sensor thus prepared,
applied a pulse voltage of +0.5 V toward the anode on the measuring
electrode relative to the counter electrode as the reference 3
minutes after the supply, and measured the electric current 5
seconds after the application. The results are shown in the graph
of FIG. 4.
EXAMPLE 2
[0064] Example 2 is a cholesterol sensor having the structure of
FIG. 3, wherein the reaction layer 8a includes the electron
mediator and the reaction layer 8b includes cholesterol oxidase,
cholesterol esterase, the surface active agent, and 0.125% by
weight of CMC. This cholesterol sensor was prepared in the
following manner.
[0065] The CMC layer 7 was formed on the electrode system upon the
electrically insulating base plate 1 in the same manner as
discussed in Example 1. The procedure of Example 2 dissolved
potassium ferricyanide in water to prepare an aqueous solution,
dropped 1 .mu.l of the aqueous solution on the CMC layer 7, and
dried the dropped solution in the drying apparatus with warm blast
of 50.degree. C. for 10 minutes. This gave the reaction layer 8a
including potassium ferricyanide. The procedure, on the other hand,
prepared a mixed aqueous solution by dissolving ChOD, ChE, the
surface active agent, and CMC in water, dropped 2.5 .mu.l of the
mixed aqueous solution in the recess defined by the slit 15 of the
cover member including the cover and the spacer, froze the dropped
solution with liquid nitrogen, and dried the frozen solution in a
Kjeldahl flask set in the freeze-drying apparatus for 3 hours. This
gave the reaction layer 8b including ChOD, ChE, the surface active
agent, and CMC. The quantities of potassium ferricyanide, the
surface active agent, ChOD, and ChE used in Example 2 as well as
those in Examples 3 and 4 and Comparative Example discussed below
were identical with those of Example 1.
[0066] The cholesterol sensor was then prepared in the same manner
as discussed in Example 1. While the time period between a supply
of the sample solution and application of a voltage is varied, the
electric current was measured as the response with regard to each
concentration of the sample solution. The results are shown in the
graph of FIG. 4.
EXAMPLE 3
[0067] Example 3 is a cholesterol sensor having the structure of
FIG. 3, wherein the reaction layer 8b includes the electron
mediator and the reaction layer 8a includes cholesterol oxidase,
cholesterol esterase, and the surface active agent. This
cholesterol sensor was prepared in the following manner.
[0068] The CMC layer 7 was formed on the electrode system upon the
electrically insulating base plate 1 in the same manner as
discussed in Example 1. The procedure of Example 3 prepared a mixed
aqueous solution by dissolving ChOD, ChE, and the surface active
agent in water, dropped 2.5 .mu.l of the mixed aqueous solution on
the CMC layer 7, froze the dropped solution with liquid nitrogen,
and dried the frozen solution in a Kjeldahl flask set in the
freeze-drying apparatus for 3 hours. This gave the reaction layer
8a including ChOD, ChE, and the surface active agent. The
procedure, on the other hand, dissolved potassium ferricyanide in
water to prepare an aqueous solution, dropped 1 .mu.l of the
aqueous solution in the recess defined by the slit 15 of the cover
member including the cover and the spacer, froze the dropped
solution with liquid nitrogen, and dried the frozen solution in a
Kjeldahl flask set in the freeze-drying apparatus for 3 hours. This
gave the reaction layer 8b including potassium ferricyanide.
[0069] The cholesterol sensor was then prepared in the same manner
as discussed in Example 1. While the time period between a supply
of the sample solution and application of a voltage is varied, the
electric current was measured as the response with regard to each
concentration of the sample solution. The results are shown in the
graph of FIG. 4.
EXAMPLE 4
[0070] Example 4 is a cholesterol sensor having the structure of
FIG. 3, wherein the reaction layer 8a includes the electron
mediator and the reaction layer 8b includes cholesterol oxidase,
cholesterol esterase, and the surface active agent. This
cholesterol sensor was prepared in the following manner.
[0071] The CMC layer 7 was formed on the electrode system upon the
electrically insulating base plate 1 in the same manner as
discussed in Example 1. The procedure of Example 4 dissolved
potassium ferricyanide in water to prepare an aqueous solution,
dropped 1 .mu.l of the aqueous solution on the CMC layer 7, froze
the dropped solution with liquid nitrogen, and dried the frozen
solution in a Kjeldahl flask set in the freeze-drying apparatus for
3 hours. This gave the reaction layer 8a. The procedure, on the
other hand, prepared a mixed aqueous solution by dissolving ChOD,
ChE, and the surface active agent in water, dropped 2.5 .mu.l of
the mixed aqueous solution in the recess defined by the slit 15 of
the cover member including the cover and the spacer, froze the
dropped solution with liquid nitrogen, and dried the frozen
solution in a Kjeldahl flask set in the freeze-drying apparatus for
3 hours. This gave the reaction layer 8b including ChOD, ChE, and
the surface active agent.
[0072] The cholesterol sensor was then prepared in the same manner
as discussed in Example 1. While the time period between a supply
of the sample solution and application of a voltage is varied, the
electric current was measured as the response with regard to each
concentration of the sample solution. The results are shown in the
graph of FIG. 4.
COMPARATIVE EXAMPLE
[0073] The CMC layer was formed on the electrode system upon the
electrically insulating base plate 1 in the same manner as
discussed in Example 1. A mixed aqueous solution was prepared by
dissolving potassium ferricyanide, the surface active agent, ChOD,
and ChE in water. The procedure of Comparative Example dropped 4.5
.mu.l of the mixed aqueous solution on the CMC layer and dried the
mixed aqueous solution with warm blast of 50.degree. C. for 15
minutes. This gave a reaction layer including potassium
ferricyanide, the surface active agent, ChOD, and ChE.
[0074] A cholesterol sensor was then prepared in the same manner as
discussed in Example 1. While the time period between a supply of
the sample solution and application of a voltage is varied, the
electric current was measured as the response with regard to each
concentration of the sample solution. The results are shown in the
graph of FIG. 4.
[0075] The technique of the present invention forms a reaction
layer having a large surface area. This arrangement ensures the
rapid dissolution of the reaction layer in a sample solution and
gives a resultant biosensor having excellent response to a
substrate even in a high concentration range of the substrate.
[0076] Although the present invention has been described in terms
of the presently preferred embodiments, it is to be understood that
such disclosure is not to be interpreted as limiting. Various
alterations and modifications will no doubt become apparent to
those skilled in the art to which the present invention pertains,
after having read the above disclosure. Accordingly, it is intended
that the appended claims be interpreted as covering all alterations
and modifications as fall within the true spirit and scope of the
invention.
* * * * *