U.S. patent application number 11/920782 was filed with the patent office on 2009-04-23 for protein-immobilized membrane, method for immobilization of protein, enzyme-immobilized electrode, and biosensor.
This patent application is currently assigned to ARKRAY, INC.. Invention is credited to Koji Katsuki, Hideaki Yamaoka.
Application Number | 20090101499 11/920782 |
Document ID | / |
Family ID | 37431302 |
Filed Date | 2009-04-23 |
United States Patent
Application |
20090101499 |
Kind Code |
A1 |
Katsuki; Koji ; et
al. |
April 23, 2009 |
Protein-Immobilized membrane, method for immobilization of protein,
enzyme-immobilized electrode, and biosensor
Abstract
The present invention relates to a protein-immobilized membrane
(14) including a cell membrane homologous layer (14A) and a protein
(14B) immobilized to the cell membrane homologous layer (14A),
where the protein contains cytochrome or a cytochrome complex. The
present invention also relates to a method for forming a
protein-immobilized membrane (14), and an enzyme-immobilized
electrode and a biosensor (X1) provided with a protein-immobilized
membrane (14). Preferably, the cell membrane homologous layer (14A)
may contain a phospholipid polymer, and the protein (14B) may be
CyGDH including an .alpha. subunit having a glucose dehydrogenase
activity and cytochrome C having a function of electron
transfer.
Inventors: |
Katsuki; Koji; (Kyoto,
JP) ; Yamaoka; Hideaki; (Kyoto, JP) |
Correspondence
Address: |
HAMRE, SCHUMANN, MUELLER & LARSON, P.C.
P.O. BOX 2902
MINNEAPOLIS
MN
55402-0902
US
|
Assignee: |
ARKRAY, INC.
KYOTO
JP
|
Family ID: |
37431302 |
Appl. No.: |
11/920782 |
Filed: |
May 18, 2006 |
PCT Filed: |
May 18, 2006 |
PCT NO: |
PCT/JP2006/309906 |
371 Date: |
November 20, 2007 |
Current U.S.
Class: |
204/403.14 ;
435/174 |
Current CPC
Class: |
C12Q 1/006 20130101;
C12N 11/06 20130101; C12N 11/08 20130101; G01N 27/3272 20130101;
G01N 2333/80 20130101; C12N 9/0006 20130101; C07K 14/80 20130101;
C07K 14/705 20130101 |
Class at
Publication: |
204/403.14 ;
435/174 |
International
Class: |
G01N 27/26 20060101
G01N027/26; C12N 11/16 20060101 C12N011/16 |
Foreign Application Data
Date |
Code |
Application Number |
May 20, 2005 |
JP |
2005-148253 |
Claims
1. A protein-immobilized membrane comprising: a cell membrane
homologous layer; and a protein immobilized to the cell membrane
homologous layer, the protein containing cytochrome or a cytochrome
complex.
2. The protein-immobilized membrane according to claim 1, wherein
the cell membrane homologous layer contains a phospholipid
polymer.
3. The protein-immobilized membrane according to claim 2, wherein
the phospholipid polymer is 2-methacryloyloxyethyl
phosphorylcholine polymer.
4. The protein-immobilized membrane according to claim 1, wherein
the cell membrane homologous layer contains a silane coupling
agent.
5. The protein-immobilized membrane according to claim 4, wherein
the silane coupling agent is tetraethoxysilane.
6. The protein-immobilized membrane according to claim 1, wherein
the protein is CyGDH containing an .alpha. subunit having a glucose
dehydrogenase activity and cytochrome C having a function of
electron transfer.
7. A method for immobilizing a protein, the method comprising: a
first step of forming a cell membrane homologous layer at an
intended portion of an immobilization target member; and a second
step of causing a protein to self-organize with respect to the cell
membrane homologous layer, the protein containing cytochrome or a
cytochrome complex.
8. The protein immobilization method according to claim 7, wherein
the cell membrane homologous layer contains a phospholipid
polymer.
9. The protein immobilization method according to claim 8, wherein
the phospholipid polymer is 2-methacryloyloxyethyl
phosphorylcholine polymer.
10. The protein immobilization method according to claim 7, further
comprising a third step of subjecting the intended portion to
hydrophilic treatment before the first step.
11. The protein immobilization method according to claim 10,
wherein, in the second step, the cell membrane homologous layer is
formed to contain a silane coupling agent.
12. The protein immobilization method according to claim 11,
wherein the silane coupling agent is tetraethoxysilane.
13. The protein immobilization method according to claim 7, wherein
the protein is CyGDH containing an .alpha. subunit having a glucose
dehydrogenase activity and cytochrome C having a function of
electron transfer.
14. An enzyme-immobilized electrode comprising: a substrate; and an
enzyme-containing layer immobilized to the substrate; wherein the
enzyme-containing layer includes a cell membrane homologous layer
and an enzyme, the enzyme containing, as a subunit, cytochrome C
immobilized to the cell membrane homologous layer by self
organization.
15. The enzyme-immobilized electrode according to claim 14, wherein
the cell membrane homologous layer contains a phospholipid
polymer.
16. The enzyme-immobilized electrode according to claim 15, wherein
the phospholipid polymer is 2-methacryloyloxyethyl
phosphorylcholine polymer.
17. The enzyme-immobilized electrode according to claim 14, wherein
the cell membrane homologous layer contains a silane coupling
agent.
18. The enzyme-immobilized electrode according to claim 17, wherein
the silane coupling agent is tetraethoxysilane.
19. The enzyme-immobilized electrode according to claim 14, wherein
the enzyme is CyGDH containing an .alpha. subunit having a glucose
dehydrogenase activity and cytochrome C having a function of
electron transfer.
20. A biosensor comprising: a substrate; and an enzyme-containing
layer immobilized to the substrate; wherein the enzyme-containing
layer includes a cell membrane homologous layer and an enzyme, the
enzyme containing, as a subunit, cytochrome C immobilized to the
cell membrane homologous layer by self organization.
21. The biosensor according to claim 20, wherein the cell membrane
homologous layer contains a phospholipid polymer.
22. The biosensor according to claim 21, wherein the phospholipid
polymer is 2-methacryloyloxyethyl phosphorylcholine polymer.
23. The biosensor according to claim 20, wherein the cell membrane
homologous layer contains a silane coupling agent.
24. The biosensor according to claim 23, wherein the silane
coupling agent is tetraethoxysilane.
25. The biosensor according to claim 20, wherein the enzyme is
CyGDH containing an .alpha. subunit having a glucose dehydrogenase
activity and cytochrome C having a function of electron
transfer.
26. The biosensor according to claim 20, further comprising: a flow
path for moving a sample; and a reagent portion-provided in the
flow path.
27. The biosensor according to claim 26, further comprising a
working electrode and a counter electrode which are partially
exposed at the flow path and utilized for applying a voltage to a
sample.
28. The biosensor according to claim 27, at least part of the cell
membrane homologous layer is formed on the working electrode.
29. The biosensor according to claim 26, wherein the reagent
portion contains a color former.
30. The biosensor according to claim 29, wherein the reagent
portion includes a chromogenic layer containing the color former,
the cell membrane homologous layer, and an enzyme-containing layer
containing the enzyme.
Description
TECHNICAL FIELD
[0001] The present invention relates to a technique for
immobilizing a protein containing cytochrome to an immobilization
target material.
BACKGROUND ART
[0002] Biosensors designed to analyze a sample by an
electrochemical or optical method are widely used. An example of
biosensors designed to analyze a sample by an electrochemical
method (see Patent Document 1, for example) is a biosensor 9 shown
in FIG. 12 of the present application.
[0003] The illustrated biosensor 9 includes a substrate 92 formed
with a working electrode 90 and a counter electrode 91, and a cover
94 bonded to the substrate via a spacer 93. The biosensor 9 further
includes a flow path 95 defined by the substrate 92, the spacer 93
and the cover 94. The flow path 95 is used for moving a sample by
capillary force and formed with a reagent portion 96.
[0004] The reagent portion 96 connects the ends of the working
electrode 90 and the counter electrode 91 and contains
oxidoreductase. The oxidoreductase catalyzes the reaction of taking
electrons from glucose, for example. The electrons taken from the
glucose are supplied to the working electrode 90. The amount of
electrons supplied to the working electrode 90 is measured as the
responsive current by utilizing the working electrode 90 and the
counter electrode 91.
[0005] The four methods described below are typical methods for
forming a reagent portion 96, i.e., the methods for immobilizing
oxidoreductase (see Non-patent document 1, for example).
[0006] In the first method, a material liquid containing
oxidoreductase is applied to an intended portion of a target, and
then the material liquid is dried. In this way, the oxidoreductase
is immobilized to the intended portion of the target.
[0007] In the second method, oxidoreductase is immobilized to an
intended portion of a target by using a cross-linker such as
glutaraldehyde.
[0008] In a third method, oxidoreductase is contained in a polymer
such carboxymethylcellulose (CMC), and then the oxidoreductase is
immobilized together with the polymer.
[0009] In a fourth method, oxidoreductase is dispersed in a
conductive material such as a carbon paste, and the resultant paste
is applied to an intended portion of a target, to immobilize the
oxidoreductase.
[0010] However, with the conventional oxidoreductase-immobilizing
methods described above, oxidoreductase fails to be immobilized in
a manner such that the active sites are oriented (located) to
exhibit efficient activity of the oxidoreductase. In other words,
the conventional methods have a drawback that the immobilization is
not performed with the orientation of the oxidoreductase being
controlled. Specifically, with the conventional methods, active
sites of oxidoreductase existing adjacent to each other may face
each other or proteins may aggregate each other so that the active
site exists within the aggregate. As a result, the ratio of the
oxidoreductase (active site) which can be utilized efficiently is
relatively low. Accordingly, the probability that oxidoreductase
comes into contact with a substrate is relatively low, so that the
activity of the immobilized oxidoreductase as a whole is low. Thus,
to exhibit the intended function of the immobilized oxidoreductase,
the amount of oxidoreductase to be loaded needs to be increased,
which is disadvantageous in terms of cost. Particularly, since
oxidoreductases are generally expensive, the increase in the amount
of oxidoreductase to be loaded leads to a considerably
disadvantageous cost increase.
[0011] Moreover, since the orientation of oxidoreductase cannot be
controlled, the ratio of the actually usable oxidoreductase varies
among biosensors even when the same amount of oxidoreductase is
loaded. As a result, when the conventional immobilization methods
which cannot control orientation are employed, the measurement
results vary among biosensors.
[0012] Further, in the above-described biosensor 9, electrons taken
from the substrate at the active site of the oxidoreductase are
transferred to the working electrode 90. However, when the
orientation of the oxidoreductase is random, the efficiency of
electron transfer from the oxidoreductase to the working electrode
is poor. Thus, when the immobilization methods by which the
orientation of the oxidoreductase becomes random are employed, an
electron mediator heeds to be added to mediate the electron
transfer between the oxidoreductase and the working electrode 90.
Therefore, the biosensor 9 provided by immobilizing oxidoreductase
by a conventional method is disadvantageous in terms of cost,
because it requires an electron mediator. Further, as the electron
mediator, metal complexes such as potassium ferrocyanide are used
some of which have an adverse effect on the human body. Thus, it is
not desirable to use an electron mediator for such an analytical
tool as the biosensor 9.
[0013] Patent document 1: JP-B-H08-10208
[0014] Non-patent document 1: MIZUTANI Fumio, "Application of
enzyme-modified electrodes to biosensors," BUNSEKI KAGAKU, Vol. 48,
No. 9 pp. 809-821, The Japan Society for Analytical Chemistry,
September, 1999.
DISCLOSURE OF THE INVENTION
[0015] An object of the present invention is to immobilize a
protein such as oxidoreductase with good orientation and to cause
the activity to be exhibited efficiently and advantageously in
terms of cost with the use of a small amount of enzyme.
[0016] Another object of the present invention is to provide a
biosensor which is capable of properly measuring the concentration
of a substrate such as glucose without using an electron
mediator.
[0017] According to a first aspect of the present invention, there
is provided a protein-immobilized membrane comprising a cell
membrane homologous layer, and a protein immobilized to the cell
membrane homologous layer, where the protein contains cytochrome or
a cytochrome complex.
[0018] According to a second aspect of the present invention, there
is provided a method for immobilizing a protein. The method
comprises a first step of forming a cell membrane homologous layer
at an intended portion of an immobilization target member, and a
second step of causing self organization of a protein with respect
to the cell membrane homologous layer, the protein containing
cytochrome or a cytochrome complex.
[0019] Preferably, the protein immobilization method according to
the present invention further comprises a third step of subjecting
the intended portion to hydrophilic treatment before the first
step.
[0020] According to a third aspect of the present invention, there
is provided an enzyme-immobilized electrode comprising a substrate,
and an enzyme-containing layer immobilized to the substrate. The
enzyme-containing layer includes a cell membrane homologous layer
and an enzyme. The enzyme contains, as a subunit, cytochrome C
immobilized to the cell membrane homologous layer by self
organization.
[0021] According to a fourth aspect of the present invention, there
is provided a biosensor comprising a substrate, and an
enzyme-containing layer immobilized to the substrate. The
enzyme-containing layer includes a cell membrane homologous layer
and an enzyme. The enzyme contains, as a subunit, cytochrome C
immobilized to the cell membrane homologous layer by self
organization.
[0022] The biosensor according to the present invention may further
comprise a flow path for moving a sample, and a reagent portion
provided in the flow path.
[0023] The biosensor according to the present invention may further
comprise a working electrode and a counter electrode which are
partially exposed at the flow path and utilized for applying a
voltage to a sample. In this case, at least part of the cell
membrane homologous layer is formed on the working electrode.
[0024] The reagent portion may contain a color former. In this
case, the reagent portion may include a chromogenic layer
containing a color former, a cell membrane homologous layer, and a
layer containing an enzyme.
[0025] The cell membrane homologous layer in the present invention
may contain a phospholipid polymer. As the phospholipid polymer, it
is preferable to use 2-methacryloyloxyethyl phosphorylcholine
polymer.
[0026] Preferably, the cell membrane homologous layer in the
present invention contains a silane coupling agent. As the silane
coupling agent, it is preferable to use tetraethoxysilane.
[0027] The protein such as an enzyme in the present invention is
CyGDH containing an .alpha. subunit having a glucose dehydrogenase
activity and cytochrome C having a function of electron
transfer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1 is an overall perspective view showing a biosensor
according to a first embodiment of the present invention.
[0029] FIG. 2 is an exploded perspective view of the biosensor
shown in FIG. 1.
[0030] FIG. 3 is a sectional view taken along lines III-III in FIG.
1, a principal portion of which is shown as enlarged.
[0031] FIG. 4 is an overall perspective view showing a biosensor
according to a second embodiment of the present invention.
[0032] FIG. 5 is a sectional view taken along lines V-V in FIG. 4,
a principal portion of which is shown as enlarged.
[0033] FIG. 6 is an AFM image showing the observation results of
the surface condition of a carbon electrode using an AFM in Example
1.
[0034] FIG. 7 is an AFM image showing the observation results of
the condition of a phospholipid polymer layer formed on the carbon
electrode surface using an AFM in Example 1.
[0035] FIG. 8 is an AFM image showing the observation results of
CyGDH immobilized to the surface of the phospholipid polymer layer
using an AFM in Example 1.
[0036] FIG. 9 is a schematic view showing the structure of a
current measuring apparatus used in Example 2.
[0037] FIG. 10 is a graph showing the time-course measurements of
the responsive current in Example 2.
[0038] FIG. 11 is a graph showing the measurements of the
responsive current in Example 2 in relation to glucose level.
[0039] FIG. 12 is a sectional view showing a principal portion of
an example of conventional biosensor.
EXPLANATIONS OF REFERENCE SIGNS
[0040] X1, X2: Biosensors [0041] 1, 5: Substrates (of a biosensor)
[0042] 11: Working electrode (of a biosensor) [0043] 12: Counter
electrode (of a biosensor) [0044] 14, 51: Reagent portions (of a
biosensor) [0045] 14A, 50B: Cell membrane homologous layers (of a
reagent portion) [0046] 14B, 50C: CyGDH layers (of a reagent
portion) [0047] 4, 8: Capillaries (flow paths) [0048] 51A:
Chromogenic layer (of a reagent portion)
BEST MODE FOR CARRYING OUT THE INVENTION
[0049] Preferred embodiments of the present invention will be
described below, as first and second embodiments, with reference to
the accompanying drawings.
[0050] The first embodiment of the present invention will be
described below with reference to FIGS. 1-3.
[0051] The biosensor X1 shown in FIGS. 1-3 is a disposable sensor
to be mounted to a concentration measuring apparatus (not shown) to
measure a blood glucose level. The biosensor X1 is adapted to
measure the blood glucose level by an electrochemical method and
includes a substrate 1, which is in the form of an elongated
rectangle, and a cover 3 laminated on the substrate via a spacer 2.
In the biosensor X1, a capillary 4 extending in the longitudinal
direction of the substrate 1 (N1, N2 directions in the figures) is
defined by the elements 1-3. The capillary 4 is utilized for moving
the blood introduced from an introduction port 40 in the
longitudinal direction of the substrate 1 (N1, N2 directions in the
figures) utilizing capillary action and retaining the introduced
blood.
[0052] The spacer 2 defines the distance from the upper surface 10
of the substrate 1 to the lower surface 30 of the cover 3, i.e.,
the height of the capillary 4 and may comprise a double-sided tape.
The spacer 2 is formed with a slit 20 having an open end. The slit
20 defines the width of the capillary 4. The open end of the slid
20 serves as the introduction port 40 for introducing blood into
the capillary 4.
[0053] The cover 3 includes an exhaust port 30 for discharging gas
from the capillary 4. The cover 3 is made of a thermoplastic resin
having a high wettability, such as Vinylon or highly crystalline
PVA.
[0054] As shown in FIGS. 2 and 3, the upper surface 10 of the
substrate 1, which is made of an insulating resin such as PET, is
formed with a working electrode 11, a counter electrode 12, an
insulating film 13 and a reagent portion 14.
[0055] Each of the working electrode 11 and the counter electrode
12 is L-shaped as a whole. Specifically, the working electrode 11
and the counter electrode 12 mostly extend in the longitudinal
direction of the substrate 1 (N1, N2 directions in the figures) and
respectively include ends 11a and 12a extending in the width
direction (N3, N4 directions in the figures). The working electrode
11 and the counter electrode 12 further include ends 11b and 12b,
respectively, which provide terminals for coming into contact with
the terminals of the concentration measuring apparatus (not shown).
The working electrode 11 and the counter electrode 12 may be formed
by screen printing using carbon paste. The working electrode 11 and
the counter electrode 12 may be made of a conductive material other
than carbon by spin coating, thermal transfer, carbon rod slice,
vapor deposition, sputtering or CVD.
[0056] The insulating film 13 covers most part of the working
electrode 11 and the counter electrode 12 while exposing the ends
11a, 12a, 11b and 12b of the working electrode 11 and the counter
electrode 12. The insulating film 13 includes an opening 13a for
exposing the ends 11a and 12a of the working electrode 11 and the
counter electrode 12. The opening 13a defines the region for
forming the reagent portion 14 and is in the form of a rectangle
elongated in the longitudinal direction of the substrate 1 (N1, N2
directions in the figures).
[0057] The insulating film 13 may be formed by screen printing
using ink containing a material having high water repellency or
photolithography using a photosensitive resin.
[0058] The reagent portion 14 is arranged to bridge the ends 11a
and 12a of the working electrode 11 and the counter electrode 12 at
the opening 13a of the insulating film 13. The reagent portion 14
includes a cell membrane homologous layer 14A and a CyGDH layer
14B.
[0059] The cell membrane homologous layer 14A is utilized for
immobilizing CyGDH with controlled orientation. The cell membrane
homologous layer 14A may be formed by applying a solution
containing phospholipid polymer to the portion 14' of the working
electrode 11 and the counter electrode 12 which is exposed through
the opening 13a of the insulating film 13 (hereinafter, the portion
14' is referred to as "exposed portion 14'") and then drying the
solution.
[0060] As the phospholipid polymer, use may be made of
2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, for
example. As the MPC polymer, use may be made of one prepared by
polymerizing MPC alone or one prepared by copolymerizing MPC with a
hydrophobic monomer such as methacrylate (e.g. butyl
methacrylate).
[0061] As the phospholipid polymer for forming the cell membrane
homologous layer 14A, polymers other than MPC polymer may be used
as long as the polymer contains a monomeric unit having a structure
similar to phospholipid forming a cell membrane in the
molecules.
[0062] As the phospholipid polymer, it is preferable to use one to
which a silane coupling agent is added. In this case, the
phospholipid polymer is reliably bonded to the exposed portion
14'.
[0063] To form the cell membrane homologous layer 14A, it is
preferable to subject the exposed portion 14' to hydrophilic
treatment in advance. By this treatment, hydrophilic groups such as
a hydroxyl group or a carboxyl group enters the exposed portion 14'
and is bonded to the silane coupling agent. Thus, phospholipid
polymer is more strongly fixed to the exposed portion 14'.
[0064] The amount of the silane coupling agent in the polymer may
be set to 10 to 500 parts by weight relative to 100 parts by weight
of the polymer component. Examples of silane coupling agent
include: tetraethoxysilane; vinyltrichlorosilane;
vinyl-tris(2-methoxyethoxy)silane;
.gamma.-methacryloxypropyltrimethoxysilane;
.gamma.-methacryloxypropyltriethoxysilane;
.beta.-(3,4-epoxycyclohexyl)ethyltrimethoxysilane;
.gamma.-glycidoxypropyltriethoxysilane;
.gamma.-aminopropyltriethoxysilane;
N-phenyl-.gamma.-aminopropyltrimethoxysilane;
.gamma.-chloropropyltrimethoxysilane; and
.gamma.-mercaptopropyltrimethoxysilane. These silane coupling
agents may be used solely or in combination.
[0065] The hydrophilic treatment of the exposed portion 14' can be
performed by various known techniques. Examples of hydrophilic
treatment which can be employed in the present invention include
VUV treatment, UV treatment, corona discharge and plasma
treatment.
[0066] The CyGDH layer 14B is provided by immobilizing CyGDH
self-organizingly to the cell membrane homologous layer 14A.
Although FIG. 3 shows the state in which CyGDH is immobilized to
the surface of the cell membrane homologous layer 14A, this figure
is a schematic view for describing the present invention. Thus,
although the inventors of the present invention have ascertained
that CyGDH is self-organizingly immobilized to the cell membrane
homologous layer 14A, the inventors have not yet found out how
CyGDH is immobilized to the cell membrane homologous layer 14A.
Further, CyGDH derived from a microorganism belonging to the
burkhorderia cepacia, which will be described later, is a
transmembrane protein. Therefore, CyGDH may not be immobilized only
at the surface of the cell membrane homologous layer 14A, as shown
in FIG. 3, but may be immobilized to the cell membrane homologous
layer 14A while penetrating the cell membrane homologous layer
14A.
[0067] The self-organizing immobilization of CyGDH to the cell
membrane homologous layer 14A may be performed by immersing the
substrate 1 provided with the cell membrane homologous layer 14A at
the exposed portion 14' into an enzyme solution containing CyGDH or
spraying the enzyme solution to the cell membrane homologous layer
14A and then drying the solution.
[0068] As will be understood from the AFM image (see FIG. 8) to be
described later, when CyGDH is self-organizingly immobilized to the
cell membrane homologous layer 14A, CyGDH is immobilized with
controlled orientation. Specifically, CyGDH is so immobilized to
the cell membrane homologous layer 14A that the active site of the
.alpha. subunit is positioned at the surface of the reagent portion
14, whereas cytochrome C is positioned close to or in contact with
the exposed portion 14' (working electrode 11).
[0069] In the present invention, as the CyGDH, use is made of those
which at least contain an .alpha. subunit having a glucose
dehydrogenase activity and cytochrome C having a function of
electron transfer. Thus, CyGDH further containing a subunit other
than .alpha. subunit and cytochrome C may be used. Examples of such
CyGDH are disclosed in international publication WO02/36779. The
CyGDH disclosed in this international publication is derived from a
microorganism belonging to the burkholderia cepacia and includes an
.alpha. subunit having a molecular weight of about 60 kDa in
SDS-polyacrylamide gel electrophoresis under a reduced condition,
including FAD as a cofactor and having a glucose dehydrogenase
activity, and cytochrome C having a molecular weight of about 43
kDa in SDS-polyacrylamide gel electrophoresis under a reduced
condition and having a function of electron transfer. The CyGDH in
the present invention further includes one prepared by utilizing a
transformant to which a gene encoding CyGDH taken from a
microorganism belonging to the burkholderia cepacia is
transferred.
[0070] The CyGDH derived from a microorganism belonging to the
burkhorderia cepacia is a transmembrane protein. That is, the CyGDH
derived from this microorganism originally exists in a cell
membrane. Thus, when such CyGDH is used, CyGDH is immobilized to
the cell membrane homologous layer 14A by self organization with
controlled orientation similarly to that in existing in a cell
membrane. Such self-organizing immobilization of CyGDH is possible
not only when CyGDH derived from a microorganism belonging to the
burkhorderia cepacia is used but also when CyGDH originally
existing in a cell membrane is used.
[0071] When the biosensor X1 having the above-described structure
is mounted to a concentration measuring apparatus (not shown) and
blood is introduced to the capillary 4 through the introduction
port 40 of the biosensor X1, the blood glucose level is measured
automatically at the concentration measuring apparatus (not
shown).
[0072] The introduction of blood to the biosensor X1 may be
performed either before or after the biosensor is mounted to the
concentration measuring apparatus (not shown). Generally, blood is
introduced by cutting the skin of the person to be tested to cause
bleeding and then applying the blood to the introduction port 40 of
the biosensor X1.
[0073] When the biosensor X1 is mounted to the concentration
measuring apparatus (not shown), the working electrode 11 and the
counter electrode 12 of the biosensor X1 come into contact with the
terminals (not shown) of the concentration measuring apparatus. In
the biosensor X1, the blood applied to the introduction port 40
moves toward the exhaust port 30 due to capillary action at the
capillary 4 and fills the capillary 4.
[0074] In the capillary 4, CyGDH reacts specifically with the
glucose in the blood to take electrons from the glucose. When a
voltage is applied to the blood using the working electrode 11 and
the counter electrode 12, the electrons taken out by the CyGDH are
transferred to the working electrode 11. In the concentration
measuring apparatus (not shown), when a voltage is applied to the
working electrode 11 and the counter electrode 12, the amount of
electrons transferred to the working electrode 11, for example, is
measured as the responsive current. Based on the responsive
current, the blood glucose level is computed.
[0075] In the biosensor X1, CyGDH is immobilized with controlled
orientation so that the active site of the .alpha. subunit is
positioned at the surface of the reagent portion 14. Thus, in the
reagent portion 14, electrons are efficiently taken from glucose.
As a result, in the biosensor X1, intended activity is properly
exhibited even with the use of a relatively small amount of CyGDH,
which is advantageous in terms of cost.
[0076] Since CyGDH is immobilized with controlled orientation, the
amount of CyGDH contained in the reagent portion 14 and the
orientation (position) of the active site do not vary among
biosensors X1. Thus, variation in sensitivity among the biosensors
X1 does not occur, so that the blood glucose level measurement is
performed properly.
[0077] Since CyGDH is immobilized with controlled orientation in
the biosensor X1, cytochrome C exists close to or in contact with
the exposed portion 14' (working electrode 11). Thus, in the
reagent portion 14, electrons taken from the glucose are
efficiently transferred to the working electrode 11. Thus, in the
biosensor X1, proper responsive current is obtained without using
an electron mediator such as a metal complex.
[0078] A second embodiment of the present invention will be
described below with reference to FIGS. 4 and 5.
[0079] Unlike the foregoing biosensor X1 (see FIGS. 1-3), the
biosensor X2 shown in FIGS. 4 and 5 is adapted to measure the blood
glucose level by an optical method.
[0080] The biosensor X2 includes a substrate 5, which is in the
form of an elongated rectangle, and a cover 7 laminated on the
substrate via a pair of spacers 6. In the biosensor X2, a capillary
8 extending in the longitudinal direction of the substrate 5 (N1,
N2 directions in the figures) is defined by the elements 5-7. The
capillary 8 is used for moving the blood introduced from an
introduction port 80 in the longitudinal direction of the substrate
5 (N1, N2 directions in the figures) utilizing capillary action and
retaining the introduced blood.
[0081] A reagent portion 51 is provided in the capillary 8. The
reagent portion 51 includes a chromogenic layer 51A, and a cell
membrane homologous layer 51B and a CyGDH layer 51C which are
formed on the chromogenic layer 51A.
[0082] The chromogenic layer 51A includes a color former and may be
formed by applying a solution containing a color former to an
intended portion of the substrate 5 and then drying the
solution.
[0083] Examples of color former which can be used in the present
invention include:
MTT(3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide);
INT(2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazoli um
chloride);
WST-4(2-(4-Iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfop
henyl)-2H-tetrazolium,monosodium salt); and
4AA(4-Aminoantipyrine).
[0084] The cell membrane homologous layer 51B and the CyGDH layer
51C can be formed similarly to those of the foregoing biosensor X1
(see FIGS. 1-3).
[0085] In this biosensor X2 again, the reagent portion 51 includes
a cell membrane homologous layer 51B and a CyGDH layer 51C,
similarly to the biosensor X1 (see FIGS. 1-3). Further, the cell
membrane homologous layer 51B is held in contact with the
chromogenic layer 51A. Thus, in the reagent portion 51, CyGDH is
immobilized with controlled orientation, i.e., with the active site
of the .alpha. subunit positioned at the surface whereas cytochrome
C is positioned in contact with or close to chromogenic layer 51A.
Thus, the biosensor X2 has the same advantages as those of the
biosensor X1 (see FIGS. 1-3).
[0086] The present invention is not limited to the foregoing
embodiments and may be modified in various ways. For instance, the
present invention is not limited to a disposable biosensor and is
also applicable to a biosensor used for monitoring the blood
glucose level with at least the electrode portion embedded in the
human body. The invention is also applicable to a biosensor for
measuring the concentration of a substrate other than glucose or to
an enzyme electrode for measuring the concentration of a substrate
such as glucose.
EXAMPLE 1
[0087] In this example, a carbon electrode, a phospholipid polymer
layer and a CyGDH layer were formed on a surface of a PET
substrate. The conditions of the surface before and after the
formation of these layers were observed using an atomic force
microscope (AFM) (Tradename "D-3100" available from Digital
Instruments).
(Observation of the Carbon Electrode Surface)
[0088] The carbon electrode was formed by screen printing using a
carbon ink available from Acheson Japan Ltd. The AFM image of the
carbon electrode is shown in FIG. 6. As will be understood from
FIG. 6, the surface of the carbon electrode had relatively large
irregularities, with carbon particles (having average particle size
of about 100 nm) appearing on the surface.
(Observation of the Phospholipid Polymer Layer Surface)
[0089] To form the phospholipid polymer layer, the surface of the
carbon electrode was first subjected to VUV treatment (hydrophilic
treatment). Then, MPC polymer solution was applied to the surface
of the carbon electrode and then dried, whereby the phospholipid
polymer layer was formed. The VUV treatment was performed by
irradiating the surface of the carbon electrode with excimer laser
having a wavelength of 172 nm in the atmosphere for 180 seconds
with the irradiation distance of 1 mm by using "MECL-M3-750"
(available from M.D. Excimer Inc.). As the MPC polymer solution,
use was made of a solution of MPC polymer containing
tetraethoxysilane as a silane coupling agent (Tradename "LIPIDURER"
available from NOF CORPORATION).
[0090] The AFM image after the formation of the phospholipid
polymer layer was shown in FIG. 7. As will be understood from FIG.
7, although the phospholipid portion of the polymer appeared on the
surface of the phospholipid polymer layer, the surface of the
phospholipid polymer was smooth as compared with that of the carbon
electrode layer (see FIG. 6), because the diameter of the
phospholipid portion was about 2 to 3 nm which was smaller than
that of carbon particles.
(Observation of the CyGDH Layer Surface)
[0091] The CyGDH layer was formed by immersing the carbon electrode
formed, with the phospholipid polymer layer in a CyGDH solution for
ten minutes. The concentration of CyGDH in the CyGDH solution was
100 U/.mu.L on the activity basis. The AFM image after the
formation of the CyGDH layer is shown in FIG. 8.
[0092] As shown in FIG. 8, the surface of the phospholipid polymer
layer was formed with regularly arranged clusters (CyGDH) each
having a diameter of about 6 to 30 nm. That is, CyGDH was
immobilized to the phospholipid polymer in such a manner that at
least part of CyGDH appeared on the surface. From the fact that the
clusters are arranged regularly, it is presumed that CyGDH is
immobilized to the phospholipid polymer layer with controlled
orientation.
EXAMPLE 2
[0093] In this example, responsiveness was examined with respect to
an electrode (inventive electrode) to which CyGDH is immobilized
via a phospholipid polymer layer and to an electrode (comparative
electrode) to which CyGDH is immobilized without the intervention
of a phospholipid polymer layer.
[0094] The inventive electrode was prepared by forming a
phospholipid polymer layer on a carbon electrode and then
immobilizing CyGDH, similarly to Example 1.
[0095] The comparative electrode was prepared similarly to the
inventive electrode except that a phospholipid polymer layer was
not formed.
[0096] The responsiveness of the inventive electrode and the
comparative electrode was evaluated as the responsive current
obtained when a voltage was applied to a glucose solution using a
current measuring apparatus Y prepared as shown in FIG. 9.
[0097] The current measuring apparatus Y includes a working
electrode Y1, a reference electrode Y2 and a counter electrode Y2,
which are connected to a potentiostat Y4. The current measuring
apparatus Y is designed to measure the responsive current by
immersing the electrodes Y1-Y3 in a glucose solution and applying a
voltage to the glucose solution. Herein, the working electrode Y1
is the inventive electrode or the comparative electrode prepared in
the above-described manner. The reference electrode Y2 is a
silver-silver chloride electrode (Tradename "RE-1B"; available from
BAS Inc.). The counter electrode Y3 is a platinum electrode.
(Linear Sweep Voltammetry)
[0098] In this example, before the responsiveness of the inventive
electrode and the comparative electrode was evaluated, measurement
by linear sweep voltammetry was performed with respect to glucose
solutions of different concentrations using the current measuring
apparatus Y in which the inventive electrode was employed as the
working electrode Y1.
[0099] In this measurement, the sweep voltage was 100 mV/sec, and
the responsive current was measured with respect to the range of
-400 mV to +700 mV. The glucose solutions had the concentrations of
0 mg/dL, 50 mg/dL, 100 mg/dL, 200 mg/dL, 400 mg/dL and 600 mg/dL,
respectively. As a result, in the range of +100 to +700 mV,
variation in responsive current in accordance with the difference
in concentration of the glucose solutions was observed. Considering
this result, in the subsequent responsive current measurement, the
voltage to be applied to the glucose solutions was set to +600
mV.
(Responsiveness)
[0100] The responsiveness of the inventive electrode and the
comparative electrode was evaluated by measuring the time course of
the responsive current with respect to each of the glucose
solutions of different concentrations. The measurement was
performed using the above-described current measuring apparatus Y
employing the inventive electrode or the comparative electrode as
the working electrode Y1. As noted above, the voltage of +600 mV
was applied in measuring the responsive current. The concentrations
of the used glucose solutions were 0 mg/dL, 50 mg/dL, 100 mg/dL,
200 mg/dL, 400 mg/dL and 600 mg/dL, respectively. The time course
of the responsive current with respect to each of the glucose
solutions is shown in FIG. 10. The responsive current one second
after the start of the measurement is shown in FIG. 11 in relation
to the glucose level.
[0101] As will be understood from FIGS. 10 and 11, when the
inventive electrode was used, the responsive current in the p order
was measured. However, when the comparative electrode was used,
merely the responsive current in the n order was measured.
Specifically, the results obtained when the comparative electrode
was used were similar to the conventionally reported measurement
results (n order) of the responsive current obtained when use was
made of a system which does not include an electron mediator such
as a metal complex. When the inventive electrode was used, on the
other hand, the responsive current in the u order which was much
higher than the conventionally reported level was measured. Thus,
it is demonstrated that the inventive electrode has high
responsiveness (sensitivity).
[0102] Moreover, as will also be understood from FIGS. 10 and 11,
when the inventive electrode is used, the difference in glucose
level is properly reflected as the difference in responsive
current. Thus, by using the inventive electrode, the glucose level
is measured properly at least in the glucose level range (0 to 600
mg/dL) with respect to which the responsive current was measured in
this example.
[0103] As will be understood from the above, the inventive
electrode in which CyGDH is immobilized via a phospholipid polymer
layer has sufficient responsiveness (sensitivity) to properly
measure the glucose level without using an electron mediator such
as a metal complex. Thus, with the use of the inventive electrode,
proper measurement of the glucose level (e.g. blood glucose level)
without using an electron mediator is possible. Since an electron
mediator is not used, to embed the inventive electrode in the human
body for use causes no harm to the human body. Thus, the present
invention is applicable to a biosensor to be embedded in the human
body to monitor the blood glucose level.
[0104] The method for immobilizing CyGDH which is employed for the
inventive electrode, i.e., the application of a phospholipid
polymer solution and the immersion in a CyGDH solution is a very
easy work. Thus, this method is applicable to a biosensor including
minute paths such as .mu.TAS. Since the phospholipid polymer layer
and the CyGDH layer formed at the minute paths are extremely thin,
the formation of these layers does not considerably hinder the
movement of a sample in the minute paths. Thus, the provision of a
reagent portion, which is made up of a phospholipid polymer layer
and a CyGDH layer, at most part of the minute paths does not cause
any problems. Thus, by forming a reagent portion over a wide range
of the minute paths, the sensitivity of the .mu.TAS, which has been
disadvantageously low, is improved. In this way, a .mu.TAS having a
high sensitivity can be provided.
* * * * *