JP2008128905A - Active oxygen detector and active oxygen detecting method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an active oxygen detector and an active oxygen detecting method, for detecting active oxygen generated by photocatalyst reaction or the like, in various reaction environment fields including atmosphere. <P>SOLUTION: This active oxygen detector is provided with an irradiation part 10 for irradiating a product generated by a reaction of at least one molecule of the active oxygen with at least one fluorescent probe, with an excitation light 7 for exciting the product, and a detecting part 11 for detecting fluorescence 14 generated by irradiating the product with the excitation light 7. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an active oxygen detection apparatus and an active oxygen detection method for detecting active oxygen by fluorescence observation of a product obtained by a reaction between active oxygen and a fluorescent probe.

Active oxygen such as singlet oxygen ( ¹ O ₂ ) and hydroxyl radical (.OH) is an important reactive species generated in a photocatalytic reaction typified by titanium dioxide (TiO ₂ ). In addition, it is considered that active oxygen is generated by irradiating the skin with ultraviolet light and causes photoaging. Until now, a method for detecting active oxygen generated in a liquid has been established by developing a fluorescent probe that reacts selectively (see Patent Document 1, etc.) and a method for directly detecting luminescence from ¹ O _2. is there.
JP 2000-32162 A

  However, a method for detecting active oxygen diffused from the solid catalyst surface or the like into the atmosphere with high sensitivity has not yet been developed. The main reasons are that the active oxygen concentration in the atmosphere is very dilute, and that fluorescent probes that selectively react with various active oxygens in the atmosphere have not been developed.

  The present invention provides an active oxygen detection device and an active oxygen detection method for detecting active oxygen generated by a photocatalytic reaction or the like in various reaction environment fields including the atmosphere.

  The active oxygen detection device of the present invention is an active oxygen detection device that detects at least one molecule of active oxygen, and a product generated by a reaction between the at least one molecule of active oxygen and at least one fluorescent probe, An irradiation unit for irradiating excitation light for exciting the product and a detection unit for detecting fluorescence generated by irradiating the product with the excitation light are provided.

  The active oxygen detection method of the present invention is an active oxygen detection method for detecting at least one molecule of active oxygen, wherein the product produced by the reaction between the at least one molecule of active oxygen and at least one fluorescent probe is It includes an irradiation step of irradiating excitation light for exciting the product, and a detection step of detecting fluorescence generated by irradiating the product with the excitation light.

  According to the active oxygen detection apparatus and the active oxygen detection method of the present invention, for example, the product is irradiated with excitation light that does not propagate beyond a predetermined distance, so that fluorescence can be observed with low-intensity background light. Therefore, active oxygen can be detected with high sensitivity even in a reaction environment field where the concentration of active oxygen is dilute. Therefore, it can be applied to various reaction environment fields including the atmosphere.

  The active oxygen detection device of the present invention is an active oxygen detection device that detects at least one molecule of active oxygen, and a product generated by a reaction between the at least one molecule of active oxygen and at least one fluorescent probe, An irradiation unit that irradiates excitation light that excites the product, and a detection unit that detects fluorescence generated by irradiating the product with the excitation light.

  For example, the active oxygen detection device of the present invention is a detection device that detects active oxygen by fluorescence observation of a product obtained by a reaction between active oxygen and a fluorescent probe. By irradiating with excitation light that does not propagate beyond the distance, the fluorescence emitted from the product is observed by the detection unit, and active oxygen is detected in situ. Therefore, active oxygen can be detected with high sensitivity even in a reaction environment field where the concentration of active oxygen is dilute. Therefore, it can be applied to various reaction environment fields including the atmosphere.

  As said irradiation part, a laser irradiation apparatus etc. can be used, for example. The irradiation device that irradiates “excitation light that does not propagate beyond a predetermined distance” may be an irradiation device that directly emits excitation light that does not propagate beyond a predetermined distance. An irradiation device that generates excitation light that does not propagate beyond a predetermined distance by irradiation may be used.

  As the detection unit, for example, a fluorescence observation apparatus including a fluorescence microscope and a high-sensitivity CCD camera can be used. In particular, by using a total reflection fluorescence microscope (TIRFM), fluorescence observation can be performed with extremely low intensity background light, so that detection sensitivity can be improved. Further, since the fluorescence observation apparatus includes a spectroscope, it is possible to measure a fluorescence spectrum at a single molecule level. Examples of the emission characteristics to be observed include a change in fluorescence intensity and a spectral shift. Furthermore, the fluorescence observation apparatus includes a measuring unit that measures the number of fluorescent bright spots, whereby the number of active oxygens can be detected. For example, C3077-70 manufactured by Hamamatsu Photonics Co., Ltd. can be used as the high sensitivity CCD camera. As the total reflection fluorescent microscope, for example, Olympus IX71 can be used. Further, as the spectroscope, for example, SP-2356 manufactured by Acton Research, Inc. can be used.

  The excitation light is preferably light that does not propagate beyond a predetermined distance and can excite the product. Here, the “predetermined distance” need only be a level that does not hinder the fluorescence observation of the product, and may be, for example, a distance equal to or shorter than the wavelength of the excitation light. Usually, when the distance is several hundred nm or less, there is almost no influence of the excitation light on the fluorescence observation.

  Specific examples of the excitation light include evanescent light, plasmon, confocal laser light, and the like. In particular, since evanescent light stays in the vicinity of the excited product as a standing wave, fluorescence can be observed with extremely low intensity background light.

  The active oxygen in the present invention includes a substance that releases active oxygen. According to the present invention, for example, reactive oxygen species such as superoxide anion radical, hydroxyl radical, hydroperoxy radical, hydrogen peroxide, singlet oxygen, nitric oxide, nitrogen dioxide, ozone and lipid peroxide can be detected. The active oxygen in the present invention includes these active oxygen species. Examples of the process of generating active oxygen include an energy transfer reaction from an excited triplet sensitizer such as porphyrin and fullerene to triplet oxygen, a photocatalytic reaction, and the like.

  The fluorescent probe used in the present invention needs to have a high reaction activity with respect to a specific active oxygen. Examples of the fluorescent probe include fluorescent probe species such as polycyclic aromatic compounds, xanthene dyes, and cyanine dyes. Among them, it is known that polycyclic aromatic compounds undergo a cycloaddition reaction selectively with singlet oxygen, and their light emission characteristics change greatly. Examples of polycyclic aromatic compounds include anthracenes, pentacenes, and terylenes. In particular, since the reaction product with singlet oxygen exhibits very strong fluorescence in the vicinity of 605 nm, the singletylene imide derivative can easily detect singlet oxygen.

  In the present invention, multiple types of fluorescent probes may be used. This is because a plurality of types of active oxygen can be detected simultaneously. Also, indirect detection of active oxygen using energy transfer or electron transfer between different fluorescent probes can be realized.

  In the present invention, the fluorescence emitted from the product may be observed at a single molecule level. This is because a spatial distribution of active oxygen can be obtained. Specific examples of the method of observing at the single molecule level include a method of observing fluorescence emitted from the product with the detection unit after placing a fluorescent probe on one main surface of a substrate such as a glass substrate, or an inorganic compound. A method in which active oxygen generated inside or outside a single tube-like, bowl-like or layered structure made of organic compounds or organic compounds is reacted with a fluorescent probe, and fluorescence emitted from the reaction product is observed by the detection unit Etc. can be exemplified. The present invention can be used not only when detecting active oxygen at a single molecule level but also when detecting an aggregate of active oxygen.

  In the present invention, when the fluorescent probe is disposed on one main surface of the substrate, light (laser light or the like) that generates the excitation light is irradiated from the main surface of the substrate opposite to the one main surface. It is preferable. This is because part of the light serving as the generation source passes through the substrate and is fixed as evanescent light on the one main surface, so that fluorescence observation can be performed with extremely low intensity background light. The thickness of the substrate may be about 0.12 to 0.17 mm, for example.

  Although the method of arrange | positioning a fluorescent probe on one main surface of a board | substrate is not specifically limited, For example, the method of apply | coating a fluorescent probe to the said main surface of the said board | substrate can be illustrated. In this case, it is preferable that the film made of the fluorescent probe is applied so that the thickness thereof is, for example, about 10 to 50 nm because fluorescence observation at the single molecule level of the product becomes easy. In addition, you may apply | coat a fluorescent probe on said one main surface through other materials (for example, resin etc.).

  As a method of arranging the fluorescent probe on one main surface of the substrate, the fluorescent probe may be chemically modified on the one main surface of the substrate. For example, when a glass substrate such as a cover glass whose surface is modified with an amino group or a thiol group is used as the substrate, a fluorescent probe into which a substituent such as a succinimidyl ester group or a maleimide group is introduced is used. After being dissolved in a solvent such as the above, the fluorescent probe can be chemically modified on the one main surface by bringing one main surface of the glass substrate into contact with this solution (temperature: 20 to 25 ° C.). In this case, since the fluorescent probe is arranged on the substrate in the form of a monomolecular film, it is easier to observe the fluorescence of the product at the single molecule level. In this case, it is also possible to detect active oxygen indirectly from the dissociation of the fluorescent probe from the substrate or the diffusion motion in the reaction environment field.

  In the present invention, the reactive environment in which active oxygen is generated or diffused is a solid medium, a liquid medium, a gas medium, a polymer, a supramolecule, a biological tissue, or the like, or the surface of the medium, or the above-mentioned difference. Examples include an interface between media. For example, the detection device of the present invention detects active oxygen in a gas diffused from the surface of the solid catalyst into the atmosphere.

  Examples of the solid medium include nanomaterials and micromaterials (for example, nanoparticles, nanotubes, nanofibers, microcrystals, and the like) made of inorganic compounds and organic compounds, and glass. Examples of the liquid medium include fluid liquids such as various organic solvents, water, ionic liquids, and micelle solutions. The gas medium is not particularly limited as long as oxygen is present in the atmosphere. Examples of the polymer include various resins (for example, polymethyl methacrylate (PMMA), polyvinyl alcohol (PVA), Nafion, etc.). Examples of the supramolecule include dendrimers, DNA, and RNA. Examples of the biological tissue include cells, lipid bilayers, skin and the like.

  Next, the active oxygen detection method of the present invention will be described. The active oxygen detection method of the present invention is an active oxygen detection method using the above-described active oxygen detection device of the present invention. Therefore, the description overlapping with the above is omitted.

  The active oxygen detection method of the present invention includes an irradiation step of irradiating a product generated by a reaction between at least one molecule of active oxygen and at least one fluorescent probe with excitation light that excites the product, and the product. And a detection step of detecting fluorescence generated by irradiating the excitation light to the active light. For example, the active oxygen detection method of the present invention is a detection method for detecting active oxygen by fluorescence observation of a product obtained by a reaction between active oxygen and a fluorescent probe, and is a predetermined distance or more away from the product. By irradiating excitation light that does not propagate to the surface, the fluorescence emitted from the product is observed, and active oxygen is detected in situ.

  Embodiments of the present invention will be described below with reference to the drawings. In addition, this invention is not limited to this Example. In the drawings to be referred to, components having substantially the same function are denoted by the same reference numerals, and redundant description may be omitted.

(Example 1)
Example 1 is an example of a method for detecting singlet oxygen ( ¹ O ₂ ) generated in the photocatalytic reaction of TiO ₂ at a single molecule level using a terrylene imide derivative (TDI). 1A to 1D to be referred to are explanatory diagrams for explaining a ¹ O ₂ detection mechanism in the detection method of the ^first embodiment, and FIG. 2 is a schematic diagram of a detection apparatus that realizes the detection method of the first embodiment. is there.

The TDI before the reaction (see FIG. 1A) shows weak fluorescence at about 670 nm by irradiation with a laser (here, wavelength 532 nm). On the other hand, TDI endoperoxide obtained by cyclic addition of ¹ O ₂ to TDI is thermally or photochemically converted into a diepoxide (TDI diepoxide) (see FIG. 1B). The final product, TDI diepoxide, exhibits very strong fluorescence near 605 nm, and can be easily detected at the single molecule level. That is, when the fluorescence is observed with a total reflection fluorescence microscope and a high-sensitivity CCD camera before and after the reaction between TDI and ¹ O ₂ , bright spots increase from the state of FIG. 1C to the state of FIG. 1D. Since each of the bright spots indicates fluorescence from a single molecule of TDI diepoxide, detection at a single molecule level can be performed. The above reaction is an inherent reaction observed in a polycyclic aromatic compound and singlet oxygen. Hereinafter, the details of the detection method (detection device) of Example 1 will be described with reference to FIG.

First, the cover glass 1 having a thickness of 0.12 to 0.17 mm was immersed in a cleaning liquid (Clean Ace manufactured by AS ONE), and the surface was ultrasonically cleaned for 3 hours. The surface of the cover glass 1 was spin-coated with a toluene solution of polymethyl methacrylate (PMMA) to form a PMMA thin film 2 having a thickness of about 14 nm and an area of 18 × 18 mm ² . Next, a chloroform solution (3 nM) of TDI was spin-coated on the PMMA thin film 2 to prepare an observation sample (hereinafter referred to as a sample thin film). In the sample thin film, TDI is placed in the concave and convex portions formed on the surface of the PMMA thin film 2, so that the convex portion on the surface of the PMMA thin film 2 captures hydroxyl radicals (generated by a photocatalytic reaction described later). By doing so, the reaction between TDI and hydroxyl radicals can be suppressed.

Next, an ethanol dispersion solution of TiO ₂ nanoparticles was spin-coated on the slide glass 3 to form a TiO ₂ thin film 4. By irradiating this TiO ₂ thin film 4 with ultraviolet light 5, a photocatalytic reaction was induced to generate ¹ O ₂ . The produced ¹ O ₂ diffuses from the TiO ₂ thin film 4 into the atmosphere, reaches a sample thin film having a certain gap D (12.5 to 2000 μm), and undergoes cycloaddition reaction with TDI. At the same time, the main surface of the cover glass 1 opposite to the PMMA thin film 2 is irradiated with a continuous wavelength laser 6 (wavelength 532 nm, intensity 50 mW) from the laser irradiation device 10 at an incident angle of 50 to 60 degrees. ¹ O ₂ could be detected by generating light 7 and exciting TDI diepoxide as a reaction product with this evanescent light 7 and observing the fluorescence at a single molecule level with a fluorescence observation device 11. . Here, as the fluorescence observation apparatus 11, a fluorescence observation apparatus including a fluorescence microscope 12 and a high sensitivity CCD camera 13 was used.

Note that the maximum reach distance of ¹ O ₂ can be determined by adjusting the gap D with a spacer made of resin or the like. Moreover, by providing the holes 3a in the slide glass 3, an arbitrary oxygen concentration gas can be introduced between the TiO ₂ thin film 4 and the sample thin film.

(Example 2)
A sample thin film was prepared under the same conditions as in Example 1, and 1,4-diazabicyclo [2.2.2] octane was applied onto PMMA thin film 2 (see FIG. 2) of this sample thin film, and then the same as in Example 1 The fluorescence was observed under the conditions. As a result, it was confirmed that the generation of TDI diepoxide was significantly suppressed. This is considered to be due to the capture of ¹ O ₂ by 1,4-diazabicyclo [2.2.2] octane. Moreover, it was confirmed that the formation of TDI diepoxide was significantly suppressed by introducing argon gas into the reaction system instead of applying 1,4-diazabicyclo [2.2.2] octane. This is thought to be due to argon gas expelling oxygen out of the reaction system and suppressing the generation of ¹ O ₂ .

(Example 3)
Example 3 is an example of a method for detecting ¹ O ₂ generated by photoexcitation of fullerene nanoparticles (C ₆₀ NP) at a single molecule level using TDI. FIG. 3 to be referred to is a schematic diagram of a detection apparatus that realizes the detection method of the third embodiment.

First, the cover glass 1 having a thickness of 0.12 to 0.17 mm was immersed in a cleaning liquid (Clean Ace manufactured by AS ONE), and the surface was ultrasonically cleaned for 3 hours. The surface of this cover glass 1 is spin-coated with 40 μL of a chloroform solution containing TDI and C ₆₀ NP (TDI concentration: 3 nM, C ₆₀ NP concentration: 0.01 gmL ⁻¹ ), and an observation sample (hereinafter referred to as a sample thin film). .) Was produced. The continuous irradiation laser 6 (wavelength 532 nm, intensity 50 mW) from the laser irradiation device 10 at an incident angle of 50 to 60 degrees with respect to the main surface of the cover glass 1 opposite to the main surface on which the chloroform solution is spin-coated. To generate evanescent light 7, and TDI and C ₆₀ NP were simultaneously excited by the evanescent light 7. As a result, energy was transferred from the excited triplet state of C ₆₀ NP to triplet oxygen in the atmosphere to generate ¹ O ₂ , and the generated ¹ O ₂ reacted with TDI. Then, ¹ O ₂ could be detected by exciting the TDI diepoxide as a reaction product with the evanescent light 7 and observing the fluorescence at the single molecule level with the fluorescence observation device 11. In addition, ¹ O ₂ could be detected in the same manner even when carbon nanotubes were used instead of C ₆₀ NP.

  The present invention is useful for detecting extremely low concentrations of active oxygen in various reaction systems. Therefore, this invention is directly related to performance evaluation and product development of cosmetics as well as photocatalysts. In addition, the present invention can be implemented not only in the air but also in various reaction environment fields such as various solid media, liquid media, gas media, polymers, supramolecules, internal surfaces of biological tissues, and their interfaces. Yes, it can be applied to many academic fields and industries.

A~D is an explanatory diagram for explaining a detection mechanism of ¹ O ₂ in the detection method of the first embodiment of the present invention. It is a schematic diagram of the detection apparatus which implement | achieves the detection method of Example 1 of this invention. It is a schematic diagram of the detection apparatus which implement | achieves the detection method of Example 3 of this invention.

Explanation of symbols

1 cover glass 2 PMMA thin film 3 slides 3a holes 4 TiO ₂ thin film 5 ultraviolet light 6 continuous wavelength laser 7 evanescent light 10 the laser irradiation device 11 fluorescence observation device 12 fluorescence microscope 13 highly sensitive CCD camera 14 fluorescent

Claims (14)

An active oxygen detector that detects at least one molecule of active oxygen,
An irradiation unit that irradiates excitation light that excites the product with respect to a product generated by a reaction between the at least one molecule of active oxygen and at least one fluorescent probe;
An active oxygen detection device comprising: a detection unit that detects fluorescence generated by irradiating the product with the excitation light.   The active oxygen detection device according to claim 1, wherein the excitation light is light that does not propagate beyond a predetermined distance.   The active oxygen detection device according to claim 1, wherein the excitation light is one of evanescent light, plasmon, and confocal laser light.   The active oxygen detection device according to claim 1, wherein the at least one molecule of active oxygen is a gas.   The active oxygen detection apparatus according to claim 1, wherein the detection unit detects the fluorescence at a single molecule level.   The active oxygen detector according to claim 1, wherein the fluorescent probe is a polycyclic aromatic compound.   The active oxygen detection device according to claim 6, wherein the polycyclic aromatic compound is a terylene diimide derivative.   The active oxygen detection apparatus according to claim 1, wherein the detection unit includes a measurement unit that measures the number of bright spots of the fluorescence. The at least one molecule of active oxygen is composed of at least one active oxygen species,
The at least one reactive oxygen species is at least one selected from the group consisting of superoxide anion radical, hydroxyl radical, hydroperoxy radical, hydrogen peroxide, singlet oxygen, nitric oxide, nitrogen dioxide, ozone and lipid peroxide. The active oxygen detection device according to claim 1, which is a seed. The active oxygen detection device further includes a substrate,
The active oxygen detector according to claim 1, wherein the at least one fluorescent probe is disposed on one main surface of the substrate. The active oxygen detection device further includes a substrate,
The at least one fluorescent probe is disposed on one main surface of the substrate;
The active oxygen detection apparatus according to claim 1, wherein the irradiation unit irradiates the excitation light from a main surface opposite to the one main surface of the substrate.   The active oxygen detection device according to claim 1, wherein the at least one fluorescent probe includes at least one fluorescent probe species.   The active oxygen detection device according to claim 1, wherein the detection unit includes a total reflection fluorescence microscope. An active oxygen detection method for detecting at least one molecule of active oxygen,
Irradiating an excitation light that excites the product to a product generated by a reaction between the at least one molecule of active oxygen and at least one fluorescent probe;
And a detection step of detecting fluorescence generated by irradiating the product with the excitation light.

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