U.S. patent application number 09/764142 was filed with the patent office on 2001-12-13 for biosensor.
Invention is credited to Ikeda, Shin, Nankai, Shiro, Watanabe, Motokazu, Yamamoto, Tomohiro.
Application Number | 20010050227 09/764142 |
Document ID | / |
Family ID | 18540677 |
Filed Date | 2001-12-13 |
United States Patent
Application |
20010050227 |
Kind Code |
A1 |
Yamamoto, Tomohiro ; et
al. |
December 13, 2001 |
Biosensor
Abstract
The present invention provides a biosensor which enables prompt
quantification by facilitating introduction of a sample solution
even when the amount of the sample solution is small and improving
dissolution of reagents. The biosensor comprises an insulating base
plate; an electrode system including at least a measuring electrode
and a counter electrode formed on the base plate; a cover member
integrated into the base plate to form a sample solution supply
pathway for supplying a sample solution to the electrode system
between the cover member and the base plate; a reaction reagent
system including at least an electron mediator and an
oxidoreductase; and a carrier supporting at least the electron
mediator or the oxidoreductase of the reaction reagent system,
wherein the carrier is partially or wholly disposed outside the
sample solution supply pathway.
Inventors: |
Yamamoto, Tomohiro;
(Hirakata-shi, JP) ; Watanabe, Motokazu;
(Katano-shi, JP) ; Ikeda, Shin; (Katano-shi,
JP) ; Nankai, Shiro; (Hirakata-shi, JP) |
Correspondence
Address: |
MCDERMOTT WILL & EMERY
600 13TH STREET, N.W.
WASHINGTON
DC
20005-3096
US
|
Family ID: |
18540677 |
Appl. No.: |
09/764142 |
Filed: |
January 19, 2001 |
Current U.S.
Class: |
204/403.04 ;
204/403.08; 204/403.11; 204/403.14; 435/25; 435/287.9 |
Current CPC
Class: |
C12Q 1/005 20130101;
G01N 27/3272 20130101 |
Class at
Publication: |
204/403 ; 435/25;
435/287.9 |
International
Class: |
G01N 027/327 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 21, 2000 |
JP |
2000-013025 |
Claims
1. A biosensor comprising: an insulating base plate; an electrode
system including at least a measuring electrode and a counter
electrode formed on said base plate; a cover member integrated into
said base plate to form a sample solution supply pathway for
supplying a sample solution to said electrode system between said
cover member and said base plate; a reaction reagent system
including at least an electron mediator and an oxidoreductase; and
a carrier supporting at least the electron mediator or the
oxidoreductase of said reaction reagent system, wherein said
carrier is partially or wholly disposed outside said sample
solution supply pathway.
2. The biosensor in accordance with claim 1, wherein said carrier
has a region supporting at least the electron mediator or the
oxidoreductase disposed closer to said electrode system than a
region supporting none of said reaction reagent system.
3. The biosensor in accordance with claim 1, wherein said
oxidoreductase is cholesterol oxidase.
4. The biosensor in accordance with claim 1, wherein said reaction
reagent system contains an enzyme capable of hydrolyzing
cholesterol ester.
5. The biosensor in accordance with claim 4, wherein said enzyme
capable of hydrolyzing cholesterol ester is cholesterol
esterase.
6. The biosensor in accordance with claim 4, wherein said reaction
reagent system contains a surfactant.
7. The biosensor in accordance with claim 1, wherein said carrier
is porous.
8. The biosensor in accordance with claim 7, wherein said carrier
is fibrous.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates to a biosensor facilitating
rapid and highly accurate quantification of a measuring subject (a
subject to be measured) contained in a sample in a simplified
manner.
[0002] A biosensor has conventionally been proposed in the Japanese
Laid-Open Patent Publication Hei 2-062952 as a system for
simplified quantification of a specific component in a sample
without diluting or stirring a sample solution.
[0003] This biosensor is produced by first forming an electrode
system comprising a measuring electrode, a counter electrode and a
reference electrode on an electrically insulating base plate by
using a screen printing method or the like, and then forming an
enzyme reaction layer comprising a hydrophilic polymer, an
oxidoreductase and an electron mediator. If occasion demands, a
buffer is added to this enzyme reaction layer.
[0004] When a sample solution containing a substrate is dropped on
the enzyme reaction layer of the biosensor thus formed, dissolution
of the enzyme reaction layer takes place, which in turn triggers a
reaction between the enzyme and the substrate, causing reduction of
the electron mediator. Upon completion of the enzyme reaction, the
reduced electron mediator is oxidized electrochemically. The
concentration of the substrate in the sample solution can be
determined from the oxidation current value measured in this
oxidizing step.
[0005] This biosensor can be used theoretically for measurements of
various materials if an appropriate enzyme for each of the
materials as the substrate is selected. For example, the use of
glucose oxidase as the oxidoreductase can realize a biosensor for
measurement of blood glucose level. This sensor is widely applied
practically as a glucose sensor. The use of cholesterol oxidase as
the oxidoreductase can realize a biosensor for measurement of serum
cholesterol.
[0006] Serum cholesterol level which serves as the diagnostic
standard is the sum of serum cholesterol and cholesterol ester
concentrations. Since cholesterol ester cannot serve as a substrate
for oxidation by cholesterol oxidase, an additional step of
converting cholesterol ester into cholesterol becomes necessary in
order to determine serum cholesterol concentrations as the
diagnostic standard. Cholesterol esterase is known as an enzyme for
catalyzing this process.
[0007] Inclusion of this cholesterol esterase together with
cholesterol oxidase in the enzyme reaction layer can realize a
biosensor for measurement of the total cholesterol concentration in
serum.
[0008] In the biosensor having such constitution, the enzyme
reaction layer is formed by dropping an aqueous reagent solution
containing at least an oxidoreductase onto the electrode system and
drying the dropped solution. Such biosensor has a problem, when the
amount of reagents is large, that the reaction layer is not
dissolved quickly after dropping of the sample solution thereonto
so that the measurement takes a long time.
[0009] Particularly in the cholesterol sensor, two kinds of
enzymes, cholesterol oxidase and cholesterol esterase, must be
contained in the enzyme reaction layer, as described previously.
Therefore, the enzyme reaction layer contains a considerably large
amount of reagents, so that dissolution of this layer takes a
significantly long time after dropping of the sample solution,
which makes it impossible to make rapid measurement.
[0010] In order to solve the above-mentioned problem, a biosensor
has been proposed in Japanese Laid-Open Patent Publication
2000-039416. The biosensor comprises an insulating base plate, an
electrode system including at least a measuring electrode and a
counter electrode formed on the base plate, a cover member which is
integrated into the base plate and forms a sample solution supply
pathway for supplying a sample solution to the electrode system
between the cover member and the base plate, and a carrier
comprising a fiber for supporting reagents containing at least an
oxidoreductase, wherein the carrier is disposed in the sample
solution supply pathway.
[0011] Also, this biosensor according to another embodiment
comprises an insulating base plate, an electrode system including
at least a measuring electrode and a counter electrode formed on
the base plate, and a carrier comprising a fiber for supporting
reagents containing at least an oxidoreductase, wherein the carrier
is fixed with an adhesive in the vicinity of the electrode
system.
[0012] In this biosensor, however, the height of the sample
solution supply pathway is required to be at least equal to or
greater than the thickness of the carrier, since the carrier is
disposed in the sample solution supply pathway. Moreover, the
biosensor actually has a structure that the carrier and the
electrode system are disposed to face each other, thereby requiring
a certain space between the electrode system and the surface of the
carrier in order to prevent the contact between them. Thus, the
height of the sample solution supply pathway is required to be
further greater.
[0013] For these reasons, when the carrier is disposed in the
sample solution supply pathway, the biosensor requires a sample
solution in a relatively large amount in order to fill the sample
solution supply pathway with the sample solution. The amount is
determined by multiplying the carrier volume calculated from the
external dimensions thereof by the porosity of the carrier to
obtain a product and by adding to the product the above-mentioned
space between the electrode system and the surface of the
carrier.
[0014] Meanwhile, it is more preferable that the porosity of the
carrier is higher in order to improve dissolution of the
reagents.
[0015] From such characteristics as described, the above-described
biosensor tends to need a larger amount of the sample solution for
the measurement than the biosensor without a carrier.
[0016] Also, especially when the sample solution is blood and the
biosensor is provided with a function of filtering blood corpuscle
components with a porous material such as a filter paper, the
porous material for filtering the blood corpuscle components has
sometimes made it difficult for the filtered sample solution to
invade into the sample solution supply pathway, since the carrier
is disposed in the sample solution supply pathway.
BRIEF SUMMARY OF THE INVENTION
[0017] In view of the above problems, an object of the present
invention is to provide a biosensor in which dissolution of
reagents is improved to enable prompt quantification and reduction
of the amount of a sample solution. Further, another object of the
present invention is to provide a biosensor in which the sample
solution can be introduced readily.
[0018] A biosensor in accordance with the present invention
comprises an electrically insulating base plate; an electrode
system including at least a measuring electrode and a counter
electrode formed on the base plate; a cover member, which is
integrated into the base plate and forms a sample solution supply
pathway for supplying a sample solution to the electrode system
between the cover member and the base plate; a reaction reagent
system including at least an electron mediator and an
oxidoreductase; and a carrier supporting at least the electron
mediator or the oxidoreductase of the reaction reagent system,
wherein the carrier is partially or wholly disposed outside the
sample solution supply pathway.
[0019] In the biosensor in accordance with the present invention,
it is effective that the carrier has a region supporting at least
the electron mediator or the oxidoreductase disposed closer to the
electrode system than a region supporting none of the reaction
reagent system.
[0020] Also, it is effective that the oxidoreductase is cholesterol
oxidase.
[0021] Further, it is preferable that the reaction reagent system
contains an enzyme capable of hydrolyzing cholesterol ester.
[0022] It is effective that the enzyme capable of hydrolyzing
cholesterol ester is cholesterol esterase.
[0023] Furthermore, it is preferable that the reaction reagent
system contains a surfactant.
[0024] It is effective that the carrier is porous and, further,
that the carrier is fibrous or comprises a fiber.
[0025] While the novel feature 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
[0026] FIG. 1 is an exploded perspective view of a biosensor in
accordance with one example of the present invention.
[0027] FIG. 2 is a perspective view of a cover member formed by
integrating a cover and a spacer of the biosensor shown in FIG. 1,
being arranged upside down against the view of FIG. 1.
[0028] FIG. 3 is a longitudinal sectional view of the main parts of
the biosensor shown in FIG. 1.
[0029] FIG. 4 is a longitudinal sectional view of the main parts of
a biosensor in accordance with another example of the present
invention.
[0030] FIG. 5 is a longitudinal sectional view of the main parts of
a biosensor in accordance with still another example of the present
invention.
[0031] FIG. 6 is a longitudinal sectional view of the main parts of
a biosensor in accordance with another example of the present
invention.
[0032] FIG. 7 is a longitudinal sectional view of the main parts of
a biosensor in accordance with still another example of the present
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0033] In the biosensor according to the present invention, a
reagent including at least one of an electron mediator and an
oxidoreductase is supported on a carrier comprising a porous
material.
[0034] The reagent is supported on the carrier in such a manner
that it is adhered to the surface of the porous material
constituting the carrier. Thus, the surface area of the reagent in
contact with a sample solution becomes larger, resulting in
improvement of the dissolution of the reagent into the sample
solution.
[0035] As the carrier, various ones may be used if it has a
function of supporting the reagent and is inactive to the enzyme
reaction and electrochemical reaction occurring in the biosensor.
For example, a sheet made by laminating a cellulose fiber, a glass
fiber or a polymeric compound fiber into a fleece or felt form is
preferable.
[0036] With regard to the layout of the carrier in the biosensor,
various changes are possible.
[0037] In one embodiment, the whole carrier is disposed outside the
sample solution supply pathway of the cover member while an end of
the carrier is brought in contact with the tip end of the sample
solution supply pathway.
[0038] In another embodiment, a part of the carrier is inserted in
the sample solution supply pathway of the cover member.
[0039] As in these embodiments, by exposing at least a part of the
carrier to outside of the sample solution supply pathway,
permeation of the sample solution into the carrier is facilitated,
thereby enabling prompt dissolution of the reagent supported on the
carrier.
[0040] Also, in the case of using a sample solution such as blood
containing a solid component which may adversely influence the
electrode reaction or the enzyme reaction, at least a part of the
carrier may be provided with a region where the reagent is not
supported, through which region the sample solution is introduced
for permeation in order to filter the solid component.
[0041] When the carrier is brought in contact with the sample
solution supply pathway of the cover member, it is desirable that
the carrier is adhered and secured to either a part of the cover
member or a part of the insulating base plate having the electrode
system formed thereon, or both thereof.
[0042] As the adhesive, it is preferable to use one having such a
high viscosity as to prevent permeation into the carrier under the
environment of sensor production, one having a poor dissolution
into water after adhesion, or one having an electrochemical
inactivity in an aqueous solution even if it is soluble in water.
For example, it is preferable to use a woodworking adhesive such as
the adhesive commercially available under the trade name of
Cemedine C from Cemedine Co., Ltd.
[0043] As the oxidoreductase to be supported on the carrier,
various compounds can be used. For example, glucose oxidase,
lactate oxidase, cholesterol oxidase and the like are listed.
[0044] When serum cholesterol level is measured, cholesterol
oxidase and an enzyme capable of hydrolyzing cholesterol ester are
used. As the enzyme capable of hydrolyzing cholesterol ester,
cholesterol esterase, lipoprotein lipase and the like are listed.
Particularly, cholesterol esterase is advantageous since it can
convert cholesterol ester into cholesterol quickly by using a
suitable surfactant.
[0045] When the enzyme capable of hydrolyzing cholesterol ester is
used, it is preferable that a surfactant having the effect of
improving the activity of this enzyme is contained in the reagent,
which is supported on the whole carrier or a part of the carrier,
since the duration of time required for the enzyme reaction can be
reduced.
[0046] For example, as the surfactant for improving the activity of
cholesterol esterase, 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-gluconeamidopropyl)ch- oleamide,
N,N-bis(3-D-gluconeamidopropyl)deoxycholeamide,
polyoxyethylene-p-t-octyl phenyl ether (for example, TritonX-100
produced by SIGMA Co., Ltd) and the like.
[0047] If the electrode system of the biosensor is formed by using
an electrochemically stable metal such as platinum, the obtained
oxidation current value is free from an error. However, since such
metal is expensive, the electrode system of a disposable sensor is
prepared by forming a silver electrode with a silver paste and the
like and subsequently coating it with a carbon paste.
[0048] However, when the surfactant is contained in the sample
solution, the sample solution permeates between carbon particles by
the action of the surfactant. As a result, the activity of the
carbon electrode may decrease, and the sample solution comes in
contact with the silver electrode. Thus, when a voltage is applied
on a measuring electrode under such condition, the silver electrode
causes an oxidation reaction to generate a current, so that a
positive error may be included in the measured current value.
[0049] For suppressing such a phenomenon, there is a method that
the surface of the electrode system is coated with a hydrophilic
polymer. This hydrophilic polymer forms a viscose layer even upon
introduction of the sample solution, suppressing the contact of the
sample solution with the electrode.
[0050] Examples of such hydrophilic polymer include
carboxymethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol,
ethylcellulose, hydroxypropylcellulose, gelatin, polyacrylic acid
and salts thereof, starch and derivatives thereof, maleic anhydride
and salts thereof, polyacrylamide, methacrylate resin, poly
2-hydroxyethyl methacrylate and the like.
[0051] An enzyme such as an oxidoreductase or cholesterol esterase
may be carried so as to cover the above-described hydrophilic
polymer layer. In this case, an electron mediator is carried on the
above-mentioned carrier.
[0052] It is preferable to further form an amphipathic substance
layer so as to cover the above-mentioned layer over the electrode
system, i.e., the hydrophilic polymer layer or the enzyme layer
covering the hydrophilic polymer layer. The amphipathic substance
layer is formed by dropping a solution prepared by dissolving an
amphipathic substance such as lecithin in an organic solvent and
subsequently drying it.
[0053] Although such amphipathic substance layer is not necessary
for the enzyme reaction and the electrode reaction, formation of
this layer allows for smooth introduction of the sample solution.
Preferable examples of such amphipathic substance include
phospholipid such as lecithin.
[0054] Further, a surfactant layer may be provided on the portion
of the cover member facing the sample solution supply pathway or
the surface of the electrode system. This layer allows the sample
solution, which has completed to react or is reacting with the
reaction reagent system contained in the carrier, to smoothly
permeate the sample solution supply pathway, thereby to reach the
electrode system.
[0055] The surfactant used for such purpose is desirably selected
from the above-listed surfactants for improving the activity of
cholesterol esterase, but any surfactant other than these may be
used if it does not disturb the reaction system.
[0056] When the surfactant layer is provided, it is essential to
form the above-mentioned hydrophilic polymer layer covering the
surface of the electrode system. In this case, the surfactant layer
must be formed by dissolving a surfactant in a solvent which does
not dissolve the hydrophilic polymer layer to obtain a solution,
dropping the solution prepared so as to cover the hydrophilic
polymer layer, and subsequently drying it.
[0057] As the electron mediator to be supported on the carrier, any
water-soluble compound which can mediate electron transfer between
the enzyme and the electrode such as potassium ferricyanide,
p-benzoquinone, phenazine methosulfate or ferrocene derivative
(oxidized form) may be used.
[0058] As the measuring method of the oxidation current, a
two-electrode system composed only of a measuring electrode and a
counter electrode or a three-electrode system further comprising a
reference electrode is applicable, and the three-electrode system
can give more accurate measurement results.
[0059] In the following, the present invention will be described
more specifically, referring to concrete examples, although the
present invention is not limited to these examples. FIG. 1 is an
exploded perspective view of the biosensor according to one example
of the present invention.
[0060] As shown in FIG. 1, numeral 1 represents an electrically
insulating base plate made of polyethylene terephthalate, and leads
2 and 3 and the ground for an electrode system are formed on this
base plate 1 by printing a silver paste using a screen printing
method. On the base plate 1, an electrically conductive carbon
paste containing a resin binder is further printed to form the
electrode system containing a measuring electrode 4 and a counter
electrode 5, and an electrically insulating paste is printed to
form an electrically insulating layer 6. The measuring electrode 4
is connected to the lead 2, and the counter electrode 5 is
connected to the lead 5, respectively. The electrically insulating
layer 6 allows the areas of exposed portions of the measuring
electrode 4 and the counter electrode 5 to be constant and covers
the leads partially.
[0061] The electrically insulating base place 1 with the electrode
system formed thereon, a cover 9 having an air vent 11, a spacer 8
and a carrier 13 supporting a reagent are adhered under the
positional relation as shown by the dotted chain lines in FIG. 1 to
form a biosensor.
[0062] In the biosensor having such constitution, a space
constituting a sample solution supply pathway is formed in a slit
12 of the spacer 8 between the base plate 1 and the cover 9.
[0063] A sample solution is introduced into the sensor from an
opening 10 forming a port of the sample solution supply pathway. In
this example, the length from the opening 10 to the edge of the air
vent 11, which is closer to the opening, is 4.5 mm, the width of
the slit 2.0 mm, and the depth of the slit 0.1 to 0.3 mm.
[0064] These dimensions are provided in order to define the
dimensions of carriers which will be described below, but the
dimensions of the carriers are not limited to those given
above.
[0065] FIG. 2 is a perspective view of a cover member formed by
laying the spacer 8 on top of the cover 9, which is arranged upside
down against the view of FIG. 1. By combining this cover member
with the base plate, the space constituting the sample solution
supply pathway is formed.
[0066] FIG. 3 is a longitudinal sectional view of the biosensor
according to one example of the present invention. In the same
manner as in FIG. 1, the electrically insulating base plate 1 is
provided with the electrode system, over which a hydrophilic
polymer layer 7 is formed, an enzyme layer 18 comprising
cholesterol oxidase and cholesterol esterase is further formed so
as to coat this hydrophilic polymer layer 7, and a surfactant layer
19 comprising Triton X-100 is further formed thereon.
[0067] Furthermore, a lecithin layer 17 is disposed on the inner
surface of the cover 9 constituting the sample solution supply
pathway, and the carrier 13 supporting a reagent is adhered to the
cover 9 with an adhesive 14 so as to come in contact with the
sample solution supply port 10.
[0068] FIG. 4 is a longitudinal sectional view of the biosensor
according to another example of the present invention. In the same
manner as in FIG. 3, the electrically insulating base plate 1 is
provided with the electrode system, over which a hydrophilic
polymer layer 7 and a lecithin layer 17 are formed. Further, the
carrier 13 supporting reagent is fixed with an adhesive 14 to the
cover 9 from the vicinity of the sample solution supply port 10 to
outside of the sample solution supply pathway in such a manner that
a part of the carrier is inserted into the sample solution supply
pathway.
[0069] FIGS. 5 to 7 are a longitudinal sectional view of a part of
the biosensor according to another example of the present
invention, with the omission of the electrode system, the
hydrophilic polymer layer and the surfactant layer.
[0070] In the biosensor illustrated in FIG. 5, a part of the
carrier 13 is fixed with an adhesive 14 to the inner surface of the
cover constituting the sample solution supply pathway. The end of
the carrier 13 exposed to outside extends beyond the end of the
insulating base plate at the sample solution supply port side. This
structure allows the carrier to be supplied with a sample from the
upper side (i.e., the cover side) or the lower side (i.e., the
insulating base plate side).
[0071] In the biosensor illustrated in FIG. 6, the end of the
insulating base plate at the sample solution supply port side
extends beyond the end of the carrier 13 exposed to outside, as
opposed to the layout in FIG. 1. This structure allows the sample
to be introduced and carried on the insulating base plate, which is
advantageous when permeation of the sample such as blood into the
carrier takes a relatively long time.
[0072] In the biosensor illustrated in FIG. 7, the carrier 13 is
secured by at least one or more protrusion(s) 15 formed on the
insulating base plate 1 constituting the sample solution supply
pathway. This structure enables the carrier 13 to be secured
without the use of an adhesive. Also, formation of at least one
protrusion 15 in a sleeper-like shape makes it possible to prevent
the phenomenon that the sample solution directly reaches the
electrode system through only the periphery of the carrier so that
it does not dissolve the reagent supported on the carrier
sufficiently.
EXAMPLE 1
[0073] In this example, a biosensor having the constitution in FIG.
3 was manufactured as described below.
[0074] First, on the electrode system on the base plate 1 in FIG.
1, a 0.5 wt % aqueous solution of sodium salt of
carboxymethylcellose (hereinafter referred to as "CMC") which is a
hydrophilic polymer was dropped and dried in a hot air drier for 10
minutes at 50.degree. C. to form a CMC layer 7.
[0075] Subsequently, 2 .mu.l of a mixed solution obtained by mixing
400 unit/ml cholesterol oxidase (hereinafter referred to as "ChOx")
and 2 kilo unit cholesterol esterase (hereinafter referred to as
"ChE") was dropped to cover the CMC layer 7 to form an enzyme layer
18.
[0076] Further, 1 .mu.l of a 5.0% ethanol solution of Triton X-100
was dropped to cover the enzyme layer 18, and dried to form a
surfactant layer 19. The surfactant layer 19 was provided to
facilitate the smooth dispersion of the sample solution throughout
the electrode system, and TritonX-100 was used to improve the
activity of ChE. Furthermore, a lecithin layer 17 was provided on
the inner surface of the cover constituting a sample solution
supply pathway.
[0077] Also, a felt-form glass filter of 0.2 mm in thickness, 2 mm
in width and 10 mm in length was prepared. The glass filter was
cooled by liquid nitrogen, and an aqueous solution containing
potassium ferricyanide as an electron mediator was then dropped on
one tip end of the glass filter. This solution froze immediately
after the dropping thereof and therefore did not disperse
throughout the glass filter, since the glass filter had been cooled
by the liquid nitrogen.
[0078] Accordingly, the potassium ferricyanide solution was dropped
and allowed to permeate about 2 mm from the tip end of the glass
filter. Thereafter, the glass filter was dried under reduced
pressure for about 4 hours in a freeze dryer. As a result, 0.33
.mu.mol of potassium ferricyanide was carried per 1 mm.sup.2 in the
area of 2 mm from the tip end of the glass filter.
[0079] As illustrated in FIG. 1, the glass filter supporting
potassium ferricyanide in a portion thereof in the above-described
manner was adhered and fixed to the cover 9 as a carrier. In this
process, the carrier was disposed in such a manner that the end
portion supporting potassium ferricyanide was brought in contact
with the tip end of the sample solution supply pathway. Then, this
cover member and the base plate 1 were adhered under the positional
relation as shown by the dotted chain lines in FIG. 1 to form a
biosensor.
[0080] Herein, the depth of a slit 12 of the sample solution supply
pathway was made 0.1 mm.
[0081] 10 .mu.l of a sample solution was supplied to the biosensor
thus formed from the other end of the carrier 13 not supporting
potassium ferricyanide such that it was absorbed therefrom. As the
sample solution, blood or a mixed solution prepared by adding blood
corpuscle to a standard serum solution, which is diluted while
maintaining the osmotic pressure at a constant level, was used.
[0082] As a result, before the blood corpuscle component of the
sample solution reached the portion of the carrier supporting
potassium ferricyanide, the filtered liquid component of the sample
solution dissolved potassium ferricyanide supported on the carrier
and invaded into the sample solution supply pathway from the sample
solution supply port, so that the surface of the electrode system
was filled with the sample solution. Three minutes after the supply
of the sample solution, a pulse voltage of +0.5 V was applied to
the measuring electrode in the anodic direction, using the counter
electrode as a reference. Five seconds after this voltage
application, the current value flowing between the measuring
electrode and the counter electrode was measured.
[0083] As a result, the measured value showed a current response
depending on the total cholesterol concentration in the sample
solution.
EXAMPLE 2
[0084] In this example, a biosensor having the constitution shown
in FIG. 4 was manufactured as described below.
[0085] In the same manner as in Example 1, a CMC layer 7 and a
lecithin layer 17 were formed over the electrode system on the base
plate 1 in FIG. 1. The lecithin layer 17 was formed also on an
inner surface of the cover constituting the sample solution supply
pathway.
[0086] Next, potassium ferricyanide, ChOx, ChE and TritonX-100 were
allowed to be supported on a felt-form glass filter of 0.2 mm in
thickness, 2 mm in width and 4.5 mm in length. Herein, TritonX-100,
which is a surfactant, was used in order to improve the activity of
ChE. The supported amount of potassium ferricyanide was 0.33
.mu.mol per 1 mm.sup.2 of the glass filter. The supported amount of
ChOx was 0.1 unit per 1 mm.sup.2 of the glass filter. Also, the
supported amounts of ChE and TritonX-100 were 1 unit and 0.15 mg,
respectively.
[0087] A woodworking adhesive (the adhesive commercially available
under the trade name of Cemedine C from Cemedine Co., Ltd.) as an
adhesive 14 was applied to the inner surface of the cover, to which
the glass filer was then adhered and fixed as a carrier such that
one tip end of the carrier was disposed at a position, which was 1
mm inner from the sample solution supply port. As a result, a
biosensor was produced so as to have a constitution that most of
the carrier was exposed to outside of the sample solution supply
pathway.
[0088] A standard serum or a solution prepared by diluting a
standard serum with physiological saline was supplied as a sample
solution to the biosensor thus produced by introducing it to a
portion of the carrier which was exposed to outside of the sample
solution supply pathway. Then, the response value was measured in
the same manner as in Example 1. As a result, the measured value
showed a favorable current response depending on the total
cholesterol concentration. In this example, the dissolution of the
reagents in the carrier was excellent. Moreover, the invasion of
the sample solution into the sample solution supply pathway was
also very smooth.
[0089] As described above, the present invention allows the sample
solution to be introduced smoothly even when the amount of the
sample solution is small and improves the dissolution of the
reagents. Therefore, it is possible to make prompt quantification
according to the present invention.
[0090] Although the present invention has been described in terms
of the presently preferred embodiments, it is to be understood that
such disclosure is not to be interpreted as limiting. Various
alterations and modifications will no doubt become apparent to
those skilled in the art to which the present invention pertains,
after having read the above disclosure. Accordingly, it is intended
that the appended claims be interpreted as covering all alterations
and modifications as fall within the true spirit and scope of the
invention.
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