JP2012078185A - Optical waveguide type biosensor chip and optical waveguide type biosensor chip manufacturing method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an optical waveguide type biosensor chip enabling light detection measurement with high accuracy while maintaining high sensitivity, and a manufacturing method thereof.SOLUTION: An optical waveguide type biosensor chip comprises: a substrate having light transmissivity; an optical waveguide layer formed on the substrate and having higher refractive index than the substrate; a first grating that makes light enter the optical waveguide layer so as to propagate the light in the optical waveguide layer; a second grating that emits the light propagated in the optical waveguide layer to the outside of the optical waveguide layer; and a metal oxide film formed on the optical waveguide layer; and a sensing film comprising at least a coloring agent fixed on the metal oxide film using a crosslinking agent.

Description

  The present invention relates to an optical waveguide biosensor chip and a method for manufacturing an optical waveguide biosensor chip.

  In the blood test, for example, in order to examine the liver function or the like, the activity of an enzyme in blood or serum serving as an index value of liver function is mixed with a dedicated reagent, and the change is measured from the change in absorbance of the dye that develops color at that time. The dedicated reagent includes a substance that reacts with the enzyme to be measured in blood (hereinafter referred to as substrate), an enzyme that is different from the enzyme to be measured that reacts with the substance produced by the reaction between the enzyme and the substrate, and a dye. , Reaction accelerators (surfactants, coenzymes, buffers) are included. A color reaction occurs when blood or serum is mixed with a dedicated reagent. The enzyme activity value in the blood is calculated from the change in absorbance per unit time of the specific wavelength of the colored dye, and this is used as the test result.

  Conventionally, as a small and highly sensitive biosensor chip, there is a planar optical waveguide biosensor chip that includes a grating coupler and a sensing film having a biomolecule recognition function and an information conversion function and uses an evanescent wave generated on the surface of the optical waveguide layer. Proposed. (For example, see Patent Document 1)

JP 2006-208359 A

  The optical waveguide biosensor described in Patent Document 1 described above uses a water-soluble polymer as a film-forming substance for the sensing film, whereby an enzyme and a dye are immobilized on the surface of the optical waveguide layer. On the other hand, in order to promote the enzyme reaction, a surfactant that prevents aggregation of enzymes is added to the sample solution used in the blood test. When this solution is dropped on the sensing film of the flat optical waveguide sensor of Patent Document 1 and detected, the water-soluble polymer as a film-forming substance is dissolved by the surfactant, and the enzyme and the dye are detached from the optical waveguide layer interface. And the sensitivity decreases. In addition, when the water-soluble polymer is dissolved, it becomes a diffuse reflection factor at the interface, and diffused light enters the detection unit, which causes a problem that high-precision light detection measurement cannot be performed.

  An object of the present invention is to provide an optical waveguide biosensor chip that enables highly accurate photodetection measurement while maintaining high sensitivity, or a method for manufacturing the same.

  An optical waveguide biosensor chip according to the present invention includes a light-transmitting substrate, an optical waveguide layer having a refractive index higher than that of the substrate, and light transmitted through the optical waveguide layer. A first grating for entering light into the optical waveguide layer; a second grating for emitting light propagated through the optical waveguide layer to the outside of the optical waveguide layer; and a metal oxide film formed on the optical waveguide layer And a sensing film having at least a color former fixed on the metal oxide film via a crosslinking agent.

  Further, the method for manufacturing an optical waveguide biosensor chip according to the embodiment includes a step of forming a pair of gratings on the main surface of a light-transmitting substrate for entering or emitting light into the substrate, A step of forming an optical waveguide layer having a higher refractive index than the substrate on a main surface of a substrate including a grating, a step of forming a metal oxide film having a lower refractive index than the optical waveguide layer on the optical waveguide layer, and A step of applying a solution having at least a crosslinking agent to a predetermined region on the metal oxide film and drying; a step of applying a solution having at least a color former to a region where the crosslinking agent is applied and dried; and a step of drying; It is characterized by having.

A sectional view of an optical waveguide type biosensor chip concerning an embodiment. Sectional drawing which shows the manufacturing process of the optical waveguide type biosensor chip which concerns on embodiment. Sectional drawing which shows the manufacturing process of the optical waveguide type biosensor chip which concerns on embodiment.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

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

  A pair of gratings 2 (incident side grating 2a, output side grating 2b) are provided in a region near both ends of a flat main surface of a transparent substrate 1 made of glass (for example, alkali-free glass) or quartz. Each is formed to allow light to enter and exit from the substrate 1. These gratings 2 are formed of a material (for example, titanium oxide) having a higher refractive index than the material constituting the substrate 1.

  The optical waveguide layer 3 is made of a polymer resin having a higher refractive index than that of the substrate 1 and is a film body having a uniform thickness set in a range of 3 to 300 μm. Adjacent to the main surface of the substrate 1 on which the grating 2 is formed.

  The metal oxide film 4 is made of a material having a refractive index lower than that of the material constituting the optical waveguide layer 3 and is formed adjacent to the main surface of the optical waveguide layer 3 so as to be in close contact therewith. Examples of the material of the metal oxide film 4 include silicon dioxide (refractive index 1.45), aluminum oxide (refractive index 1.63), beryllium oxide (refractive index 1.82), magnesium oxide (refractive index 1.70), and the like. However, since the refractive index of the high refractive index resin that guides light is about 1.49 to 1.70, silicon oxide having the lowest refractive index among the above materials is desirable in the embodiment.

  The protective film 5 is made of a material that does not react with all the reagents put into the sensor chip and has a liquid repellency (for example, a fluororesin material). It is formed adjacent to the surface of the metal oxide film 4 so as to cover both ends of the metal oxide film 4 corresponding to the region where the grating 2 is formed, that is, the region corresponding to the grating 2.

  The dye film 6 is formed of a color former fixed on a surface of the metal oxide film 4 where a protective film is not formed via a crosslinking agent. Examples of the crosslinking agent include a silane coupling agent and a diisocyanate agent, and the color former and the metal oxide film 4 are bonded by covalent bonding of a functional group of the crosslinking agent. The film thickness including the dye film 6 and the metal oxide film 4 is determined by the refractive index of the metal oxide film 4 and the optical waveguide layer 3, the wavelength of light propagating through the optical waveguide, and the evanescent wave stain calculated by the incident angle. It is desirable to make it less than the distance. For example, if the refractive index of the optical waveguide resin is 1.56, the refractive index of the metal oxide film formed on it is 1.45, and the angle of light incident on the interface between the optical waveguide resin and the metal oxide film is 77.8 °, evanescent The wave seepage distance is calculated to be 212 nm. Since this becomes a light absorption region, the thickness of the metal oxide film and the crosslinked dye film is preferably within 212 nm.

  The sensing film 7 is formed on the surface of the dye film 6 in a region surrounded by the protective film 5 on the line segment connecting the gratings 2. The sensing film 7 has, for example, a biomolecule recognition function and an information conversion function. The sensing film 7 in the optical waveguide biosensor chip is a film that generates a reaction product having a predetermined concentration in accordance with a sample having a predetermined concentration introduced on the film. The reaction product has the property of consuming energy by acting on the light guided in the optical waveguide type biosensor chip or the evanescent wave generated from this light, and absorbs or emits fluorescence.

  In order to function as such a membrane, the membrane body is a porous tissue, a labeled antibody that binds to the specimen by an antigen-antibody reaction, a reagent that reacts with the label to generate a reaction product, a label and a reagent Catalysts that promote the reaction are appropriately combined depending on the type of chemicals and are individually stored in the pores in the porous structure. When the solvent of the sample solution permeates through the pores of the porous membrane, the membrane swells, and the sensing membrane constituent material is movably released to promote reaction with the sample.

When the sensing membrane 7 is a glucose sensing membrane, the glucose sensing membrane includes a glucose oxidase or reductase, a film-forming polymer resin, and, if necessary, a water permeability promoter such as polyethylene glycol. Examples of the oxidase, the reagent that develops color by reacting with the product of the reaction between the sample and the enzyme, and the color former in glucose sensing include combinations shown in Table 1 below.

Examples of the film-forming polymer resin in the glucose sensing membrane include cellulose polymer resins such as carboxymethyl cellulose and hydroxyethyl cellulose.

  In the case of the glucose sensing membrane, since the color former in the glucose sensing membrane needs to be immobilized on a metal film, the color former of the dye film 6 has extremely low toxicity to a living body and can be immobilized on the metal film. It is desirable to use the possible N, N′-bis (2-hydroxy-3-sulfopropyl) tolidine.

  The operation of the optical waveguide biosensor chip shown in FIG. 1 will be described.

  A specimen containing a biomolecule is brought into contact with the sensing film 7 of the biosensor chip, and the biomolecule in the specimen is extracted to the sensing film 7. This biomolecule causes a biochemical reaction with the sensing film 7.

  At this time, since the metal oxide film 4 and the color former are bonded by the cross-linking agent, the color former is detached from the surface of the metal oxide film 4 even if the film-forming substance is dissolved by the surfactant contained in the specimen. There is nothing. That is, the color former can carry out a color reaction while maintaining a certain distance from the optical waveguide layer 3.

  In this state, as shown in FIG. 1, a light source (for example, a laser diode) 8 and a light receiving element (for example, a photodiode) 9 are respectively disposed on the left side and the right side of the back surface of the substrate 1 of the biochemical sensor chip. When light is incident on the back side of the substrate 1 of the biosensor chip, the laser light is refracted through the substrate 1 at the interface between the incident side grating 2a and the optical waveguide layer 3, and further, the optical waveguide layer 3, the substrate 1, and the metal oxide film 4 It propagates while being refracted multiple times at the interface. At this time, the evanescent wave of the light propagating in the optical waveguide layer 3 responds to a change (for example, a change in absorbance) based on a biochemical reaction of a biomolecule in the specimen in the sensing film 7 upon refraction at the interface of the metal oxide film 4. Absorbed.

  The light propagated through the optical waveguide layer 3 is emitted from the output side grating 2 b from the back surface of the substrate 1 and received by the light receiving element 9. The received laser light intensity is a value that is lower than the light intensity (initial light intensity) received when the sensing film 7 does not biochemically react with the biomolecule, and the amount of the biomolecule is detected from the decrease rate. It becomes possible.

  Next, a method for manufacturing the optical waveguide biochemical sensor chip according to the first embodiment will be described.

  2 and 3 are schematic views showing a method for manufacturing the optical waveguide type biochemical sensor chip of the first embodiment.

  First, as shown in FIG. 2A, a material film (refractive index higher than this substrate) is formed on the main surface of the substrate 1 which is a light-transmitting flat plate made of alkali-free glass or quartz having a wafer-like shape. For example, a titanium oxide film) is formed by sputtering. Next, the titanium oxide film formed in the previous step is partially removed by lithography and dry etching so as to form a lattice pattern with a predetermined pitch, and the incident side grating 2a and the emission side grating 2b are formed.

  All the gratings 2 are formed at an equal pitch with the same line length. If light is incident on the grating 2 from the outside, the grating functions as a coupler, and if light propagated in the optical waveguide layer is incident on the grating 2, the grating functions as a decoupler.

  Next, as shown in FIG. 2B, the entire surface of the main surface of the translucent substrate 1 on which the grating 2 is formed has a refractive index higher than that of the material constituting the translucent substrate 1 and is similarly translucent. An optical waveguide layer film 3 made of a polymer resin material and having a thickness of 3 to 300 μm is formed by applying a polymer resin material having a property with a spin coater or the like and drying it.

  Next, as shown in FIG. 2C, a metal oxide such as silicon dioxide having a refractive index lower than that of the optical waveguide layer 3 is applied to the surface portion of the optical waveguide layer film 3 by sputtering or spin coating. An oxide film 4 is formed. The range of the thickness of the metal oxide film 4 is, for example, that the refractive index of the optical waveguide resin is 1.56, the refractive index of the metal oxide film formed thereon is 1.45, and is incident on the interface between the optical waveguide resin and the metal oxide film. When the angle of light to be emitted is 77.8 °, the seepage distance of the evanescent wave that becomes the light absorption region is calculated to be 212 nm. Therefore, the metal oxide film and the dye film formed on it must be within 212 nm. There is. Since the thickness of the dye film is about 5 to 10 nm, the metal oxide film is preferably within 200 nm.

  Next, as shown in FIG. 3D, the surface portion of the metal oxide film 4 corresponding to the region where the grating 12 is formed is lower than the material constituting the metal oxide film 4 such as a fluorine-based resin material. A material that does not react with the refractive index and the reagent is screen-printed and dried to form the protective film 5. In this case, the region where the dye film 6 and the sensing film 7 are formed later is operated so that the protective film 5 is not formed. Thereby, the protective film 5 becomes a film having a frame structure surrounding a region where the dye film 6 and the sensing film 7 are formed.

  Next, the metal oxide film 4 and the protective film 5 are washed. Specifically, the surfaces of the metal oxide film 4 and the protective film 5 are irradiated with excimer ultraviolet light (for example, wavelength 172 nm), immersed in a base solution, and washed with pure water. Since the surface of the region not protected by the protective film 5 contains impurities such as a fluorine resin material, it is removed.

  Next, as shown in FIG. 3E, a crosslinking agent such as a silane coupling agent is applied and dried on the surface of the metal oxide film 4 where the protective film 5 is not formed. Further, a color former is applied and dried thereon to form a dye film 6. As a result, the color former is fixed to the main surface of the metal oxide film 4 via the crosslinking agent. The dye film 6 may be formed by simultaneously applying and drying a solution containing a cross-linking agent and a color former. However, since the color formers may be bonded to each other by the cross-linking agent, it is desirable to treat them separately.

Next, as shown in FIG. 3 (f), a film-forming coating solution having components and compositions corresponding to Table 1 is dropped onto the region where the protective film 5 is not formed. The coating liquid is dried to form a sensing film 7 in a region located between the gratings 2 where the protective film 5 is not formed.

  The optical waveguide biosensor chip is completed through the above steps.

  According to the first embodiment described above, since the color former is fixed to the metal oxide film 4 on the optical waveguide layer 3, the film-forming substance is dissolved by the surfactant contained in the specimen. However, it is possible to prevent the color former from detaching from the surface of the metal oxide film 4. That is, the color former can carry out a color reaction while maintaining a certain distance from the optical waveguide layer 3. This makes it possible to perform highly accurate photodetection measurement.

  The metal oxide film 4 also functions as a protective film for preventing light propagating in the optical waveguide layer 3 from being lost. Conventionally, if the metal oxide film 4 is not formed, when the sample solution is introduced into the sensing film, the sensing film that has absorbed the solution swells and a refractive index change occurs. As the refractive index changes, the evanescent wave oozing distance that occurs when light propagates through the optical waveguide layer 3 fluctuates. This variation causes variations in the light detection measurement. Furthermore, due to swelling of the sensing film, it becomes a scattering component at the interface between the waveguiding layer and the sensing film and diffuse reflection occurs. Due to this diffuse reflection, variations occur in the photodetection measurement. By forming the metal oxide film 4 on the optical waveguide layer 3 as in this embodiment, it is possible to suppress the change in refractive index and the diffuse reflection, and to absorb the evanescent wave due to the coloring of the color former. Photodetection measurement can be performed by measuring a change in the intensity of propagating light due only to scattering, and therefore, highly accurate photodetection measurement can be performed.

  The embodiments of the present invention have been described above with reference to specific examples. However, the present invention is not limited to these specific examples. In other words, those specific examples that have been appropriately modified by those skilled in the art are also included in the scope of the present invention as long as they have the characteristics of the present invention. Each element included in each of the specific examples described above and their arrangement, material, condition, shape, size, and the like are not limited to those illustrated, and can be appropriately changed.

In addition, each element included in each of the above-described embodiments can be combined as long as technically possible, and combinations thereof are also included in the scope of the present invention as long as they include the features of the present invention.
In addition, in the category of the idea of the present invention, those skilled in the art can conceive of various changes and modifications, and it is understood that these changes and modifications also belong to the scope of the present invention. .

  Also, these embodiments are presented as examples and are not intended to limit the scope of the invention. These novel embodiments can be implemented in various other forms, and various omissions, replacements, and changes can be made without departing from the scope of the invention. These embodiments and modifications thereof are included in the scope and gist of the invention, and are included in the invention described in the claims and the equivalents thereof.

  DESCRIPTION OF SYMBOLS 1 ... Board | substrate, 2a ... Incident side grating, 2b ... Outgoing side grating, 3 ... Optical waveguide layer, 4 ... Metal oxide film, 5 ... Protective film, 6 ... Dye film, 7 ... Sensing film, 8 ... Light source, 9 ... Light reception element

Claims (5)

  A substrate having translucency, an optical waveguide layer formed on the substrate and having a higher refractive index than the substrate, and a first light incident on the optical waveguide layer for propagating light through the optical waveguide layer A grating, a second grating that emits light propagating in the optical waveguide layer to the outside of the optical waveguide layer, a metal oxide film formed on the optical waveguide layer, and a crosslinking agent on the metal oxide film And a sensing film having at least a color former immobilized via the optical waveguide biosensor chip.   The optical waveguide biosensor chip according to claim 1, wherein the metal oxide film is silicon dioxide.   The optical waveguide biosensor chip according to claim 1, wherein the crosslinking agent is a silane coupling agent.   A step of forming a pair of gratings on the main surface of the light-transmitting substrate for entering or emitting light into the substrate; and a light guide having a higher refractive index than the substrate on the main surface of the substrate including the grating. A step of forming a waveguide layer; a step of forming a metal oxide film having a lower refractive index than the optical waveguide layer on the optical waveguide layer; and a solution having at least a crosslinking agent in a predetermined region on the metal oxide film A method for producing an optical waveguide type biosensor chip comprising: a step of applying and drying, and a step of applying and drying a solution having at least a color former in a region where the crosslinking agent is applied and dried. .   A step of forming a grating on the main surface of the light-transmitting substrate for entering or emitting light into the substrate; and an optical waveguide layer having a higher refractive index than the substrate on the main surface of the substrate including the grating. A step of forming a metal oxide film having a lower refractive index than the optical waveguide layer on the optical waveguide layer, and a predetermined region on the metal oxide film having at least a crosslinking agent and a color former. A method of manufacturing an optical waveguide biosensor chip, comprising: applying a solution and drying the solution.

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