WO2014188621A1

WO2014188621A1 - Examination method and sensor

Info

Publication number: WO2014188621A1
Application number: PCT/JP2013/081663
Authority: WO
Inventors: 三津夫川▲崎▼; 哲郎高松; 義規原田; 丈夫南川; 昂司竹田; 祐山崎; 昌博川▲崎▼
Original assignee: ウシオ電機株式会社; 国立大学法人京都大学; 京都府公立大学法人
Priority date: 2013-05-20
Filing date: 2013-11-25
Publication date: 2014-11-27
Also published as: JP2014228323A

Abstract

This invention provides an examination method whereby cell membranes in a biological material can be examined using optics that do not require rigorously precise design. Said examination method, which is used to examine specimens containing cell membranes, has the following steps: a step (a) in which a light-intensifying element that has a substrate, an intensifying-electromagnetic-field formation layer in which a large number of separate metal microparticles are dispersed across the surface of the substrate, and a protective layer formed on top of the substrate and the intensifying-electromagnetic-field formation layer is prepared; a step (b) in which a specimen to be examined is placed on the top surface of the protective layer; a step (c) in which the light-intensifying element is exposed to excitation light; and a step (d) in which light emitted from the specimen is received and the emission spectrum thereof is measured.

Description

Inspection method, sensor

The present invention relates to an inspection method, and more particularly to an inspection method for a test piece including a cell membrane, which can analyze the spectrum of the cell membrane surface with high sensitivity. The present invention also relates to a sensor used for such an inspection method.

Cell membranes are composed of various phospholipids, proteins, or sugar chains. Not only functions as a partition between cells inside and outside, but also signaling between cells inside and outside through receptors, selection of specific molecules by channels It plays important functions for cells, such as general permeation, sensing outside the cell, and uptake of external substances by endocytosis. Therefore, if it is possible to visualize the molecular localization and functions of phospholipids, proteins, sugar chains, etc. present in the cell membrane, the possibility of leading to the elucidation of functions important for these cells widens (see Non-Patent Document 1). .

As a conventional method for observing cell membranes, a method using a fluorescent dye as a label, an observation method using an electron microscope, a TIRF (Total Internal Reflection Fluorescence) method, a structured illumination microscope, a photoactivated localization microscope, or stimulated emission fluorescence Examples thereof include an observation method using a super-resolution microscope such as a microscope.

Since the electron microscope has high spatial resolution (on the order of nm), it is possible to observe a fine structure such as a cell membrane (thickness of about 10 nm).

Since the analysis depth of the TIRF method is as shallow as about 100 nm, the surface and the vicinity thereof can be observed, and only the cell membrane can be observed (see Patent Document 1).

The super-resolution microscope uses light-specific phenomena such as light interference, single-molecule excitation / measurement and high-accuracy position measurement, and stimulated emission to achieve resolution that exceeds the conventional diffraction limit. A fine structure such as a cell membrane can be observed.

Special table 2010-525313 gazette

The method using a fluorescent dye as a label requires introducing a fluorescent dye exogenously according to the protein or sugar chain component to be observed, preparing an antibody, or introducing a gene into a cell to express the fluorescent protein. was there. Conventionally, the only fluorescent dyes that can be used for such applications are dyes that have a very high emission quantum yield, and thus there are restrictions on the types of dyes that can be used.

In addition, the state of cells may change during the process of introducing a fluorescent dye, binding of an antibody, or expression of a fluorescent protein, and the observed information may not be in a normal state. Furthermore, since it was necessary to carry out the above process, it could not be used for clinical purposes.

Since the TIRF method has a very low fluorescence detection efficiency, this method is limited to observation using a fluorescent dye exhibiting a very high emission quantum yield.

Observation methods using a super-resolution microscope perform complicated interference optical systems for realizing structured illumination, fluorescent dyes that emit light with high brightness that enables photoactivation and single-molecule measurement, and stimulated emission. In particular, a fluorescent dye that does not fade even under strong laser light intensity is required. For this reason, as in the above two methods, the selectivity of the dye is extremely narrow and a complicated optical system is required as an inspection apparatus.

In addition to the above method, there is a conventional method for measuring the Raman spectrum and fluorescence spectrum of the outermost surface using the localized surface plasmon effect. In this method, a device for realizing a localized surface plasmon effect is prepared, light is irradiated in a state where a test piece to be inspected is placed on the device, and light emission from the test piece is spectrally analyzed. It is. The light enhancement effect by the localized surface plasmon effect enhances light emission from the test piece.

However, according to this method, silver fine particles or the like are coated on the surface of the element for realizing the localized surface plasmon effect. Therefore, when a biological material is to be inspected, if this biological material is placed on the device, halide ions contained in the biological material and silver fine particles etc. are chemically reacted and altered, and the silver fine particles are detached and dissolved. Occurs, and the light enhancement effect cannot be obtained. In other words, conventionally, it has been thought that a biological material cannot be inspected using the localized surface plasmon effect.

In view of the above problems, an object of the present invention is to provide an inspection method capable of inspecting a cell membrane of a biological material under an optical system that does not require strict accuracy design. Moreover, an object of this invention is to provide the sensor utilized for such an inspection method.

The inspection method of the present invention is an inspection method for a test piece including a cell membrane,
An optical enhancement element comprising: a substrate; an enhanced electromagnetic field forming layer in which a large number of metal fine particles are dispersed and arranged independently of each other on the surface of the substrate; and a protective layer formed on the substrate and the enhanced electromagnetic field forming layer Preparing step (a),
Placing the test piece to be inspected on the upper surface of the protective layer (b),
Irradiating the light enhancement element with excitation light (c),
And (d) measuring light emission spectrum by receiving light emitted from the test piece.

In the method of the present invention, the localized surface plasmon effect can be used because the structure includes an enhanced electromagnetic field forming layer in which a large number of metal fine particles are dispersed and arranged independently from each other on the surface of the substrate. For this reason, unlike the surface plasmon effect using evanescent waves, there is no strict restriction on the incident angle of light with respect to the substrate, and the degree of freedom is ensured.

Further, in the method of the present invention, a test piece to be inspected is placed on the upper surface of the protective layer formed on the enhanced electromagnetic field forming layer. For this reason, even when a biological material is included as a test piece, the test piece is only in direct contact with the protective layer, and the metal fine particles are not in direct contact with the test piece for a long time. For this reason, when the metal fine particles are continuously exposed to the halide ions for a long time, the halide ions and the metal fine particles do not undergo chemical reaction and change in quality. As a result, Ag having a high light enhancement effect can be used as the metal fine particles. In the configuration of the present invention, Ag is preferably used as the metal fine particles, but other metal materials (for example, Au) may be used.

When utilizing the localized surface plasmon effect as in the present invention, the light enhancement effect acts only in the vicinity of a large number of metal fine particles constituting the enhanced electromagnetic field forming layer. Although the test piece is placed on the upper surface of the protective layer formed on the upper layer of the enhanced electromagnetic field forming layer, the strong enhancing effect reaches the surface through the protective layer formed on the upper layer of the enhanced electromagnetic field forming layer. The light-enhancing effect can be exerted on the test piece placed on the upper surface of the protective layer far from the metal fine particles, particularly on the surface layer portion of the test piece. Therefore, strong luminescence derived from the surface layer portion of the biological material as the test piece, that is, the cell membrane can be obtained.

That is, even if the output of the excitation light to be irradiated is kept low, high Raman scattered light from the test piece can be received by the light enhancement effect, and can be used for spectrum analysis. And as a result of not having to irradiate strong excitation light at the time of inspection, it is possible to prevent light (thermal) decomposition of the biological material. In addition, since the response time can be increased without applying a time constant to the electronic circuit, it is possible to observe rapid changes such as cell differentiation, morphological changes in the cell membrane, and biochemical changes in the cell membrane. Specific examples include observation of phenomena such as cell membrane protein turnover, cell membrane rip-flop, and signaling by receptors in the cell membrane.

In addition, because spectrum analysis can be performed using Raman scattered light, it is not necessary to prepare antibodies that match the components of the protein or sugar chain to be observed or to introduce a gene that expresses a specific fluorescent protein. Label-free observation is possible. Moreover, selective observation is possible even for lipids for which there has conventionally not been a selectively detected dye.

In this case, since the pretreatment operation for introducing the fluorescent substance as the labeling dye is eliminated, the composition can be specified by measuring the Raman spectrum of the living cell membrane and the surface of the living tissue simultaneously with the collection.

The method of the present invention can be used not only for Raman scattered light from a test piece but also for fluorescence spectrum analysis.

In this case, since the localized surface plasmon effect can be used, it is possible to use a non-luminous dye (luminescence quantum yield of about 0.01 or less) that does not emit light in a normal state as a dye for labeling the test piece. Become. That is, when light is incident on the light enhancement element on which the test piece is placed, the light enhancement effect by the enhanced electromagnetic field forming layer is exerted through the protective layer, and thus the non-luminescent pigment labeled on the test piece emits light. The test piece can be inspected by receiving the emitted light at the light receiving unit and performing spectrum analysis. And according to this method, it becomes possible to use a non-light-emitting dye that does not shine in a normal state as a labeling dye, so that the selectivity of the dye is extremely widened.

When labeling with a non-luminescent pigment, for example, a carotenoid, flavonoid, or quinoid pigment can be used. Examples of self-luminous substances that can be used include collagen, riboflavin, NADH (reduced nicotinamide adenine dinucleotide), FDH (flavin adenine dinucleotide), and colored proteins. Further, it may be labeled with an autoluminescent substance (for example, autoluminescent protein).

Furthermore, when the test piece itself is self-luminous, the label itself is not necessary. According to this method, since the light enhancement effect is high, it is possible to detect the light emission utilizing the self-luminous property of the test piece by the light receiving unit.

The step (c) may be a step of irradiating the light enhancement element with excitation light from the side opposite to the side on which the test piece is placed. Thereby, the cell membrane can be observed in-situ while the cells are cultured. In addition, the cell membrane can be observed even for a biological tissue sample having a thickness that does not transmit light.

The protective layer may be composed of an inorganic substance having an orientation in connection with the large number of metal fine particles or a polymer of an organic substance having an orientation.

With this configuration, it is possible to transmit the enhanced electromagnetic field due to the localized surface plasmon effect in the enhanced electromagnetic field forming layer to the surface of the protective layer with high efficiency, and the test piece placed on the surface of the protective layer can be highly efficient. Can emit light.

The protective layer may contain a halogen element.

With this configuration, it is possible to transmit the enhanced electromagnetic field caused by the localized surface plasmon effect in the enhanced electromagnetic field forming layer to the surface of the protective layer with higher efficiency. Note that this configuration is different from the case where a biological substance (biological sample) containing halide ions is directly brought into contact with exposed metal fine particles in a configuration having no protective layer, and a material containing a halogen element is used as the protective layer. Therefore, the function which protects the damage to the metal fine particle by halide ion is ensured similarly to said structure.

The sensor of the present invention is a sensor used for inspection of a test piece including a cell membrane,
An optical enhancement element comprising: a substrate; an enhanced electromagnetic field forming layer in which a large number of metal fine particles are dispersed and arranged independently of each other on the surface of the substrate; and a protective layer formed on the substrate and the enhanced electromagnetic field forming layer Comprising
The light enhancement element includes a first region in which the enhanced electromagnetic field forming layer is formed on the substrate, the protective layer is formed on the enhanced electromagnetic field forming layer, and the enhanced electromagnetic field forming layer on the substrate. And a second region where the protective layer is formed.

According to this configuration, when the test piece is placed so as to straddle the first region and the second region on the sensor and irradiated with light, the portion of the test piece placed on the first region has an enhanced electromagnetic field. While the light enhancement effect by the formation layer extends, the light enhancement effect by the enhanced electromagnetic field formation layer does not reach the location of the test piece placed on the second region. Since the light enhancement effect extends only in the vicinity of a large number of metal fine particles constituting the enhanced electromagnetic field forming layer and in the vicinity of the surface of the protective layer on the upper layer, it is applied to the surface layer portion of the test piece placed on the first region. On the other hand, the effect of enhancing light is exerted. For this reason, the light which has the spectrum derived from the constituent material of the surface layer part of a test piece is obtained from the part of the test piece mounted on the 1st field.

On the other hand, since the enhanced electromagnetic field forming layer does not exist on the second region, the light enhancing action of the enhanced electromagnetic field forming layer does not reach the part of the test piece placed on the second region. For this reason, the portion of the test piece placed on the second region emits light having a spectrum derived from the substance constituting the inside of the surface layer as well as the surface layer of the test piece. When the test piece is a biological material, the volume of the cytoplasm that forms the inner surface of the surface layer is extremely large compared to the cell membrane that forms the surface layer, and thus light emission having a cytoplasm-derived spectrum is obtained.

Therefore, according to this sensor, by irradiating light, it is possible to obtain the spectrum of the substance constituting the surface layer of the test piece and the substance constituting the inside of the surface layer from one test piece. For example, when the test piece is a biological material-derived adipocyte, it is possible to easily obtain both the cell membrane constituting the surface layer and the spectrum data of the adipocyte covered with the cell membrane.

According to the present invention, an inspection method capable of inspecting a biological material can be realized under an optical system that does not require strict accuracy design. Further, according to the sensor of the present invention, a sensor suitable for inspection using the above inspection method can be realized.

It is drawing which shows the structure of the sensor of this invention typically. It is a conceptual diagram for demonstrating the inspection method of this invention. It is a conceptual diagram of the measuring apparatus for verifying the performance of the sensor of this invention. It is a graph which shows the result of having plotted the relationship between the thickness of a protective layer, and photointensity | strength when a rhodamine 6G pigment | dye is carry | supported with respect to a sensor. It is a graph which shows the result of having plotted the relationship between the thickness of a protective layer when a fuchsin pigment | dye is carry | supported with respect to a sensor, and photointensity. 6 is a graph comparing spectral distributions of light received by the light receiving units of Example 2 and Comparative Example 1. FIG. It is a graph which shows the Raman spectrum received by the light-receiving part when each sensor of Example 3 and Example 1 was irradiated with excitation light. It is a graph which shows the Raman spectrum light-received by the light-receiving part, when a fat tissue is mounted on a sensor (or board | substrate) and light is irradiated. It is a schematic diagram at the time of mounting a cell sample such as adipose tissue as a test piece on a light enhancement element (on a sensor). It is the graph which compared the fluorescence intensity received in Example 5 and Comparative Example 3. FIG. It is drawing which shows typically the structure of the sensor of 2nd Embodiment of this invention. It is the graph which compared the spectrum distribution of the light received by the light-receiving part when each sensor of Example 6 and Comparative Example 4 was irradiated with excitation light. It is the graph which compared the spectral distribution of the light received by the light-receiving part when each sensor of Example 7 and Comparative Example 5 was irradiated with excitation light. It is a graph which shows the Raman spectrum light-received by the light-receiving part when each sensor of Example 8 and Example 9 was irradiated with excitation light.

[First Embodiment]
A first embodiment of the present invention will be described with reference to the drawings. In each figure, the dimensional ratio in the drawing does not necessarily match the actual dimensional ratio.

<Sensor structure>
FIG. 1 is a drawing schematically showing the structure of a sensor used in the inspection method of the present invention. The sensor 1 includes a substrate 7, an enhanced electromagnetic field forming layer 9, and a protective layer 11 to constitute a light enhancement element. Then, the test piece 5 to be inspected is placed on the upper surface of the protective layer 11 and inspected.

The material of the substrate 7 is not particularly limited, and for example, glass, ceramics, resin, or the like can be used. As will be described later, when heat treatment (for example, heating at 100 ° C. or higher) is performed in the manufacturing process of the sensor 1, it is preferable to have heat resistance such as glass or polyimide resin.

The enhanced electromagnetic field forming layer 9 is configured by a large number of metal fine particles 10 being dispersed and arranged on the surface of the substrate 7 independently of each other. The arrangement method of the metal fine particles 10 on the surface of the substrate 7 may be a two-dimensional random arrangement or a regular arrangement.

As the metal fine particles 10 constituting the enhanced electromagnetic field forming layer 9, for example, Ag can be suitably used, but Au, Al, and the like can be used as long as they can be excited by irradiation with excitation light to realize the localized surface plasmon effect. Other materials such as Cu can also be used. In addition, as the shape of the metal fine particles 10, for example, those having shape anisotropy such as a flat hemispherical shape or a flat plate shape can be suitably used. Note that it is desirable that all the metal fine particles 10 have a uniform size and shape, but there may be some variation in size and shape.

Further, the particle diameter of the metal fine particles 10 is preferably smaller than the wavelength of the excitation light. Here, in the present specification, “particle size” refers to a projected area equivalent circle diameter measured by microscopy. Specifically, it is obtained as follows. Field of view of a scanning electron microscope (for example, 1.5 μm × 2 μm) in which a 2 μm-long line segment is observed to be enlarged to a length of 6 cm (magnification: 30000 times) in an arbitrarily selected region on the surface of the sensor 1 As an imaging region, a secondary electron image of the region in the sensor 1 is obtained. At this time, for each of the metal fine particles having a brightness index (256 steps) of about 100 or more, the diameter of a perfect circle having the same area as the metal fine particle 10 is obtained as the particle diameter of the metal fine particle 10.

The particle size of the metal fine particles 10 is in the range of 5 to 300 nm, for example, and the thickness is in the range of 5 to 70 nm, for example. Further, the density of the metal fine particles 10 constituting the enhanced electromagnetic field forming layer 9 can be, for example, about 10 ⁸ to 10 ¹¹ particles / cm ² .

As an example of a method for forming such an enhanced electromagnetic field forming layer 9, a method in which a dispersion liquid in which a precursor of metal fine particles 10 is dispersed in an appropriate solvent is applied to the surface of the substrate 7 by a spin coating method and heated. be able to. Further, as another method, a method of dipping the precursor of the metal fine particles 10 on the surface of the substrate 7 and heating, a method of vacuum-depositing the metal fine particles 10 on the surface of the substrate 7, and a method of depositing the metal fine particles 10 on the surface of the substrate 7 A sputter deposition method or the like can be used.

In the sensor 1, a protective layer 11 is formed on the enhanced electromagnetic field forming layer 9 including the surface of the substrate 7 exposed between adjacent metal fine particles 10. Examples of the material constituting the protective layer 11 include silicon oxide, titanium oxide, cerium oxide, boron oxide, phosphorus oxide, magnesium oxide, calcium oxide, aluminum oxide, gallium oxide, germanium oxide, and zinc oxide.

The average thickness of the protective layer 11 is preferably 50 to 250 nm, for example. In particular, since the thickness of the protective layer 11 is 85 nm or more, even when the test piece 5 contains a halide ion (for example, Cl ⁻ ) such as a biological material, sufficient resistance to the metal fine particles 10 ( Protection function).

Such a sensor 1 is manufactured by the following method, for example. First, a metal nanoparticle film is formed on the surface of the substrate 7, and the graininess is changed by heat-treating the film, whereby an enhanced electromagnetic field forming layer 9 made of metal fine particles 10 having a particle diameter within a predetermined range is formed. Form. At this time, the particle diameter of the metal fine particles 10 to be formed can be adjusted by appropriately changing the heat treatment conditions.

Next, a protective layer is grown in the thickness direction from the metal fine particle 10 as a starting point on the surface of the enhanced electromagnetic field forming layer 9 including the surface portion of the substrate 7 exposed between the adjacent metal fine particles 10 by vapor deposition. Thus, the protective layer 11 having a columnar structure is formed. The thickness of the protective layer 11 can be adjusted by appropriately changing the growth conditions and time. Moreover, as a formation method of the protective layer 11, methods, such as a radio frequency (RF) sputter vapor deposition method, an electron beam vapor deposition method, or an electron cyclotron resonance (ECR) sputter vapor deposition method, can be selected suitably and can be utilized.

<How to use sensor 1>
Next, a method for inspecting the test piece 5 using the sensor 1 of the present embodiment will be described. FIG. 2 is a conceptual diagram for explaining the inspection method. Here, a method for performing spectrum analysis by irradiating the sensor 1 on which the test piece 5 is placed with excitation light and receiving Raman scattered light emitted from the test piece 5 will be described.

In the present embodiment, in the inspection, the light source 41 for irradiating excitation light from the back side of the substrate 7 of the sensor 1 (the side opposite to the side on which the test piece 5 is placed) and the light emission from the test piece 5 are emitted. A light receiving unit 43 for receiving light is used. The light source unit 41 includes an excitation light source 50, a filter 51, and a mirror 61. The light receiving unit 43 includes a filter 53, a spectroscope 55, and a photodetector 56. Further, in FIG. 2, a configuration is provided in which a half mirror 62 that transmits excitation light from the light source unit 41 and reflects light emitted from the test piece 5 is provided. Further, the condenser lens 52 is installed for the purpose of condensing excitation light from the light source unit 41 and light emission from the test piece 5. The apparatus configuration shown in FIG. 2 is merely an example, and the present invention is not limited to this configuration.

In a state where the test piece 5 is placed on the protective layer 11, excitation light is incident from the light source unit 41 toward the sensor 1. Due to the incident light, the plasmon electric field generated in the enhanced electromagnetic field forming layer 9 of the sensor 1 propagates to the test piece 5 placed on the upper surface of the protective layer 11. Thereby, strong excitation light is exerted on the test piece 5, and high-output Raman scattered light is emitted from the test piece 5. The Raman scattered light is reflected by the half mirror 62 and received by the light receiving unit 43. Wavelength decomposition is performed by the spectroscope 55 of the light receiving unit 43, and a spectral distribution of light received by the photodetector 56 is obtained.

Suppose a biological material including a cell membrane as the test piece 5. At this time, the enhanced electromagnetic field forming layer 9 formed by a large number of metal fine particles 10 suppresses the excitation light from entering the inside of the test piece 5, that is, the inside of the cell membrane, and the excitation light partially penetrates to the inside of the cell. It also acts as a filter for non-enhanced Raman scattered light due to, thereby simultaneously playing an important role in preventing unenhanced Raman signals from being doubled.

Further, the enhanced electromagnetic field formed by the enhanced electromagnetic field forming layer 9 selectively excites the surface layer of the biological material, that is, the molecular species in the vicinity of the cell membrane, through the protective layer 11 having electromagnetic field propagating property, thereby the cell surface ( The Raman signal of the cell membrane is selectively obtained. Since the enhanced Raman scattered light generated in this way is optically strongly coupled to the enhanced electromagnetic field forming layer 9, the enhanced electromagnetic field forming layer 9 efficiently transmits the scattered light to the light receiving portion 43 disposed on the back side of the test piece 5. It leads well and gives a strong Raman signal due to these synergistic effects.

<Description of sensor 1 performance>
Hereinafter, the fact that the light enhancement effect can be realized by the enhanced electromagnetic field forming layer 9 provided in the sensor 1 of the present invention will be described with reference to examples.

(Example 1)
First, a method for manufacturing the sensor 1 of Example 1 will be described. A glass slide having a size of several centimeters was used as the substrate 7, and an Ag film for forming the metal fine particles 10 was formed on the surface of the glass slide by depositing Ag with a thickness of about 10 nm. Thereafter, the substrate 7 on which the Ag film is formed is heated for several minutes on a hot plate at about 100 ° C., thereby changing the granularity of the Ag film and thereby increasing the number of metals (Ag) as the enhanced electromagnetic field forming layer 9. An Ag fine particle monolayer film of fine particles 10 was formed. The particle diameter of the metal fine particles 10 in the obtained Ag fine particle monolayer film is in the range of 50 to 150 nm, the thickness is about 20 nm on average, and the density of the metal fine particles 10 is approximately 5 × 10 ⁹ particles / cm 3. ² .

Thereafter, using an RF sputtering apparatus “RFS-200 type” (manufactured by Ulvac), sputtering is performed under the following conditions using silicon oxide (SiO ₂ ) as a target, thereby exposing the substrate 7 exposed between the adjacent metal fine particles 10. The sensor 1 was produced by forming the protective layer 11 on the surface of the enhanced electromagnetic field forming layer 9 including the surface portion of the above. The thickness of the protective layer 11 was adjusted by appropriately changing the sputtering time.

The sputtering conditions are as follows.
-Distance from the target to the surface of the enhanced electromagnetic field forming layer 9: 45 mm
・ Atmosphere: Ar 3.0Pa (during discharge)
・ Discharge output: 100W
・ RF frequency: 13.6MHz
-Growth rate of the protective layer 11: 8.5 nm / min

Next, a performance verification method for the sensor 1 manufactured as Example 1 will be described. Here, in order to show that the sensor 1 has a light enhancement effect, the fluorescence enhancement effect is verified. That is, the dye molecule is coated on the surface of the protective layer 11 by spin-coating a diluted ethanol solution of rhodamine 6G dye (Rh6G: emission quantum yield of about 1) at 3000 revolutions on the surface of the protective layer 11 on the sensor 1. It was made to carry on. The relationship between the density of the dye molecules carried on the surface of the sensor 1 and the dye concentration of the solution used for spin coating is that the amount of dye molecules carried is 3 × 10 ¹¹ when the concentration of rhodamine 6G dye is 1 μM. / Cm ² .

The emission quantum yield Φ is defined by the ratio between the number of photons absorbed by the molecule and the number of photons emitted by fluorescence (see Equation 1). Here, k _f is a fluorescence transition rate constant of a molecule in an electronically excited state, and _knr is a non-radiative transition rate constant (speed at which quenching occurs per unit time).
(Equation 1)
_{_{Φ = k f / (k f}} + k nr)

If all excited molecules return to the ground state by fluorescence, the emission quantum yield becomes 1, and a substance having an emission quantum yield close to this value is called a “substance with a high emission quantum yield”. However, in practice, it is not 1 due to non-radiative transition. The non-radiative transition is a transition that returns to the ground state without emitting fluorescence. A dye having a low emission quantum yield is a dye having k _f <kn _nr . Note that a dye that hardly emits light under the condition of k _f << k _nr is called a non-light emitting dye.

The intensity of light emitted from the sample (dye molecule) by irradiating the sensor 1 with excitation light was measured by a measuring apparatus shown in FIG. FIG. 3 is a conceptual diagram of a measuring device for verifying the performance of the sensor 1. A light source unit 41 is configured by the diode laser as the excitation light source 50 and the filter 51, light is incident on the sensor 1 from the light source unit 41, and light from the dye molecules carried on the surface of the protective layer 11 is received by the light receiving unit. 43 receives light. The light receiving unit 43 is composed of a condenser lens 52, a filter 53, a light receiving head 54, and a photodetector 55 (electronically cooled diode array detector).

More specifically, a green diode laser (wavelength of 532 nm) having an output of less than 1 mW is used as the excitation light source 50, and light emitted from the excitation light source 50 is not condensed through the filter 51 (energy density is about 30 mW / cm). ² ) or anti-condensation (energy density of about 10 mW / cm ² or less) is made incident on the sensor 1 at an angle of about 45 ° as excitation light. Then, the light scattered in the 90 ° angle direction by the dye molecules carried on the sensor 1 was condensed by the condenser lens 52 onto the light receiving head 54 of the photodetector 55 through the filter 53.

FIG. 4 shows the measurement results of the measurement apparatus shown in FIG. In FIG. 4, the vertical axis represents the increase in luminescence intensity (unit: times), corresponding to the relative ratio to the luminescence intensity measured by the same method for the same amount of dye supported on the glass substrate without the enhancement effect. To do.

In addition, a non-luminescent fuchsine dye (luminescence quantum yield << 0.01) is used instead of rhodamine 6G dye as a sample, and the sample is supported on the surface of the protective layer 11 at a density of 3 × 10 ¹² particles / cm ^2. The emission intensity was measured by the same method as described above. The measurement results are shown in FIG.

4 and 5, it can be seen that a high light emission enhancement rate is maintained even when the thickness of the protective layer 11 is 200 nm or more regardless of the light emission quantum yield of the dye itself. In particular, it can be seen that a light enhancement factor of several thousand times or more is ensured for a dye having an emission quantum yield of less than 0.01, that is, a non-luminescent pigment. Thereby, even if the protective layer 11 having a predetermined thickness is formed on the upper layer of the metal fine particles 10, it can be seen that the light emission enhancing effect can be propagated to the upper surface.

That is, according to the sensor 1, due to the interaction between the molecular light-emitting dipole and the dipole-type surface plasmon (localized surface plasmon), the radiation transition speed k _f of the dye (see Equation 1 above) is increased as a result. Thereby, since k _f >> kn _nr , the above-mentioned light emission quantum yield becomes large, and originally, it is a non-light emitting substance (k _f << k _nr ) whose light emission quantum yield is 0.01 or less. It can be seen that even pigments emit light strongly.

As another verification, the Raman scattering intensity of rhodamine 6G dye with the same arrangement as in FIG. 3 except that a He—Ne laser (wavelength 632.8 nm) with an output of less than 1 mW was used as the excitation light source 50 instead of the diode laser. Was measured as a function of the thickness of the protective layer 11. As a result, even if the film thickness of the protective layer 11 exceeds 200 nm, an enhanced Raman signal (intensity is about 10) which is not different from the signal obtained when the dye molecules are directly adsorbed on the surface of the metal fine particles 10. ⁵ times) was obtained.

As one of the reasons why the enhancement effect propagates about the thickness of the protective layer 11 as described above, the following points can be considered. In the columnar protective layer 11 formed of a dielectric having orientation by vapor deposition, the plasmon electric field is not disturbed and reaches the surface of the protective layer 11 without loss. Therefore, even when the thickness of the protective layer 11 is increased to some extent, the electromagnetic field (localized surface plasmon) generated in the metal fine particles 10 is transmitted to the surface of the protective layer 11.

That is, as shown in FIG. 2, by placing the test piece 5 made of a biological material on the surface of the protective layer 11 and irradiating the excitation light, the influence of the enhanced electromagnetic field is exerted on the surface layer portion of the test piece 5. As a result, a substance that constitutes the surface layer portion of the test piece 5, that is, light that strongly contains a spectrum derived from the cell membrane is emitted. The cell membrane can be analyzed by receiving the emitted light at the light receiving unit 43 and obtaining a spectral distribution.

(Example 2)
An aqueous solution of antibody IgG labeled with a fuchsin dye was added dropwise to the sensor 1 produced by the same method as in Example 1. More specifically, an aqueous solution of antibody IgG labeled with a fuchsin dye was dropped onto the upper surface of the protective layer 11.

(Comparative Example 1)
An aqueous solution of antibody IgG labeled with a fuchsin dye was dropped onto the substrate 7 in the same manner as in Example 2.

Then, FIG. 6 shows the result of examining the spectral distribution of the light obtained by the light receiving unit 43 by causing excitation light to enter the respective elements of Example 2 and Comparative Example 1 using the measuring device shown in FIG. . In FIG. 6, the horizontal axis represents the wavelength of light, and the vertical axis represents the emission intensity.

Referring to FIG. 6, according to the configuration of Example 2, the fuchsin dye, which is a non-luminescent labeling substance, emits luminescence quantum by the interaction through the protective layer 11 with the localized plasmon generated by the enhanced electromagnetic field forming layer 9. It can be seen that the yield is improved and the amount of light emitted by the fuchsin dye is significantly increased. On the other hand, according to the configuration of Comparative Example 1, there is no light emission enhancement effect. This shows that the sensor 1 of the present invention has a very high light emission enhancement effect.

<Description of Performance of Sensor 1 with Different Configuration>
The protective layer 11 of the sensor 1 may contain a halogen element in advance. As a method of containing the halogen element in the protective layer 11, a method of immersing the sensor 1 in which the protective layer 11 is formed in an aqueous solution of a halide salt can be used. For example, when the concentration of the halide salt aqueous solution is 0.1 to 0.3 mol / L, the maximum effect can be obtained by room temperature immersion for about 5 to 30 minutes, but the concentration of the aqueous solution is higher than that. On the other hand, if it is low, the same effect can be obtained by adjusting the immersion time according to the concentration. The halogen element content in the protective layer 11 is preferably 0.002% by mass to 0.05% by mass.

Specific examples of the halide salt include alkali metal salts such as sodium chloride (NaCl), potassium chloride (KCl), sodium bromide (NaBr), and potassium iodide (KI), and calcium chloride (CaCl ₂ ). And alkaline earth metal salts.

Thus, even when the sensor 1 is immersed in the halide salt aqueous solution in advance, since many metal fine particles 10 are covered with the protective layer 11 around and above the metal fine particles 10, halide ions directly contact the metal fine particles 10. Thus, the metal fine particles 10 do not react with the halide ions and leave the substrate 7. Then, by adding a halogen element to the protective layer 11 in advance as described above, the light enhancement effect can be further enhanced as shown in Examples described later.

(Example 3)
Using the same method as in Example 1, the processing time by the RF sputtering apparatus was adjusted, and the sensor 1 having the protective layer 11 having a thickness of 100 nm was produced. Thereafter, the substrate 7 on which the enhanced electromagnetic field forming layer 9 and the protective layer 11 are formed is immersed in a sodium chloride aqueous solution having a chloride ion concentration of 0.2 to 0.3 mol / L for about 30 minutes. By washing with water and drying, halide ions were contained in the protective layer 11 to obtain the sensor 1 of this example. Although it is difficult to accurately determine the content ratio of the halogen element contained in the protective layer in the light enhancement element obtained by this method, it is estimated to be about 0.01% by mass. Then, a thin ethanol solution of rhodamine 6G dye is spin-coated at 3000 revolutions on the upper surface of the protective layer 11 of the sensor 1, so that the dye molecules have a density of 3 × 10 ¹¹ particles / cm ² on the surface of the protective layer 11. It was made to carry by.

For comparison, the sensor 1 was also produced in the same manner except that it was not immersed in an aqueous sodium chloride solution. The sensor 1 corresponds to the element of the first embodiment.

Then, the intensity of Raman scattered light emitted from the sample (dye molecule) by irradiating excitation light to the sensor 1 of Example 3 and Example 1 is changed to a He—Ne laser whose output is less than 1 mW instead of the diode laser 50. The measurement was performed with the same measuring apparatus as in FIG. 3 except that (wavelength 632.8 nm) was used as the excitation light source. The result is shown in FIG. In the graph of FIG. 7, the vertical axis indicates the Raman scattering intensity (cps), and the horizontal axis indicates the Raman shift (cm ⁻¹ ).

In FIG. 7, the intensity of Raman scattered light was compared for each sensor 1 of Example 3 and Example 1. This is because the intensity of Raman scattered light itself is smaller than the intensity of fluorescence, so that it is easy to compare in comparing the light enhancement effects of both. In Example 3 in which halide ions were immersed, a remarkable Raman signal was observed compared to Example 1 in which halide ions were not immersed, suggesting that the light enhancement effect was even higher.

In the sensor 1 of Example 1 in which the halide ions in FIG. 7 are not immersed, as described above with reference to FIGS. 4 and 5, the effect of extremely increasing the emission intensity is obtained. Further, it is suggested that Example 3 in which halide ions are immersed has a higher light emission enhancement effect than Example 1.

In Example 3, a sodium chloride aqueous solution having a chloride ion concentration of 0.2 to 0.3 mol / L was immersed, but instead, the chloride ion concentration was 0.2 mol / L. An element formed by immersing a certain potassium chloride aqueous solution, an element formed by immersing a sodium bromide aqueous solution having a bromide ion concentration of 0.2 mol / L, an iodine having a iodide ion concentration of 0.2 mol / L The same measurement was performed on the element formed by immersing the potassium chloride aqueous solution and the element formed by immersing the calcium chloride aqueous solution having a chloride ion concentration of 0.2 mol / L. High Raman scattered light was received. On the other hand, when the same measurement was performed on an element formed by immersing a sodium sulfate aqueous solution having a sulfate ion concentration of 0.2 mol / L instead of a sodium chloride aqueous solution, the intensity of the received Raman scattered light was low. there were. This shows that the light enhancement effect can be further enhanced when a halogen element is added to the protective layer 11.

<Inspection example using sensor 1>
Hereinafter, the test result of the test piece 5 performed based on the usage method of the sensor 1 mentioned above is demonstrated.

Example 4
Using the sensor 1 of the present invention, the fatty tissue of a Wistar rat (8 weeks old, female) as the test piece 5 is placed on the upper surface of the protective layer 11 and excited by the light source unit 41 in the same manner as in FIG. The Raman spectrum from the test piece 5 was measured by irradiation with light.

The measurement conditions are as follows.
・ Measuring device: DXR-Smart Raman
Excitation light wavelength: 632.8 nm
Excitation light intensity: 4mW
Excitation light diameter: φ10μm
Excitation light illuminance: 50 μW / μm ² = 5 KW / cm ²
Measurement conditions: Exposure for 2 seconds each time is intermittently performed 16 times.

(Comparative Example 2)
A fatty tissue of a Wistar rat (8 weeks old, female) as the test piece 5 was placed on the upper surface of the substrate 7, and the excitation light was irradiated from the light source unit 41 in the same manner as in Example 4 to find the Raman from the test piece 5. The spectrum was measured.

The Raman spectrum measurement results in Example 4 and Comparative Example 2 are shown in FIG. In the graph of FIG. 8, the vertical axis indicates the Raman scattering intensity (cps), and the horizontal axis indicates the Raman shift (cm ⁻¹ ). In addition, the spectrum measured by the micro Raman spectroscope for the section sample of the adipose tissue is also listed as “document value”. The measurement conditions and the like are described, for example, in T. Minamikawa et al., Histochem Cell Biol. 139 (1), 181-93 (2013).

The spectrum in Comparative Example 2 in which the test piece 5 is directly placed on the substrate 7 has a relatively good match with the spectrum described in the literature. On the other hand, the spectrum in Example 4 in which the test piece 5 is placed on the sensor 1 of the present invention, more specifically, on the protective layer 11, is different from that in Comparative Example 2 and literature values in a different position. A shift peak was observed. That is, in the same manner, the adipose tissue was placed on the upper surface of the protective layer 11 of the sensor 1 even though the sliced sample of the adipose tissue was placed and irradiated with excitation light and the spectrum of Raman scattered light was measured. In Example 4 and Comparative Example 2 in which the adipose tissue is placed on the upper surface of the substrate 7, there is a difference in the position of the spectrum peak. From this, the following contents are inferred.

FIG. 9 is a schematic view when a cell sample such as adipose tissue as the test piece 5 is placed on the light enhancement element (on the sensor 1). The adipocyte 71 constituting the adipose tissue has a cell membrane 73 of about 10 nm. The fat cells 71 have a cytoplasm 74 containing fat droplets inside the cell membrane 73. Actually, the inside of the cell membrane 73 includes a cell nucleus and the like in addition to the cytoplasm 74. However, since the occupied area is smaller than the cytoplasm 74, only the cytoplasm 74 will be described here for convenience. The cytoplasm 74 has a diameter of about φ100 μm, and the cell membrane 73 covering the cytoplasm 74 has a thickness of about 10 nm.

The light emission enhancement effect by the enhanced electromagnetic field forming layer 9 is propagated to the region near the surface of the protective layer 11 through the protective layer 11, but does not propagate to a location far away from the surface of the protective layer 11. As shown in FIG. 9, the fat cell 71 has a cell membrane 73 having a thickness of about 10 nm in the vicinity of the surface of the protective layer 11. For this reason, in Example 4, as shown in FIG. 8, the Raman scattered light derived from the cell membrane 73 was remarkably enhanced as compared with the Raman scattered light derived from the cytoplasm 74 inside thereof, and as a result, almost the same peak position was shown. It is thought that.

On the other hand, in the case of Comparative Example 2 in which the test piece 5 is directly placed on the substrate 7, Raman scattered light derived from the cell membrane 73 and Raman scattered light derived from cells inside the cell film 73 are obtained. Here, in consideration of the degree of concentration of excitation light by the condenser lens 52, the degree of diffusion of light within the test piece 5, or the degree of condensation of Raman scattered light by the condenser lens 52, it is placed on the substrate 7. In the obtained cell sample, it is considered that Raman scattered light from an optical path of about 10 μm is measured.

As described above, since most of the cytoplasm 74 occupies the inner side of the

cell membrane

73, 10 nm corresponds to the cell membrane 73 and the remaining 9990 nm corresponds to the cytoplasm 74 when considering the optical path of 10 μm in the cell sample. It can be considered that it corresponds. Therefore, the Raman scattered light intensity of the cell membrane 73 is about 1/10000 with respect to the Raman scattered light intensity of the cytoplasm 74. Thereby, the Raman scattered light obtained in Comparative Example 2 is considered to be mainly composed of the Raman scattered light of the cytoplasm 74. This is consistent with the fact that the peak value of Comparative Example 2 is different in FIG. 8 and that the peak value and the document value are substantially equal.

In FIG. 8, the reason why the peak level of Example 4 and the peak level of Comparative Example 2 show the same value is considered as follows.

It is considered that the light emission enhancing effect due to the enhanced electromagnetic field forming layer 9 propagates at a position of about 10 nm from the surface of the protective layer 11. This is also understood from the fact that in Example 4, the Raman spectrum derived from the cell membrane 73 is strongly confirmed in FIG. In this case, the enhancement of Raman scattered light is about 10 ⁵ to 10 ⁶ times.

As described above with reference to FIG. 2, excitation light (wavelength: 632.8 nm) is irradiated from the back side of the substrate 7 from the light source unit 41. And since the sensor 1 of this invention has the augmented electromagnetic field formation layer 9 and the protective layer 11 on the board | substrate 7, light transmittance falls compared with the case where it comprises only the board | substrate 7. FIG. The transmittance of light at a wavelength of 600 to 800 nm of the sensor 1 of the present invention is about 10% of the transmittance of the substrate 7.

In the case of Comparative Example 2, the analysis depth is about 10 μm as described above. From this, the enhancement of Raman scattered light obtained in Example 4 relative to Comparative Example 2 is (difference in analysis depth) × (intensification) × (transmittance) = (10 nm / 10000 nm) × (10 ⁵ to 10 ⁶ times) × (1/10) = about 10 times.

Further, in the actual experiment, the upper surface of the sensor 1 and the test piece 5 are not completely adhered to each other, and the distance between the upper surface of the protective layer 11 and the cell membrane 73 is about several nanometers away. The value is smaller than “double”. As a result, it is presumed that the intensity of the Raman spectrum of the test piece 5 placed on the upper surface of the sensor 1 is approximately the same as the intensity of the Raman spectrum of the test piece 5 placed on the upper surface of the substrate 7.

However, this does not indicate that the sensor 1 of the present invention including the enhanced electromagnetic field forming layer 9 does not have a light emission enhancing effect as compared with the case where the test piece 5 is directly placed on the substrate 7. . The sensor 1 of the present invention has an effect of enhancing the Raman scattered light in the vicinity of the surface of the protective layer 11 extremely high, whereby the surface layer of the test piece 5 is obtained by performing spectral analysis of the Raman scattered light. It can be used for part inspection.

(Example 5)
Using the sensor 1 of the present invention, the MCF-7 cell line (derived from human breast cancer) as the test piece 5 was placed on the upper surface of the protective layer 11, and DiI (Molecular probes, Cell Tracker (registered trademark) CM-DiI) Labeled with dye. In FIG. 3, an erecting confocal laser microscope (FV1000, Olympus) is used as the photodetector 56, and the light source unit 41 is passed through a water immersion objective lens (Olympus, LUMFL N, 60 times, NA 1.10). The test piece 5 was irradiated with He—Ne laser light (543 nm) to receive light emitted from the dye labeled on the test piece 5.

More specifically, MCF-7 cells were cultured on a culture dish using DME medium (Dulbecco's modified Eagle's medium) until they became subconfluent. Thereafter, 2 × 10 ⁵ cells in total were passaged to sensor 1 placed on a plastic culture dish (BD Falcon, 353001, diameter 35 nm).

After 48 hours, the culture solution was washed with PBS (phosphate buffered saline), and then the DiI dye was added dropwise to a culture dish filled with 2 mL of PBS to a concentration of 1 μmol and incubated for 15 minutes (37 °, 5% CO ₂ ).

Then, after leaving still in a refrigerator (under 4 degreeC) for 5 minutes, the excitation light was irradiated by the said method and the light emission from the labeled pigment | dye of the test piece 5 was measured. For the measurement, an upright confocal laser microscope was used, irradiated with excitation light through a water immersion objective lens, and scanned in the height direction. Of the generated fluorescence, it was transmitted through a confocal pinhole (aperture diameter: 110 μm), and a wavelength range of 560 nm to 660 nm was detected by the filter 53.

(Comparative Example 3)
Except for the point that the test piece 5 is placed on the upper surface of the substrate 7, it is the same as the fifth embodiment.

FIG. 10 shows the results of Example 5 and Comparative Example 3. In FIG. 10, the horizontal axis represents the scanning displacement (μm) and indicates how far away from the surface of the protective layer 11 (Example 5) or the surface of the substrate 7 (Comparative Example 3). The vertical axis represents the emission (fluorescence) intensity. According to FIG. 10, the fluorescence intensity of Example 5 is about twice that of Comparative Example 3, which suggests that the fluorescence spectrum can be easily measured.

[Second Embodiment]
A second embodiment of the present invention will be described with reference to the drawings.

FIG. 11 is a plan view schematically showing the structure of the sensor 1a according to the second embodiment of the present invention. In FIG. 11, the protective layer 11 is not shown for convenience of explanation. Similar to the sensor 1 described above in the first embodiment, the sensor 1a includes a substrate 7, an enhanced electromagnetic field forming layer 9, and a protective layer 11. However, the difference is that a region (first region 20) where the enhanced electromagnetic field forming layer 9 is formed and a region (second region 21) where the enhanced electromagnetic field forming layer 9 is not formed exist on the substrate 7. That is, on the first region 20, as in the sensor 1 described in the first embodiment, an enhanced electromagnetic field forming layer 9 composed of a large number of metal fine particles 10 is formed on the substrate 7, and the protective layer 11 is formed thereon. Is formed. On the other hand, on the second region 21, the enhanced electromagnetic field forming layer 9 does not exist on the substrate 7, and only the protective layer 11 is formed.

Also in the case of performing an inspection using such a sensor 1a, the test piece 5 is placed on the surface of the sensor 1a as in FIG. The Raman scattered light or fluorescence from 5 is received and analyzed. Here, the test piece 5 is placed on the surface of the protective layer 11 so as to straddle the first region 20 and the second region 21 of the sensor 1a.

By placing the test piece 5 in this manner, as described above, the emission intensity in the vicinity of the surface of the protective layer 11 is enhanced on the first region 20, and as a result, mainly derived from the surface layer portion of the test piece 5. Light emission is received by the light receiving unit 43. On the other hand, on the second region 21, since there is no effect of increasing the light emission intensity, light emitted mainly from the internal tissue of the test piece 5 is received by the light receiving unit 43. Therefore, according to such a sensor 1a, it is possible to inspect both the surface layer portion of the test piece 5 and the internal tissue from one test piece 5. Specifically, as shown in FIG. 9A, when fat cells 71 are used as the test piece 5, the emission spectrum mainly derived from the cell membrane 73 is received from the portion placed on the first region 20. The emission spectrum derived mainly from the cytoplasm 74 is received from the portion placed on the second region 21. Thereby, the test object with respect to the test piece 5 can be expanded.

[Third Embodiment]
A third embodiment of the present invention will be described with reference to the drawings.

In the case of obtaining the fluorescence intensity from the test piece 5 using the sensor 1 of the present invention, it is assumed that in Examples 1 to 3 and 5 described above, the labeling dye is used to receive light emitted from the dye. Explained. However, when the test piece 5 itself has a self-luminous property, the dye labeling as described above may not be performed.

(Example 6)
An aqueous solution of a self-luminous biopolymer collagen having a low luminescence quantum yield was spin-coated on the upper surface of the protective layer 11 of the sensor 1 produced by the same method as in Example 1.

(Comparative Example 4)
As in Example 6, the substrate 7 was spin-coated with an aqueous solution of self-luminous biopolymer collagen having a low luminescence quantum yield.

(Example 7)
An aqueous solution of self-luminous riboflavin having a low emission quantum yield was spin-coated on the upper surface of the protective layer 11 of the sensor 1 produced by the same method as in Example 1.

(Comparative Example 5)
Similarly to Example 7, the substrate 7 was spin-coated with an aqueous solution of self-luminous riboflavin having a low emission quantum yield.

FIG. 12 shows the results of examining the spectral distribution of the luminescence obtained by the light receiving unit 43 by causing excitation light to enter the elements of Example 6 and Comparative Example 4 using the measurement apparatus shown in FIG. Similarly, FIG. 13 shows the results of examining the spectral distribution of the light emission obtained by the light receiving unit 43 when the excitation light is incident on the respective elements of Example 7 and Comparative Example 5 using the measuring apparatus shown in FIG. Show. 12 and 13, the horizontal axis represents the wavelength of light, and the vertical axis represents the emission intensity.

Referring to FIG. 12, according to the configuration of Example 6, the self-luminous biopolymer collagen having a low luminescence quantum yield passes through the protective layer 11 with the localized plasmon generated by the enhanced electromagnetic field forming layer 9. It can be seen that the quantum yield of light emission is improved by the interaction, and the amount of light emitted from the biopolymer collagen is significantly increased. On the other hand, according to the configuration of Comparative Example 4, there is no luminescence enhancement effect, and it is suggested that analysis by receiving light emitted from the self-luminous biopolymer collagen is difficult.

Similarly, referring to FIG. 13, according to the configuration of Example 7, the self-luminous riboflavin having a low emission quantum yield passes through the protective layer 11 with the localized plasmon generated by the enhanced electromagnetic field forming layer 9. It can be seen that the quantum yield of light emission is improved by the interaction, and the amount of light emitted by riboflavin is significantly increased. On the other hand, according to the configuration of Comparative Example 5, there is no effect of enhancing luminescence, and it is suggested that analysis by receiving light emitted from self-luminous riboflavin is difficult.

[Fourth Embodiment]
A fourth embodiment of the present invention will be described with reference to the drawings.

The sensor in this embodiment is different only in that the protective layer 11 provided in the sensor 1 of each embodiment described above is composed of an organic polymer having crystallinity (orientation), and the others are common.

As the organic polymer, an acrylic polymer such as polymethyl methacrylate, polyvinyl alcohol, or the like can be used. In this case, the protective layer 11 is formed by dropping or applying the protective layer forming liquid onto the substrate 7 using a spin coat method, a dip coat method, a spray coat method, a slit coat method, a bar coat method, or the like. However, the spin coating method can be suitably used as a method for forming the protective layer 11 having the most uniform thickness.

When the protective layer 11 contains a halogen element, the protective layer forming liquid may be prepared by dissolving a predetermined polymer and a metal salt of a halogen in a solvent, or dissolving a predetermined polymer in the solvent. Can be prepared by mixing a polymer solution obtained by mixing a metal salt solution in which a halogen metal salt is dissolved in a solvent.

The solvent for preparing the protective layer forming liquid is appropriately selected according to the polymer and metal salt used. Specifically, any polymer that can dissolve the polymer and metal salt used may be used. For example, when a water-soluble polymer such as polyvinyl alcohol is used as the polymer, water can be used as the solvent. When a polymer insoluble in water such as polymethyl methacrylate is used as the polymer, for example, cyclohexanone is used as a solvent for preparing the polymer solution, and water and acetone are used as the solvent for preparing the metal salt solution. A protective layer forming solution can be prepared by mixing a polymer solution and a metal salt solution using a mixed solvent.

The content ratio of the polymer in the protective layer forming liquid is determined by the combination of the above coating method and the target protective film thickness. For example, when a polyvinyl alcohol film is formed from the aqueous solution by using a spin coating method (3000 rpm), the content of the polymer necessary for adjusting the film thickness to 100 nm is about 4.5% by mass. The ratio of the metal salt in the protective layer forming liquid is set according to the content ratio of the halogen element in the target protective layer 11 and the content ratio of the polymer in the protective layer forming liquid.

(Example 8)
In the same manner as in Example 1, an Ag fine particle monolayer film composed of a large number of metal (Ag) fine particles 10 as the enhanced electromagnetic field forming layer 9 was formed on the substrate 7.

Also, a protective layer forming solution was prepared by dissolving 5% by mass of polyvinyl alcohol (manufactured by Wako Pure Chemical Industries, Ltd., degree of polymerization of about 500) and 0.2 mmol / L of sodium chloride in pure water. . Thereafter, the prepared protective layer forming solution is applied to the surface of the substrate 7 by spin coating, and dried at about 60 ° C., and crystallization is promoted, thereby enhancing the electromagnetic field forming layer 9 including the surface portion of the substrate 7. The sensor 1 was produced by forming the protective layer 11 on the surface. The protective layer 11 had a crystallinity of 50% or more and a thickness of 110 nm.

The upper surface of the protective layer 11 is spin-coated with a diluted ethanol solution of rhodamine 6G dye at 3000 revolutions so that the dye molecules are supported on the surface of the protective layer 11 at a density of 3 × 10 ¹¹ particles / cm ^2. It was.

Example 9
A light enhancement element was produced in the same manner as in Example 6 except that the protective layer forming solution was changed to one prepared as follows. That is, a polymer solution in which 3% by mass of polymethyl methacrylate (Wako Pure Chemical Industries, Ltd.) was dissolved in cyclohexanone (Wako Pure Chemical Industries, Ltd.) was prepared. On the other hand, a metal salt solution in which 20 mmol / L of sodium chloride was dissolved in a mixed solvent in which pure water and acetone were mixed at a mass ratio of 1: 1 was prepared. Then, a protective layer forming solution was prepared by mixing the polymer solution and the metal salt solution at a volume ratio of 9: 1.

Then, the intensity of Raman scattered light emitted from the sample (dye molecule) was measured by irradiating the sensor 1 of Example 8 and Example 9 with excitation light by the same method as in FIG. The result is shown in FIG. Similarly to FIG. 7, in the graph of FIG. 14, the vertical axis indicates the Raman scattering intensity (cps), and the horizontal axis indicates the Raman shift (cm ⁻¹ ). From the graph of FIG. 14, even when the protective layer 11 is composed of an organic polymer, a strong Raman scattering signal can be confirmed, and it can be seen that the light enhancement effect can be propagated to the upper surface of the protective layer 11. .

That is, as in the present embodiment, the sensor 1 includes a protective layer 11 made of an organic polymer, and a biological material having a cell membrane as a test piece 5 is placed on the upper surface of the protective layer 11 to provide a light source. By irradiating the excitation light from the part 41 and analyzing the spectrum of Raman scattered light or fluorescence from the test piece 5, it is possible to inspect the cell membrane of the test piece 5 as in the first embodiment.

In each of Examples 8 and 9, the protective layer 11 contains a halogen element. However, the protective layer 11 may be made of an organic polymer without containing a halogen element. .

[Another embodiment]
In each of the above embodiments, the sensor 1 may be configured to have a multilayer structure further including a highly reflective layer and a dielectric layer. In such a structure, the highly reflective layer and the dielectric layer are formed in this order on the surface of the substrate 7, and the enhanced electromagnetic field forming layer 9 is formed on the surface of the dielectric layer.

DESCRIPTION OF SYMBOLS 1, 1a: Sensor 5: Test piece 7: Board | substrate 9: Enhanced electromagnetic field formation layer 10: Metal fine particle 11: Protection layer 20: 1st area | region 21: 2nd area | region 41: Light source part 43: Light receiving part 50: Excitation light source 51: Filter 52: Condensing lens 53: Filter 54: Light receiving head 55: Spectrometer 56: Photo detector 61: Mirror 62: Half mirror 71: Fat cell 73: Cell membrane 74: Cytoplasm

Claims

A test method for a test piece including a cell membrane,
An optical enhancement element comprising: a substrate; an enhanced electromagnetic field forming layer in which a large number of metal fine particles are dispersed and arranged independently of each other on the surface of the substrate; and a protective layer formed on the substrate and the enhanced electromagnetic field forming layer Preparing step (a),
Placing the test piece to be inspected on the upper surface of the protective layer (b),
Irradiating the light enhancement element with excitation light (c),
And an inspection method comprising the step (d) of measuring light emission spectrum by receiving light emitted from the test piece.
2. The inspection method according to claim 1, wherein the step (c) is a step of irradiating the light enhancement element with excitation light from a side opposite to the side on which the test piece is placed.
3. The inspection method according to claim 2, wherein light emitted from the test piece is Raman scattered light or fluorescence.
4. The protective layer according to claim 1, wherein the protective layer is composed of an inorganic substance having an orientation or an organic polymer having an orientation in association with the large number of metal fine particles. Inspection method described in 1.
4. The inspection method according to claim 1, wherein the protective layer contains a halogen element.
A sensor used to inspect a specimen including a cell membrane,
An optical enhancement element comprising: a substrate; an enhanced electromagnetic field forming layer in which a large number of metal fine particles are dispersed and arranged independently of each other on the surface of the substrate; and a protective layer formed on the substrate and the enhanced electromagnetic field forming layer Comprising
The light enhancement element includes a first region in which the enhanced electromagnetic field forming layer is formed on the substrate, the protective layer is formed on the enhanced electromagnetic field forming layer, and the enhanced electromagnetic field forming layer on the substrate. And a second region in which the protective layer is formed.