JP5387131B2 - Surface plasmon enhanced fluorescence sensor, chip structure used in surface plasmon enhanced fluorescence sensor, and specimen detection method using surface plasmon enhanced fluorescence sensor - Google Patents

Description

  The present invention relates to a surface plasmon enhanced fluorescence sensor based on the principle of surface plasmon excitation enhanced fluorescence spectroscopy (SPFS), a chip structure used for the surface plasmon enhanced fluorescence sensor, and the surface plasmon enhancement. The present invention relates to a specimen detection method using a fluorescence sensor.

Conventionally, for example, detection of minute analytes in a living body has been performed based on the principle of surface plasmon excitation enhanced fluorescence spectroscopy (SPFS).
Surface plasmon excitation-enhanced fluorescence spectroscopy (SPFS) is a method in which a rough wave (surface plasmon) is generated on the surface of a metal thin film under the condition that the laser light (excitation light) irradiated from a light source attenuates total reflection (ATR) on the surface of the metal thin film. ) To increase the photon amount of the laser light (excitation light) emitted from the light source by several tens to several hundreds times (electric field enhancement effect of surface plasmons), thereby improving the efficiency of the fluorescent material near the metal thin film. It is a method for detecting an extremely small amount and / or an extremely low concentration of analyte by exciting well.

  In recent years, surface plasmon enhanced fluorescence sensors based on the principle of surface plasmon excitation enhanced fluorescence spectroscopy (SPFS) have been developed. For example, Patent Literature 1 and Patent Literature 2 disclose the technology thereof. .

  Such a surface plasmon enhanced fluorescence sensor 100 has a basic structure as shown in FIG. 6 and is first formed of a metal thin film 102, a reaction layer 104 formed on one side surface of the metal thin film 102, and the other side surface. A chip structure 108 having a dielectric member 106.

  The chip structure 108 includes a light source 112 that is incident on the dielectric member 106 and irradiates the excitation light 110 toward the metal thin film 102 on the dielectric member 106 side. The light receiving means 116 for receiving the metal thin film reflected light 114 reflected by the light is provided.

  On the other hand, on the reaction layer 104 side of the chip structure 108, a light detection means 120 that receives fluorescence 118 emitted from a fluorescent substance labeled with the analyte captured by the reaction layer 104 is provided.

  In addition, between the reaction layer 104 and the light detection means 120, the condensing member 122 for condensing the fluorescence 118 efficiently and the light contained other than the fluorescence 118 are removed, and only the necessary fluorescence is selected. A wavelength selection function member 124 is provided.

  When the surface plasmon enhanced fluorescence sensor 100 is used, the reaction layer 104 in which the analyte labeled with the fluorescent material is captured in advance is formed on the metal thin film 102, and in this state, the dielectric member from the light source 112 is formed. 106 is irradiated with excitation light 110, and the excitation light 110 is incident on the metal thin film 102 at a specific angle (resonance angle) θ1, thereby generating a dense wave (surface plasmon) on the metal thin film 102. Yes.

  In addition, when a close-packed wave (surface plasmon) is generated on the metal thin film 102, the excitation light 110 and the electronic vibration in the metal thin film 102 are coupled, resulting in a phenomenon that the light amount of the metal thin film reflected light 114 is reduced.

  For this reason, if the point where the signal of the metal thin film reflected light 114 received by the light receiving means 116 changes (the amount of light decreases) can be found, the resonance angle θ1 at which rough waves (surface plasmons) occur can be obtained.

  Then, due to the phenomenon of generating the rough wave (surface plasmon), the fluorescent material of the reaction layer 104 on the metal thin film 102 is efficiently excited, and thereby the light amount of the fluorescence 118 emitted from the fluorescent material is increased.

  By receiving the increased fluorescence 118 by the light detection means 120 via the light collecting member 122 and the wavelength selection function member 124, it becomes possible to detect an extremely small amount and / or extremely low concentration of the analyte. ing.

  Thus, the surface plasmon enhanced fluorescence sensor 100 is a high-sensitivity measurement sensor that enables observation of minute molecular activities, particularly between biomolecules.

Japanese Patent No. 3294605 JP 2006-208069 A

  However, in the conventional surface plasmon enhanced fluorescence sensor 100 as described above, when the detection sensitivity of the light detection means 120 is increased, in addition to the “fluorescence 118 generated by the fluorescent material in the reaction layer 104” which is the detection target, this “ It has been confirmed by the present inventors that “light having a wavelength close to the wavelength of fluorescence 118” or “light having a wavelength overlapping with the wavelength of fluorescence 118” has also been detected, and true fluorescence detection has been performed. Not found out.

That is, in such a conventional surface plasmon enhanced fluorescence sensor 100, light other than the fluorescence 118 is reduced by the wavelength selection function member 124, but the wavelength close to the fluorescence 118 and the wavelength overlapping the fluorescence wavelength of the fluorescence 118. In the case of light having a wavelength, it cannot be removed sufficiently by the wavelength selection function member 124 provided between the conventional light detection means 120 and the reaction layer 104. In particular, an extremely small amount and / or extremely low concentration of analyte is detected. To detect fluorescence with high accuracy and high sensitivity.

  Further, if the conventional wavelength selection function member 124 tries to completely remove unnecessary light having a wavelength overlapping with the fluorescence wavelength of the fluorescence 118, it leads to a reduction in the fluorescence 118 of the fluorescent substance to be detected, There is also a problem that improvement in detection sensitivity cannot be expected.

  Such light having a wavelength close to the fluorescence wavelength of the fluorescence 118 and light having a wavelength overlapping with the fluorescence wavelength have an adverse effect on the accuracy of the detection value as the detection sensitivity of the fluorescence 118 increases. Therefore, there is a demand for a surface plasmon-enhanced fluorescence sensor that can reliably detect fluorescence and accurately detect fluorescence even when detection sensitivity is increased.

  The present invention has been made in view of such problems of the prior art, and reliably detects the fluorescence emitted by the fluorescent substance in the reaction layer that is the detection target, and detects the fluorescence with extremely high accuracy even when the detection sensitivity is increased. It is an object of the present invention to provide a surface plasmon-enhanced fluorescent sensor capable of performing the above-described steps, a chip structure used for the surface plasmon-enhanced fluorescent sensor, and a specimen detection method using the surface plasmon-enhanced fluorescent sensor.

The present invention was invented to solve the problems in the prior art as described above,
The chip structure used in the surface plasmon enhanced fluorescence sensor of the present invention is:
A fluorescent material for labeling the analyte trapped in the reaction layer formed on the metal thin film by irradiating the metal thin film with excitation light through a dielectric member to enhance the electric field on the metal thin film. A chip structure used in a surface plasmon enhanced fluorescence sensor that is excited and detects fluorescence emitted from the fluorescent material ,
The chip structure is
The metal thin film;
Wherein a reaction layer formed on one side surface of the metal thin film,
And the dielectric member formed on the other side surface of the metal thin film,
Comprising at least
Further, between the dielectric member and the metal thin film,
It has an excitation light selective transmission layer that allows light having an excitation wavelength to excite the fluorescent material to reach the metal thin film, and reduces that light having a fluorescence wavelength of the fluorescent material reaches the metal thin film .

If the excitation light selective transmission layer is provided in this way, the excitation light can reach the metal thin film to generate surface plasmons to excite the fluorescent substance in the reaction layer, and the fluorescence of the fluorescent substance in the reaction layer. Since unnecessary light having a wavelength overlapping with the wavelength can be reduced, a very small amount of fluorescence in the reaction layer can be detected with extremely high accuracy.

  In particular, in the excitation light selective transmission layer formed between the base material of the dielectric member and the metal thin film, such as extremely weak fluorescence (autofluorescence) generated from the dielectric member when the excitation light is incident on the dielectric member. Even if there is an unnecessary component with a wavelength that overlaps with the fluorescence wavelength of the fluorescent substance in the reaction layer, the light can be reduced before reaching the metal thin film, so that the excitation light can further reduce the metal thin film. Fluorescence, which is a very small amount of detection target generated in the rear reaction layer, can be detected with extremely high accuracy.

Further, the chip structure of the present invention is
The excitation light selective transmission layer is
The film is formed by sputtering and / or vacuum deposition.
Thus, if it is a sputtering method and / or a vacuum evaporation method, an excitation light selective transmission layer can be formed especially easily.

Further, the chip structure of the present invention is
A fluorescent material for labeling the analyte trapped in the reaction layer formed on the metal thin film by irradiating the metal thin film with excitation light through a dielectric member to enhance the electric field on the metal thin film. A chip structure used in a surface plasmon enhanced fluorescence sensor that is excited and detects fluorescence emitted from the fluorescent material,
The chip structure is
The metal thin film;
The reaction layer formed on one side of the metal thin film;
The dielectric member formed on the other side surface of the metal thin film;
Consisting of at least
The dielectric member is
Excitation light that excites the fluorescent material reaches the metal thin film, and has an excitation light selective transmission part that reduces the fluorescence wavelength light of the fluorescent material reaching the metal thin film,
The excitation light selective transmission part is
The dielectric member includes an additive having a characteristic of absorbing light having a fluorescent wavelength of the fluorescent material.

If the excitation light selective transmission part is provided in this way, the excitation light can reach the metal thin film and generate surface plasmons to excite the fluorescent substance in the reaction layer, and the fluorescence of the fluorescent substance in the reaction layer. Since unnecessary light having a wavelength overlapping with the wavelength can be reduced, a very small amount of fluorescence in the reaction layer can be detected with extremely high accuracy.
Furthermore, if the excitation light selective transmission part does not contain in the dielectric member an additive having a characteristic of absorbing unnecessary light that overlaps the fluorescence wavelength of the fluorescent substance in the reaction layer, the dielectric member itself is Light with a wavelength that overlaps the fluorescence wavelength of the fluorescent substance, especially fluorescence (autofluorescence) generated from the dielectric member when incident excitation light is incident on the dielectric member can be absorbed. The extremely small amount of fluorescence in the layer can be detected with very high accuracy.

Further, the chip structure of the present invention is
The additive is
It is contained in the whole dielectric member.

  In this way, if the entire dielectric member is an excitation light selective transmission part, this additive is added to the raw material of the base material of the dielectric member, and the dielectric material is made using the raw material containing this additive. By molding the body member, the entire dielectric member can be easily and easily used as the excitation light selective transmission portion.

  In addition, the excitation light selective transmission part can reduce the light with a wavelength that overlaps the fluorescence wavelength of the fluorescent material in the reaction layer, so that the extremely small amount of fluorescence in the reaction layer can be detected with high accuracy when detecting the fluorescence that is the detection target. can do.

Further, the chip structure of the present invention is
The additive is
It is contained in a part of the dielectric member.

  In this way, even when only a part of the dielectric member is an excitation light selective transmission part, it is possible to reduce light having a wavelength overlapping with the fluorescence wavelength of the fluorescent material in the reaction layer. In addition, a very small amount of fluorescence in the reaction layer can be detected with extremely high accuracy.

  In the case of manufacturing a dielectric member in which only a part of the excitation light selective transmission part is manufactured, when forming the dielectric member, for example, an injection molding method such as a two-color molding method or an excitation light of a part of the dielectric member in advance. It can be easily manufactured by using a method of forming only the selective transmission part separately and bonding it with a normal dielectric member with an adhesive or the like.

Further, the chip structure of the present invention is
The excitation light is
The excitation light is incident on the metal thin film at a resonance angle.

Further, the surface plasmon enhanced fluorescence sensor of the present invention,
The chip structure according to any one of the above is provided.
In the case of the surface plasmon enhanced fluorescence sensor having the above-described chip structure, the excitation light reaches the metal thin film due to the structure of the dielectric member of the chip structure to generate the surface plasmon, and the reaction layer. Because it is possible to excite the fluorescent material of the reaction layer and to reduce unnecessary light with a wavelength that overlaps the fluorescence wavelength of the fluorescent material in the reaction layer, it is possible to detect a very small amount of fluorescence in the reaction layer with extremely high accuracy. Can do.

Moreover, the specimen detection method of the present invention comprises:
It is performed using the above-described surface plasmon enhanced fluorescence sensor.
As described above, since the specimen detection method of the present invention is performed with the chip structure using the dielectric member having the excitation light selective transmission part described above, a desired analyte can be detected with high accuracy.

  According to the present invention, a surface plasmon-enhanced fluorescent sensor and a surface plasmon-enhanced fluorescence sensor capable of reliably detecting fluorescence emitted from a fluorescent substance in a reaction layer as a detection target and performing ultra-high-precision fluorescence detection even when the detection sensitivity is increased. It is possible to provide an analyte detection method using a chip structure used in a fluorescence sensor and a surface plasmon enhanced fluorescence sensor.

FIG. 1 is a schematic view of a surface plasmon enhanced fluorescence sensor of the present invention. FIG. 2 is a schematic view of the chip structure in the first embodiment used in the surface plasmon enhanced fluorescence sensor of the present invention. FIG. 3 is a schematic view of the chip structure in the second embodiment used in the surface plasmon enhanced fluorescence sensor of the present invention. FIG. 4 is a schematic view of a chip structure in a third embodiment used in the surface plasmon enhanced fluorescence sensor of the present invention. FIG. 5 is a schematic view of a chip structure in a fourth embodiment used in the surface plasmon enhanced fluorescence sensor of the present invention. FIG. 6 is a schematic view of a conventional surface plasmon enhanced fluorescence sensor.

Hereinafter, embodiments of the present invention will be described in more detail based on the drawings.
FIG. 1 is a schematic view of a surface plasmon enhanced fluorescence sensor of the present invention, and FIGS. 2 to 5 are schematic views of Examples 1 to 4 of a chip structure used in the surface plasmon enhanced fluorescence sensor of the present invention. .

  The surface plasmon-enhanced fluorescence sensor and the chip structure used in the surface plasmon-enhanced fluorescence sensor according to the present invention irradiates excitation light to a metal thin film to generate a dense wave (surface plasmon) and generate fluorescence generated by an excited phosphor. Even if it detects correctly and raises a detection sensitivity, it enables it to perform fluorescence detection with super high accuracy.

<Surface plasmon-enhanced fluorescence sensor 10 and specimen detection method using this surface plasmon-enhanced fluorescence sensor 10>
As shown in FIG. 1, the surface plasmon enhanced fluorescence sensor 10 of the present invention includes a metal thin film 12, a reaction layer 14 formed on one side surface of the metal thin film 12, and a dielectric member 16 formed on the other side surface. And a chip structure 18 having the following.

  The chip structure 18 includes a light source 22 that is incident on the dielectric member 16 and irradiates the excitation light 20 toward the metal thin film 12 on the dielectric member 16 side. The light receiving means 26 for receiving the metal thin film reflected light 24 reflected on the light is provided.

  Here, the excitation light 20 emitted from the light source 22 is preferably laser light, and a gas laser or solid-state laser having a wavelength of 200 to 1000 nm and a semiconductor laser having a wavelength of 385 to 800 nm are preferable.

On the other hand, on the reaction layer 14 side of the chip structure 18, a light detection means 30 that receives the fluorescence 28 generated in the reaction layer 14 is provided.
As the light detection means 30, it is preferable to use an ultra-sensitive photomultiplier tube or a CCD image sensor capable of multipoint measurement.

  In addition, between the reaction layer 14 of the chip structure 18 and the light detection means 30, a light collecting member 32 for efficiently collecting light, and transmission of light having a wavelength different from that of the fluorescence 28 in the light. And a wavelength selection function member 34 formed so as to selectively transmit the fluorescence 28.

  As the condensing member 32, any condensing system may be used as long as it aims at efficiently condensing the fluorescent signal on the light detecting means 30. As a simple condensing system, a commercially available objective lens used in a microscope or the like may be used. The magnification of the objective lens is preferably 10 to 100 times.

On the other hand, as the wavelength selection function member 34, an optical filter, a cut filter, or the like can be used.
Examples of the optical filter include a neutral density (ND) filter and a diaphragm lens.

  Furthermore, the cut filter includes external light (illumination light outside the device), excitation light (excitation light transmission component), stray light (excitation light scattering component at various points), and plasmon scattering light (excitation light originated from plasmon A filter that removes various types of noise light such as scattered light generated due to the influence of structures or deposits on the surface of the excitation sensor) and autofluorescence of the enzyme fluorescent substrate, such as an interference filter and a color filter.

  In such a specimen detection method using the surface plasmon enhanced fluorescence sensor 10, for example, a reaction layer 14 in which an analyte labeled in advance with a fluorescent substance is captured is provided on the metal thin film 12. 22, the dielectric member 16 is irradiated with the excitation light 20, and the excitation light 20 is applied to the metal thin film 12 at a specific angle (resonance angle (angle formed by the excitation light 20 and the perpendicular of the metal thin film 12 when the electric field is enhanced) θ1). Incident incidence causes a dense wave (surface plasmon) on the metal thin film 12.

  Note that when a close-packed wave (surface plasmon) is generated on the metal thin film 12, the excitation light 20 and the electronic vibration in the metal thin film 12 are coupled, and the signal of the metal thin film reflected light 24 changes (the amount of light decreases). Therefore, it is only necessary to find a point where the signal of the metal thin film reflected light 24 received by the light receiving means 26 changes (the amount of light decreases).

  Then, due to this dense wave (surface plasmon), the fluorescent material generated in the reaction layer 14 on the metal thin film 12 is efficiently excited, thereby increasing the amount of the fluorescent light 28 emitted from the fluorescent material and condensing the fluorescent light 28. By receiving light by the light detection means 30 via the member 32 and the wavelength selection function member 34, it is possible to detect an extremely small amount and / or extremely low concentration of the analyte.

  The metal thin film 12 of the chip structure 18 is preferably made of at least one metal selected from the group consisting of gold, silver, aluminum, copper, and platinum, more preferably gold, It consists of a metal alloy.

Such a metal is suitable for the metal thin film 12 because it is stable against oxidation and the electric field enhancement due to the dense wave (surface plasmon) increases.
Examples of the method for forming the metal thin film 12 include sputtering, vapor deposition (resistance heating vapor deposition, electron beam vapor deposition, etc.), electrolytic plating, electroless plating, and the like. Among these, the sputtering method and the vapor deposition method are preferable because the thin film formation conditions can be easily adjusted.

  Further, the thickness of the metal thin film 12 is in the range of gold: 5 to 500 nm, silver: 5 to 500 nm, aluminum: 5 to 500 nm, copper: 5 to 500 nm, platinum: 5 to 500 nm, and alloys thereof: 5 to 500 nm. It is preferable to be within.

  From the viewpoint of the electric field enhancement effect, within the range of gold: 20-70 nm, silver: 20-70 nm, aluminum: 10-50 nm, copper: 20-70 nm, platinum: 20-70 nm, and alloys thereof: 10-70 nm More preferably.

  If the thickness of the metal thin film 12 is within the above range, close-packed waves (surface plasmons) are easily generated, which is preferable. Moreover, if it is the metal thin film 12 which has such thickness, a magnitude | size (length x width) will not be specifically limited.

  On the other hand, the reaction layer 14 contains a sample in which a fluorescent substance is bound to an analyte, and examples of such a sample include blood, serum, plasma, urine, nasal fluid, saliva, stool, and body cavity. Fluid (spinal fluid, ascites, pleural effusion, etc.).

  The analyte contained in the sample is, for example, a nucleic acid (DNA, RNA, polynucleotide, oligonucleotide, PNA (peptide nucleic acid), which may be single-stranded or double-stranded, or nucleoside. , Nucleotides and their modified molecules), proteins (polypeptides, oligopeptides, etc.), amino acids (including modified amino acids), carbohydrates (oligosaccharides, polysaccharides, sugar chains, etc.), lipids, or modified molecules thereof, Specific examples thereof include a complex, and may be a carcinoembryonic antigen such as AFP (α-fetoprotein), a tumor marker, a signal transduction substance, a hormone, and the like, and is not particularly limited.

  Furthermore, the fluorescent substance is not particularly limited as long as it is a substance that emits fluorescence 28 by being irradiated with predetermined excitation light 20 or excited by using a field effect. The fluorescence 28 in this specification includes various types of light emission such as phosphorescence.

As the dielectric member 16, various optically transparent inorganic substances, natural polymers, and synthetic polymers can be used. From the viewpoint of chemical stability, manufacturing stability, and optical transparency, silicon dioxide (SiO 2 ₂ ) or titanium dioxide (TiO ₂ ).

  Further, such a surface plasmon enhanced fluorescence sensor 10 adjusts the optimum angle (resonance angle θ1) of the surface plasmon resonance by the excitation light 20 irradiated from the light source 22 to the metal thin film 12, so that an angle variable unit (not shown). ), A computer (not shown) for processing information input to the light detection means 30, and the like.

  Here, the angle variable unit (not shown) synchronizes the light receiving means 26 and the light source 22 in order to obtain the total reflection attenuation (ATR) condition with a servo motor, and enables an angle change of 45 to 85 °, and the resolution. Is preferably 0.01 ° or more.

The surface plasmon enhanced fluorescence sensor 10 of the present invention having the above-described configuration has a characteristic configuration particularly in the dielectric member 16 of the chip structure 18.
One typical embodiment of such a dielectric member 16 is to reduce the light of the fluorescent wavelength of the fluorescent material of the reaction layer 14 at the interface with the metal thin film 12 so that it does not reach the metal thin film 12, and the excitation light. An excitation light selective transmission layer 38 is formed as an excitation light selective transmission part 36 that transmits at least the main wavelength 20 and reaches the metal thin film 12.
Hereinafter, an example of the chip structure 18 having the dielectric member 16 having such a configuration will be described.

<Chip structure 18>
In the development of the chip structure 18, in the fluorescence detection of the trace amount and / or extremely low concentration of the analyte by the light detection means 30, extremely weak fluorescence (autofluorescence) generated from the dielectric member 16 when the excitation light 20 is incident. It has been confirmed by the present inventors that extremely weak light having a wavelength other than the main wavelength emitted from the light source 22 of the excitation light 20 decreases the sensitivity of fluorescence detection.

  For this reason, in the chip structure 18 according to the first embodiment shown in FIG. 2, the excitation light selective transmission layer 38 is formed at the interface between the dielectric member 16 and the metal thin film 12, and this excitation light selective transmission layer 38 is formed. Thus, only the excitation light 20 irradiated from the light source 22 to the dielectric member 16 and incident at a resonance angle θ1 that generates a dense wave (surface plasmon) reaches the metal thin film 12.

  Such an excitation light selective transmission layer 38 prevents light having a wavelength overlapping with the fluorescence wavelength of the fluorescent material of the reaction layer 14 (for example, extremely weak fluorescence (autofluorescence) generated by the dielectric member 16) from reaching the metal thin film 12. Can be.

  As such an excitation light selective transmission layer 38, for example, a layer configuration such as an optical filter or a cut filter used in the wavelength selection function member 34 disposed between the reaction layer 14 and the light detection means 30 is used. be able to.

The excitation light selective transmission layer 38 may be formed by any method as long as it is a known film formation method, but is preferably formed by using, for example, a sputtering method or a vacuum evaporation method.
The excitation light selective transmission layer 38 is greatly different from the wavelength selection functional member 34 because the wavelength selection functional member 34 performs wavelength selection after the fluorescence 28 to be detected and other light are mixed, and therefore the fluorescence 28 of the detection target. While unnecessary light having a wavelength overlapping with the fluorescence wavelength cannot be separated from the fluorescence 28 to be detected, the excitation light selective transmission layer 38 is provided before the excitation light 20 reaches the metal thin film 12 before the reaction layer 14. Thus, before the fluorescence 28 emitted from the reaction layer 14 and other light are mixed, unnecessary light can be eliminated.

  Usually, an optical filter or a cut filter, which is an example of the wavelength selection function member 34, is configured to exhibit characteristics such as transmission, absorption, and reflection with respect to vertically incident light having a specific wavelength.

  On the other hand, the excitation light selective transmission layer 38 is configured to transmit the excitation light 20 incident at the resonance angle θ1, and the light incident perpendicularly to the surface of the excitation light selective transmission layer 38, particularly the reaction. Light having a wavelength overlapping with the fluorescence wavelength of the fluorescence 28 of the layer 14 can be reduced.

  The excitation light selective transmission layer 38 may be configured to absorb or reflect light having a wavelength that overlaps the fluorescence wavelength of the fluorescent material of the reaction layer 14, and is incident at the resonance angle θ1. Any configuration may be used as long as at least the main wavelength light of the excited excitation light 20 reaches the metal thin film 12.

  As described above, in the chip structure 18 according to the first embodiment, the excitation light selective transmission layer 38 does not allow the light having the fluorescence wavelength of the reaction layer 14 to reach the metal thin film 12, and unnecessary light having a wavelength overlapping the fluorescence wavelength. Can be reduced.

  For this reason, if the surface plasmon enhanced fluorescence sensor 10 using such a chip structure 18 is used, the electric field on the metal thin film 12 is enhanced to excite the fluorescent material in the reaction layer 14 formed on the metal thin film 12. In this case, the fluorescence 28 generated from the fluorescent substance of the reaction layer 14 that is the detection target can be detected more accurately by the light detection means 30, and ultrahigh-precision fluorescence detection can be performed.

Therefore, if analyte detection is performed using the surface plasmon enhanced fluorescence sensor 10 using such a chip structure 18, a desired analyte can be detected with high accuracy.
Next, the chip structure 18 shown in FIG. 3 is a schematic view in the second embodiment.

  The chip structure 18 shown in FIG. 3 has basically the same configuration as the chip structure 18 of the first embodiment shown in FIG. Detailed description is omitted.

The chip structure 18 shown in FIG. 3 is different from the first embodiment in that the excitation light selective transmission layer 38 is formed on the excitation light incident surface 40 of the dielectric member 16.
In this case, extremely weak light having a wavelength overlapping with the fluorescence wavelength of the fluorescent substance in the reaction layer 14 emitted from the light source 22 of the excitation light 20 can be removed.

  That is, extra light having a wavelength that overlaps the fluorescence wavelength of the fluorescent material of the reaction layer 14 can be reduced, so that the fluorescence 28 generated from the fluorescent material of the reaction layer 14 that is the detection target can be detected more accurately. It has become.

  Further, by arranging the wavelength selection function member 34 used conventionally between the light source 22 and the dielectric member 16, it is possible to have the same action as the excitation light selective transmission layer 38 described above. In this case, however, it is necessary to prepare the wavelength selection function member 34 separately from the chip structure 18, which is not desirable because the number of parts increases, the cost increases, and the installation space increases.

  On the other hand, as shown in FIG. 3, if the excitation light selective transmission layer 38 is formed on the excitation light incident surface 40 of the dielectric member 16, it is integrated as the chip structure 18. This is desirable because it does not increase the installation space and requires no installation space.

  The method for forming the excitation light selective transmission layer 38 may be any method as long as it is a known film formation method, as in the case of Example 1 described above. For example, a sputtering method or a vacuum evaporation method is used. It is preferable to form them.

Next, the chip structure 18 shown in FIG. 4 is a schematic view in the third embodiment.
The chip structure 18 shown in FIG. 4 has basically the same configuration as the chip structure 18 of the first embodiment shown in FIG. Detailed description is omitted.

  In the chip structure 18 shown in FIG. 4, the excitation light selective transmission part 36 in the dielectric member 16 adds an additive into the dielectric member 16 having a characteristic that the fluorescent light of the fluorescent material of the reaction layer 14 is absorbed. It is different from Example 1 in that it is contained.

In the dielectric member 16 of such a chip structure 18, the entire dielectric member 16 serves as the excitation light selective transmission portion 36.
When manufacturing the dielectric member 16 which is the excitation light selective transmission part 36 as a whole, an additive having a characteristic of absorbing light of the fluorescent wavelength of the fluorescent material of the reaction layer 14 is added to the raw material of the dielectric member 16 in advance. It can be easily manufactured by adding and molding the dielectric member 16 using this.

In addition, as an additive which has a characteristic which absorbs the light of the fluorescence wavelength of the fluorescent substance of the reaction layer 14 in advance, for example, a near infrared absorber such as a phthalocyanine compound can be used.
As described above, in the chip structure 18 according to the third embodiment, the dielectric member 16 itself serves as the excitation light selective transmission portion 36 instead of the excitation light selective transmission layer 38 described above. Since it is not necessary to newly form the excitation light selective transmission layer 38, manufacturing is easy.

  Even in this case, unnecessary light having a wavelength overlapping with the fluorescence wavelength of the fluorescent substance in the reaction layer 14 can be reduced. Therefore, if the surface plasmon enhanced fluorescence sensor 10 using the chip structure 18 in Example 3 is used, When the electric field on the metal thin film 12 is enhanced to excite the fluorescent material of the reaction layer 14 formed on the metal thin film 12, the fluorescence 28 generated from the fluorescent material of the reaction layer 14 to be detected is more accurately detected. It can be detected by the means 30, and ultra-high-precision fluorescence detection can be performed.

Next, the chip structure 18 shown in FIG. 5 is a schematic view in the fourth embodiment.
Since the chip structure 18 shown in FIG. 5 has basically the same configuration as the chip structure 18 of the third embodiment shown in FIG. 4, the same components are designated by the same reference numerals. Detailed description is omitted.

  The chip structure 18 shown in FIG. 5 is made of an additive having a characteristic that the dielectric member 16 absorbs light of the fluorescence wavelength of the fluorescent material of the reaction layer 14 in the same manner as the chip structure 18 shown in FIG. Although contained in the dielectric member 16, the dielectric member 16 is different from the chip structure 18 shown in FIG. 4 in that a part of the dielectric member 16 is an excitation light selective transmission portion 36.

About the additive used here, the same thing as Example 3 can be used.
Further, when manufacturing the dielectric member 16, part of which is the excitation light selective transmission part 36, at the time of forming the dielectric member 16, for example, an injection molding method such as a two-color molding method, or a part of the dielectric member 16 in advance. A method of forming only the excitation light selective transmission part 36 separately and bonding it with an ordinary dielectric member with an adhesive or the like can be used, and is not particularly limited.

  Even in this case, since it is possible to reduce unnecessary light of the fluorescence wavelength of the fluorescent material of the reaction layer 14, if the surface plasmon enhanced fluorescence sensor 10 using the chip structure 18 in Example 4 is used, the metal thin film 12 is used. When the fluorescent material of the reaction layer 14 formed on the metal thin film 12 is excited by enhancing the electric field above, the fluorescence 28 generated from the fluorescent material of the reaction layer 14 to be detected is more accurately detected by the light detection means 30. It is possible to detect and detect fluorescence with ultra-high accuracy.

  The surface plasmon enhanced fluorescence sensor 10 according to the present invention, the chip structure 18 used in the surface plasmon enhanced fluorescence sensor 10 and the specimen detection method using the surface plasmon enhanced fluorescence sensor have been described in the preferred embodiments (examples). It is not limited to (Example).

  For example, any combination, such as appropriately combining the first to fourth embodiments, may be used, and various modifications can be made without departing from the object of the present invention.

[Example 1]
<Conditions>
・ Excitation light wavelength 635nm
-Fluorescent material of reaction layer Alexa Fluor (registered trademark) 647 (Molecular Probes) (fluorescence peak wavelength of about 670 nm)
-Material of dielectric member BK7 (manufactured by SCHOTT)
-Material of metal thin film Gold (Au)
・ Metal film thickness 50nm
Excitation light selective transmission layer Alternating multilayer film of silicon dioxide (SiO ₂ ) and titanium dioxide (TiO ₂ ) (general band-pass filter structure having characteristics shown in Table 1 for blocking vertically incident light of 750 nm or less )

＜試験＞
誘電体部材と金属薄膜との界面に、上記条件を満たした励起光選択透過層を設けたチップ構造体を作成し、チップ構造体の誘電体部材側から波長６３５ｎｍの励起光を照射し、反応層で励起された蛍光を光検出手段で検出した（図１の構成）。

<Test>
A chip structure in which an excitation light selective transmission layer satisfying the above conditions is provided at the interface between the dielectric member and the metal thin film, and excitation light having a wavelength of 635 nm is irradiated from the dielectric member side of the chip structure to react. The fluorescence excited by the layer was detected by the light detection means (configuration in FIG. 1).

  As a result of the detection, it was confirmed that the light incident perpendicularly to the metal thin film showed the characteristics of the bandpass filter shown in Table 1 and was able to reduce light having a wavelength overlapping with the fluorescence wavelength of the reaction layer. It was.

  In addition, it was confirmed by simulation that the excitation light incident at the resonance angle localizes the electric field enhancement area on the metal thin film as shown in Table 2.

［比較例１］
＜条件＞
・励起光の波長 ６３５ｎｍ
・反応層の蛍光材料 Ａｌｅｘａ Ｆｌｕｏｒ（登録商標）６４７（Ｍｏｌｅｃｕｌａｒ Ｐｒｏｂｅｓ社製）（蛍光ピーク波長６７０ｎｍ程度）
・誘電体部材の材質 ＢＫ７（ＳＣＨＯＴＴ社製）
・金属薄膜の材質 金（Ａｕ）
・金属薄膜の膜厚 ５０ｎｍ

[Comparative Example 1]
<Conditions>
・ Excitation light wavelength 635nm
-Fluorescent material of reaction layer Alexa Fluor (registered trademark) 647 (Molecular Probes) (fluorescence peak wavelength of about 670 nm)
-Material of dielectric member BK7 (manufactured by SCHOTT)
-Material of metal thin film Gold (Au)
・ Metal film thickness 50nm

<Test>
Fluorescence was detected in the same manner as in Example 1 except that no excitation light selective transmission layer was provided between the dielectric member and the metal thin film (configuration in FIG. 6).
As a result of the detection, it was confirmed that the light incident on the metal thin film was reduced only by the transmittance of the metal thin film, and the light transmitted through the gold film was detected mixed with the fluorescence of the reaction layer.
Moreover, as shown in Table 3, it was confirmed by simulation that the electric field enhancement effect was lower than that in Example 1.

実施例１と比較例１の検出結果から、比較例１のように励起光選択透過層が設けられていない従来のチップ構造体を用いて蛍光検出を行うよりも、実施例１のように励起光選択透過層が設けられているチップ構造体を用いて蛍光検出を行う方が、電場増強効果が高まることが確認された。

From the detection results of Example 1 and Comparative Example 1, excitation is performed as in Example 1 rather than performing fluorescence detection using a conventional chip structure having no excitation light selective transmission layer as in Comparative Example 1. It was confirmed that the electric field enhancement effect is enhanced when fluorescence detection is performed using the chip structure provided with the light selective transmission layer.

DESCRIPTION OF SYMBOLS 10 ... Surface plasmon enhancement fluorescence sensor 12 ... Metal thin film 14 ... Reaction layer 16 ... Dielectric member 18 ... Chip structure 20 ... Excitation light 22 ... Light source 24 ... Metal thin film reflected light 26... Light receiving means 28... Fluorescence 30 .. light detecting means 32... Condensing member 34 .. wavelength selection function member 36. Excitation light selective transmission layer 40... Excitation light incident surface .theta.1..resonance angle (angle formed by excitation light and normal of metal thin film when electric field is enhanced).
DESCRIPTION OF SYMBOLS 100 ... Surface plasmon enhancement fluorescence sensor 102 ... Metal thin film 104 ... Reaction layer 106 ... Dielectric member 108 ... Chip structure 110 ... Excitation light 112 ... Light source 114 ... Metal thin film reflected light 116 ... light receiving means 118 ... fluorescence 120 ... light detecting means 122 ... light collecting member 124 ... wavelength selection function member

Claims (8)

A fluorescent material for labeling the analyte trapped in the reaction layer formed on the metal thin film by irradiating the metal thin film with excitation light through a dielectric member to enhance the electric field on the metal thin film. A chip structure used in a surface plasmon enhanced fluorescence sensor that is excited and detects fluorescence emitted from the fluorescent material ,
The chip structure is
The metal thin film;
Wherein a reaction layer formed on one side surface of the metal thin film,
And the dielectric member formed on the other side surface of the metal thin film,
Comprising at least
Further, between the dielectric member and the metal thin film,
A surface having an excitation light selective transmission layer that causes light having an excitation wavelength to excite the fluorescent material to reach the metal thin film and reduces light having the fluorescence wavelength of the fluorescent material from reaching the metal thin film. A chip structure used in a plasmon enhanced fluorescence sensor. The excitation light selective transmission layer is
2. The chip structure used in the surface plasmon enhanced fluorescence sensor according to claim 1 , wherein the chip structure is formed by sputtering and / or vacuum deposition. A fluorescent material for labeling the analyte trapped in the reaction layer formed on the metal thin film by irradiating the metal thin film with excitation light through a dielectric member to enhance the electric field on the metal thin film. A chip structure used in a surface plasmon enhanced fluorescence sensor that is excited and detects fluorescence emitted from the fluorescent material,
The chip structure is
The metal thin film;
The reaction layer formed on one side of the metal thin film;
The dielectric member formed on the other side surface of the metal thin film;
Consisting of at least
The dielectric member is
Excitation light that excites the fluorescent material reaches the metal thin film, and has an excitation light selective transmission part that reduces the fluorescence wavelength light of the fluorescent material reaching the metal thin film,
The excitation light selective transmission part is
A chip structure used in a surface plasmon enhanced fluorescence sensor, wherein the dielectric member contains an additive having a property of absorbing light having a fluorescence wavelength of the fluorescent material . The additive is
The chip structure used in the surface plasmon enhanced fluorescence sensor according to claim 3 , wherein the chip structure is contained in the entire dielectric member. The additive is
The chip structure used in the surface plasmon enhanced fluorescence sensor according to claim 3 , wherein the chip structure is contained in a part of the dielectric member. The excitation light is
Chip structure used for the surface plasmon enhanced fluorescence sensor according to any one of claims 1-5, characterized in that the excitation light incident at the resonance angle to the metal thin film. A surface plasmon enhanced fluorescence sensor comprising the chip structure according to any one of claims 1 to 6 . A specimen detection method, which is performed using the surface plasmon enhanced fluorescence sensor according to claim 7 .

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