CN1828278A - Optical Glucose Sensor Chip - Google Patents

Abstract

Provided is an optical glucose sensor chip which can maintain good diffusion of glucose and can inhibit elution of an enzyme or the like from a sensor film even at a temperature of at least 40 ℃. The glucose sensor is characterized by comprising a glass substrate, a pair of optical elements formed on the main surface of the glass substrate, which emit light to the outside of the glass substrate when light enters the substrate, and a glucose sensor film formed on the main surface of the substrate forming the optical elements, wherein a color-developing agent, a 1 st enzyme that oxidizes or reduces glucose, and a 2 nd enzyme that generates a substance that develops a color of the color-developing agent by a reaction with a product of the enzyme are held on a thin film formed of a film-forming polymer compound and a crosslinkable polymer compound.

Description

Optical Glucose Sensor Chip

technical field

The present invention relates to an optical glucose sensing chip.

Background technique

As an optical glucose sensor chip, for example, a low-invasive blood glucose measurement chip that indirectly checks blood glucose levels by extracting body fluid from subcutaneous tissue has been developed. The sensor chip has a structure with the following components: a glass substrate, and a pair of gratings formed on the surface of the substrate for incident and emission of light in the substrate, and a pair of gratings formed on the surface of the substrate between the gratings. Glucose sensing membrane. The glucose sensing membrane contains a chromogen (such as 3,3', 5,5'-tetramethylbenzidine (TMBZ)), an enzyme that oxidizes or reduces glucose (such as glucose oxidase (GOD)), and the The product formed by the first enzyme reacts to produce a second enzyme (such as peroxidase (POD)) that produces a color-developing substance and a film-forming polymer compound (such as cellulose such as carboxymethylcellulose (CMC) derivative).

In the glucose sensor chip having such a structure, when a film-like gel is placed between the skin and the sensor membrane and an electric field is applied, glucose in the subcutaneous tissue fluid passes through the gel from the skin to the sensor membrane. At this time, TMBZ as a color developing agent in the above-mentioned sensing membrane develops color due to the reaction of glucose, GOD, and POD. In this state, when light is incident on the above-mentioned substrate, and the light is refracted by the surface of the substrate and the above-mentioned grating, the light will propagate at the interface between the above-mentioned substrate and the sensor film containing the colored TMBZ, and pass through the substrate and the sensor film. The interface of another grating is refracted, for example by a photodetector. The intensity of the laser light after receiving the light becomes lower than the light intensity (initial intensity) received by the photodetector at the time of non-color development by the color development of the chromogenic agent of the glucose sensing membrane. According to the decrease rate Measure the concentration of the above glucose.

As a method for allowing glucose in the subcutaneous tissue fluid to reach the sensor membrane from the skin, reverse ion tophoresis can also be considered. This counter iontophoresis method is a method as follows: an adapter having a through hole (well) is connected to the skin, a sensing chip is mounted on the adapter so that the sensing film is located on the side of the well, The interior is filled with water-containing extraction medium, and a microvoltage is applied from the outside to extract the glucose in the subcutaneous tissue fluid from the skin using the extracted medium, and further make the glucose reach the above-mentioned sensing membrane to detect the amount of glucose. This counter iontophoresis method has the following problems due to the use of an aqueous extraction medium.

That is, the sensing membrane of the above-mentioned glucose sensor chip contains a film-forming polymer compound such as CMC, which is high molecular weight and poorly soluble in water, as a binder. Inhibition while maintaining the sensitivity of the chip. However, if the above-mentioned extraction medium is heated, the dissolution of the sensing membrane is promoted, so that the chromogenic agent or enzyme is eluted from the sensing membrane, and there is a problem that the sensitivity of the chip decreases.

Contents of the invention

The object of the present invention is to provide an optical glucose sensor chip that maintains good glucose diffusion and can suppress the elution of color reagents or enzymes from the sensor membrane at least at a temperature of 40°C.

According to the present invention, there is provided an optical glucose sensor chip, which is characterized in that it includes a substrate, a pair of optical components formed on the main surface of the substrate, inject light into the interior of the substrate, and radiate light to the outside of the substrate. Formed on the main surface of the above-mentioned substrate between the above-mentioned optical components, the first enzyme that holds the color developing agent and the first enzyme that oxidizes or reduces glucose on the film formed by the film-forming polymer compound and the cross-linkable polymer compound, and the above-mentioned The reaction of the product of the enzyme produces the second enzyme optical glucose sensing membrane that produces the substance that causes the above-mentioned chromogen to develop color.

According to the present invention, there is provided an optical glucose sensor chip, characterized by comprising a glass substrate, and a pair of optical sensors formed on the main surface of the glass substrate for injecting light into the glass substrate and emitting light to the outside of the glass substrate. components, formed on the main surface of the above-mentioned substrate on which the above-mentioned optical components are formed, a light reflection path layer made of a resin with a higher refractive index than the above-mentioned substrate, and formed between the above-mentioned optical components on the above-mentioned reflection path layer, The film formed by the film-forming polymer compound and the cross-linking polymer compound holds a color-developing agent and a first enzyme that oxidizes or reduces glucose, and reacts with the product of the above-mentioned enzyme to produce the color-developing agent. Substance 2 Enzyme Glucose Sensing Membrane.

According to the present invention, it is possible to provide an optical glucose sensor chip capable of quantitatively detecting the amount of glucose in a sample while maintaining good diffusion of glucose and suppressing dissolution of the sensing membrane.

Brief description of the drawings

[ Fig. 1] Fig. 1 is a cross-sectional view showing a glucose sensor chip according to Embodiment 1.

[ Fig. 2] Fig. 2 is a cross-sectional view showing a glucose sensor chip according to Embodiment 2.

[ Fig. 3] Fig. 3 is a graph showing the measurement sensitivity of the glucose sensor chip of Example 1 at different glucose amounts at 25°C and 37°C.

[ Fig. 4] Fig. 4 is a graph showing the measurement sensitivity of each glucose sensor chip in Example 2 with respect to the change in NaCl concentration.

【Symbol Description】

1, 11... glass substrate 2... SiO ₂ surface layer, 3, 12... grating, 4, 14... glucose sensing film, 5, 15... laser source (laser diode), 6, 16... Photodetectors (Light Emitting Diodes).

Detailed ways

The optical glucose sensor chip according to the present invention will be described in detail below with reference to the accompanying drawings.

(Embodiment 1)

FIG. 1 is a cross-sectional view showing an optical glucose sensor chip according to Embodiment 1. FIG.

The glass substrate 1 has, for example, 3 nm or more on the main surface. Thick SiO ₂ surface layer 2. The optical components are respectively formed on the surface near both edges of the above-mentioned SiO ₂ surface layer 2 to inject light into the substrate 1 or to emit light from the substrate 1, and a pair of gratings 3 are used. In addition, optical components can also be replaced by prisms. These gratings 3 consist of titanium oxide, for example, which has a higher refractive index than the above-mentioned SiO ₂ surface layer 2 . A protective film having a lower refractive index than the above grating 3 is formed to cover the above grating 3 . The material of the protective film is made of, for example, a fluorine-based resin that does not react with the chemical solution or sample used.

A glucose sensing film 4 is formed on the SiO ₂ surface layer 2 of the above-mentioned substrate 1 between the above-mentioned gratings 3 . The glucose sensing film 4 is composed of a thin film body formed of a film-forming polymer compound and a cross-linkable polymer compound, and the thin film holds a color developing agent, a first enzyme for oxidizing or reducing glucose, and a product produced by the enzyme. The second enzyme that reacts to produce a substance that causes the chromogenic agent to develop color remains active.

The enzymes and chromogenic reagents in the above-mentioned glucose sensing membrane 4 can be used in combinations shown in Table 1 below, for example.

Table 1 Glucose A reagent that produces a substance that causes a chromogenic agent to develop color Reagent Oxidase glucose oxidase Peroxidase 3,3',5,5'-Tetramethylbenzidine N,N'-bis(2-hydroxy-3-sulfopropyl)benzylidine 3,3'-Diaminobenzidine Hexokinase Glucose-6-phosphate dehydrogenase 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide 2-(4-Crimson phenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazole 3,3'-[3,3'-Dimethoxy-(1,1'-biphenyl)-4,4'-diyl]bis(2,5-diphenyl)-2H-chloride tetrazole Reductase glucose dehydrogenase Phosphomolybdic acid Anthranilate

Examples of the film-forming polymer compound used for the glucose sensor membrane 4 include cellulose-based polymer compounds. As the cellulosic polymer compound, an ionic cellulose derivative or a nonionic cellulose derivative can be used.

Ionic cellulose derivatives include, for example, anionic cellulose derivatives such as carboxymethyl cellulose, cellulose sulfate or salt compounds thereof, and cationic cellulose derivatives such as chitin and chitosan, or salts thereof. Salt compounds such as acid salts and the like can be used alone or in admixture. Among these, sodium salt, potassium salt, etc. are mentioned as a salt compound.

Nonionic cellulose derivatives, for example, alkyl celluloses such as methyl cellulose and ethyl cellulose; hydroxyalkyl celluloses such as hydroxyethyl cellulose and hydroxypropyl cellulose; hydroxypropyl methylcellulose; Hydroxyalkylalkylcellulose such as hydroxypropylethylcellulose, hydroxypropylethylcellulose, hydroxydiethylcellulose, and hydroxyethylmethylcellulose; and microfibrillated cellulose, etc., these substances can be used alone, Or use it in a mixture.

As the crosslinkable polymer compound used in the above-mentioned glucose sensing membrane 4, for example, hydrophilic monomers and hydrophobic monomers having at least one group selected from hydroxyl, carboxyl, amino, and ionic functional groups can be cited. copolymer. It is confirmed through experiments that the copolymer of the hydrophilic monomer and the hydrophobic monomer is particularly preferably a copolymer of 2-methacryloyloxyethylphosphorylcholine and butyl methacrylate.

In the above-mentioned glucose sensing membrane, the above-mentioned cross-linkable polymer compound is preferably contained in an amount of 10 ⁻⁴ to 10% by weight in a weight ratio relative to the entire composition of the glucose sensing membrane. If the content of the crosslinkable polymer compound is less than ^10-4 % by weight based on the weight ratio of the entire composition, it is difficult to prevent the film structure of the film from being dissolved and broken in a heated state, or to prevent voids remaining in the film structure. Chromogenic reagent or enzyme spilled to the external medium. On the other hand, if the content of the crosslinkable polymer compound exceeds 10% by weight, the amount of the chromogenic agent or enzyme in the glucose sensing membrane may be relatively reduced, thereby possibly reducing the sensitivity of the chip.

The glucose sensing membrane 4 described above is allowed to further contain polyethylene glycol or ethylene glycol that imparts water permeability in the pores of the membrane structure. In this way, the hydrophilicity is improved, and when water is used as a medium for introducing glucose, the reaction sensitivity is improved.

Next, the operation of the optical glucose sensor chip shown in FIG. 1 above will be described.

An adapter (not shown) having a through hole is connected to a sample, such as human skin, and the sensor chip is mounted on the adapter so that the glucose sensing membrane 4 is located on the side of the through hole. The adapter prevents the glucose sensing membrane 4 from directly contacting the sample, which helps to improve the reproducibility of the sensor. In the space formed in this way, the above-mentioned through hole is filled with extraction medium (such as liquids such as water, physiological saline, which do not directly react with the sample or the sensing membrane, well known), and by applying a microvoltage to the sample from the outside, the liquid in the subcutaneous tissue fluid Glucose is extracted from the skin to the extraction medium, and further penetrates into the above-mentioned sensing membrane 4 from the extraction medium. When the combination of the enzyme (oxidation or reductase) and the chromogenic agent constituting the glucose sensing membrane 4 is, for example, glucose oxidase (GOD), peroxidase (POD) and 3, 3', 5 shown in Table 1 above, , in the case of 5'-tetramethylbenzidine (TMBZ), the glucose infiltrated into the sensing membrane 4 is decomposed by GOD to generate hydrogen peroxide, and the hydrogen peroxide is decomposed by POD to release active oxygen, and the active oxygen is used to make TMBZ color. That is, the degree of color development of TMBZ changes according to the amount of glucose.

In such a state, through a polarizing filter not shown, laser light is incident on the back surface of the above-mentioned substrate 1 from the above-mentioned laser source 5 (such as a laser diode), and the laser light passes through the _SiO2 surface layer 2 of the substrate 1 and the left grating. The interface refraction of 3 further passes through the interface refraction between the SiO ₂ surface layer 2 and the glucose sensing film 4 containing the colored developer, and propagates on the substrate 1 containing the SiO ₂ surface layer 2 . At this time, evanescent waves (evanescent waves) of propagating light are absorbed in accordance with the degree of color development based on the amount of glucose in the glucose sensor membrane 4 described above. The light propagating on the substrate 1 is emitted by the right grating 12 and received by a photodetector (such as a photodiode) 6 . The received laser light intensity is lower than the received light intensity (initial intensity) when the sensor film 4 does not develop color, and the glucose amount can be detected based on this decrease rate.

In the process of detecting the amount of glucose using the optical glucose sensor chip of Embodiment 1, the glucose sensing membrane 4 contains a cross-linked polymer compound and has high resistance to membrane dissolution, so that the glucose in the subcutaneous tissue fluid is extracted from the skin to When the water-containing extraction medium further penetrates into the glucose sensing membrane 4, even if heated (for example, about 36° C.) water penetrates into the sensing membrane 4 together with glucose, it will not dissolve, and the dissolution of enzymes, etc. in the membrane can be inhibited. .

In particular, as a crosslinkable polymer compound, by using, for example, a copolymer of a hydrophilic monomer and a hydrophobic monomer having at least one group selected from hydroxyl, carboxyl, amino, and ionic functional groups, the above-mentioned hydrophilicity can be utilized. The monomer can improve water retention performance and maintain high water permeability in the glucose sensing membrane, and high resistance to membrane dissolution can be imparted by the hydrophobic monomer. Therefore, in the above-mentioned sensor membrane, the presence of the above-mentioned glucose enables sufficient color development and good sensitivity to be maintained, and at the same time, the dissolution of the membrane structure, the dissolution of the support under heating, and the elution of enzymes and the like can be more reliably suppressed.

Therefore, according to Embodiment 1, it is possible to provide an optical glucose sensor chip capable of detecting the amount of glucose in a sample with high sensitivity even after a long period of time in a heated state.

(Embodiment 2)

FIG. 2 is a cross-sectional view showing an optical glucose sensor chip according to Embodiment 2. FIG.

A pair of gratings 12 serving as optical components are respectively formed near both edges of the main surface of the glass substrate 11 in order to enter and emit light on the glass substrate 11 . These gratings 12 are made of, for example, titanium oxide having a higher refractive index than the above-mentioned substrate 11 . On the main surface of the substrate 11 including the grating 12, a light reflection path layer 13 made of a thermosetting or photosetting resin having a higher refractive index than the substrate 11 is formed. The light reflection path layer 13 is formed such that its main surface is parallel to the main surface of the above-mentioned substrate 11 including the above-mentioned grating 12 . Glucose sensing film 14 is formed on the part of the above-mentioned light reflection path layer 13 corresponding to the space between the above-mentioned gratings 12 . The glucose sensing membrane 14 contains an enzyme that oxidizes or reduces glucose, an enzyme that reacts with a product produced by the enzyme to produce a substance that causes the color developer to develop color, a color developer, a film-forming polymer compound, and a crosslinkable polymer compound. .

The above-mentioned light reflection path layer 13 has a smooth surface, preferably has a thickness of 10 μm or more, and more preferably has a thickness of 10 to 200 μm. The light reflection passage layer having a thickness of 10 μm or more can suppress attenuation of light intensity during light propagation, for example, an LED light source may be used in addition to a laser light source.

The enzymes and color-developing agents in the above-mentioned glucose sensor membrane 14 can be used in combinations shown in Table 1 above, for example.

Examples of the film-forming polymer compound in the glucose sensor membrane 14 include cellulosic polymer compounds such as carboxymethylcellulose and hydroxycellulose. It can be confirmed through experiments that the copolymer of the hydrophilic monomer and the hydrophobic monomer is particularly preferably a copolymer of 2-methacryloyloxyethylphosphorylcholine and butyl methacrylate.

Examples of the crosslinkable polymer compound in the above-mentioned glucose sensing membrane 14 include hydrophilic monomers having at least one group selected from the group consisting of hydroxyl, carboxyl, amino, and ionic functional groups and Copolymers of hydrophobic monomers.

For the reasons described in Embodiment 1, the glucose sensing membrane preferably contains 10 ⁻⁴ to 10% by weight of the crosslinkable polymer compound.

The above-mentioned glucose sensing membrane 4 may further contain polyethylene glycol for imparting water permeability.

Next, the operation of the optical glucose sensor chip shown in FIG. 2 above will be described.

An adapter (not shown) having a through hole is connected to a sample, such as human skin, and the sensor chip is mounted on the adapter so that the glucose sensing membrane 14 is located on the side of the through hole. The above-mentioned through hole is filled with an extraction medium containing water, and by applying a microvoltage from the outside, the glucose in the subcutaneous tissue fluid is extracted from the skin to the medium, and further penetrates into the above-mentioned sensing membrane 14 . When the combination of the enzyme (oxidizing or reductase) and the color developing agent constituting the glucose sensing membrane 14 is, for example, glucose oxidase (GOD), peroxidase (POD) and 3, 3', 5, In the case of 5'-tetramethylbenzidine (TMBZ), the glucose permeated into the sensing membrane 14 is decomposed by GOD to generate hydrogen peroxide, and the hydrogen peroxide is decomposed by POD to release active oxygen, and TMBZ is displayed by the active oxygen. color. That is, the degree of color development of TMBZ changes according to the amount of glucose.

In such a state, through a polarizing filter not shown, laser light is incident on the back surface of the above-mentioned substrate 11 from the above-mentioned laser source 15 (such as a laser diode), and the laser light passes through the substrate 11 and is captured by the main surface and the left grating. 12 is refracted at the interface, enters the light guiding layer 13 , is further refracted at the interface between the light guiding layer 13 and the glucose sensing film 14 containing the colored developer, and propagates on the light guiding layer 13 . At this time, the evanescent wave of the propagating light is absorbed according to the degree of coloration based on the amount of glucose in the glucose sensor membrane 14 described above. The light propagating on the above-mentioned photoconductive layer 13 is emitted by the right grating 12 and received by a photodetector (such as a photodiode) 16 . The received laser light intensity is lower than the received light intensity (initial intensity) when the sensor film 14 is not developing color, and the glucose amount can be detected based on this drop rate.

In the process of detecting the amount of glucose using the optical glucose sensor chip of Embodiment 2, the glucose sensing membrane 14 contains a cross-linked polymer compound and has high resistance to membrane dissolution, so that the glucose in the subcutaneous tissue fluid is extracted from the skin to the When the water-containing extraction medium further penetrates into the glucose sensing membrane 14, even if heated (for example, about 36° C.) water penetrates into the sensing membrane 14 together with the glucose, the membrane structure of the film will not dissolve, and the retention in the membrane can be suppressed. Dissolution of enzymes, etc.

In particular, as a crosslinkable polymer compound, by using, for example, a copolymer of a hydrophilic monomer and a hydrophobic monomer having at least one group selected from hydroxyl, carboxyl, amino, and ionic functional groups, the above-mentioned hydrophilicity can be utilized. The monomer can improve water retention performance and maintain high water permeability in the glucose sensing membrane, and high resistance to membrane dissolution can be imparted by the hydrophobic monomer. Therefore, in the above-mentioned sensor membrane, by the presence of the above-mentioned glucose, sufficient color development and good sensitivity can be maintained, and the rupture of the membrane structure of the thin film in a heated state and the loss of the color-developing agent or enzyme supported by the thin film can be more reliably suppressed. Dissolution.

Therefore, according to Embodiment 2, it is possible to provide an optical glucose sensor chip capable of detecting the amount of glucose in a sample with high sensitivity even after a long period of time in a heated state.

In Embodiments 1 and 2 above, it is useful to use a nonionic cellulose derivative such as hydroxyethyl cellulose as the film-forming polymer compound mixed in the sensor film.

That is, if an ionic cellulose derivative such as carboxymethyl cellulose is mixed as a film-forming polymer compound in the sensing membrane, the physical properties such as viscosity will fluctuate as the salt concentration of the extraction medium changes, so the glucose in the sample The detection sensitivity of the amount changes. For the above-mentioned nonionic cellulose derivatives, even if the salt concentration of the extraction medium changes, the physical properties such as viscosity do not change. Therefore, it is possible to design the detection sensitivity that does not show the amount of glucose in the sample to the salt contained in the extraction medium. Concentration-dependent sensing membrane.

Therefore, by mixing a nonionic cellulose derivative as a film-forming macromolecular compound in the sensing film, it is possible to provide even if the salt concentration contained in the extraction medium changes (for example, the NaCl concentration changes from 0.00001% by weight to 1% by weight), An optical glucose sensor chip that can also detect the amount of glucose in a sample with stable sensitivity.

Hereinafter, embodiments of the present invention are described.

(Example 1)

Mix 1436 μL of isopropanol (IPA), 956 μL of pure water, 210 μL of 0.01 mol/L, pH 6.0 phosphate buffer, 60 μL of 1 volume % polyethylene glycol (PEG) in isopropanol, 600 μL of 1 mg /ml of 3,3',5,5'-tetramethylbenzidine (TMBZ) in isopropanol, 640 μL of 2 wt% carboxymethylcellulose (CMC) aqueous solution, 8 μL of 1 wt% Linked polymer compound (copolymer of 2-methacryloyloxyethylphosphorylcholine and butyl methacrylate) aqueous solution, 0.67 mg/mL peroxidase (POD) solution (dissolved in 0.01 mol/L Phosphate buffer (pH: 6.0)) and 5.33 mg/mL glucose oxidase (GOD) solution (dissolved in 0.01 mol/L phosphate buffer (pH: 6.0)), stirred, and prepared 4000 μL for the generation of glucose sensor film coating solution.

Next, an alkali-free glass substrate with a refractive index of 1.52 having a _SiO2 surface layer with a thickness of 10 nm on the main surface was prepared, and a titanium oxide film with a refractive index of 2.2 to 2.4 and a thickness of 50 nm was formed on the _SiO2 surface layer of the substrate by sputtering. Next, a resist is applied on the oxide film, dried, and a resist pattern is formed by lithography. Next, using the resist pattern as a mask, the titanium oxide film was selectively removed by reactive ion etching, and after gratings were formed on the surface near both edges of the above-mentioned SiO ₂ surface layer, the resist pattern was removed by grinding.

Next, the substrate was dry-cleaned by oxygen RIE, and then cut into a size of 17 mm×6.5 mm by dicing to form a chip shape. Then, 8 μL of the above-mentioned coating solution for forming a glucose sensor film was dropped on the surface of the sensor film forming region located between the gratings of the above-mentioned substrate. Drying was carried out by purging with inert gas and vacuum drying to form a porous (water-permeable) film with a thickness of 0.8 μm, and the optical glucose sensor chip shown in FIG. 1 above was manufactured. In addition, the droplet of the coating liquid for glucose sensor membrane formation which was added dropwise had the following composition.

Phosphate buffer: 0.000525 mol/L

PEG: 0.15% by volume

TMBZ: 0.15mg/dL

POD: 0.0015mg/mL

GOD: 0.012mg/mL

CMC: 0.32% by weight

Copolymer of 2-methacryloyloxyethylphosphorylcholine and butyl methacrylate: 0.002% by weight

Connect an adapter with a well to an appropriate flat plate (such as a glass plate), mount the sensor chip on the adapter, place the glucose sensing membrane on the side of the well, and divide the well. Into each through hole, inject various aqueous solutions containing glucose 0mg/dL (not included), 0.05mg/dL, 0.2mg/dL, 0.5mg/dL, 1mg/dL, at a temperature of 25°C and 37°C, make The above-mentioned aqueous solution penetrates into the above-mentioned sensing membrane. At this time, the membrane structure of the glucose sensing membrane has glucose oxidase (GOD), peroxidase (POD) and 3,3',5,5'-tetramethylbenzidine (TMBZ), which retain activity, therefore, GOD decomposes the exuded glucose to generate hydrogen peroxide, and POD decomposes the hydrogen peroxide to release active oxygen, and TMBZ develops color with this active oxygen. In fact, it was confirmed that the degree of color development of TMBZ changes according to the amount of glucose.

In the way that the through hole is filled with glucose-free water (temperature 25°C, 37°C), as shown in FIG. The interface between the _SiO2 surface layer 2 of the substrate 1 and the grating 3 on the left refracts the laser light, and further refracts the laser light at the interface between the _SiO2 surface layer 2 and the glucose sensing film 4 containing the colored developer, and the laser light is refracted on the SiO2-containing surface layer 2. ₂ The laser light propagating on the substrate 1 of the surface layer 2 and refracted through the interface between the grating 3 on the right and the substrate 1 is received by the photodetector 6 to detect its light intensity (initial light intensity).

In addition, in the way that the through hole is filled with glucose-containing water (temperature 25°C, 37°C), the same method is used to pass the _SiO2 surface layer 2 and the glucose sensor membrane 4 containing the colored developer. After the laser light is refracted at the interface and propagates on the substrate 1 containing the _SiO2 surface layer 2, the intensity of the laser light is detected (measurement of light intensity)

The decrease rate (sensitivity) was obtained from the initial light intensity at 25°C and 37°C obtained from the glucose sensor chip and the measured light intensity from the following formula.

Declining rate (%) = [(initial light intensity - measured light intensity) / initial light intensity] × 100

The results are shown in Fig. (3).

It can be clearly seen from Fig. 3 that the sensitivity of the sensor chip of Example 1 shows glucose concentration dependence in the range of glucose concentration 0.05-1.0 mg/dL, and the sensitivity is shown when the temperature of the measurement is between 25°C and 37°C. a certain sensitivity. That is, it can be seen that the amount of glucose in the sample can be detected with high sensitivity even in a heated state.

(Example 2)

In the same manner as in Example 1, except that hydroxyethyl cellulose (HEC) was mixed in the droplets of the coating liquid for forming a glucose sensing membrane instead of the CMC of Example 1, and the concentration was 0.32% by weight, As a sensing film, the optical glucose sensor chip (hereinafter referred to as sensor chip A) as shown in FIG. 1 was manufactured.

Using the obtained sensor chip A and the same glucose sensor chip as in Example 1 (hereinafter referred to as sensor chip B), fill the through holes with aqueous solutions containing 0.25 mg/dL glucose and 0 to 154 mmol of different NaCl concentrations respectively. (Temperature 37 degreeC), except for this, the sensitivity with respect to NaCl concentration was calculated|required by the method similar to Example 1. The results are shown in Fig. 4 .

It is clear from Fig. 4 that the sensitivity of the sensor chip B having a sensor film containing condensed methyl cellulose (CMC) as a film-forming polymer compound changes concentration-dependently in the NaCl concentration range of 0 to 154 mmol .

In contrast, it can be seen that the sensitivity of the sensor chip A having a sensor film containing hydroxyethyl cellulose (HEC) as a film-forming polymer compound does not depend on the concentration of NaCl in the range of 0 to 154 mmol. keep it steady. That is, it can be known that the sensor chip A can detect the amount of glucose in the sample with stable sensitivity even if the NaCl concentration of the extraction medium changes.

In addition, for the glucose sensor chip shown in FIG. 2 having a reflection path layer made of a thermosetting resin or a photosetting resin having a higher refractive index than the substrate, it is possible to detect with high sensitivity in a heated state as in Example 1. As for the amount of glucose in the sample, as in Example 2, even if the NaCl concentration (salt concentration) changes, the amount of glucose in the sample can be detected with stable sensitivity.

In addition, in the above-mentioned embodiments and examples, only one material is selected for each of the first enzyme, the second enzyme, and the color developing agent contained in one glucose sensing membrane, but various materials may be mixed according to the purpose of use. Even the crosslinkable polymer compound and the film-forming polymer compound can be similarly mixed with various materials according to the purpose of use within the scope of the present invention.

In addition, in the above-described embodiments, although glass is used as the substrate, the material is not limited as long as it propagates the reference beam and has light-transmitting properties. Various resin materials such as thin films formed of single crystals, thermosetting resin materials, thermoplastic resin materials, and photocurable resin materials can also be used.

Claims (10)

1. An optical glucose sensor chip, characterized in that it has a substrate, a pair of optical components formed on the main surface of the substrate, for injecting light into the interior of the substrate, and emitting light to the outside of the substrate. Formed on the main surface of the above-mentioned substrate between the above-mentioned optical components, the first enzyme that holds the color developing agent and the first enzyme that oxidizes or reduces glucose on the film formed by the film-forming polymer compound and the cross-linkable polymer compound, and the above-mentioned The glucose sensing membrane of the second enzyme that reacts the product of the enzyme to produce a substance that causes the above-mentioned chromogen to develop color. 2. An optical glucose sensor chip, characterized in that it has a glass substrate, and a pair of optical sensors formed on the main surface of the glass substrate for injecting light into the inside of the glass substrate and emitting light to the outside of the glass substrate. components, formed on the main surface of the above-mentioned substrate on which the above-mentioned optical components are formed, a light reflection path layer made of a resin with a higher refractive index than the above-mentioned substrate, and formed between the above-mentioned optical components on the above-mentioned reflection path layer, A color-developing agent and a first enzyme that oxidizes or reduces glucose is held on a thin film formed of a film-forming polymer compound and a cross-linking polymer compound, and a substance that causes the color-developing agent to develop color is produced by reacting with the product of the enzyme The 2nd enzyme glucose sensing membrane. 3. The optical glucose sensor chip according to claim 1 or 2, characterized in that the cross-linkable polymer compound is a copolymer of a hydrophilic monomer and a hydrophobic monomer. 4. The optical glucose sensor chip according to claim 1 or 2, characterized in that: the above-mentioned cross-linking polymer compound is hydrophilic having at least one group selected from hydroxyl, carboxyl, amino, and ionic functional groups. Copolymers of monomers and hydrophobic monomers. 5. The optical glucose sensor chip according to claim 3, characterized in that: the copolymer of the above-mentioned hydrophilic monomer and hydrophobic monomer is 2-methacryloyloxyethylphosphorylcholine and methacrylic acid Copolymers of butyl esters. 6. The optical glucose sensor chip according to claim 4, characterized in that: the copolymer of the above-mentioned hydrophilic monomer and hydrophobic monomer is 2-methacryloyloxyethylphosphorylcholine and methacrylic acid Copolymers of butyl esters. 7. The optical glucose sensing chip according to claim 1 or 2, wherein the glucose sensing membrane further contains polyethylene glycol for imparting water permeability. 8. The optical glucose sensor chip according to claim 1 or 2, wherein the first enzyme is glucose oxidase, the second enzyme is peroxidase, and the chromogenic agent is 3,3',5, At least one of 5'-tetramethylbenzidine or N,N'-bis(2-hydroxy-3-sulfopropyl)benzidine. 9. The optical glucose sensor chip according to claim 1 or 2, characterized in that the film-forming polymer compound is a nonionic cellulose derivative. 10. The optical glucose sensor chip as claimed in claim 9, characterized in that: said non-ionic cellulose derivative is at least one selected from the group consisting of alkyl cellulose, hydroxyalkyl cellulose and hydroxyalkyl alkyl cellulose. kind of substance.

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