JP2012026924A - Optical fiber type surface plasmon enhanced fluorescence measurement device and plasmon excitation sensor used in the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an optical fiber type surface enhanced fluorescence measurement device which is capable of detecting a desired analyte with high precision by applying optical fiber technology to surely collect fluorescent light and is capable of easily detecting the desired analyte without controlling a resonance angle of excitation light, and to provide a plasmon excitation sensor used in the same.SOLUTION: A plasmon excitation sensor used in an optical fiber type surface enhanced fluorescence measurement device has an optical fiber structure comprising at least a core part made of a columnar dielectric member having metal fine particles dispersed therein and a clad part provided on the outside of the core part in a concentric-circle state and having, on an inner surface, a reaction layer for capturing an analyte labeled by a fluorochrome and is configured so that an electric field of the metal fine particles is enhanced by irradiation of excitation light from a light source to one side end part of the core part to excite the fluorochrome in the reaction layer and thereby enhanced fluorescent light is detected by optical detection means.

Description

  The present invention relates to an optical fiber type surface plasmon enhanced fluorescence measuring apparatus based on the principle of surface plasmon excitation enhanced fluorescence spectroscopy (SPFS) and plasmon excitation used in the optical fiber type surface plasmon enhanced fluorescence measuring apparatus. It relates to sensors.

  Conventionally, a phenomenon (surface plasmon resonance (SPR) phenomenon) in which high light output is obtained by resonating electrons and light in a minute region such as a nanometer level is applied. A surface plasmon resonance device (hereinafter referred to as an SPR device) that detects a small amount of analyte is used.

  In such an SPR device 100, as shown in FIG. 5, a metal thin film 102 is formed on the upper surface of a dielectric member 106, and a fixed layer 104 on which a predetermined ligand is fixed is formed on the metal thin film 102. ing.

  Further, a light source 108 that irradiates the excitation light 110 toward the metal thin film 102 is provided below the dielectric member 106, and the metal thin film reflected light 114 that is irradiated from the light source 108 and reflected by the metal thin film 102 is received. A light receiving means 112 is provided.

  In use of the SPR device 100, a specimen sample solution containing a specific analyte is passed through the fixed layer 104, and in this state, the excitation light 110 is directed toward the metal thin film 102 from below the dielectric member 106 at the resonance angle 116. The metal thin film reflected light 114 that has been irradiated and reflected by the metal thin film 102 is received by the light receiving means 112.

  When the excitation light 110 is irradiated toward the metal thin film 102 at the resonance angle 116, a close-packed wave (surface plasmon) is generated on the metal thin film 102. At this time, the excitation light 110 and the electronic vibration in the metal thin film 102 are cupped. As a result, the light amount of the metal thin film reflected light 114 is reduced.

  In this phenomenon, since the resonance angle 116 changes depending on the presence or absence of the analyte, if the resonance angle 116 when the sample solution not containing the analyte is passed through the fixed layer 104 is examined in advance, the resonance angle 116 is different. In this case, it can be determined that the predetermined analyte is included. Thereby, it is possible to know whether or not a predetermined analyte is contained in the specimen sample solution.

  On the other hand, based on the principle of surface plasmon excitation enhanced fluorescence spectroscopy (SPFS) applying such a surface plasmon resonance (SPR) phenomenon, the surface plasmon enhancement that enables the analyte detection with higher accuracy than the SPR device 100. A fluorescence measuring apparatus (hereinafter referred to as SPFS apparatus 200) has also been developed (for example, Patent Documents 1 and 2).

  In such an SPFS apparatus 200, as shown in FIG. 6, a metal thin film 202 is first formed on the upper surface of a dielectric member 206, and an analyte labeled with a fluorescent dye is further formed on the metal thin film 202. A plasmon excitation sensor 218 having a reaction layer 204 to be captured is provided.

  The dielectric member 206 side of the plasmon excitation sensor 218 receives the light source 208 that irradiates the excitation light 210 toward the metal thin film 202 and the metal thin film reflected light 214 that is irradiated from the light source 208 and reflected by the metal thin film 202. A light receiving means 212 is provided.

  On the other hand, on the reaction layer 204 side of the plasmon excitation sensor 218, a light detection means 222 that receives fluorescence 220 emitted from a fluorescent dye labeled with the analyte captured by the reaction layer 204 is provided.

  In addition, between the reaction layer 204 and the light detection means 222, the light collecting member 224 for efficiently condensing the fluorescent light 220 and the light included in the portion other than the fluorescent light 220 are removed, and only the necessary fluorescent light 220 is selected. A wavelength selection function member 226 is provided.

  When the SPFS apparatus 200 is used, a reaction layer 204 in which an analyte labeled with a fluorescent dye is captured in advance is formed on the metal thin film 202, and in this state, the light source 208 enters the dielectric member 206. The excitation light 210 is irradiated, and the excitation light 210 is incident on the metal thin film 202 at the resonance angle 216, thereby generating a dense wave (surface plasmon) on the metal thin film 202.

  In addition, when a close-packed wave (surface plasmon) is generated on the metal thin film 202, the excitation light 210 and the electronic vibration in the metal thin film 202 are coupled to reduce the light amount of the metal thin film reflected light 214. If a point where the signal of the metal thin film reflected light 214 received at 212 changes (the amount of light decreases) is found, a resonance angle 216 at which a dense wave (surface plasmon) occurs can be obtained.

  Then, due to the phenomenon of generating the dense wave (surface plasmon), the fluorescent dye of the reaction layer 204 on the metal thin film 202 is efficiently excited, and thereby the amount of the fluorescent light 220 emitted from the fluorescent dye is increased.

  By receiving the increased fluorescence 220 by the light detection means 222 via the light collecting member 224 and the wavelength selection function member 226, it is possible to detect an extremely small amount and / or an extremely low concentration of the analyte. ing.

In recent years, technological development for further improving the accuracy of the SPR device 100 and the SPFS device 200 has been actively performed.
For example, in the SPR device 100, optical fiber technology is applied, a mechanism for changing the incident angle, or a light source including various angle components is included, and there is no need to use a dielectric member (prism), and a small and simple SPR. An apparatus 100 is disclosed (for example, Patent Documents 3 to 5).

  Also disclosed is a localized plasmon sensor technology that allows fine particles to be bonded to the surface of an optical fiber, does not require a resonance angle shift structure, does not require the use of a dielectric member (prism), and can detect trace components with a small and simple structure. (For example, Patent Document 6).

  As shown in FIG. 7, the optical fiber type SPR device 300 using such optical fiber technology totally reflects the excitation light 310 and the core portion 318 made of the dielectric member 306 as a transmission part of the excitation light 310. An optical fiber 322 having a double structure including a clad portion 320 that prevents the excitation light 310 from leaking to the outside is provided.

  At one end of the optical fiber 322, the core portion 318 is exposed, a metal thin film 302 is provided on the outer surface of the core portion 318, and a fixed layer 304 for fixing a specific ligand is provided on the outer side. Is formed.

  A light source 308 for irradiating the core 318 with the excitation light 310 is provided at the other end of the optical fiber 322, and the metal thin film reflected light 314 reflected by the metal thin film 302 is received at one end. A light receiving means 312 is provided.

  In using such an optical fiber type SPR device 300, first, a specimen sample solution containing a predetermined analyte is passed through the fixed layer 304, and the predetermined analyte is captured by the fixed layer 304.

  In this state, the dielectric member 306 of the core portion 318 is irradiated with the excitation light 310 at the resonance angle 316, and the metal thin film reflected light 314 reflected on the metal thin film 302 outside the core portion 318 is received by the light receiving means 312. Thus, it is possible to know whether or not the analyte is contained in the specimen sample solution.

  As with the optical fiber type SPR device 300, the development of an optical fiber type SPFS device using optical fiber technology is also desired.

Japanese Patent No. 3294605 JP 2006-208069 A Japanese Patent No. 29389973 Japanese Patent No. 3231675 Japanese Patent No. 3592065 JP 2005-181296 A

However, since the measurement method of the SPFS device is completely different from that of the SPR device, it is difficult to apply the optical fiber technology.
In an SPR device, the change in the dielectric constant and abundance of an analyte can be measured basically by determining the resonance angle, that is, the angle at which the component that has become localized field light without being reflected from the metal thin film is maximized. .

  In the optical fiber type SPR device, by measuring the change in the amount of transmitted light that the light incident at various angles from one side end portion reaches the light receiving means installed on the opposite side end surface through the core portion, Similarly, the resonance angle can be measured, and is in principle close to ATR (total reflection attenuation).

  In addition, when the structure in which the metal thin film 302 is formed on the outer surface of the dielectric member 306 is applied to the SPFS device as in the optical fiber type SPR device 300 shown in FIG. 7, the fluorescence generated when the analyte is detected. The light detection means for detecting the light is required outside the optical fiber, and the system cannot be a small and simple system that is characteristic of the optical fiber.

Furthermore, it is known that a localized plasmon sensor using fine particles does not increase sensitivity because the conversion efficiency from propagating light to localized field light does not increase.
Further, in the optical fiber type SPR device 300, when the excitation light 310 is irradiated into the dielectric member 306 of the core portion 318, angle control for setting the resonance angle 316 is necessary, but this control is very difficult. There was a need for improvement.

  The present invention has been made in view of the current situation, and is an optical fiber type surface plasmon enhanced fluorescence measurement that can apply optical fiber technology to reliably collect fluorescence and detect a desired analyte with high accuracy. An object is to provide an apparatus and a plasmon excitation sensor used in the apparatus.

  Another object of the present invention is to provide an optical fiber type surface plasmon enhanced fluorescence measuring apparatus capable of easily detecting a desired analyte without controlling the resonance angle of excitation light, and a plasmon excitation sensor used therefor. And

The present invention was invented to solve the problems in the prior art as described above,
The plasmon excitation sensor of the present invention is
A plasmon excitation sensor used in an optical fiber type surface plasmon enhanced fluorescence measuring device,
The plasmon excitation sensor is
A core portion made of a cylindrical dielectric member in which metal fine particles are dispersed;
A clad part provided concentrically outside the core part and having a reaction layer on the inner surface for capturing an analyte labeled with a fluorescent dye;
An optical fiber structure comprising at least
One end of the core is irradiated with excitation light from a light source, the electric field of the metal fine particles is enhanced to excite the fluorescent dye of the reaction layer, and the enhanced fluorescence is detected by the light detection means. It is configured.

  With this configuration, since the excitation light does not leak out of the plasmon excitation sensor, the use efficiency of the excitation light is high, the S / N ratio is improved, and the analyte is detected with high accuracy. Can do.

  In addition, in the above sensor structure, the metal fine particles are in close proximity so as to be substantially conductive, and the structure for ensuring light transmission eliminates the need for incident angle control as in a normal localized plasmon sensor. On the other hand, the electric field is enhanced at the interface of the metal fine particles while passing through the metal fine particles, so that the light path (core) capable of propagating the local field light that cannot be achieved by the local plasmon and the sensed fluorescence path ( Can be separated. Furthermore, since the optical path length can be increased in the core, the light utilization efficiency can be remarkably increased.

Therefore, it is not necessary to find the resonance angle of the excitation light, and desired analyte detection can be performed with a simple structure and high sensitivity.
Furthermore, with such a configuration, resin molding can be performed integrally with a mold, so that the manufacturing is easy and the manufacturing cost can be reduced.

The plasmon excitation sensor of the present invention is
The metal fine particles are nanowires or nanocolloids.
In this way, nanowires or nanocolloids do not interfere with the transmission of light when the core member is irradiated with excitation light, so surface plasmons are reliably generated on the surface of the nanowires or nanocolloids, and fluorescence is emitted. It can be excited to perform the desired analyte detection.

  In particular, in the case of nanowires, since the nanowires overlap in the length direction in the dielectric member and the transmittance per metal particle density can be kept high, surface plasmons can be reliably generated.

The plasmon excitation sensor of the present invention is
A gap is formed at the interface between the core portion and the cladding portion,
The specimen sample solution is filled in the gap.

  If the sample sample solution is filled in the gap at the interface between the core part and the clad part in this way, this gap becomes a substitute for the flow path. is there.

The plasmon excitation sensor of the present invention is
A coating layer is formed concentrically outside the clad portion.
Thus, if the coating layer is provided outside the cladding portion, noise components other than the fluorescence generated in the reaction layer can be prevented, so that desired analyte detection can be performed with high accuracy.

The plasmon excitation sensor of the present invention is
A dichroic mirror is disposed at one end of the core portion and the cladding portion irradiated with excitation light.

  If the dichroic mirror is provided in this way, it is possible to prevent the fluorescence generated in the reaction layer from returning to the light source side, so that it is possible to reliably detect the fluorescence and perform desired analyte detection with high accuracy. it can.

In addition, the optical fiber type surface plasmon enhanced fluorescence measuring device of the present invention,
A plasmon excitation sensor according to any of the above,
A light source that is provided at one end of the core part and the clad part in the plasmon excitation sensor and that emits excitation light;
A light detecting means provided at the other end of the core and the clad and for detecting the enhanced fluorescent dye;
At least.

  With such an optical fiber type surface plasmon enhanced fluorescence measuring device, the above-described plasmon excitation sensor does not require angle control when irradiating excitation light from a light source, has high light utilization efficiency, and has an S / N ratio. The desired analyte can be detected with high accuracy.

  According to the present invention, the optical fiber technology is applied, and metal fine particles are contained in the core member so as not to disturb the excitation light, and surface plasmons are generated in the metal fine particles to enhance the fluorescence of the reaction layer. Therefore, it is possible to provide an optical fiber type surface plasmon enhanced fluorescence measuring apparatus and a plasmon excitation sensor used therefor that can reliably collect fluorescence and detect a desired analyte with high accuracy.

  In addition, it is possible to provide an optical fiber type surface plasmon enhanced fluorescence measuring apparatus capable of easily detecting a desired analyte without controlling the resonance angle of excitation light, and a plasmon excitation sensor used therefor.

FIG. 1 is a schematic view of an embodiment of the optical fiber type surface plasmon enhanced fluorescence measurement (SPFS) apparatus of the present invention. 2 is a cross-sectional view taken along line AA of the plasmon excitation sensor in the optical fiber type surface plasmon enhanced fluorescence measurement (SPFS) apparatus shown in FIG. FIG. 3 is a schematic view of another embodiment of the optical fiber type surface plasmon enhanced fluorescence measurement (SPFS) apparatus of the present invention. 4 is a cross-sectional view of the plasmon excitation sensor in the optical fiber type surface plasmon enhanced fluorescence measurement (SPFS) apparatus shown in FIG. FIG. 5 is a schematic diagram showing the basic structure of a surface plasmon resonance (SPR) device. FIG. 6 is a schematic view showing a basic structure of a surface plasmon enhanced fluorescence measurement (SPFS) apparatus. FIG. 7 is a schematic diagram showing the basic structure of an optical fiber type surface plasmon resonance (SPR) apparatus.

Hereinafter, embodiments of the present invention will be described in more detail based on the drawings.
FIG. 1 is a schematic view of an embodiment of an optical fiber type surface plasmon enhanced fluorescence measuring apparatus according to the present invention, and FIG. 2 is a cross section taken along line AA of a plasmon excitation sensor in the optical fiber type surface plasmon enhanced fluorescence measuring apparatus shown in FIG. FIG.

  The optical fiber type surface plasmon enhanced fluorescence measuring apparatus of the present invention and the plasmon excitation sensor used therefor can reliably collect fluorescence by applying optical fiber technology, and can detect a desired analyte with high accuracy. It is possible to easily detect a desired analyte without controlling the resonance angle of the excitation light.

<Optical fiber type surface plasmon enhanced fluorescence measurement (SPFS) apparatus 10>
As shown in FIG. 1, an optical fiber type surface plasmon enhanced fluorescence measuring apparatus (hereinafter referred to as an optical fiber type SPFS apparatus) 10 of the present invention is a core composed of a cylindrical dielectric member 16 in which metal fine particles 12 are dispersed. An optical fiber structure comprising at least a portion 38 and a clad portion 40 provided concentrically outside the core portion 38 and having a reaction layer 14 for capturing an analyte carrying a fluorescent dye on the inner surface. A plasmon excitation sensor 18 is provided.

Here, as the material of the dielectric member 16 constituting the core portion 38, various optically transparent inorganic substances, natural polymers, and synthetic polymers can be used, and chemical stability, manufacturing stability, and optical transparency can be used. In view of the above, it is preferable to contain silicon dioxide (SiO ₂ ) or titanium dioxide (TiO ₂ ).

Further, it is preferable to use nanowires or nanocolloids as the metal fine particles 12 dispersed in the dielectric member 16.
Among these, the nanowires overlap in the dielectric member 16 in the length direction, and thus are suitable for reliably generating surface plasmons.

Here, the particle diameter of the nanocolloid is preferably in the range of 20 to 500 nm.
On the other hand, the long side length of the nanowire is preferably in the range of 1 to 100 μm, and the short side length is preferably in the range of 10 to 500 nm. The aspect ratio between the long side and the short side is preferably in the range of 5 to 100.

  Further, the material of the metal fine particles 12 is made of a metal that generates plasmon, preferably at least one metal selected from the group consisting of gold, silver, aluminum, copper, and platinum, more preferably silver. Furthermore, it is made of an alloy of these metals. The above metals are suitable for the metal fine particles 12 because they are stable against oxidation and increase in electric field due to dense waves (surface plasmons) becomes large.

  Further, as a method of manufacturing the core part 38 in which the metal fine particles 12 are dispersed in the dielectric member 16, for example, a resin material that is a raw material of the dielectric member 16 is melted, and the metal fine particles 12 are put in and mixed therewith. What is necessary is just to shape | mold with a metal mold | die.

  On the other hand, the clad portion 40 can be basically made of the same material except that it is set lower than the refractive index of the dielectric member 16 constituting the core portion 38. As the plastic material, for example, polyethylene Polyesters such as terephthalate (PET) and polyethylene naphthalate, polyolefins such as polyethylene (PE), polypropylene (PP), polystyrene and cyclic olefin resins, vinyl resins such as polyvinyl chloride and polyvinylidene chloride, polyether ethers Ketone (PEEK), polysulfone (PSF), polyethersulfone (PES), polycarbonate (PC), polyamide, polyimide, acrylic resin, triacetyl cellulose (TAC), and the like can be used.

As the inorganic material, silicon dioxide (SiO ₂ ) or titanium dioxide (TiO ₂ ) can be used.
Further, from the viewpoints of chemical stability, production stability, and optical transparency, silicon dioxide (SiO ₂ ) or titanium dioxide (TiO ₂ ) is preferable. From the viewpoint of ease of handling and cost, polyethylene terephthalate, acrylic resin, It is preferable to contain triacetyl cellulose.

Such a clad portion 40 can be easily manufactured by forming the tubular clad portion 40 and providing the reaction layer 14 on the inner surface thereof.
Then, by inserting the core portion 38 into the clad portion 40, the plasmon excitation sensor 18 in which the clad portion 40 is concentrically arranged with respect to the core portion 38 as shown in FIG.

  At this time, a gap 34 is formed between the core portion 38 and the clad portion 40. For positioning of both portions, a positioning member (not shown) is separately provided and fixed. Any fixing method may be used, such as forming flange portions (not shown) at both ends of the core portion 38 and connecting the flange portion and the clad portion 40.

  Here, the gap 34 provided between the core portion 38 and the cladding portion 40 serves as a flow path as will be described later, and is filled with a specimen sample solution containing a desired analyte. The analyte is captured by the ligand of the reaction layer 14.

Examples of the specimen used for the specimen sample solution include blood, serum, plasma, urine, nasal fluid, saliva, feces, body cavity fluid (spinal fluid, ascites, pleural effusion, and the like).
Analytes contained in the sample solution include, for example, nucleic acids (DNA, RNA, polynucleotides, oligonucleotides, PNA (peptide nucleic acid), which may be single-stranded or double-stranded), Alternatively, nucleosides, nucleotides and their modified molecules), proteins (polypeptides, oligopeptides, etc.), amino acids (including modified amino acids), carbohydrates (oligosaccharides, polysaccharides, sugar chains, etc.), lipids, or modifications thereof Examples thereof include molecules, complexes, and the like, and specifically, carcinoembryonic antigens such as AFP (α-fetoprotein), tumor markers, signal transduction substances, hormones, and the like may be used without particular limitation.

  Further, the fluorescent dye used for labeling the analyte is not particularly limited as long as it is a substance that emits fluorescence 26 by being irradiated with predetermined excitation light 20 or excited by using a field effect. In addition, the fluorescence 26 as used in this specification includes various light emission, such as phosphorescence.

  In the present invention, the fluorescent dye has a function of emitting fluorescence by irradiating with predetermined excitation light or by exciting using the electric field effect. Here, “fluorescence” includes various types of light emission such as phosphorescence.

  There is no restriction | limiting in particular in the kind of such fluorescent dye, Any of well-known fluorescent dye may be sufficient. In general, fluorescent dyes with large Stokes shifts that allow the use of a fluorometer with a filter rather than a monochromator and also increase the efficiency of detection are preferred.

  Examples of the fluorescent dye include a fluorescent dye of the fluorescein family (manufactured by Integrated DNA Technologies), a fluorescent dye of the polyhalofluorescein family (manufactured by Applied Biosystems Japan Co., Ltd.), and a fluorescent dye of the hexachlorofluorescein family (applied biotechnology). Systems Japan Co., Ltd.), Coumarin Family Fluorescent Dye (Invitrogen), Rhodamine Family Fluorescent Dye (GE Healthcare Bioscience), Cyanine Family Fluorescent Dye, Indocarbocyanine Family fluorescent dyes, oxazine family fluorescent dyes, thiazine family fluorescent dyes, squarain family fluorescent dyes, chelated lanthanide family fluorescent dyes , BODIPY (registered trademark) family fluorescent dye (manufactured by Invitrogen), naphthalenesulfonic acid family fluorescent dye, pyrene family fluorescent dye, triphenylmethane family fluorescent dye, Alexa Fluor (registered trademark) ) Dye series (manufactured by Invitrogen Corporation).

  Further, U.S. Pat. Nos. 6,406,297, 6,221,604, 5,994,063, 5,808,044, 5,880,287, The fluorescent dyes described in US Pat. Nos. 5,556,959 and 5,135,717 can also be used in the present invention.

  Table 1 shows the absorption wavelength (nm) and emission wavelength (nm) of typical fluorescent dyes included in these families.

また蛍光色素は、上記有機蛍光色素に限られない。例えばＥｕ、Ｔｂ等の希土類錯体系の蛍光色素も本発明に用いられ得る。

The fluorescent dye is not limited to the organic fluorescent dye. For example, rare earth complex fluorescent dyes such as Eu and Tb can also be used in the present invention.

In general, rare earth complexes have a large wavelength difference between an excitation wavelength (about 310 to 340 nm) and an emission wavelength (about 615 nm for an Eu complex and 545 nm for a Tb complex), and a long fluorescence lifetime of several hundred microseconds or more. is there. An example of a commercially available rare earth complex-based fluorescent dye is ATBTA-Eu ³⁺ .

A light source 22 for irradiating the excitation light 20 into the core portion 38 is provided at one end of the plasmon excitation sensor 18.
The excitation light 20 emitted from the light source 22 is preferably a laser beam, and an LD laser having a wavelength of 200 to 900 nm and 0.001 to 1,000 mW, or a semiconductor laser having a wavelength of 230 to 800 nm and 0.01 to 100 mW is suitable. .

On the other hand, at the other end of the plasmon excitation sensor 18, a light detection means 28 for detecting the fluorescence 26 emitted from the fluorescent dye enhanced in the reaction layer 14 is provided.
It is also possible to have a computer (not shown) for processing information input to the light detection means 28.

  The optical fiber type SPFS device 10 configured as described above is prepared by first preparing a plasmon excitation sensor 18 having a reaction layer 14 on which a predetermined ligand is fixed.

  Then, a specimen sample solution containing a desired analyte is filled in the gap 34 provided between the core portion 38 and the cladding portion 40 of the plasmon excitation sensor 18, and the analyte is captured by the ligand of the reaction layer 14. To be. The captured analyte is then attached and fluorescently labeled.

When the analyte is captured by the ligand, if the analyte is labeled with a fluorescent dye, the analyte labeled with the fluorescent dye is captured in the reaction layer 14 at a time.
A light source 22 for irradiating excitation light 20 to one end of the plasmon excitation sensor 18 is disposed, and light detection means 28 for detecting fluorescence 26 generated in the reaction layer 14 at the other end. The optical fiber type SPFS apparatus 10 is prepared.

  In this state, when one end of the core portion 38 is irradiated with the excitation light 20 from the light source 22, surface plasmons are generated on the surface of the metal fine particles 12 in the dielectric member 16 of the core portion 38, and thereby the anatomical reaction layer 14 is analyzed. The fluorescence 26 captured by the light is excited, and the light of the fluorescence 26 is increased.

  In the optical fiber type SPR device 300, it is necessary to irradiate the excitation light 310 emitted from the light source 308 at the resonance angle 316. However, in the optical fiber type SPFS device 10 of the present invention, the structure is a localized plasmon. The excitation light 20 does not need to be incident at a resonance angle, and the work procedure is small and the handling is easy.

Then, by detecting the increased fluorescence 26 by the light detection means 28, desired analyte detection can be performed.
When detecting the fluorescence 26, it is preferable to dispose only the fluorescence 26 by disposing a mask 30 at the other end of the core portion 38.

Further, if the coating layer 32 is formed concentrically outside the reaction layer 14, it is possible to prevent light other than the fluorescence 26 from being detected by the light detection means 28 as noise.
Furthermore, if the dichroic mirror 36 is provided at one end of the plasmon excitation sensor 18, it is possible to prevent the fluorescence 26 generated in the reaction layer 14 from returning to the light source 22 side.

  As described above, the optical fiber type surface plasmon enhanced fluorescence measuring apparatus 10 and the plasmon excitation sensor 18 used in the optical fiber type of the present invention apply the optical fiber technology as described above and do not interfere with the excitation light 20 as described above. Since the metal fine particles 12 are contained therein and surface plasmons are generated in the metal fine particles 12 to enhance the fluorescence 26 of the reaction layer 14, the fluorescence 26 is surely collected and the desired analyte is highly accurate. Can be detected.

Further, it is possible to easily detect a desired analyte without controlling the resonance angle of the excitation light 20.
Next, the optical fiber type SPFS apparatus 10 shown in FIG. 3 and FIG. 4 is a schematic diagram in the second embodiment of the present invention.

  The optical fiber type SPFS apparatus 10 shown in FIGS. 3 and 4 has basically the same configuration as the optical fiber type SPFS apparatus 10 of the first embodiment shown in FIGS. Reference numerals are assigned and detailed description thereof is omitted.

  In the optical fiber type SPFS device 10 shown in FIGS. 3 and 4, instead of creating the gap 34 between the core part 38 and the clad part 40, a flow path 42 is formed at the boundary surface between the core part 38 and the clad part 40. This is different from the first embodiment in that it is formed.

  Since the plasmon excitation sensor 18 having such a configuration is fixed in contact with the portions other than the flow path 42 provided on the boundary surface between the core portion 38 and the cladding portion 40, a member for fixing both portions is provided. It is unnecessary and easy to manufacture.

  In this case, the dielectric member 16 which is the core portion 38 is kneaded by adding metal nanowires after the plastic material is fluidized at a temperature equal to or higher than the glass transition point or by adding an organic solvent. After being uniformly dispersed, the core portion 38 is produced by molding with a cylindrical mold or frame.

  A plasmon excitation sensor 18 of the present invention is manufactured by separately manufacturing a molded product of a dielectric material that is hollow and has an inlet and an outlet of a flow path and has a joint with the core 38 and is connected to the core 38 at the end face. Can be manufactured.

<Production example of silver nanowire>
Referring to the method described in Non-Patent Document 1 (Adv. Mater. 2002, 14, 833 to 837), ethylene glycol (EG) as a reducing agent and polyvinylpyrrolidone (PVP: PVP: An average molecular weight of 1.3 million (manufactured by Aldrich) was used, and the nucleation step and the particle growth step were separated to form particles to prepare a silver nanowire dispersion.

<Nucleation process>
While stirring 100 ml of the EG solution maintained at 160 ° C. in the reaction vessel, 2.0 ml of an EG solution of silver nitrate (silver nitrate concentration: 1.0 mol / l) was added at a constant flow rate over 1 minute.

Thereafter, the silver ions were reduced while being held at 160 ° C. for 10 minutes to form silver core particles. Subsequently, 10.0 ml of PVP EG solution (PVP concentration: 3.0 × 10 ⁻¹ mol / l) was added at a constant flow rate over 10 minutes.

<Particle growth process>
The reaction liquid containing the core particles after the nucleation step is held at 160 ° C. with stirring, 100 ml of an EG solution of silver nitrate (silver nitrate concentration: 1.0 × 10 ⁻¹ mol / l), and PVP 100 ml of EG solution (PVP concentration: 3.0 × 10 ⁻¹ mol / l) was added over 120 minutes at a constant flow rate using the double jet method.

  In the particle growth process, the reaction solution was sampled every 30 minutes and confirmed with an electron microscope. As a result, the core particles formed in the nucleation process grew into a wire-like form over time, and the particle growth Formation of new fine particles in the process was not observed.

  About the silver nanowire finally obtained, the electron micrograph was image | photographed and the average particle diameter was calculated | required by measuring the particle size of the short-axis direction of a 300 silver nanowire particle image and a long-axis direction. The average particle size in the minor axis direction was 150 nm, and the average length in the major axis direction was 70 μm.

<Demineralized water washing process>
After cooling the reaction solution after the particle formation step to room temperature, it was subjected to a desalted water washing treatment using an ultrafiltration membrane having a fractional molecular weight of 0.2 μm, and the solvent was replaced with ethanol. Finally, the liquid volume was concentrated to 100 ml to prepare a silver nanowire EtOH dispersion.

  Disperse this silver nanowire EtOH dispersion and urethane acrylate (PR-202: manufactured by Mitsubishi Kasei Co., Ltd., refractive index 1.58) in a 25% methyl isobutyl ketone solution so that the concentration of the silver nanowire is 1%. A liquid was prepared.

<Example of production of core part (38)>
The silver nanowire dispersion liquid was poured into a cylindrical stainless steel container having a diameter of 5 mm and a length of 30 cm, and the solvent was removed by placing the container in an 80 ° C. dryer overnight. A plastic core (38) containing silver nanowires was molded.

<Manufacture example of a clad part (40)>
PMMA (Mitsubishi Rayon Acrypet, Refractive Index 1.49) has an inner diameter of 6 mm, an outer diameter of 7 mm, an end face having a connecting portion with a core portion (38), and a cylinder having a length of 5 cm is molded, and a flow path The holes at the inlet and outlet were processed.

<Example of manufacturing optical fiber type SPFS device (10)>
The core part (38) and the clad part (40) are combined, the connecting part on one end face is sealed and fixed with a plastic member, and a dichroic mirror (36) is provided on the other end face, and the light source (22) An optical fiber type SPFS device (10) of the present invention was produced by connecting the light detection means (28).

<Immobilization of primary antibody>
A primary antibody was fixed by physical adsorption by circulating a 1.4 mg / ml anti-α-fetoprotein (AFP) monoclonal antibody (6D2, manufactured by Mikuli Immuno Laboratory Co., Ltd.) for 1 hour in the flow path.

<Preparation of labeled secondary antibody>
Preparation of “FITC” Labeled Anti-AFP Monoclonal Antibody An anti-α fetoprotein (AFP) monoclonal antibody (6D2, 2.5 mg / ml, manufactured by Mikuri Immuno Laboratory Co., Ltd.) was prepared using a commercially available FITC7 labeling kit.

[Measurement example using optical fiber type SPFS device (10)]
A predetermined amount of AFP shown in Table 2 was allowed to flow for 30 minutes, and then a labeled secondary antibody was allowed to flow for 30 minutes, followed by washing and replacement with a buffer, and signal measurement was performed.
On the other hand, a signal measured without AFP was used as a blank.

[Measurement example using LPFS (localized plasmon) device]
After adding 2 g of polyvinyl alcohol PVA117 (manufactured by Kuraray Co., Ltd.) and 0.1 g of a curing agent triazine to 10 g of colloidal gold (manufactured by Tanaka Kikinzoku Co., Ltd.) solution having a particle size of 40 nm, it is coated on a transparent glass substrate and dried at 80 ° C. A plasmon sensor substrate was prepared.

A flow path was attached to this substrate, the primary antibody was immobilized by amine coupling using 3-aminopropyltriethoxysilane (manufactured by Shin-Etsu Silicone), and reacted with a predetermined amount of AFP shown in Table 3. After washing, it was further reacted with a labeled secondary antibody, followed by washing. The sample was exposed vertically with a 630 nm LD through a polarizing plate, attached with a cut filter, and detected with a photomultiplier.
On the other hand, a signal measured without AFP was used as a blank.

  As described above, the optical fiber type SPFS device 10 of the present invention is a simple and small useful biosensor without adjusting the incident angle of the excitation light. From the results of Tables 2 and 3, for example, LPFS ( It was confirmed that the biosensor is highly sensitive and capable of highly accurate detection compared to the localized plasmon device.

  Furthermore, the optical fiber type SPFS apparatus 10 of the present invention has a limitation that the dynamic range of measurement cannot be ensured unless a high refractive index glass is used in the wavelength region where the excitation light is in the near-infrared range as in the conventional SPFS apparatus. The design freedom can be improved.

  The preferred embodiments of the optical fiber type SPFS device 10 and the plasmon excitation sensor 18 used in the optical fiber type SPFS device 10 according to the present invention have been described above. However, the present invention is not limited to the above-described embodiments and does not depart from the object of the present invention. Various changes are possible.

DESCRIPTION OF SYMBOLS 10 ... Optical fiber type surface plasmon enhanced fluorescence measurement (SPFS) apparatus 12 ... Metal fine particle 14 ... Reaction layer 16 ... Dielectric member 18 ... Plasmon excitation sensor 20 ... Excitation light 22 .... · Light source 26 · · · Fluorescence 28 · · · Light detection means 30 · · · Mask 32 · · · Coating layer 34 · · · Clearance 36 · · · Dichroic mirror 38 · · · Core portion 40 · · · Cladding portion 42 · · · ..Flow path 100 ... Surface plasmon resonance (SPR) device 102 ... Metal thin film 104 ... Fixed layer 106 ... Dielectric member 108 ... Light source 110 ... Excitation light 112 ... Light reception Means 114 ... Metal thin film reflected light 116 ... Resonance angle 200 ... Surface plasmon enhanced fluorescence measurement (SPFS) device 202 ... Metal thin film 204 ... Reaction layer 206 ... Dielectric part 208 ... light source 210 ... excitation light 212 ... light receiving means 214 ... metal thin film reflected light 216 ... resonance angle 218 ... plasmon excitation sensor 220 ... fluorescence 222 ... light detection means 224 ... Condensing member 226 ... Wavelength selection function member 300 ... Optical fiber type surface plasmon resonance (SPR) device 302 ... Metal thin film 304 ... Fixed layer 306 ... Dielectric member 308 ... Light source 310 ... excitation light 312 ... light receiving means 314 ... reflected light 316 ... resonance angle 318 ... core part 320 ... clad part 322 ... optical fiber

Claims (6)

A plasmon excitation sensor used in an optical fiber type surface plasmon enhanced fluorescence measuring device,
The plasmon excitation sensor is
A core portion made of a cylindrical dielectric member in which metal fine particles are dispersed;
A clad part provided concentrically outside the core part and having a reaction layer on the inner surface for capturing an analyte labeled with a fluorescent dye;
An optical fiber structure comprising at least
One end of the core is irradiated with excitation light from a light source, the electric field of the metal fine particles is enhanced to excite the fluorescent dye of the reaction layer, and the enhanced fluorescence is detected by the light detection means. A plasmon excitation sensor characterized by comprising.   The plasmon excitation sensor according to claim 1, wherein the metal fine particles are nanowires or nanocolloids. A gap is formed at the interface between the core portion and the cladding portion,
The plasmon excitation sensor according to claim 1 or 2, wherein the gap is filled with a specimen sample solution.   The plasmon excitation sensor according to any one of claims 1 to 3, wherein a coating layer is formed concentrically outside the clad portion.   The plasmon excitation sensor according to any one of claims 1 to 4, wherein a dichroic mirror is disposed at one end of the core portion and the clad portion where the excitation light is irradiated. A plasmon excitation sensor according to any one of claims 1 to 5,
A light source for irradiating excitation light, provided at one end of the core part and the clad part in the plasmon excitation sensor;
A light detection means for detecting the enhanced fluorescent dye, provided at the other end of the core portion and the clad portion;
An optical fiber type surface plasmon enhanced fluorescence measuring apparatus comprising:

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