JP2014202574A - Optical sensor and method for manufacturing sensor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To solve such a problem that a sensor using a photonic crystal that immobilizes a recognition element to a surface to measure a refractive index variation caused by binding/dissociation with respect to a specimen has a small variation, thus making it difficult to detect a low-molecular specimen.SOLUTION: The photonic crystal is prepared using a material with molecular recognition ability. Extraction of a specimen to the interior of the photonic crystal and a change in physical properties of the material are used for detection and quantitative determination so as to detect and quantitatively determine the specimen such as ion.

Description

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

(Biosensor)
Various biosensors used for the purpose of gene analysis, clinical diagnosis, detection of harmful substances, etc. have been developed in medical institutions, research institutions, and environmental analysis institutions.

(Photonic crystal)
Photonic crystal (PhC) is a nanometer-sized dielectric periodic structure that reflects light of a specific wavelength depending on the period and size. Its reflection characteristics change sharply depending on the refractive index around the PhC, so it is expected to be applied to sensors.

(Sensor using PhC)
The following are reported as optical sensors using PhC.
Japanese Patent Laid-Open No. 2007-271609 (Patent Document 1) states that “a photonic crystal (C) composed of a periodic structure (N) composed of a solid substance (M) that is insoluble in an aqueous medium is a main constituent element, and a periodic structure The specific binding substance (X) is immobilized on the surface of the body (N) or the specific binding substance (X) is immobilized on the surface of the structural unit (L) of the periodic structure (N). Disclosed is a featured biosensor (S).
However, unlike the optical sensor of the present invention, the biosensor has a specific binding substance (X) immobilized on the surface of the periodic structure (N).

WO2010 / 044274 (Patent Document 2) is “an optical sensor for detecting a predetermined object to be detected, in which a predetermined shape is periodically arranged two-dimensionally and functions as a photonic crystal; An optical sensor comprising: a metal layer formed on the surface of an uneven structure and capable of exciting localized surface plasmon resonance ”is disclosed.
However, the biosensor has a measurement principle different from that of the optical sensor of the present invention, and a metal layer is formed on the surface of the concavo-convex structure that functions as a photonic crystal.

JP 2011-80848 (Patent Document 3) states that “a photonic crystal (C) composed of periodically arranged particles and a specific binding substance (X) that specifically binds or reacts with a target substance. A stimulus-responsive polymer material (M) that changes in volume when the target substance and the specific binding substance bind or react with each other, and the photonic crystal comprises the stimulus-responsive polymer material The biosensor (S) ”is included.
However, in the biosensor, unlike the optical sensor of the present invention, the photonic crystal is made into particles by silica or the like and has a multilayer structure. Due to this structure, it is difficult to produce a periodic structure with good reproducibility (uniformly).

JP2007-271609 WO2010 / 044274 JP2011-80848

The reported sensor using PhC immobilizes the recognition element on the surface and measures the change in refractive index caused by binding / dissociation with the test substance, so the change is small and low molecular weight test is performed. It is difficult to detect substances, and the PhC itself used is made of metal oxides such as Si and TiO ₂ by techniques such as electron beam lithography and etching. There is a problem that the cost is high and the structure of the sensor is complicated and difficult to manufacture.

  In order to solve the above-mentioned problems, the present inventors create PhC using a material having molecular recognition ability, and use extraction of a test substance into PhC and change in physical properties of the material for detection and quantification. As a result, it was confirmed that analytes such as ions can be detected and quantified, and the above problems can be solved.

That is, the present invention is as follows.
1. An optical sensor for detecting a test substance,
It has a detection site with a concavo-convex structure that functions as a photonic crystal,
The detection site includes a recognition element,
Optical sensor.
2. The detection sites are polyvinyl chloride (PVC), polymethyl methacrylate, polyethylene (glycol) diacrylate (PEGDA), polyvinylidene chloride (PVDC), acrylamide, agarose, polystyrene, polypropylene, polyethylene, collagen, gelatin, cellulose, poly 2. The optical sensor according to item 1, which is composed of dimethylsiloxane (PDMS), polyester (PET), and / or polyethylene glycol.
3. 3. The optical sensor according to item 1 or 2, wherein the recognition element is an ionophore, a chelate, an enzyme, DNA, RNA, a sugar chain, and / or a protein.
4). 4. The optical sensor according to any one of items 1 to 3, wherein a film thickness of the detection site is 0.1 μm to 8.0 μm.
5. The manufacturing method of the optical sensor of any one of preceding clauses 1-4 including the following processes,
(1) A step of preparing a plasticized resin solution by dissolving a resin, a plasticizer, and a recognition element in a solvent,
(2) The process of producing the detection site | part which consists of plasticizing resin which has an uneven structure which contains a recognition element and functions as a photonic crystal using this solution using an array mold.
6). 6. The method for producing an optical sensor according to item 5 above, wherein the array mold is a PVA pillar array mold.
7). An optical sensor substrate made of plasticized polyvinyl chloride having a concavo-convex structure that functions as a photonic crystal.
8). The manufacturing method of the board | substrate for optical sensors of preceding clause 7 including the following processes,
(1) A step of preparing a plasticized polyvinyl chloride solution by dissolving polyvinyl chloride and a plasticizer in a solvent;
(2) A step of producing plasticized polyvinyl chloride having an uneven structure from the solution using an array mold.
9. 9. The method for producing a substrate for an optical sensor according to 8 above, wherein the array mold is a PVA pillar array mold.

  Compared with the conventional PhC sensor, the optical sensor of the present invention can reproduce (1) the periodic structure with good reproducibility (uniformly) without the need to produce a multilayer structure, and (2) It can be detected with high sensitivity and specificity, (3) PhC can be produced with an inexpensive resin material, and (4) it can be easily produced using nanoimprint lithography.

Plasticized resin (PVC) hole array PhC fabrication process. External view of PVA pillar array mold. (A) SEM image of COP-made PhC used as the produced mold, (b) SEM image of PVA-made pillar array mold. (A) Appearance photograph of the produced plasticized PVC PhC from the vertical direction, (b) Appearance photograph from the direction of 30 degrees from the vertical direction. SEM image of the surface of the plasticized PVC-made PhC. Reflection spectra of plasticized PVC PhC and plasticized PVC smooth film. Relationship between thickness of PVC PhC and reflection spectrum intensity. A flow cell for measurement produced with a PP sheet {(a) PP sheet for flow cell, (b) Flow cell cross section during measurement}. Response measurement results of ion-responsive plasticized PVC PhC. Measurement results of response speed to 10 mM KOH / 100 mM HCl introduction. Measurement results of ion selectivity of ion-responsive plasticized PVC PhC. Comparison result of response change of ion-responsive plasticized PVC PhC and response change of ion-responsive plasticized PVC film. The conceptual diagram of the optical sensor of this invention.

  The optical sensor of the present invention (see FIG. 13) has a detection part having an uneven structure that functions as a photonic crystal (PhC), and the detection part includes a recognition element.

(PhC)
A photonic crystal (PhC) is a structure having a certain regularity, that is, a periodicity. As the periodic structure, a hexagonal close-packed structure, which is a crystal structure similar to that of a crystalline inorganic solid, a cubic structure is used. Examples thereof include a closely packed structure, a body-centered cubic packed structure, a log pile structure, a diamond structure, and a wood pile structure. Of these periodic structures, a hexagonal close-packed structure is preferable.

(Uneven structure that functions as PhC)
The concavo-convex structure functioning as PhC in the present invention is not particularly limited as long as it is a material that functions as PhC and can include the recognition element described below. For example, resin-based materials include polyvinyl chloride (PVC), polymethyl methacrylate, polyethylene (glycol) diacrylate (PEGDA), polyvinylidene chloride (PVDC), acrylamide, agarose, polystyrene, polypropylene, polyethylene, collagen, gelatin, Cellulose, polydimethylsiloxane (PDMS), polyester (PET, etc.), polyethylene glycol and the like can be mentioned, with polyvinyl chloride (PVC) being particularly preferred.

(Recognition element)
The recognition element in the present invention is an ionophore for ion detection (valinomycin, CCCP, FCCP, ionomycin, gramicidin A, nigericin, monensin, etc.), heavy metal detection chelate (EDTA, NTA, DTPA, GLDA, HEDTA, GEDTA, TTHA, HIDA, DHEG and the like), disease marker detection enzymes (glucose oxidase and the like), DNA, RNA, sugar chains, proteins and the like, but are not particularly limited.

(Detection site)
The detection site in the present invention has a concavo-convex structure functioning as PhC on at least the surface in contact with the test substance, and further includes a recognition element.
The thickness (film thickness) of the detection site maintains the necessary strength as an optical sensor, and is 0.1 μm to 8.0 μm, preferably 0.15 μm to 7.0 μm, more preferably 0.2 μm to 3.0 μm according to Example 1 below. Most preferably, it is 0.3 μm to 1.5 μm.

(Test substance)
The test substance in the present invention is not particularly limited as long as it is a substance recognized by the recognition element. For example, ions, heavy metals, proteins serving as disease markers, DNA, RNA, sugar chains and the like can be mentioned.

(Optical sensor manufacturing method)
The optical sensor of the present invention preferably uses a nanoimprint method as exemplified in the following steps. This is because the nanoimprint method can process a minute structure precisely and easily, so that a manufacturing cost is low, and a sensor with extremely high sensitivity and high reliability can be provided.
In addition, the conventional optical sensor using PhC has low reproducibility, and the error between the produced sensors is very large. Therefore, it is difficult to compare optical characteristics between different optical sensors. However, since the same optical sensor of the present invention formed by nanoimprint technology can be mass-produced with good reproducibility at low cost, the optical characteristics can be compared even between different optical sensors.

(Production of array mold)
(1) After cleaning the surface of the pillar array type PhC or the hole array type PhC with ethanol or the like, it is placed on a spin coater and dried by spinning.
(2) Using a Pasteur pipette, drop the PVA aqueous solution or PVP aqueous solution onto the pillar array type PhC or hole array type PhC placed on the spin coater with the surface with the nano-periodic structure facing up, spin and coat do.
(3) Dry pillar array type PhC or hole array type PhC coated with PVA aqueous solution or PVP aqueous solution.
(4) Pillar array type PhC or hole array type PhC coated film-like PVA is placed on a mold release support (water-soluble double-sided tape is placed on a PET film), and pillar array type using tweezers Release PhC or hole array type PhC by mechanical peeling from the edge.
If necessary, it is confirmed using a scanning electron microscope (SEM) that the PhC nano-periodic structure is transferred to the released PVA film or PVP film.
A preferred array mold of the present invention is a PVA pillar array mold.

{Preparation of a detection site (a plastic array hole array including a recognition element) including a recognition element and including a concavo-convex structure}
(1) A resin, a plasticizer, and a recognition element are dissolved in a solvent to prepare a plasticized resin solution.
(2) Place the PVA pillar array mold, PVA hole array mold, PVP pillar array mold, or PVP hole array mold prepared above in a spin coater, and paste the plasticizing resin solution of (1) with a Pasteur pipette. The solution is dropped on the mold and spun to form a film and dried.
(3) A PET support is installed, the water-soluble double-sided tape is peeled off from the release support, and the mold is dissolved and released in water by immersing in distilled water.
(4) After dipping, the plasticized resin film containing the recognition element is pulled up from the water, washed with distilled water, and a PET support is placed on the other surface.
If necessary, confirm that the nano-periodic structure is transferred to the surface of the plasticized resin film containing the released recognition element by SEM.
The “plasticizer” used in the present invention includes NPOE (2-Nitro phenyl octyl ether), DOP (Dioctyl phthalate, dioctyl phthalate), DOS (Dioctyl sebacate, di-2-ethylhexyl sebacate), Examples include, but are not limited to, DOA (Dioctyl adipate), DOPP (Di-n-octyl phenylphosphonate), dioctyl trimellitic acid, and citric acid triester.
The “solvent” used in the present invention is not particularly limited as long as it is a solvent that dissolves the resin and has high volatility. For example, THF, cyclohexane, ethyl acetate and the like can be mentioned.

(Optical sensor substrate composed of plasticized polyvinyl chloride with concavo-convex structure that functions as PhC)
In the present invention, “a substrate for an optical sensor composed of plasticized polyvinyl chloride having an uneven structure functioning as PhC” is also an object.
In the conventional method, an optical sensor substrate made of plasticized polyvinyl chloride having a concavo-convex structure functioning as PhC could not be produced. However, in the present invention, the substrate could be produced by the following method. The substrate can be used as a normal biosensor substrate.

(Manufacturing method of optical sensor substrate composed of plasticized polyvinyl chloride with concavo-convex structure functioning as PhC)
A method for manufacturing a substrate for an optical sensor composed of plasticized polyvinyl chloride having a concavo-convex structure functioning as PhC includes the following steps.
(1) A step of preparing a plasticized polyvinyl chloride solution by dissolving polyvinyl chloride and a plasticizer in a solvent.
(2) Using this solution, PVA pillar array mold, PVA hole array mold, PVP pillar array mold or PVP hole array mold is used to produce plasticized polyvinyl chloride having a concavo-convex structure that functions as PhC. Process.

(Method of using the optical sensor of the present invention)
The method for using the optical sensor of the present invention is exemplified below.
(1) A sample not containing a test substance or the like is brought into contact with the concavo-convex structure of the optical sensor of the present invention, irradiated with light, and the optical characteristics of the reflected light, particularly the peak wavelength and intensity of the reflected light. Get information. Any light source may be used. For example, a tungsten / halogen light source, a light emitting diode, an LED, a deuterium lamp, an organic EL, or a laser may be used. In addition, for example, a multichannel spectroscope, a CCD image sensor, a CMOS sensor, or the like can be used as the light detection means for detecting the reflected light.
(2) Next, after the sample containing the test substance is brought into contact with the concavo-convex structure of the optical sensor of the present invention, information on the optical characteristics of the reflected light is obtained in the same manner.
(3) Finally, the presence / absence or amount of the test substance contained in the sample is detected by comparing the optical characteristic of the reflected light of (2) with the optical characteristic of (1).

  The optical characteristics of the reflected light when the concentration of the test substance contained in the sample is changed in advance are detected by an optical sensor, converted into a database, and reflected from the optical sensor after contacting this data with the sample. The presence / absence or amount of the test substance contained in the sample may be detected by comparing the optical characteristics of the light.

In addition, information on optical characteristics of transmitted light may be used by irradiating the optical sensor with light. In this case, it may be used as follows.
(1) A sample not containing a test substance or the like is brought into contact with the concavo-convex structure of the optical sensor 1, irradiated with light, and information on the optical characteristics of transmitted light, particularly on the peak wavelength and intensity of reflected light. get.
(2) Next, after the sample containing the test substance is brought into contact with the concavo-convex structure of the optical sensor of the present invention, information on the optical characteristics of the transmitted light is obtained in the same manner.
(3) Finally, the presence / absence or amount of the test substance contained in the sample is detected by comparing the optical characteristics of the transmitted light of (2) with the optical characteristics of the transmitted light of (1).

  Hereinafter, the present invention will be described in detail with specific examples, but the present invention is not limited to these examples.

(Production and characterization of plasticized PhC)
In producing PhC made of plasticized resin, it is necessary to meet the requirements from the PhC side and the optode membrane side. That is, in order to exhibit sufficient performance as PhC, it is necessary to make the base material portion supporting the nano-periodic structure thin, and at the same time, it is preferable to make it thin in order to speed up the response as an optode membrane.
Therefore, in this example, a nano-periodic structure (PhC) was produced on a film of the order of several hundred nanometers.
The imprint method was selected as a method for producing the nano-periodic structure. With this method, it is easy to spin-coat a plasticizing resin {in this embodiment, using polyvinyl chloride (PVC) as an example of the resin} on the mold without complicated steps such as etching. Fabrication of nano-periodic structures was possible.
This time, the mold release method was selected by dissolving the mold. Making a mold with a material that is insoluble in the organic solvent in which the plasticized PVC is dissolved, and insoluble in the plasticized PVC, puts a mechanical load on the film-like plasticized PVC. Mold release was possible without any problems.
Furthermore, in the production of plasticized PVC PhC, the film thickness that greatly contributes to both PhC performance and ion sensor performance was investigated. In addition, about film thickness, conditions were examined by adjusting a viscosity by changing the solvent dilution rate of a plasticized PVC polymer solution. Details are as follows.

(Production of plasticized plastic PhC by nanoimprint method)
A plasticized resin hole array PhC was produced by the following steps (see FIG. 1).

1. Creation of PVA (polyvinyl alcohol) pillar array mold Cycloolefin polymer (COP) hole array type PhC (hole size 230 nm, lattice constant 400 nm, hole depth 200 nm, provided by SCIVAX Corporation) using punch And cut out to a diameter of 12 mm.
The cut COP PhC was sufficiently dried by washing the surface with ethanol, placing it on a spin coater, and spinning for 10 seconds at 8000 rpm.
A 10% (w / w) PVA aqueous solution was prepared as a water-soluble polymer solution. When preparing, weigh 10 g of PVA (165-17915, manufactured by Wako Pure Chemical Industries) and 90 g of distilled water, mix well in a 110 mL screw jar, and mix at 90 ° C with a constant-temperature oven. After heating and complete dissolution, the mixture was allowed to cool to room temperature.
Using a Pasteur pipette, drop the prepared aqueous PVA solution (about 1 mL) dropwise onto COP PhC placed on a spin coater with the surface with the nano-periodic structure facing up, and quickly spin at 8000 rpm for 3 seconds. Coated.
COP-made PhC coated with an aqueous PVA solution was dried at 70 ° C. for 10 minutes by a constant air constant temperature oven. Then, it took out from the ventilation constant temperature thermostat, and stood to cool to room temperature.
Prepared release support with water-soluble double-sided tape on PET film (Black & White copier Transparency Film, manufactured by 3M) cut into 20 mm squares, and PVA in film form coated on COP PhC Was placed on the mold release support, and the mold was released by mechanically peeling the COP PhC from the end using tweezers.
It was confirmed by SEM that the nanoperiodic structure of COP-made PhC was transferred to the released PVA film.

2. Production of plasticized PVC hole array PhC As plasticized PVC polymer solution, PVC (high molecular weight 81387, manufactured by Fluka) 40 mg, as plasticizer 2-Nitro phenyl octyl ether (NPOE, S038, manufactured by Tokyo Chemical Industry Co., Ltd.) 80 mg and Tetrahydrofuran (THF) 1080 mg were weighed and stirred in a screw tube. THF was prepared separately by weighing 480 mg and 280 mg.
Place the PVA pillar array mold prepared above on a spin coater, drop the plasticized PVC polymer solution (about 1 mL) onto the mold with a Pasteur pipette, and quickly spin at 1500 rpm for 10 seconds. To form a film. After drying for 1 minute, a PET support was placed, the water-soluble double-sided tape was peeled off from the release support, and the mold was dissolved and released in water by immersing in distilled water for about 2 hours.
After soaking for 2 hours, the plasticized PVC film was pulled up from the water, washed with distilled water, and a PET support was placed on the other side.
The demolded plasticized PVC film was confirmed to have a nano-periodic structure transferred to the surface by SEM after drying.
Furthermore, a plastic petri dish sample holder was installed on the movable stage, and the reflection spectrum in water was measured.
Finally, the prepared plasticized PVC solution was spin-coated on a Si substrate {N (100), manufactured by Niraco) under the same conditions, and the plasticized PVC film thickness was measured using an ultra-deep microscope.

3. Evaluation of PVA Pillar Array Mold The appearance of the manufactured PVA pillar array mold is shown in FIG. FIG. 2A shows the light source irradiated from the vertical direction, and FIG. 2B shows the light source irradiated from the 30 degree direction. By irradiating light obliquely, the structural color produced by the surface nano-periodic structure acting as a reflective diffraction grating was confirmed.
FIG. 3 shows SEM images of COP-made PhC and PVA-made pillar array molds used as the produced mold. FIG. 3A is a surface SEM image of COP PhC used as a mold, and FIG. 3B is a surface SEM image of a PVA pillar array mold.
As apparent from the SEM image of FIG. 3B, it was confirmed that the manufactured PVA mold had a structure in which pillars having a diameter of 230 nm were regularly arranged. This indicates that the PVA solution was adhered along the surface structure even on the hydrophobic COP. Furthermore, incompletely transcribed portions could not be confirmed.
From the above, it was confirmed that a PVA pillar array mold could be produced.

4). Evaluation of plasticized PVC-made PhC Fig. 4 shows the appearance of the plasticized PVC-made PhC. 4A is an image when the light source is irradiated from the vertical direction, and FIG. 4B is an image when irradiated obliquely from the direction of 30 degrees from the PhC plane. The structural color due to the behavior of the nanoperiodic structure on the PhC surface as a reflective diffraction grating was confirmed.
FIG. 5 shows an SEM image of the surface of the plasticized PVC-made PhC.
From the SEM image in FIG. 5, it was confirmed that a periodic structure having a hole diameter of about 230 nm was transferred. It was also confirmed that there was no residual PVA and no disruption of the periodic structure.
From the above, it was shown that the PVA mold was completely dissolved in water and was resistant to THF, which is a plasticized PVC solution solvent.
The PVA used this time had a saponification rate of 80%, but when the saponification rate was changed to 95%, the vinyl acetate, which is a hydrophobic part in the PVA, decreased, so the resistance to THF further increased and good transfer performance was achieved. can get.

(Evaluation of reflection spectrum of plasticized PVC PhC in water)
The reflection spectrum of plasticized PVC PhC in water was evaluated.
The measurement was performed in a state where PhC made of plasticized PVC was fixed to a plastic petri dish using polyimide tape and distilled water was added until the PhC was completely immersed in water. For comparison, the reflection spectrum of a smooth film made of plasticized PVC having no nano-periodic structure on the surface was also measured.
The measurement results are shown in FIG.
As is apparent from FIG. 6, the produced plasticized PVC PhC confirmed a reflection spectrum having a large peak at 570 nm. On the other hand, the reflection spectrum intensity of the smooth film was weak and strongly reflected the spectrum distribution of the light source. The reflection spectrum of the smooth film is considered to be due to Fresnel reflection. Assuming that the reflection spectrum of plasticized PVC PhC is the sum of the Fresnel reflection of the nano-periodic structure forming surface and the smooth surface behind it, the reflection intensity of plasticized PVC PhC greatly exceeds that of a smooth film. Don't be.
Therefore, it was confirmed that the reflection spectrum of plasticized PVC PhC was extracted by diffractive radiation after light was scattered by the nano-periodic structure and guided to the opposite smooth surface.

(Evaluation of reflection spectrum intensity by difference in thickness of plasticized PVC PhC)
The reflection spectrum intensity due to the difference in thickness of plasticized PVC PhC was evaluated.
First, the relationship between the amount of THF in the plasticized PVC polymer solution and the film thickness of the plasticized PVC PhC was confirmed. The confirmation results are shown in Table 1 below.
As apparent from Table 1 below, it can be seen that as the weight ratio of THF increases, the viscosity of the polymer solution decreases and the film thickness decreases accordingly.

Furthermore, the measurement results of the reflection spectrum intensity of plasticized PVC PhC having different thicknesses are shown in FIG.
As is clear from the measurement results of FIG. 7, it was observed that the reflection intensity increased as the PhC thickness decreased. This is because the diffraction of the light guided in the PhC plane direction increases as the PhC thickness decreases, and the light extraction efficiency by diffracted radiation increases accordingly.

From the results of this example, it was confirmed that PhC made of plasticized PVC through a PVA mold could be produced.
More specifically, it was confirmed that the plasticized PVC PhC produced was able to accurately transfer the structure (hole diameter 230 nm, lattice constant 400 nm) of the COP hole array PhC used as the primary mold. This indicated that the imprint method via PVA mold is useful.
Since PVA used as a secondary mold has low adhesiveness with many hydrophobic materials, it can be produced from a mold made of silicon, glass, metal material, or Teflon ^{(registered trademark)} other than COP.
Furthermore, from the reflection spectrum measurement result of the plasticized PVC-made PhC, it was confirmed that when the polymer-made PhC having a concavo-convex structure was irradiated with light, it did not function as a reflective diffraction grating but functioned as a grating coupler.
As described above, when the plasticized PVC-made PhC is used as an optode membrane ion sensor, both the response speed and the response change amount can be achieved by reducing the thickness.

(Conclusion)
Formation of nano-periodic structure on plasticized PVC, which is a soft material with rubber-like elasticity, has been realized by imprinting via PVA mold.
In this example, a plasticized PVC polymer solution in which PVC, a plasticizer, an ion recognition element, and a dye are dissolved in THF in a PVA pillar array mold prepared by spin coating a PVA aqueous solution into a COP hole array mold. The film was formed by spin-coating, and the mold was dissolved in water and released to produce PhC made of plasticized PVC.
The PhC succeeded in producing a nano-periodic structure with a hole diameter of 230 nm on a thin film having a thickness of 0.35 μm.

(Production and evaluation of plasticized PVC PhC capable of detecting test substances)
By adding an ion recognition element and a fat-soluble dye to the plasticized PVC PhC produced in Example 1, a PhC capable of detecting a test substance (having ion responsiveness) was produced.
This time, we used valinomycin (ionophore with high selectivity for potassium ion) as an ion recognition element, KD-M11, a merocyanine type dye as a fat-soluble dye, and an acidic solution as a counter ion. TFPB {(Tetrakis [3,5-bis (trifluoromethyl) phenyl] borate, sodium salt)}, which is a fat-soluble anion that does not decompose even, was used.
Ion responsiveness was evaluated by the reflection spectrum of plasticized PVC PhC measured when a solution containing various concentrations of potassium and sodium at a constant pH was introduced. Since the absorption of the dye is determined by the ratio of hydrogen ions / potassium / sodium ions in the solution, the selectivity to ions was also evaluated from the response to each ion under a constant pH condition. Details are as follows.

(Production of ion-responsive plasticized PVC PhC)
According to the PVC PhC manufacturing method of Example 1, ion-responsive plasticized PVC PhC was manufactured.
PVC (40 mg), NPOE (80 mg), THF (1174.77 mg), valinomycin (4.51 mg, manufactured by Wako Pure Chemical Industries, Ltd.), KD-M11 {1- (dodecyl) -4-[(3 ', 5'-dibromo-4'-oxocyclohexa-2',5'-dienylidene) ethylidene] -1,4-dihydroquinoline, 2.28 mg, see Analytica Chimica Acta 373 (1998) 271-289}, TFPB (3.74 mg, Tokyo Chemical Industry) (Manufactured by Kogyo Co., Ltd.) and weighed well in a screw tube.
Since valinomycin, KD-M11, and TFPB are in small quantities and accurate weighing is difficult as they are, weigh 5 times, dissolve completely in 10 mL of THF, and dispense 2 mL. When THF was evaporated, it was redissolved in THF when used.
For comparison, a smooth plasticized PVC film having no nano-periodic structure was also prepared by the same method. The produced ion-responsive plasticized PVC PhC and smooth film were immersed in 100 mM hydrochloric acid and stored.

(Preparation of ion response evaluation solution)
Glycine (0.7507 g) was weighed and dissolved in 1 L of distilled water to prepare a 10 mM glycine aqueous solution. 6 M HCl was added dropwise to this 10 mM glycine aqueous solution while measuring the pH with a pH meter to adjust the pH to 2.5. The pH 2.5 glycine / HCl buffer thus prepared was stored refrigerated and returned to room temperature before measurement.
Potassium chloride 745.5 mg were weighed into pH 2.5 glycine / HCl Buffer of about 30 mL was completely dissolved, up to volume with pH 2.5 glycine / HCl Buffer with 100 mL volumetric flask, pH 2.5 10 ^-1 M KCl solution Was prepared.
Next, weigh 10 mL of the pH2.5 10 ^-1 M KCl solution, and use a 100 mL volumetric flask to make up with pH 2.5 glycine / HCl buffer and dilute 10 times to obtain a pH2.5 10 ^-2 M KCl solution. Was prepared. A pH 2.5 10 ⁻³ M KCl to pH 2.5 10 ⁻⁶ M KCl solution was prepared in the same procedure.
Furthermore, 584.4 mg of sodium chloride weighed in pH 2.5 glycine / HCl buffer was completely dissolved, and a pH 2.5 10 ⁻¹ M NaCl to pH 2.5 10 ⁻⁶ M NaCl solution was prepared in the same procedure.

(Ion responsiveness evaluation method)
An ion-responsive plasticized PVC PhC was placed in a measurement flow cell made of a PP sheet (see FIG. 8).
The solution to be introduced is the above-described pH 2.5 10 ⁻¹ M to pH 2.5 10 ⁻⁶ M KCl / NaCl solution, 100 mM HCl, and 10 mM KOH solution.
When measuring, 5 mL of various solutions were measured and introduced with a pipette, the solution was removed using a pipette, and then the 5 mL solution was introduced again using a pipette.
The reflection spectrum was measured 30 seconds after introduction of the solution.

(Ion response evaluation)
FIG. 9 shows changes in spectrum when ion-responsive plasticized PVC PhC is immersed in 100 mM HCl and 10 mM KOH.
As is clear from the results in FIG. 9, when 100 mM HCl is introduced, there is a reflection peak around 590 nm, and when 10 mM KOH is introduced, the reflection intensity decreases by 40%, and the reflection peak shifts to around 580 nm. did. The change in the reflection intensity is due to the absorption of the dye, and the reflection peak shift does not match the reflection peak of plasticized PVC PhC with no dye added and the maximum absorption wavelength of KD-M11. Since the maximum absorption wavelength of M11 is longer than the reflection peak of PhC, the shift is considered to have occurred.
In addition to the change in the absorption wavelength of the dye contained in the plasticized PVC PhC, it is considered that the change in the reflection spectrum is caused by the extraction of potassium ions. From the result of this response, it can be seen that the change due to the absorption of the dye is more dominant than the change due to the refractive index.

The result of measuring the response speed is shown in FIG.
As is clear from the results of FIG. 10, both the process of extracting K ⁺ into plasticized PVC PhC (KOH introduction) and the process of extracting K ⁺ from the plasticized PVC into the aqueous phase (HCI introduction) Confirmed to complete within 10 seconds. That is, it was confirmed that the optical sensor of the present invention has a very fast response speed.
This very fast response speed is considered to be due to the thin film thickness of 0.35 μm. In addition, even if the solution was repeatedly exchanged, the same reflection intensity change was exhibited with the same solution, so it was confirmed that the response could be performed reversibly.

The measurement result of ion selectivity is shown in FIG.
As is clear from the results of FIG. 11, when the potassium ion concentration in the introduced solution increases, the reflection intensity decreases due to the change in the absorption wavelength of the dye accompanying ion extraction. Since it did not occur, the change in reflection intensity was small.
That is, the optical sensor of the present invention can specifically recognize a test substance.

FIG. 12 shows a comparison result between the response variation of the ion-responsive plasticized PVC PhC and the response variation of the ion-responsive plasticized PVC film.
As is clear from the results of FIG. 12, it was found that the amount of change in plasticized PVC PhC having a nano-periodic structure on the surface was 20 times (40/2) larger than that of a smooth film having no nano-periodic structure.
Since the composition of PhC and smooth film is the same, there is no difference in the response of the dye. Therefore, the difference in the amount of change is because the optical path length has changed.

(Conclusion)
According to the result of this example, a plasticized PVC in which PVC, a plasticizer, an ion recognition element, and a dye are dissolved in THF in a PVA pillar array mold prepared by using a PVA aqueous solution as a spin coat on a COP hole array mold. A film was formed by spin-coating a polymer solution, and the mold was dissolved in water and released, whereby the optical sensor of the present invention could be produced.
It was confirmed that the optical sensor of the present invention can detect a test substance with high sensitivity and specificity. The sensor was able to observe a change in the reflection spectrum due to a change in the absorption wavelength of the dye accompanying ion extraction. It was also confirmed that this change included a change in the refractive index of plasticized PVC accompanying ion extraction and a change in the spectrum accompanying a change in the refractive index of the solution.
In addition, it was confirmed that the ion-responsive plasticized PVC PhC produced in this example had selectivity derived from the ion selectivity of the added ionophore and valinomycin.
Furthermore, when comparing the amount of response change between plasticized PVC PhC with nano-periodic structure and plasticized PVC smooth film without nano-periodic structure, PhC is 20 times the amount of response change with respect to smooth film. Turned out to have.
As described above, the optical sensor of the present invention can detect a test substance specifically and about 20 times more sensitively than a conventional sensor using PhC.

  A new optical biosensor can be provided.

Claims (9)

An optical sensor for detecting a test substance,
It has a detection site with a concavo-convex structure that functions as a photonic crystal,
The detection site includes a recognition element,
Optical sensor.
The detection sites are polyvinyl chloride (PVC), polymethyl methacrylate, polyethylene (glycol) diacrylate (PEGDA), polyvinylidene chloride (PVDC), acrylamide, agarose, polystyrene, polypropylene, polyethylene, collagen, gelatin, cellulose, poly The optical sensor according to claim 1, comprising dimethylsiloxane (PDMS), polyester (PET), and / or polyethylene glycol.
The optical sensor according to claim 1, wherein the recognition element is an ionophore, a chelate, an enzyme, DNA, RNA, a sugar chain, and / or a protein.
The optical sensor according to claim 1, wherein a film thickness of the detection site is 0.1 μm to 8.0 μm.
The manufacturing method of the optical sensor of any one of Claims 1-4 including the following process,
(1) A step of preparing a plasticized resin solution by dissolving a resin, a plasticizer, and a recognition element in a solvent,
(2) The process of producing the detection site | part which consists of plasticizing resin which has an uneven structure which contains a recognition element and functions as a photonic crystal using this solution using an array mold.
The method for producing an optical sensor according to claim 5, wherein the array mold is a PVA pillar array mold.
An optical sensor substrate made of plasticized polyvinyl chloride having a concavo-convex structure that functions as a photonic crystal.
The method for producing a substrate for an optical sensor according to claim 7, comprising the following steps:
(1) A step of preparing a plasticized polyvinyl chloride solution by dissolving polyvinyl chloride and a plasticizer in a solvent;
(2) A step of producing plasticized polyvinyl chloride having an uneven structure from the solution using an array mold.
  The method for producing a substrate for an optical sensor according to claim 8, wherein the array mold is a PVA pillar array mold.