U.S. patent application number 11/780033 was filed with the patent office on 2008-01-24 for optical glucose sensor chip and method of manufacturing the same.
Invention is credited to Masaaki Hirakawa, Kayoko Oomiya, Ikuo Uematsu.
Application Number | 20080020454 11/780033 |
Document ID | / |
Family ID | 38971916 |
Filed Date | 2008-01-24 |
United States Patent
Application |
20080020454 |
Kind Code |
A1 |
Uematsu; Ikuo ; et
al. |
January 24, 2008 |
OPTICAL GLUCOSE SENSOR CHIP AND METHOD OF MANUFACTURING THE
SAME
Abstract
An optical glucose sensor chip includes a substrate, a pair of
optical elements formed on a surface of the substrate for
introducing light into the substrate and for emitting the light
from the substrate, and a glucose sensing membrane formed on the
surface of the substrate at a position between the optical
elements. The sensing membrane includes a color reagent substrate,
a first enzyme which oxidizes or reduces glucose, a second enzyme
that generates a material which makes the color reagent substrate
exhibit color by a reaction with a product obtained by oxidation or
reduction of glucose, a nonionic cellulose derivative, and an ionic
polymer into which a buffer is incorporated. At least one of the
first and second enzymes is coated with the ionic polymer, and the
color reagent substrate. The first and second enzymes, the buffer
and the ionic polymer are supported by the nonionic cellulose
derivative.
Inventors: |
Uematsu; Ikuo;
(Yokohama-shi, JP) ; Hirakawa; Masaaki;
(Yokohama-shi, JP) ; Oomiya; Kayoko;
(Yokohama-shi, JP) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND MAIER & NEUSTADT, P.C.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Family ID: |
38971916 |
Appl. No.: |
11/780033 |
Filed: |
July 19, 2007 |
Current U.S.
Class: |
435/288.7 |
Current CPC
Class: |
G01N 21/552 20130101;
C12Q 1/002 20130101; C12Q 1/006 20130101; G01N 21/78 20130101 |
Class at
Publication: |
435/288.7 |
International
Class: |
C12M 1/00 20060101
C12M001/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 20, 2006 |
JP |
2006-198173 |
Claims
1. An optical glucose sensor chip comprising: a substrate; a pair
of optical elements formed on a principal surface of the substrate
for introducing light into the substrate and for emitting the light
from the substrate; and a glucose sensing membrane formed on the
principal surface of the substrate at a position between the
optical elements; wherein the sensing membrane includes a color
reagent substrate, a first enzyme which oxidizes or reduces
glucose, a second enzyme that generates a material which makes the
color reagent substrate exhibit color by a reaction with a product
obtained by oxidation or reduction of glucose, a nonionic cellulose
derivative, and an ionic polymer into which a buffer is
incorporated, at least one of the first and second enzymes is
coated with the ionic polymer, and the color reagent substrate, the
first and second enzymes, the buffer and the ionic polymer are
supported by the nonionic cellulose derivative.
2. The sensor chip according to claim 1, wherein the first enzyme
is glucose oxidase, the second enzyme is peroxidase and the color
reagent substrate is at least one of 3,3,5,5-tetramethylbenzidine
and N,N'-bis(2-hydroxy-3-sulfopropyl)tridine.
3. The sensor chip according to claim 1, wherein the ionic polymer
is a negative ionic polymer.
4. The sensor chip according to claim 3, wherein the negative ionic
polymer is a polymer containing at least one anionic group selected
from the group consisting of a phosphate, a carboxylate and a
sulfonate.
5. The sensor chip according to claim 1, wherein the nonionic
cellulose derivative is at least one selected from the group
consisting of an alkyl cellulose, a hydroxyalkyl cellulose and a
hydroxyalkylalkyl cellulose.
6. The sensor chip according to claim 1, wherein the sensing
membrane further contains a crosslinking high-molecular
compound.
7. The sensor chip according to claim 6, wherein the crosslinking
high-molecular compound is a copolymer of a hydrophobic monomer and
a hydrophilic monomer having at least one group selected from the
group consisting of a hydroxyl group, a carboxyl group, an amino
group and an ionic functional group.
8. The sensor chip according to claim 7, wherein the copolymer of a
hydrophilic monomer and a hydrophobic monomer is a copolymer of
2-methacryloyloxyethylphosphorylcholine and butylmethacrylate.
9. The sensor chip according to claim 1, wherein the glucose
sensing membrane further contains polyethylene glycol or ethylene
glycol for endowing the membrane with water permeability.
10. An optical glucose sensor chip comprising: a glass substrate; a
pair of optical elements formed on a principal surface of the glass
substrate for introducing light into the glass substrate and for
emitting the light from the glass substrate; a light-reflecting
path layer formed on the principal surface of the substrate on
which the optical elements are formed and made of a resin having a
higher refractive index than the substrate; and a glucose sensing
membrane formed on the light-reflecting path layer at a position
between the optical elements; wherein the sensing membrane includes
a color reagent substrate, a first enzyme which oxidizes or reduces
glucose, a second enzyme that generates a material which makes the
color reagent substrate exhibit color by a reaction with a product
obtained by oxidation or reduction of glucose, a nonionic cellulose
derivative, and an ionic polymer into which a buffer is
incorporated, at least one of the first and second enzymes is
coated with the ionic polymer, and the color reagent substrate, the
first and second enzymes, the buffer and the ionic polymer are
supported by the nonionic cellulose derivative.
11. The sensor chip according to claim 10, wherein the first enzyme
is glucose oxidase, the second enzyme is peroxidase and the color
reagent substrate is at least one of 3,3,5,5-tetramethylbenzidine
and N,N'-bis(2-hydroxy-3-sulfopropyl)tridine.
12. The sensor chip according to claim 10, wherein the ionic
polymer is a negative ionic polymer.
13. The sensor chip according to claim 12, wherein the negative
ionic polymer is a polymer containing at least one anionic group
selected from the group consisting of a phosphate, a carboxylate
and a sulfonate.
14. The sensor chip according to claim 10, wherein the nonionic
cellulose derivative is at least one selected from the group
consisting of an alkyl cellulose, a hydroxyalkyl cellulose and a
hydroxyalkylalkyl cellulose.
15. The sensor chip according to claim 10, wherein the sensing
membrane further contains a crosslinking high-molecular
compound.
16. The sensor chip according to claim 15, wherein the crosslinking
high-molecular compound is a copolymer of a hydrophobic monomer and
a hydrophilic monomer having at least one group selected from the
group consisting of a hydroxyl group, a carboxyl group, an amino
group and an ionic functional group.
17. The sensor chip according to claim 16, wherein the copolymer of
a hydrophilic monomer and a hydrophobic monomer is a copolymer of
2-methacryloyloxyethylphosphorylcholine and butylmethacrylate.
18. The sensor chip according to claim 10, wherein the glucose
sensing membrane further contains polyethylene glycol or ethylene
glycol for endowing the membrane with water permeability.
19. A method of manufacturing an optical glucose sensor chip,
comprising: preparing a glucose sensing membrane-forming coating
solution by using any of: (a) a method in which at least one of a
first enzyme that oxidizes or reduces glucose and a second enzyme
that generates a material which makes a color reagent substrate
exhibit color by a reaction with a product obtained by oxidation or
reduction of glucose is mixed in advance with an aqueous solution
containing an ionic polymer and a buffer, and the mixed solution is
added to and mixed with the other enzyme, a color reagent substrate
and a nonionic cellulose derivative; (b) a method in which the
first and second enzymes are respectively mixed in advance with an
aqueous solution containing an ionic polymer and a buffer, and the
respective prepared mixed solutions are added to and mixed with a
color reagent substrate and a nonionic cellulose derivative; or (c)
a method in which both the first and second enzymes are mixed in
advance with an aqueous solution containing an ionic polymer and a
buffer, and the prepared mixed solution is added to and mixed with
a color reagent substrate and a nonionic cellulose derivative;
forming a pair of optical elements on a substrate for introducing
light into the substrate and for emitting the light from the
substrate; and applying the glucose sensing membrane-forming
coating solution to a substrate area positioned between the optical
elements, followed by drying to form a glucose sensing
membrane.
20. A method of manufacturing an optical glucose sensor chip,
comprising: preparing a glucose sensing membrane-forming coating
solution by using any of: (a) a method in which at least one of a
first enzyme that oxidizes or reduces glucose and a second enzyme
that generates a material which makes a color reagent substrate
exhibit color by a reaction with a product obtained by oxidation or
reduction of glucose is mixed in advance with an aqueous solution
containing an ionic polymer and a buffer, and the mixed solution is
added to and mixed with the other enzyme, a color reagent substrate
and a nonionic cellulose derivative; (b) a method in which the
first and second enzymes are respectively mixed in advance with an
aqueous solution containing an ionic polymer and a buffer, and the
respective prepared mixed solutions are added to and mixed with a
color reagent substrate and a nonionic cellulose derivative; or (c)
a method in which both the first and second enzymes are mixed in
advance with an aqueous solution containing an ionic polymer and a
buffer, and the prepared mixed solution is added to and mixed with
a color reagent substrate and a nonionic cellulose derivative;
forming a pair of gratings on a substrate for introducing light
into the substrate and for emitting the light from the substrate;
forming a light-reflecting path layer made of a resin having a
higher refractive index than the substrate on a principal surface
of the substrate on which the optical elements are formed; and
applying the glucose sensing membrane-forming coating solution to
the part of the light-reflecting path layer positioned between the
optical elements, followed by drying to form a glucose sensing
membrane.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is based upon and claims the benefit of
priority from prior Japanese Patent Application No. 2006-198173,
filed Jul. 20, 2006, the entire contents of which are incorporated
herein by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to an optical glucose sensor
chip and a method of manufacturing the optical glucose sensor
chip.
[0004] 2. Description of the Related Art
[0005] As an optical glucose sensor chip, for example, a less
invasive type blood sugar level measuring chip has been developed
which indirectly measures a blood sugar level by extracting body
fluid of subcutaneous tissues. This sensor chip has a structure
including a glass substrate, a pair of gratings which are formed on
a surface of the substrate and introduce light to or emit the light
from the substrate, and a glucose sensing membrane which is
positioned between the gratings and formed on the surface of the
substrate. This glucose sensing membrane contains a color reagent
substrate (for example, 3,3'5,5'-tetramethylbenzidine [TMBZ]), a
first enzyme (for example, glucose oxidase [GOD]) that oxidizes or
reduces glucose, a second enzyme (for example, peroxidase [POD])
generating a material that reacts with a product obtained by the
oxidation or reduction of glucose to make the color reagent
substrate exhibit color, and a film forming high-molecular compound
(for example, a cellulose derivative such as carboxymethyl
cellulose [CMC]).
[0006] When a sheet gel is disposed between the skin and the above
sensing membrane to apply an electric field in a glucose sensor
chip having such a structure, glucose in a subcutaneous tissue
solution penetrates the gel from the skin and reaches the above
sensing membrane. At this time, the above TMBZ as a color reagent
substrate in the sensing membrane makes to exhibit color due to the
reaction between glucose and GOD or POD. When light is made to be
incident to the above substrate and to be refracted on the surface
of the substrate and on one of the above gratings, the light
propagates through an interface between the above substrate and the
sensing membrane containing color-formed TMBZ, is refracted on the
interface between the substrate and the other grating and is
received by, for example, a light receiving element. The intensity
of the received laser light is less than the intensity (initial
intensity) of the laser light received by the light receiving
element when the above sensing membrane has not exhibited color.
For this reason, the concentration of the above glucose can be
detected from the ratio of reduction of the above light
intensity.
[0007] However, when the above sensing membrane is stored and used
for a long time, the activity of the first and second enzymes in
the membrane rapidly deteriorates. Examples of the cause of the
deterioration include a variation in the pH of the sensing
membrane, a variation in the ionic strengths of the first and
second enzymes and hydrolysis of the first and second enzymes. When
the first and second enzymes deteriorate, the first enzyme reacts
insufficiently with glucose which is the subject to be measured.
The reduction in the reactivity with glucose reduces the generation
of the material that aids the color-forming material obtained by
the subsequent reaction with the second enzyme in exhibiting color,
with the result that this causes a reduction in the reactivity with
the color-forming material and a reduction in the degree of the
color to be exhibited, bringing about a deterioration in the
sensitivity of the glucose sensor chip.
BRIEF SUMMARY OF THE INVENTION
[0008] The present invention is to provide an optical glucose
sensor chip which can limit or prevent the deterioration of the
first and second enzymes in the sensing membrane with time and to
provide a method of manufacturing the optical glucose sensor
chip.
[0009] According to a first aspect of the present invention, there
is provided an optical glucose sensor chip comprising:
[0010] a substrate;
[0011] a pair of optical elements which formed on a principal
surface of the substrate for introducing light into the substrate
and for emitting the light from the substrate; and
[0012] a glucose sensing membrane formed on the principal surface
of the substrate at a position between the optical elements;
[0013] wherein the sensing membrane includes a color reagent
substrate, a first enzyme which oxidizes or reduces glucose, a
second enzyme that generates a material which makes the color
reagent substrate exhibit color by a reaction with a product
obtained by oxidation or reduction of glucose, a nonionic cellulose
derivative, and an ionic polymer into which a buffer is
incorporated,
[0014] at least one of the first and second enzymes is coated with
the ionic polymer, and
[0015] the color reagent substrate, the first and second enzymes,
the buffer and the ionic polymer are supported by the nonionic
cellulose derivative.
[0016] According to a second aspect of the present invention, there
is provided an optical glucose sensor chip comprising:
[0017] a glass substrate;
[0018] a pair of optical elements formed on a principal surface of
the glass substrate for introducing light into the glass substrate
and for emitting the light from the glass substrate;
[0019] a light-reflecting path layer formed on the principal
surface of the substrate on which the optical elements are formed
and made of a resin having a higher refractive index than the
substrate; and
[0020] a glucose sensing membrane formed on the light-reflecting
path layer at a position between the optical elements;
[0021] wherein the sensing membrane includes a color reagent
substrate, a first enzyme which oxidizes or reduces glucose, a
second enzyme that generates a material which makes the color
reagent substrate exhibit color by a reaction with a product
obtained by oxidation or reduction of glucose, a nonionic cellulose
derivative, and an ionic polymer into which a buffer is
incorporated,
[0022] at least one of the first and second enzymes is coated with
the ionic polymer, and
[0023] the color reagent substrate, the first and second enzymes,
the buffer and the ionic polymer are supported by the nonionic
cellulose derivative.
[0024] According to a third aspect of the present invention, there
is provided a method of manufacturing an optical glucose sensor
chip, comprising:
[0025] preparing a glucose sensing membrane-forming coating
solution by using any of: (a) a method in which at least one of a
first enzyme that oxidizes or reduces glucose and a second enzyme
that generates a material which makes a color reagent substrate
exhibit color by a reaction with a product obtained by oxidation or
reduction of glucose is mixed in advance with an aqueous solution
containing an ionic polymer and a buffer, and the mixed solution is
added to and mixed with the other enzyme, a color reagent substrate
and a nonionic cellulose derivative; (b) a method in which the
first and second enzymes are respectively mixed in advance with an
aqueous solution containing an ionic polymer and a buffer, and the
respective prepared mixed solutions are added to and mixed with a
color reagent substrate and a nonionic cellulose derivative; or (c)
a method in which both the first and second enzymes are mixed in
advance with an aqueous solution containing an ionic polymer and a
buffer, and the prepared mixed solution is added to and mixed with
a color reagent substrate and a nonionic cellulose derivative;
[0026] forming a pair of optical elements on a substrate for
introducing light into the substrate and for emitting the light
from the substrate; and
[0027] applying the glucose sensing membrane-forming coating
solution to a substrate area positioned between the optical
elements, followed by drying to form a glucose sensing
membrane.
[0028] According to a fourth aspect of the present invention, there
is provided a method of manufacturing an optical glucose sensor
chip, comprising:
[0029] preparing a glucose sensing membrane-forming coating
solution by using any of: (a) a method in which at least one of a
first enzyme that oxidizes or reduces glucose and a second enzyme
that generates a material which makes a color reagent substrate
develop color by a reaction with a product obtained by oxidation or
reduction of glucose is mixed in advance with an aqueous solution
containing an ionic polymer and a buffer, and the mixed solution is
added to and mixed with the other enzyme, a color reagent substrate
and a nonionic cellulose derivative; (b) a method in which the
first and second enzymes are respectively mixed in advance with an
aqueous solution containing an ionic polymer and a buffer, and the
respective prepared mixed solutions are added to and mixed with a
color reagent substrate and a nonionic cellulose derivative; or (c)
a method in which both the first and second enzymes are mixed in
advance with an aqueous solution containing an ionic polymer and a
buffer, and the prepared mixed solution is added to and mixed with
a color reagent substrate and a nonionic cellulose derivative;
[0030] forming a pair of gratings on a substrate for introducing
light into the substrate and for emitting the light from the
substrate;
[0031] forming a light-reflecting path layer made of a resin having
a higher refractive index than the substrate on a principal surface
of the substrate on which the optical elements are formed; and
[0032] applying the glucose sensing membrane-forming coating
solution to the part of the light-reflecting path layer positioned
between the optical elements, followed by drying to form a glucose
sensing membrane.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING
[0033] FIG. 1 is a sectional view showing a glucose sensor chip
according to a first embodiment;
[0034] FIG. 2 is a sectional view showing a glucose sensor chip
according to a second embodiment; and
[0035] FIG. 3 is a view showing a variation in the absorbance
(sensitivity) of a glucose sensor chip of each of Examples 1 and 2
and Comparative Example 1 with the passage of a storage time.
DETAILED DESCRIPTION OF THE INVENTION
[0036] An optical glucose sensor chip according to an embodiment of
the present invention will be explained in detail with reference to
the drawings.
First Embodiment
[0037] FIG. 1 is a sectional view showing an optical glucose sensor
chip according to a first embodiment.
[0038] A glass substrate 1 is provided with a SiO.sub.2 surface
layer 2 having a thickness of 3 nm or more on its principal
surface. A pair of optical elements, for example, a pair of
gratings 3 is formed in the vicinities of both ends of the
SiO.sub.2 surface layer 2 respectively to introduce light into the
substrate 1 and to emit the light from the inside of the substrate
1. In this case, the optical elements may be substituted with a
prism or the like. These gratings 3 are formed of, for example,
titanium oxide having a higher refractive index than the above
SiO.sub.2 surface layer 2. A protective film having a lower
refractive index than the above gratings 3 may be formed so as to
coat the gratings 3. This protective film is made of a material,
for example, a fluororesin, inert to a chemical solution and a
specimen to be used.
[0039] A glucose sensing membrane 4 is formed at a position between
the gratings 3 on a part of the surface of the SiO.sub.2 surface
layer 2 of the substrate 1. This glucose sensing membrane 4
includes a color reagent substrate, a first enzyme that oxidizes or
reduces glucose, a second enzyme that reacts with a product
obtained by oxidizing or reducing glucose to generate a material
making the color reagent substrate exhibit color, a nonionic
cellulose derivative, and an ionic polymer into which a buffer is
incorporated. In the glucose sensing membrane 4, at least one of
the above first and second enzymes is coated with the ionic polymer
into which the buffer is incorporated. The above color reagent
substrate, first and second enzymes, ionic polymer and buffer are
supported by the above nonionic cellulose derivative.
[0040] Here, the standard for coating at least one of the first and
second enzymes with the ionic polymer into which the buffer is
incorporated, is determined according to the degree of
deterioration with time as explained below.
[0041] Specifically, a product obtained by adding glucose to the
first enzyme to react is made to act on a specified color reagent
substrate, thereby causing the color reagent substrate exhibit
color to measure the absorbance at this time. Then, after this
first enzyme is exposed to an atmosphere of a fixed temperature and
a fixed humidity for a fixed time, a product obtained by adding
glucose to the first enzyme to react is made to act on a specified
color reagent substrate, thereby causing the color reagent
substrate exhibit color to measure the absorbance at this time. The
ratio of absorbance reduction between the former and the latter is
calculated.
[0042] Also, the first and second enzymes are added to glucose and
a material produced by a reaction with a product of the first
enzyme is made to act on a specified color reagent substrate to
cause the color reagent substrate to exhibit color, thereby
measuring the absorbance at this time. After the second enzyme is
exposed to an atmosphere of a fixed temperature and a fixed
humidity for a fixed time in the same manner as in the case of
measuring the absorbance of the first enzyme, this second enzyme is
added to glucose together with the first enzyme and a material
produced by a reaction with a product of the first enzyme is made
to act on a specified color reagent substrate to cause the color
reagent substrate to exhibit color, thereby measuring the
absorbance at this time. The ratio of absorbance reduction between
the former and the latter is calculated.
[0043] The absorbance reduction ratio due to the deterioration of
the first enzyme with time is compared with the absorbance
reduction ratio due to the deterioration of the second enzyme with
time, to select the one having the larger absorbance reduction
ratio, and the selected one is coated with the ionic polymer into
which the buffer is incorporated. Also, when the absolute value of
the absorbance reduction ratio is large in either the first or
second enzyme, it is preferable to coat both with the ionic polymer
into which the buffer is incorporated.
[0044] The enzyme and color reagent substrate in the glucose
sensing membrane 4 are used in the combinations shown in the
following Table 1.
TABLE-US-00001 TABLE 1 First enzyme Second enzyme Color reagent
substrate Oxidizing Glucose Peroxidase
3,3',5,5'-tetramethylbenzidine enzyme oxidase
N,N'-bis(2-hydroxy-3-sulfopropyl)tolidine 3,3'-diaminodenzidine
Hexokinase Glucose-6- 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-
phosphoric acid 2H-tetrazolium bromide dehydrogenase
2-(4-rhodophenyl)-3-(2,4-dinitrophenyl)-5-
(2,4-disulfophenyl)-2H-tetrazolium
3-3'-[3,3'-dimethoxy-(1,1'-biphenyl)-4,4'-
diyl]bis(2,5-diphenyl)-2H-tetrazolium choloride Reducing Glucose
Phosphorus Aminobenzoic acid enzyme dehydrogenase molybdate
[0045] The nonionic cellulose derivative used in the glucose
sensing membrane 4 is a high-molecular compound participating in
the formation of a film. Examples of the nonionic cellulose
derivative may include alkyl celluloses such as methyl cellulose
and ethyl cellulose; hydroxyalkyl celluloses such as hydroxyethyl
cellulose and hydroxypropyl cellulose; hydroxyalkylalkyl celluloses
such as hydroxypropylmethyl cellulose, hydroxypropylethyl
cellulose, hydroxydiethyl cellulose and hydroxyethylmethyl
cellulose; and microfibrous celluloses. These materials may be used
either singly or in the form of a mixture.
[0046] The above ionic polymer has the function of limiting the
precipitation of a salt from the above first and second enzymes
during long-term storage and use. This ionic polymer includes a
positive ionic polymer and a negative ionic polymer. Examples of
the positive ionic polymer include polymers containing a cationic
group such as an amino group, guanidino group or biguanide group.
Specific examples of the positive ionic polymer include
polyallylamine hydrochloride, polyvinylpyridine and polylysine.
Examples of the negative ionic polymer include polymers containing
an anionic group such as phosphates, carboxylates or sulfonates.
Specific examples of the negative ionic polymer include
polystyrenesulfonic acid, polyvinylsulfuric acid, polyaspartic
acid, polyacrylic acid, polymethacrylic acid, polymaleic acid,
polyfumaric acid or cellulose derivatives such as carboxymethyl
cellulose and cellulose acetate. Among these ionic polymers,
negative ionic polymers are preferable.
[0047] The above buffer has the function of controlling the pH and
ionic strength of the first and second enzymes to suppress
variations in the forms and structures of these enzymes during
long-term storage and use. As this buffer, for example, a
phosphoric acid buffer, acetic acid buffer, citric acid buffer,
boric acid buffer, tartaric acid buffer, trishydrochloric acid
buffer or carbonic acid buffer may be used.
[0048] At least one of the first and second enzymes is coated with
the ionic polymer having incorporated therein such a buffer,
whereby not only the precipitation of a salt from the enzyme during
long-term storage and use but also variations in the forms and
structures of these enzymes are limited to keep these enzymes in
highly activated condition.
[0049] The above glucose sensing membrane 4 is permitted to contain
a crosslinking high-molecular compound. Examples of the
crosslinking high-molecular compound may include copolymers of a
hydrophilic monomer having at least one group selected from a
hydroxyl group, carboxyl group, amino group and ionic functional
groups and a hydrophobic monomer. The copolymer of a hydrophilic
monomer and a hydrophobic monomer is preferably a copolymer of
2-methacryloyloxyethylphosphorylcholine and butylacrylate.
[0050] The above crosslinking high-molecular compound is preferably
contained in the above glucose sensing membrane in an amount of
10.sup.-4 to 10% by weight with respect to all the components of
the glucose sensing membrane. When the content of the crosslinking
high-molecular compound is less than 10.sup.-4% by weight with
respect to all the components of the glucose sensing membrane, it
is difficult to prevent the phenomena that the structure of the
film is dissolved and broken under heating condition and that the
color reagent substrate and enzymes retained in voids in the
structure of the film are eluted in an external medium. When the
content of the crosslinking high-molecular compound exceeds 10% by
weight, on the other hand, there is a fear that the amounts of the
color reagent substrate and enzymes in the glucose sensing membrane
are relatively reduced and therefore, the sensitivity of the chip
is lowered.
[0051] The above glucose sensing membrane 4 is permitted to further
contain polyethylene glycol or ethylene glycol that provides water
permeability in voids in the structure of the film. The glucose
sensing membrane 4 containing such polyethylene glycol is increased
in hydrophilic properties and it is therefore possible to raise
reaction sensitivity when water is used as the medium for
introducing glucose.
[0052] Next, the action of the optical glucose sensor chip shown in
FIG. 1 will be explained.
[0053] An adapter (not shown) having a through-hole (well) is
brought into contact with a specimen, for example, the skin of a
human body and the aforementioned sensor chip is attached to the
adapter in such a manner that the glucose sensing membrane 4 is
positioned on the well side. This adapter avoids the direct contact
of the glucose sensing membrane 4 with the specimen to contribute
to the promotion of the reproducibility of the sensing. An
extracting medium (for example, a liquid such as water or
physiological brine, which does not react directly with the
specimen or sensing membrane but has an affinity to them) is filled
in the well of the adapter on the side on which the glucose sensing
membrane is positioned. Glucose in a skin tissue solution is
extracted to the extracting medium from the skin by applying
microvoltage to the specimen from the outside and further
penetrates the sensing membrane 4 from the extracting medium. When
the combination of the first and second enzymes (oxidizing or
reducing enzyme) and the color reagent substrate constituting the
glucose sensing membrane 4 is, for example, a combination of
glucose oxidase (GOD), peroxidase (POD) and 3,3',5,5'-tetramethyl
benzidine (TMBZ) shown in the above Table 1, the glucose that has
penetrated the sensing membrane 4 is decomposed by GOD to generate
hydrogen peroxide, which is then decomposed by POD to emit active
oxygen which causes TMBZ to exhibit color. In other words, the
chromaticity of TMBZ varies according to the amount of glucose.
[0054] In this situation, laser light is made to be incident to the
backside of the substrate 1 from the laser light source 5 (for
example, a laser diode) through a polar screen (not shown). The
incident laser light propagates in the substrate 1 including the
SiO.sub.2 surface layer 2 while it is refracted at an interface
between the SiO.sub.2 surface layer 2 of the substrate 1 and the
grating 3 on the left and also at an interface between the
SiO.sub.2 surface layer 2 and the glucose sensing membrane 4
containing the color reagent substrate which has exhibited color.
At this time, the evanescent wave of the propagated light is
absorbed according to the chromaticity correlated with the amount
of glucose contained in the glucose sensing membrane 4. The light
propagated in the substrate 1 is emitted from the grating 3 on the
right and is received by a light receiving element 6 (for example,
a photodiode). The intensity of the received laser light is lower
than the intensity (initial intensity) of the laser light received
when the color of the sensing membrane 4 is not exhibited. As a
result, it is possible to detect the amount of glucose from the
ratio of intensity reduction of the laser light.
[0055] Next, explanations will be given of a method of
manufacturing the optical glucose sensor chip shown in FIG. 1.
[0056] First, a coating solution for formation of a glucose sensing
membrane is prepared by the following method.
[0057] (1) At least one of a first enzyme that oxidizes or reduces
glucose and a second enzyme that generates a material which makes a
color reagent substrate exhibit color by a reaction with a product
obtained by oxidation or reduction of glucose is mixed in advance
with an aqueous solution containing an ionic polymer and a buffer.
In this mixing process, the above one of the first and second
enzymes is coated with the ionic polymer into which the buffer is
incorporated. In succession, the mixed solution is added to and
mixed with the other enzyme, a color reagent substrate and a
nonionic cellulose derivative to prepare a glucose sensing
membrane-forming coating solution in which the above one enzyme
coated with the ionic polymer is dispersed together with the other
enzyme and color reagent substrate in the nonionic cellulose
derivative that is a film forming high-molecular compound.
[0058] (2) The aforementioned first and second enzymes are
respectively mixed in advance in an aqueous solution containing an
ionic polymer and a buffer. In this mixing process, the first and
second enzymes are coated with the ionic polymer into which the
buffer is incorporated. In succession, the prepared two mixed
solutions are added to and mixed with a color reagent substrate and
a nonionic cellulose derivative to prepare a glucose sensing
membrane-forming coating solution in which the above first and
second enzymes respectively coated with the ionic polymer are
dispersed together with the above color reagent substrate in the
nonionic cellulose derivative that is a film forming high-molecular
compound.
[0059] (3) Both the aforementioned first and second enzymes are
mixed in advance in an aqueous solution containing an ionic polymer
and a buffer. In this mixing process, the first and second enzymes
are coated with the ionic polymer into which the buffer is
incorporated. In succession, the prepared mixed solution is added
to and mixed with a color reagent substrate and a nonionic
cellulose derivative to prepare a glucose sensing membrane-forming
coating solution in which the above first and second enzymes coated
with the ionic polymer are dispersed together with the above color
reagent substrate in the nonionic cellulose derivative that is a
film forming high-molecular compound.
[0060] Then, a pair of optical elements, for example, gratings, is
formed on the substrate. The pair of gratings is formed by the
formation of a titanium oxide film on the substrate and by
patterning. Successively, the foregoing glucose sensing
membrane-forming coating solution is applied to a substrate area
positioned between the gratings, followed by drying to form a
glucose sensing membrane, thereby manufacturing an optical glucose
sensor chip.
[0061] As mentioned above, when detecting the amount of glucose by
using the optical glucose sensor chip of the first embodiment, at
least one of the first and second enzymes in the glucose sensing
membrane is coated with the ionic polymer into which the buffer is
incorporated. For this reason, during long-term storage and use,
the precipitation of a salt from the enzyme can be suppressed and
variations in the shape and structure of the enzyme are suppressed
to keep the enzyme in highly activated conditions. As a result, it
is possible to provide an optical glucose sensor chip which can
detect glucose amount in a specimen highly sensitively and stably
for a long time.
[0062] Also, according to the method of the first embodiment, a
glucose sensing membrane-forming coating solution can be prepared
in which at least one of the first and second enzymes is coated
with the ionic polymer into which the buffer is incorporated and
dispersed together with the color reagent substrate in the nonionic
cellulose derivative that is a film forming high-molecular
compound, by using any of the following methods: (a) at least one
of the first enzyme that oxidizes or reduces glucose and the second
enzyme that generates a material which makes the color reagent
substrate develop color by a reaction with a product obtained by
oxidation or reduction of glucose is mixed in advance with an
aqueous solution containing the ionic polymer and the buffer, and
the mixed solution is added to and mixed with the other enzyme, the
color reagent substrate and the nonionic cellulose derivative; (b)
the aforementioned first and second enzymes are respectively mixed
in advance with an aqueous solution containing the ionic polymer
and the buffer, and the respective prepared mixed solutions are
added to and mixed with the color reagent substrate and the
nonionic cellulose derivative; or (c) both the aforementioned first
and second enzymes are mixed in advance with an aqueous solution
containing the ionic polymer and the buffer, and the prepared mixed
solution is added to and mixed with the color reagent substrate and
the nonionic cellulose derivative. Thereafter, a pair of gratings
is formed on the substrate and the foregoing glucose sensing
membrane-forming coating solution is applied to a substrate area
positioned between the gratings, followed by drying to form a
glucose sensing membrane, thereby manufacturing an optical glucose
sensor chip which can detect glucose amount in a specimen highly
sensitively and stably for a long time.
Second Embodiment
[0063] FIG. 2 is a sectional view showing an optical glucose sensor
chip according to a second embodiment.
[0064] A pair of gratings 12 which are optical elements is formed
on the principal surface of a glass substrate 11 in the vicinities
of both ends of the substrate 11 to introduce light into the
substrate 11 and to emit the light from the substrate 11,
respectively. These gratings 12 are made of, for example, titanium
oxide having a higher refractive index than the above substrate 11.
A light-reflecting path layer 13 formed of a heatcurable or
photocurable resin having a higher refractive index than the
substrate 11 is formed on the principal surface of the substrate 11
including the gratings 12. The principal surface of the
light-reflecting path layer 13 is formed in parallel to the
principal surface of the substrate 11 including the gratings
12.
[0065] A glucose sensing membrane 14 is formed on the part above
the light-reflecting path layer 13 positioned between the gratings
12. This glucose sensing membrane 14 has the same structure as that
of the first embodiment, that is, the structure in which at least
one of a first enzyme that oxidizes or reduces glucose and a second
enzyme that generates a material which makes a color reagent
substrate exhibit color by a reaction with a product obtained by
oxidation or reduction of glucose is coated with an ionic polymer
into which a buffer is incorporated, and these enzymes, ionic
polymer, buffer and color reagent substrate are supported by the
nonionic cellulose derivative.
[0066] Here, the standard for coating at least one of the first and
second enzymes with the ionic polymer into which the buffer is
incorporated, is the same as that explained in the above first
embodiment.
[0067] The above light-reflecting path layer 13 preferably has a
smooth surface and a thickness of 10 .mu.m or more and more
preferably 10 to 200 .mu.m. A light-reflecting path layer having a
thickness of 10 .mu.m or more makes it possible to limit a decay of
light intensity when light is propagated and to use, for example, a
LED light source in addition to a laser light.
[0068] The first and second enzymes and color reagent substrate in
the above glucose sensing membrane 14 are used in the combinations
shown in the above Table 1.
[0069] As the ionic polymer, buffer and nonionic cellulose
derivative in the glucose sensing membrane 14, the same ones
exemplified in the above first embodiment may be used.
[0070] The above glucose sensing membrane 14 may further contain a
crosslinking high-molecular compound and may also contain
polyethylene glycol or ethylene glycol as explained in the above
first embodiment.
[0071] Next, the action of the optical glucose sensor chip shown in
FIG. 2 will be explained.
[0072] An adapter (not shown) having a through-hole (well) is
brought into contact with a specimen, for example, the skin of a
human body and the aforementioned sensor chip is attached to the
adapter in such a manner that the glucose sensing membrane 14 is
positioned on the well side. An extracting medium including water
is filled in the well of the adapter on the side on which the
glucose sensing membrane is positioned. Glucose in a skin tissue
solution is extracted to the extracting medium from the skin by
applying microvoltage to the specimen from the outside and further
penetrates the sensing membrane 14. When the combination of the
first and second enzymes (oxidizing or reducing enzyme) and the
color reagent substrate constituting the glucose sensing membrane
14 is, for example, a combination of glucose oxidase (GOD),
peroxidase (POD) and 3,3',5,5'-tetramethyl benzidine (TMBZ) shown
in the above Table 1, the glucose that has penetrated the sensing
membrane 14 is decomposed by GOD to generate hydrogen peroxide,
which is then decomposed by POD to emit active oxygen which causes
TMBZ to exhibit color. In other words, the chromaticity of TMBZ
varies according to the amount of glucose.
[0073] In this situation, laser light is made to be incident to the
backside of the substrate 11 from the laser light source 15 (for
example, a laser diode) through a polar screen (not shown). The
laser light passes through the substrate 11 and is refracted at an
interface between the principal surface of the substrate 11 and the
grating 12 on the left, whereby the light is incident to an optical
waveguide layer 13. The light is also refracted at an interface
between the optical waveguide layer 13 and the glucose sensing
membrane 14 containing the color reagent substrate which has
exhibited color to propagate in the optical waveguide layer 13. At
this time, the evanescent wave of the propagated light is absorbed
according to the chromaticity correlated with the amount of glucose
contained in the glucose sensing membrane 14. The light propagated
in the optical waveguide layer 13 is emitted from the grating 12 on
the right and is received by a light receiving element 16 (for
example, a photodiode). The intensity of the received laser light
is lower than the intensity (initial intensity) of the laser light
received when the color of the sensing membrane 14 is not
exhibited. As a result, it is possible to detect the amount of
glucose from the ratio of intensity reduction of the laser
light.
[0074] Next, explanations will be given of a method of
manufacturing the optical glucose sensor chip shown in FIG. 2.
[0075] First, by using any of the same three methods that are
explained in the first embodiment, a coating solution for formation
of a glucose sensing membrane is prepared in which at least one of
the first and second enzymes is coated in advance with the ionic
polymer into which the buffer is incorporated and dispersed
together with the color reagent substrate in the nonionic cellulose
derivative that is a film forming high-molecular compound.
[0076] Then, a pair of optical elements, for example, gratings is
formed on the principal surface of the glass substrate. The pair of
gratings is formed by the formation of a titanium oxide film on the
glass substrate and by patterning. Successively, a light-reflecting
path layer made of a heatcurable or photocurable resin having a
higher refractive index than the substrate is formed on the
principal surface of the substrate on the side formed with the
grating. Then, the foregoing glucose sensing membrane-forming
coating solution is applied to the part of the light-reflecting
path layer positioned between the gratings, followed by drying to
form a glucose sensing membrane, thereby manufacturing an optical
glucose sensor chip.
[0077] As mentioned above, when detecting the amount of glucose by
using the optical glucose sensor chip of the second embodiment, at
least one of the first and second enzymes in the glucose sensing
membrane is coated with the ionic polymer into which the buffer is
incorporated in the same manner as in the first embodiment. For
this reason, during long-term storage and use, the precipitation of
a salt from the enzyme can be suppressed and variations in the
shape and structure of the enzyme are suppressed to keep the enzyme
in highly activated conditions. As a result, it is possible to
provide an optical glucose sensor chip which can detect glucose
amount in a specimen highly sensitively and stably for a long
time.
[0078] Also, according to the second embodiment, a glucose sensing
membrane-forming coating solution can be prepared in which at least
one of the first and second enzymes is coated with the ionic
polymer into which the buffer is incorporated and dispersed
together with the color reagent substrate in the nonionic cellulose
derivative that is a film forming high-molecular compound.
Thereafter, a pair of gratings is formed on the principal surface
of the glass substrate, a light-reflecting path layer having a
higher refractive index than the substrate is formed on the
principal surface of the substrate on the side formed with the
gratings, and the foregoing glucose sensing membrane-forming
coating solution is applied to between the gratings above the
light-reflecting path layer, followed by drying to form a glucose
sensing membrane, thereby manufacturing an optical glucose sensor
chip which can detect glucose amount in a specimen highly
sensitively and stably for a long time.
[0079] The present invention will be explained by way of
examples.
EXAMPLE 1
[0080] Nine .mu.L of a mixture solution of a 0.67 mg/mL peroxidase
(POD) solution (dissolved in a 0.01 mol/L phosphoric acid buffer
solution [pH: 6.0]) and a 5.33 mg/mL glucose oxidase (GOD) solution
(dissolved in a 0.01 mol/L phosphoric acid buffer solution [pH:
6.0]) was mixed in 1 .mu.L of an aqueous 1 wt % carboxymethyl
cellulose (CMC) (negative ionic polymer) solution and the mixture
was stirred. Nine .mu.L of the obtained mixture solution was added
to 143.6 .mu.L of isopropyl alcohol (IPA), 116.6 .mu.L of purified
water, 6 .mu.L of an isopropyl alcohol solution containing 1% by
volume of polyethylene glycol (PEG), 60 .mu.L of an isopropyl
alcohol solution containing 1 mg/mL of
3,3',5,5'-tetramethylbenzidine (TMBZ), 64 .mu.L of an aqueous 2 wt
% hydroxyethyl cellulose (HEC) solution and 0.8 .mu.L of an aqueous
crosslinking high-molecular compound
(2-methacryloyloxyethylphosphorylcholine/butylmethacrylate
copolymer) solution and these components were mixed and stirred to
prepare 400 .mu.L of a glucose sensing membrane-forming coating
solution.
[0081] Next, a non-alkali glass substrate which was provided with a
SiO.sub.2 surface layer having a thickness of 10 nm on its
principal surface and had a refractive index of 1.52 was prepared.
A titanium oxide film having a thickness of 50 nm and a refractive
index of 2.2 to 2.4 was formed on the SiO.sub.2 surface layer of
this substrate by sputtering. In succession, a resist was applied
to the titanium oxide film and dried, and then a resist pattern was
formed by lithography. Then, by using the resist pattern as a mask,
the titanium oxide film was selectively removed by reactive ion
etching (RIE), thereby forming gratings on the SiO.sub.2 surface
layer in the vicinity of each end of the surface. Then, the resist
pattern was removed by ashing.
[0082] Next, the above substrate was dry-cleaned by oxygen RIE and
then cut into a chip form having a size of 17 mm.times.6.5 mm.
Then, 8 .mu.L of the above glucose sensing membrane-forming coating
solution was dripped on the surface of a sensing membrane forming
area positioned between the gratings of the substrate, and dried by
purging using inert gas and vacuum drying, thereby forming a porous
(water permeable) glucose sensing membrane 0.8 .mu.m in thickness.
In this manner, an optical glucose sensor chip shown in the above
FIG. 1 was manufactured. A liquid droplet of the glucose sensing
membrane-forming coating solution to be dripped had the following
composition. [0083] Phosphoric acid buffer solution: 0.000525 mol/L
[0084] Phosphoric acid buffer solution: 0.0003 mol/L [0085] PEG:
0.15% by volume [0086] TMBZ: 0.15 mg/dL [0087] POD: 0.0015 mg/mL
[0088] GOD: 0.012 mg/mL [0089] CMC (negative ionic polymer):
0.0005% by weight [0090] HEC: 0.64% by weight [0091]
2-Methacryloyloxyethylphosphorylcholine and butylmethacrylate
copolymer: 0.002% by weight
EXAMPLE 2
[0092] An optical glucose sensor chip shown in the above FIG. 1 was
produced by forming a sensing membrane in the same manner as in
Example 1 except that polylysine as a positive ionic polymer was
blended in the droplet of the glucose sensing membrane-forming
coating solution in an amount of 0.0008 .mu.g/L in place of CMC as
a negative ionic polymer in Example 1.
COMPARATIVE EXAMPLE 1
[0093] An optical glucose sensor chip shown in the above FIG. 1 was
produced by forming a sensing membrane in the same manner as in
Example 1 by using a glucose sensing membrane-forming coating
solution prepared by one mixing operation using the same components
as in Example 1 except that the ionic polymer and the sodium
phosphate buffer were not contained.
[0094] With regard to the optical glucose sensor chips obtained in
Examples 1 and 2 and Comparative Example 1, a variation in
sensitivity (absorbance) to glucose with time was measured using
the following method.
[0095] Specifically, an adapter having a through-hole (well) was
brought into contact with a proper flat plate (for example, a glass
plate). Each sensor chip was attached to this adapter in such a
manner that the glucose sensing membrane was disposed on the well
side to partition the well. In the situation (temperature:
35.degree. C.) where an aqueous solution containing 1 mg/dL of
glucose was filled in the well, laser light was made to be incident
to the backside of the substrate 1 from a laser diode 5 through a
polar screen as shown in FIG. 1. The incident laser light was
refracted at an interface between the SiO.sub.2 surface layer 2 of
the substrate 1 and the grating 3 on the left, and also refracted
at an interface between the SiO.sub.2 surface layer 2 and the
glucose sensing membrane 4 containing the color reagent substrate
which had exhibited color to propagate in the substrate 1 including
the SiO.sub.2 surface layer 2. The laser light propagated by the
refraction at an interface between the grating 3 on the right and
the substrate 1 was received by a photodiode 6 to measure the
intensity (absorbance) of the received laser light.
[0096] The same operations as above were carried out after the
sensor chip had been stored for one day, 14 days, 40 days and 100
days in the case of the sensor chips of Examples 1 and 2
respectively and after the sensor chip had been stored for one day,
7 days and 90 days in the case of the sensor chip of Comparative
Example 1.
[0097] The results of these operations are shown in FIG. 3.
[0098] As can be understood from FIG. 3, the sensitivity of the
sensor chip of Comparative Example 1 deteriorates more after the
chip has been stored for 7 days and more significantly after the
chip has been stored for 90 days compared with the sensitivity
obtained before the sensor is stored.
[0099] It can be understood, on the other hand, that each sensor
chip of Examples 1 and 2 has a sensitivity similar to that obtained
before the sensor chip is stored even after the sensor chip has
been stored for 100 days. It is found that, particularly, the
sensor chip of Example 1 provided with a glucose sensing membrane
containing a negative ionic polymer has a higher capability of
maintaining sensitivity with the passage of storage time than that
of Example 2 provided with a glucose sensing membrane containing a
positive ionic polymer.
[0100] Similarly to Example 1, the glucose sensor chip shown in
FIG. 2 provided with a light-reflecting path layer made of a
heatcurable or photocurable resin having a higher refractive index
than the substrate could maintain high sensitivity even after being
stored for a long time.
[0101] Also, as each of the first enzyme, second enzyme and color
reagent substrate supported by the glucose sensing membrane used in
the above embodiments and examples, only one material is selected.
However, plural materials may be combined according to the purpose
of use.
[0102] Moreover, glass is used as the substrate in the above
embodiment. However, no restriction is imposed on this material as
long as it has the characteristics allowing reference light to
propagate and be transmitted. Film bodies of single crystals and
various resin materials may also be used, such as heatcurable resin
materials, thermoplastic resin materials and photocurable resin
materials.
[0103] Additional advantages and modifications will readily occur
to those skilled in the art. Therefore, the invention in its
broader aspects is not limited to the specific details and
representative embodiments shown and described herein. Accordingly,
various modifications may be made without departing from the spirit
or scope of the general inventive concept as defined by the
appended claims and their equivalents.
* * * * *