JP2011167165A - Method for manufacturing optical sensor chip - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for manufacturing optical sensor chips which can control permeability change of a sensing film in a retention period by removing bound water contained in the sensing film. <P>SOLUTION: In the method of manufacturing optical sensor chips, at least one enzyme in a first enzyme oxidizing or reducing a measured object and a second enzyme generating a substance of a coloring substance by reacting with the generated product of the first enzyme is coated with an ionic polymer containing a buffer; the first enzyme, the second enzyme, the coloring substance, and a nonionic cellulose derivative are mixed to prepare coating liquid for sensing film formation; and the sensing film is formed by applying the coating liquid for sensing film formation on an optical waveguide layer formed on a substrate and heating and drying the coating liquid. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for manufacturing an optical sensor chip.

  As an optical sensor chip, for example, a minimally invasive blood glucose measurement device has been developed in which blood glucose level is indirectly examined by extracting body fluid from subcutaneous tissue. The sensor chip is formed on a glass substrate, a pair of gratings formed on the surface of the substrate for allowing light to enter and emit in the substrate, and a glucose sensing film formed on the surface of the substrate located between the gratings. It has a structure provided with. The glucose sensing membrane includes a color former (for example, 3, 3 ′, 5, 5′-tetramethylbenzidine (TMBZ)), a first enzyme that oxidizes or reduces glucose (for example, glucose oxidase (GOD)), the first enzyme. A second enzyme (for example, peroxidase (POD)) that generates a substance that reacts with the product of the above to generate a color former, and a film-forming polymer (for example, a cellulose derivative such as hydroxyethyl cellulose (HEC)).

  In the optical sensor chip having such a structure, when a sheet-like gel is placed between the skin and the sensing membrane and an electric field is applied, glucose in the subcutaneous tissue fluid permeates the gel from the skin and reaches the sensing membrane. . At this time, TMBZ, which is a color former in the sensing film, develops color due to the reaction between glucose, GOD and POD. In this state, when light is incident on the substrate and refracted by the substrate surface and the one grading, the light propagates through the interface between the substrate and the sensing film containing colored TMBZ, and at the interface between the substrate and the other grating. The light is refracted and received by, for example, a light receiving element. The received laser light intensity is a value that is lower than the light intensity (initial intensity) received by the light receiving element during non-color development due to color development of the color former of the glucose sensing film, and the concentration of the glucose is determined from the rate of decrease. To detect.

  When detecting glucose collected in a sample solution of several μL as described above, with a conventional optical sensor chip, if stored for a long time, the film-forming substance deteriorates and the permeability of the film-forming substance is reduced. descend. As the deterioration factor, the permeability of the material forming the film itself is lowered, or moisture interacting with the film forming material when applied and dried (hereinafter referred to as “bound water”) is removed. In other words, the bound water gradually escapes from the film-forming substance during storage. When the permeability of the film-forming substance is lowered, the diffusion rate of glucose contained in a small amount of the collection solution into the sensing film is lowered, and the first enzyme, the second enzyme, and the dye in the sensing film are stepwise. The reaction is reduced. As a result, the degree of color development is lowered and the sensitivity of the sensor chip is lowered.

JP 2008-22863 A

  The present invention is intended to provide a method of manufacturing an optical sensor chip that can suppress the change in permeability of the sensing membrane during storage by removing bound water contained in the sensing membrane.

  According to the first aspect of the present invention, any one of the first enzyme that oxidizes or reduces the measurement object and the second enzyme that generates a substance that develops color by reacting with the product of the first enzyme. The enzyme is premixed with an aqueous solution of an ionic polymer and a buffer, and this mixed solution is added to the other enzyme, the color former and the nonionic cellulose derivative, or the first and second enzymes are respectively added to the ionic polymer. And the aqueous solution of the buffering agent in advance, and each mixed solution is added to the color former and the nonionic cellulose derivative, or both the first and second enzymes are premixed with the aqueous solution of the ionic polymer and the buffering agent. Adding the mixed solution to the color former and the nonionic cellulose derivative, or preparing a coating solution for forming a sensing film by either, and Forming a pair of optical elements for causing light to enter the light reflecting layer formed on the surface and emitting the light outside the optical waveguide layer; and a main surface of the substrate on which the optical elements are formed. Forming the optical waveguide layer made of a resin having a higher refractive index than the substrate; and applying the sensing film-forming coating solution between the optical elements on the optical waveguide layer, followed by drying by heating to form a sensing film. And a process for producing an optical sensor chip.

  According to the second aspect of the present invention, any one of the first enzyme that oxidizes or reduces the measurement target and the second enzyme that generates a substance that develops a color former by reacting with the product of the first enzyme. The enzyme is premixed with an aqueous solution of an ionic polymer and a buffer, and this mixed solution is added to the other enzyme, the color former and the nonionic cellulose derivative, or the first and second enzymes are respectively added to the ionic polymer. And the aqueous solution of the buffering agent in advance, and each mixed solution is added to the color former and the nonionic cellulose derivative, or both the first and second enzymes are premixed with the aqueous solution of the ionic polymer and the buffering agent. Adding the mixed solution to the color former and the nonionic cellulose derivative, or preparing a coating solution for forming a sensing film by either A step of forming a pair of optical elements for making light incident and emitting the light to the outside of the substrate, and applying the sensing film forming coating solution on the substrate region between the optical elements, heating and drying, and sensing Forming a film, and a method for manufacturing an optical sensor chip is provided.

  ADVANTAGE OF THE INVENTION According to this invention, the manufacturing method of the optical sensor chip which can suppress the permeability | transmittance change of the sensing membrane at the time of a preservation | save by removing the bound water contained in a sensing membrane can be provided.

Sectional drawing which shows the optical sensor chip which concerns on 1st Embodiment. Sectional drawing which shows the optical sensor chip which concerns on 2nd Embodiment. The diagram which shows the change of the light absorbency (sensitivity) with progress of the storage period in the optical sensor chip of Example 1 and Comparative Examples 1 and 2. FIG.

  Hereinafter, an optical sensor chip according to an embodiment of the present invention will be described in detail with reference to the drawings. In the following, an optical sensor chip used for measuring glucose will be described. However, it is not limited to glucose, and can be used for various measurements such as L-glutamic acid, ascorbic acid, sucrose, and pyruvic acid, which are food analysis target substances .

(First embodiment)
FIG. 1 is a cross-sectional view showing the optical sensor chip according to the first embodiment.

  A pair of gratings 2 as optical elements are formed in the vicinity of both ends of the main surface of the glass substrate 1 in order to make light incident on and emitted from the substrate 1. These gratings 2 are made of, for example, titanium oxide having a higher refractive index than that of the substrate 1. An optical waveguide layer 3 made of a thermosetting or photocurable resin having a higher refractive index than that of the substrate 1 is formed on the main surface of the substrate 1 including the grating 2. The main surface of the optical waveguide layer 3 is formed to be parallel to the main surface of the substrate 1 including the grating 2.

  The glucose sensing film 4 is formed on the optical waveguide layer 3 corresponding to the gap between the gratings 2. The glucose sensing membrane 4 comprises a color former, a first enzyme that oxidizes or reduces glucose, a second enzyme that generates a substance that develops color by reacting with a product of the first enzyme, and a nonionic cellulose derivative. Wherein at least one of the first and second enzymes is covered with an ionic polymer incorporating a buffer, and the enzyme, ionic polymer, buffer and color former are the nonionic cellulose. It has a structure retained by a derivative.

  Here, the criterion for covering at least one of the first and second enzymes with an ionic polymer incorporating a buffering agent is determined by the degree of deterioration with time described below.

  That is, the product produced by adding glucose to the first enzyme and reacting it is allowed to act on a specific color former to cause color development, and the absorbance at this time is measured. After the first enzyme is exposed to an atmosphere of a certain temperature and humidity for a certain period of time, glucose is added to the first enzyme and the product produced by reacting is allowed to act on a specific color former to cause color development. Measure the absorbance. The rate of decrease in absorbance of the latter with respect to the former is determined.

  In addition, the first enzyme and the second enzyme are added to glucose, and a substance produced by reacting with the product of the first enzyme is allowed to act on a specific color former to develop color, and the absorbance at this time is measured. After the second enzyme is exposed to an atmosphere having a constant temperature and humidity for the same time as the absorbance measurement of the first enzyme, the second enzyme is added to glucose together with the first enzyme and reacted with the product of the first enzyme. The substance thus produced is allowed to act on a specific color former to cause color development, and the absorbance at this time is measured. The rate of decrease in absorbance of the latter with respect to the former is determined.

  The rate of decrease in absorbance due to the time degradation of the first enzyme was compared with the rate of decrease in absorbance due to the time degradation of the second enzyme, an enzyme having a large rate of decrease in absorbance was selected, and a buffer was incorporated. Cover with ionic polymer. Moreover, when the absolute value of the rate of decrease in absorbance is large in both the first and second enzymes, it is preferable to cover both with an ionic polymer incorporating a buffer.

  The optical waveguide layer 3 preferably has a smooth surface and a thickness of 10 μm or more, more preferably 10 to 200 μm. The optical waveguide layer having a thickness of 10 μm or more can suppress the attenuation of light intensity during the propagation of light. For example, an LED light source can be used in addition to the laser light source.

The first and second enzymes and the color former in the glucose sensing membrane 4 are used in combinations shown in Table 1 below, for example.

  The nonionic cellulose derivative used for the glucose sensing membrane 4 is a polymer compound involved in membrane formation. Examples of the nonionic cellulose derivatives include alkyl celluloses such as methyl cellulose and ethyl cellulose; hydroxyalkyl celluloses such as hydroxyethyl cellulose and hydroxypropyl cellulose; hydroxypropyl methyl cellulose, hydroxypropyl ethyl cellulose, hydroxydiethyl cellulose, and hydroxyethyl methylcellulose. Examples thereof include hydroxyalkylalkyl cellulose; and microfibrobrominated cellulose. These can be used alone or in the form of a mixture.

  The ionic polymer has a function of suppressing salt precipitation from the first and second enzymes during long-term storage and use. The 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, a guanidino group, and a biguanide group. Specific examples of the positive ionic polymer include polyallylamine hydrochloride, polyvinyl pyridine, polylysine and the like. Examples of the negative ionic polymer include polymers containing an anionic group such as phosphate ester, carboxylate, and sulfonate ester. Specific examples of the negative ionic polymer include polystyrene sulfonic acid, polyvinyl sulfate, polyaspartic acid, polyacrylic acid, polymethacrylic acid, polymaleic acid, polyfumaric acid, or cellulose derivatives such as carboxymethyl cellulose and cellulose acetate. It is done. Of these ionic polymers, negative ionic polymers are preferred.

  The buffer has a function of controlling changes in the form and structure of the first and second enzymes by controlling the pH and ionic strength of the first and second enzymes during long-term storage and use. Examples of the buffer include phosphate buffer, acetate buffer, citrate buffer, borate buffer, tartaric acid buffer, Tris-HCl buffer, and carbonate buffer.

  By covering at least one of the first and second enzymes with an ionic polymer incorporating such a buffering agent, salt precipitation from the enzyme is suppressed during long-term storage and use, and the enzyme It is possible to maintain a high active state by suppressing changes in the form and structure.

  The glucose sensing membrane 4 is allowed to contain a crosslinkable polymer compound. Examples of the crosslinkable polymer compound include a copolymer of a hydrophilic monomer having at least one group selected from a hydroxyl group, a carboxyl group, an amino group, and an ionic functional group and a hydrophobic monomer. . It has been experimentally confirmed that the copolymer of the hydrophilic monomer and the hydrophobic monomer is particularly preferably a copolymer of 2-methacryloyloxyethyl phosphorylcholine and butyl methacrylate.

The crosslinkable polymer compound is preferably contained in the glucose sensing membrane 4 in an amount of 10 ⁻⁴ to 10 wt% with respect to the total composition of the glucose sensing membrane 4. When the content of the crosslinkable polymer compound is less than 10 ⁻⁴ wt% with respect to the total composition, the color former that dissolves and collapses in the heated state or is retained in the voids in the film structure It is difficult to prevent the enzyme and the enzyme from eluting into the external medium. On the other hand, if the content of the crosslinkable polymer compound exceeds 10% by weight, the permeability of the sample solution in the glucose sensing membrane may be lowered, and the chip sensitivity may be lowered.

  The glucose sensing membrane 4 is allowed to further contain polyethylene glycol or ethylene glycol for imparting water permeability in the voids of the membrane structure. As a result, the hydrophilicity increases, and the reaction sensitivity increases when water is used as a medium for introducing glucose.

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

  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 above-described sensor chip is attached to the adapter so that the glucose sensing membrane 4 is located on the well side. The adapter prevents the glucose sensing membrane 4 from coming into direct contact with the specimen and contributes to improving the reproducibility of sensing. By filling the void created by this, the extraction medium (for example, liquid such as water, physiological saline, etc., which does not react directly with the specimen or the sensing membrane, and adapts), and applying a minute voltage to the specimen from the outside The glucose in the subcutaneous tissue fluid is extracted from the skin into the extraction medium, and further penetrates the sensing membrane 4 from the extraction medium. For example, glucose oxidase (GOD), peroxidase (POD) and 3, 3 ′ shown in Table 1 above are combinations of the first and second enzymes (oxidation or reductase) and the color former constituting the glucose sensing membrane 4. In the case of 5,5′-tetramethylbenzidine (TMBZ), glucose permeated into the sensing membrane 4 decomposes by GOD to generate hydrogen peroxide, and decomposes this hydrogen peroxide by POD to generate active oxygen. And TMBZ is developed by this active oxygen. That is, the color development degree of TMBZ changes according to the amount of glucose.

  In this state, laser light is incident on the back side of the substrate 1 from the light source (for example, a laser diode) 5 through a polarization filter (not shown), so that the laser light passes through the substrate 1 to the main surface and the left grating 2. The light is refracted at the interface and incident on the optical waveguide layer 3, and further refracted at the interface between the optical waveguide layer 3 and the glucose sensing film 4 containing the color developing agent, and propagates through the optical waveguide layer 3. At this time, the evanescent wave of the propagated light is absorbed according to the degree of color development based on the amount of glucose in the glucose sensing film 4. The light propagated through the optical waveguide layer 3 is emitted from the right grating 2 and received by a light receiving element (for example, a photodiode) 6. The intensity of the received laser beam becomes a value lower than the intensity of light received when the sensing film 4 is not colored (initial intensity), and the glucose amount can be detected from the rate of decrease.

  Next, a method for manufacturing the optical sensor chip shown in FIG. 1 will be described.

  First, a glucose sensing film forming coating solution is prepared by any one of the following methods (1) to (3).

  (1) A first enzyme that oxidizes or reduces glucose, and a second enzyme that generates a substance that develops a color developing agent by reacting with the product of the first enzyme, is selected from an ionic polymer and a buffer. Premix with aqueous solution. In this mixing step, one of the first and second enzymes is coated with an ionic polymer in which a buffer is incorporated. Subsequently, this mixed solution is added to the other enzyme, the color former and the nonionic cellulose derivative and mixed, so that one enzyme covered with the ionic polymer forms a film together with the other enzyme and the color former. A coating solution for forming a glucose sensing film dispersed in a nonionic cellulose derivative that is a polymer compound is prepared.

  (2) The first and second enzymes are previously mixed with an aqueous solution of an ionic polymer and a buffering agent, respectively. In this mixing step, the first and second enzymes are each coated with an ionic polymer incorporating a buffer. Subsequently, the mixed solution is added to the color former and the nonionic cellulose derivative and mixed, whereby the first and second enzymes respectively covered with the ionic polymer together with the color former and the film-forming polymer. A coating solution for forming a glucose sensing film dispersed in a nonionic cellulose derivative as a compound is prepared.

  (3) Both the first and second enzymes are premixed with an aqueous solution of an ionic polymer and a buffer. In this mixing step, the first and second enzymes are coated with an ionic polymer in which a buffer is incorporated. Subsequently, the mixed solution is added to the color former and the nonionic cellulose derivative, and the first and second enzymes covered with the ionic polymer by mixing are the film-forming polymer compound together with the color former. A coating solution for forming a glucose sensing film dispersed in a nonionic cellulose derivative is prepared.

Next, a pair of optical elements 2, for example, gratings, for causing light to enter the optical waveguide layer 3 to be formed later and to emit light to the outside of the optical waveguide layer 3 are formed on the main surface of the glass substrate 1. It is formed by film formation and patterning. Subsequently, an optical waveguide layer 3 made of a thermosetting or photocurable resin having a higher refractive index than the substrate is formed on the main surface of the substrate 1 on which the grating 2 is formed. Subsequently, the glucose sensing film 4 is formed by applying the coating solution for forming the glucose sensing film between the gratings 2 on the optical waveguide layer 3 and heating and drying to produce an optical sensor chip. The heat drying performed here is performed in an inert gas atmosphere, for example, in a nitrogen atmosphere at about 40 ° C.

  As described above, in the detection of the amount of glucose by the optical sensor chip of the first embodiment, the glucose sensing membrane is covered with an ionic polymer in which at least one of the first and second enzymes has taken in the buffer. Therefore, precipitation of salts from the enzyme can be suppressed during long-term storage and use, and changes in the form and structure of the enzyme can be suppressed to maintain a high active state. In addition, after the glucose sensing film forming coating solution is applied onto the substrate region of the grating, the bound water interacting with the film forming substance is removed by heating and drying. Thereby, at the time of preservation | save of a sensing membrane, the temporal change of the osmosis | permeation state can be suppressed and detection sensitivity can be stabilized.

In addition, since the enzyme is covered with an ionic polymer during heat drying, the structural change of the enzyme due to heat is suppressed, and a high activity state can be maintained.

Further, in the manufacturing process, there is no variation in the amount of moisture in the sensing film due to the drying position of the sensor chip during application and drying, and the variation between chips can be improved.

(Second Embodiment)
FIG. 2 is a cross-sectional view showing an optical sensor chip according to the second embodiment.

The glass substrate 11 has a SiO ₂ surface layer 12 having a thickness of, for example, 3 nm or more on the main surface. A pair of optical elements, for example, a pair of gratings 13 are respectively formed on the surfaces near both ends of the SiO ₂ surface layer 12 so that light enters the substrate 11 or emits the light in the substrate 11. The optical element may be replaced with a prism or the like. These gratings 13 are made of, for example, titanium oxide having a higher refractive index than the SiO2 surface layer 12. A protective film having a lower refractive index than that of the grating 13 may be formed so as to cover the grating 13. This protective film is made of a material that does not react with the chemical solution or specimen to be used, for example, a fluororesin.

The glucose sensing film 14 is formed on the SiO ₂ surface layer 12 of the substrate 11 located between the gratings 13. The glucose sensing membrane 14 includes a color former, a first enzyme that oxidizes or reduces glucose, a second enzyme that generates a substance that develops the color former by reacting with a product of the first enzyme, and nonionic cellulose. A derivative, wherein at least one of the first and second enzymes is covered with an ionic polymer incorporating a buffer, and the enzyme, the ionic polymer, the buffer and the color former are nonionic. It has a structure held by a cellulose derivative.

  Here, the standard for covering at least one of the first and second enzymes with an ionic polymer incorporating a buffer is the same as that described in the first embodiment.

  The enzyme and the color former in the glucose sensing membrane 14 are used, for example, in combinations shown in Table 1 above.

  As the ionic polymer, the buffering agent and the nonionic cellulose derivative in the glucose sensing membrane 14, the same ones as those mentioned in the first embodiment can be used.

  As described in the first embodiment, the glucose sensing film 14 is allowed to further contain a crosslinkable polymer compound and further contain polyethylene glycol or ethylene glycol.

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

  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 above-described sensor chip is attached to the adapter so that the glucose sensing membrane 14 is located on the well side. By filling the well with an extraction medium containing water and applying a minute voltage from the outside, glucose in the subcutaneous tissue fluid is extracted from the skin into the medium and further permeates the sensing membrane 14. The combination of the first and second enzymes (oxidation or reductase) and the color former constituting the glucose sensing membrane 14 is, for example, glucose oxidase (GOD), peroxidase (POD) and 3, 3 ′, as shown in Table 1 above. In the case of 5,5′-tetramethylbenzidine (TMBZ), glucose permeated into the sensing membrane 14 is decomposed by GOD to generate hydrogen peroxide, and this hydrogen peroxide is decomposed by POD to generate active oxygen. Release and develop TMBZ with this active oxygen. That is, the color development degree of TMBZ changes according to the amount of glucose.

In this state, laser light is incident on the back side of the substrate 11 from the laser light source (for example, laser diode) 15 through a polarization filter (not shown), so that the laser light is on the SiO 2 surface layer 12 of the substrate 11 and the left grating 13. the refracted at the interface, to further propagate the substrate 11 including the SiO ₂ surface layer 12 is refracted at the interface of glucose sensing layer 14 containing a color former and color the SiO ₂ surface layer 12. At this time, the evanescent wave of the propagating light is absorbed according to the degree of color development based on the amount of glucose in the glucose sensing film 14. The light propagated through the substrate 11 is emitted from the right grating 13 and received by a light receiving element (for example, a photodiode) 16. The intensity of the received laser beam becomes a value that is lower than the intensity of light received when the sensing film 14 is not colored (initial intensity), and the amount of glucose can be detected from the rate of decrease.

  Next, a method for manufacturing the optical sensor chip shown in FIG. 2 will be described.

  First, at least one of the first and second enzymes is preliminarily covered with an ionic polymer in which the buffering agent is incorporated by any one of the three methods similar to the first embodiment described above, and the color former A coating solution for forming a glucose sensing film dispersed in a nonionic cellulose derivative that is a film-forming polymer compound is prepared.

  Next, a pair of optical elements 13, for example, a grating 13, for allowing light to enter the substrate 11 and emitting the light to the outside of the substrate 11, is formed by forming and patterning a titanium oxide film. Subsequently, the glucose sensing film 14 is formed by applying the glucose sensing film forming coating solution on the region of the substrate 11 between the gratings 13 and heating and drying to produce an optical sensor chip. The heat drying performed here is performed in an inert gas atmosphere, for example, in a nitrogen atmosphere at about 40 ° C.

  As described above, in the detection of the amount of glucose by the optical sensor chip of the second embodiment, the glucose sensing membrane is covered with an ionic polymer in which at least one of the first and second enzymes has taken in the buffer. Therefore, precipitation of salt from the enzyme can be suppressed during long-term storage and use, and changes in the form and structure of the enzyme can be suppressed to maintain a high active state. In addition, after the glucose sensing film forming coating solution is applied onto the substrate region of the grating, the bound water interacting with the film forming substance is removed by heating and drying. Thereby, at the time of preservation | save of a sensing membrane, the temporal change of the osmosis | permeation state can be suppressed and detection sensitivity can be stabilized.

In addition, since the enzyme is covered with an ionic polymer during heat drying, the structural change of the enzyme due to heat is suppressed, and a high activity state can be maintained.

Further, in the manufacturing process, there is no variation in the amount of moisture in the sensing film due to the drying position of the sensor chip during application and drying, and the variation between chips can be improved.

  Examples of the present invention will be described below.

Example 1
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 (0.01 9 μL of a mixed solution of a mol / L phosphate buffer (pH: 6.0) was mixed with 1 μL of a 1 wt% carboxymethylcellulose (CMC) aqueous solution as a negative ionic polymer and stirred. 9 μL of the obtained mixed solution was 143.6 μL of isopropyl alcohol (IPA), 116.6 μL of pure water, 6 μL of an isopropyl alcohol solution of 1% by volume of polyethylene glycol (PEG), and 1 mg / mL of 3,3 ′, 5,5. '-Tetramethylbenzidine (TMBZ) 60 μL of isopropyl alcohol solution, 2% by weight of hydroxyethylcellulose (HEC) 64 μL and 1% by weight of crosslinked polymer (copolymer of 2-methacryloyloxyethyl phosphorylcholine and butyl methacrylate) The solution was added to 0.8 μL of an aqueous solution, mixed and stirred to prepare 400 μL of a glucose sensing membrane forming coating solution.

Next, a non-alkali glass substrate having a refractive index of 1.52 having a SiO ₂ surface layer having a thickness of 10 nm on the main surface is prepared, and an oxide having a refractive index of 2.2 to 2.4 and a thickness of 50 nm is formed on the SiO ₂ surface layer of this substrate. A titanium film was formed by sputtering. Subsequently, a resist pattern was formed on the titanium oxide film by applying a resist, drying, and lithography. Subsequently, the titanium oxide film was selectively removed by reactive ion etching using the resist pattern as a mask to form a grating on the surface near both ends of the SiO2 surface layer, and then the resist pattern was removed by ashing.

  Next, the substrate was dry-cleaned by oxygen RIE, and then cut into a size of 17 mm × 6.5 mm by dicing into a chip shape. Subsequently, 8 μL of the glucose sensing film forming coating solution was dropped on the surface of the sensing film forming region located between the gratings of the substrate. The optical sensor chip shown in FIG. 2 was manufactured by purging with inert gas and heating and drying at 40 ° C. to form a glucose sensing film.

(Comparative Example 1)
8 μL of the coating solution for forming the glucose sensing film in Example 1 was dropped on the surface of the sensing film forming region located between the gratings on the substrate and dried at room temperature to form a glucose sensing film. The optical sensor chip shown was manufactured.

(Comparative Example 2)
8 μL of the coating solution for forming the glucose sensing film in Example 1 was dropped on the surface of the sensing film forming region located between the gratings on the substrate, and vacuum-dried to form a glucose sensing film. The optical sensor chip shown was manufactured.

  The optical glucose sensor chip prepared in Example 1 and Comparative Examples 1 and 2 was evaluated by the following method.

That is, when the optical sensor chip is attached, the glucose sensing film 14 is located in a chamber having a facing surface at a position facing the glucose sensing film 14 and forming a space between the sensing film 14 and the facing surface. It attached so that it might be located in the side, and the division holding a measurement solution was formed. In the form in which the gap between the formed sensing film 14 and the facing surface is filled with 4.5 μL of a 0.25 mg / dL glucose solution, as shown in FIG. 11 When incident on the back surface side, the laser light is refracted at the interface between the SiO ₂ surface layer 12 and the left grating 13 of the substrate 1, and further at the interface between the SiO ₂ surface layer 12 and the glucose sensing film 14 containing a color developing agent. The substrate 11 including the refracted SiO ₂ surface layer 12 was propagated, the laser beam propagated by refraction at the interface between the right grating 13 and the substrate 11 was received by the photodiode 16, and the laser beam intensity was measured.

  In the same operation as in Example 1 and Comparative Example 1, the sensor chip was stored for 1 day, and then measured using a sample stored at 37 ° C. for 5 days. The act of storing for 5 days at 37 ° C. corresponds to an accelerated test that requires one year at room temperature in an optical sensor chip. Therefore, this result shows the characteristics of the glucose sensing membrane after one year.

  These results are shown in FIG.

  As can be seen from FIG. 3, in the optical sensor chips of Comparative Examples 1 and 2, the absorbance of the ones stored for 5 days at 37 ° C. is lower than that before storage. On the other hand, it can be seen that the optical sensor chip of Example 1 has almost the same sensitivity with little change in absorbance compared with before storage even after storage at 37 ° C. for 5 days.

  In addition, even in the optical sensor chip shown in FIG. 1 having an optical waveguide layer made of a thermosetting resin or a photocurable resin having a higher refractive index than that of the substrate, high sensitivity can be obtained even after long-term storage as in Example 1. It was possible to maintain.

  In the embodiments and examples, only one kind of material is selected and added for the first enzyme, the second enzyme, and the color former held by the glucose sensing membrane, but a plurality of materials may be used depending on the purpose of use. You may mix.

  Furthermore, in the said embodiment, although glass is used as a board | substrate, if it has the characteristic which propagates and permeate | transmits reference light, this material will not be limited. Various resin materials such as a film made of a single crystal, a thermosetting resin material, a thermoplastic resin material, and a photocurable resin material can also be used.

DESCRIPTION OF SYMBOLS 1,11 ... Glass substrate, 2,13 ... Grating, 3 ... Optical waveguide layer, 4,14 ... Glucose sensing film | membrane, 5,15 ... Laser light source (laser diode), 6,16 ... Light receiving element (photodiode), 12 ... SiO ₂ surface layer

Claims (3)

Either the first enzyme that oxidizes or reduces the object to be measured, or the second enzyme that generates a substance that develops a color developing agent by reacting with the product of the first enzyme, is an aqueous solution of an ionic polymer and a buffer. And the mixture is added to the other enzyme, the color former and the nonionic cellulose derivative, or the first and second enzymes are premixed with an aqueous solution of an ionic polymer and a buffer, respectively. Each mixed solution is added to the color former and the nonionic cellulose derivative, or both the first and second enzymes are premixed with an aqueous solution of an ionic polymer and a buffer, and the mixture is added to the color former and the nonionic cellulose derivative. Adding to the ionic cellulose derivative, or preparing a sensing film forming coating solution by either,
Forming a pair of optical elements for causing light to enter a light reflecting layer formed on a main surface of the substrate and emitting the light to the outside of the optical waveguide layer;
Forming the optical waveguide layer made of a resin having a higher refractive index than the substrate on the main surface of the substrate on which the optical element is formed;
Applying the sensing film-forming coating solution between the optical elements on the optical waveguide layer, and heating and drying to form a sensing film. Either the first enzyme that oxidizes or reduces the object to be measured, or the second enzyme that generates a substance that develops a color developing agent by reacting with the product of the first enzyme, is an aqueous solution of an ionic polymer and a buffer. And the mixture is added to the other enzyme, the color former and the nonionic cellulose derivative, or the first and second enzymes are premixed with an aqueous solution of an ionic polymer and a buffer, respectively. Each mixed solution is added to the color former and the nonionic cellulose derivative, or both the first and second enzymes are premixed with an aqueous solution of an ionic polymer and a buffer, and the mixture is added to the color former and the nonionic cellulose derivative. Adding to the ionic cellulose derivative, or preparing a sensing film forming coating solution by either,
Forming a pair of optical elements for causing light to enter the substrate and emitting the light to the outside of the substrate;
Applying the sensing film-forming coating solution on the substrate region between the optical elements, and heating and drying to form a sensing film.   3. The method of manufacturing an optical sensor chip according to claim 1, wherein the step of forming the sensing film is performed by heating and drying in an inert gas atmosphere.

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