JP2008170418A - Sensor, sensing device, and sensing method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sensor capable of detecting highly efficiently fluorescence emitted from a fluorescent label with high S/N ratio, and capable of dispensing with a complicated optical system for the detection. <P>SOLUTION: This sensor 1 has a sensing surface 1s bonded only with a specific substance R to be detected, and a metal portion 20 with at least a portion exposed on the sensing surface 1s and capable of exciting a localized plasmon. The sensor 1 is used in sensing for detecting two-photon excitation fluorescence or multi-photon excitation fluorescence of the fluorescent label Lu, by labeling the substance R to be detected with the fluorescent label Lu coupled selectively with the substance R to be detected, and the sensing surface 1s is irradiated with a measurement light L1 of a wavelength that can excite the localized plasmon in the metal portion 20 and that is an absorption wavelength emitting the two-photon excitation fluorescence or the multi-photon excitation fluorescence of the fluorescent label Lu. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a sensor used for sensing in which a substance to be detected is labeled with a fluorescent label that selectively binds to the substance to be detected, and two-photon excited fluorescence or multiphoton excited fluorescence of the fluorescent label is detected, and The present invention relates to a sensing device and a sensing method using the sensor.

  In recent years, in fluorescence analysis performed by immunoassay in the biochemical field, a sensor having a sensing surface capable of binding a specific substance to be detected is used to excite a fluorescent label bound to the substance to be detected with evanescent waves or surface plasmons. Thus, a fluorescence measurement method for sensing a substance to be detected has been proposed.

  In these methods, since fluorescence is emitted only when a substance to be detected is bound to the sensing surface, real-time measurement is possible. Further, since excitation light does not reach the detector, there is little fluorescence background light to be detected, and measurement can be performed with a relatively high S / N ratio.

  However, in the above fluorescence measurement method, substances coexisting with the substance to be detected in the sample, such as water, serum proteins, and enzymes, may absorb excitation light, and depending on the substance, unnecessary fluorescence may be emitted. is there. In addition, when a fluorescent label is excited by surface plasmon, a substance to be detected is bound to a sensor formed by a prism formed with a metal thin film that causes surface plasmon, but the prism absorbs excitation light, and in some cases unnecessary fluorescence is emitted. May be emitted. Absorption and fluorescence of excitation light by a coexisting substance that is not a detection target lowers the detection accuracy of the fluorescence that is originally desired to be detected.

  As a method for suppressing the absorption and fluorescence of excitation light by a coexisting substance and performing fluorescence measurement with a higher S / N ratio, there is a two-photon excitation fluorescence measurement method using a fluorescent label capable of two-photon excitation. Since the two-photon excitation fluorescence is fluorescence using excitation light having a wavelength twice that of normal fluorescence, the wavelength of the excitation light and the absorption wavelength of the coexisting substance can be shifted, and noise is low. Fluorescence can be measured. Further, since the wavelength of the excitation light used is in the near infrared region, there is no possibility that the detected substance is destroyed during the measurement even when the detected substance is a living tissue or the like.

However, the absorption cross section of the two-photon excitation is about several tens of orders of magnitude smaller than that of the one-photon excitation. Therefore, in order to obtain sufficient fluorescence, a very expensive pulse laser with a high peak value is usually used. Yes. As a method of obtaining sufficient fluorescence intensity without using such a pulse laser, a method of exciting two-photon excitation fluorescence with surface plasmons is disclosed (Patent Document 1).
JP 2001-21565 A

  Patent Document 1 describes that, by exciting two-photon excitation fluorescence with an electric field generated by surface plasmons, the transition probability is improved by two orders of magnitude or more, and the fluorescence efficiency is remarkably increased. However, when surface plasmons are used, a sensor comprising a prism formed with a metal thin film that causes surface plasmons is required, and a complicated optical system is required for irradiation of measurement light and detection of detection light, so that the sensing device is expensive. It will be complicated. In addition, considering the absorption cross-sectional area, improvement of the transition probability of about two orders of magnitude does not mean that sufficient fluorescence efficiency is obtained, and there is still a problem of improving fluorescence efficiency.

  The present invention has been made in view of the above circumstances, and the fluorescence efficiency of the fluorescence emitted from the fluorescent label is high, the fluorescence emitted from the fluorescent label can be detected with a high S / N ratio, and the configuration of the sensor itself is provided. It is an object of the present invention to provide a simple sensor that does not require a complicated optical system for measurement light irradiation and detection light detection, and a sensing device using the sensor.

  The sensor of the present invention has a sensing surface capable of binding to a specific object to be detected, labels the substance to be detected with a fluorescent label that selectively binds to the substance to be detected, and two-photons of the fluorescent label In the sensor used for sensing to detect excitation fluorescence or multiphoton excitation fluorescence, at least a part of the sensor is exposed on the sensing surface, and has a metal part that can excite localized plasmons, It is a wavelength capable of exciting localized plasmons in the metal part, and is irradiated with measurement light having an absorption wavelength that emits two-photon excitation fluorescence or multiphoton excitation fluorescence of the fluorescent label. .

  In the present specification, “two-photon excitation fluorescence” is fluorescence using excitation light having a wavelength twice that of ordinary fluorescence excitation light, and “multiphoton excitation fluorescence” is three times or more of ordinary fluorescence excitation light. Fluorescence using excitation light having an integral multiple of the wavelength.

In the sensor according to the present invention, it is preferable that the metal part has an uneven structure smaller than the wavelength of the measurement light, and the surface of the metal part is subjected to surface modification that selectively binds to the substance to be detected. It is preferable.
In this specification, “an uneven structure smaller than the wavelength of the measurement light” means an average size of convex portions and concave portions (here, “size” indicates a maximum width) and an average pitch. It means smaller than the wavelength of the measurement light. There may or may not be metal in the recess.

  As a preferred aspect of the sensor of the present invention, the base of the metal part is a dielectric having a plurality of fine holes therein, and at least a plurality of the fine holes opened on the surface on the sensing surface side. The metal part is composed of a filling part filled in the fine holes of the dielectric, and a protruding part formed on the filling part so as to protrude from the surface of the dielectric and larger than the diameter of the filling part. What consists of a some metal part is mentioned.

  The dielectric is made of a metal oxide obtained by anodizing at least a part of a metal to be anodized, and the plurality of micropores are formed in the metal oxide during the anodization process. It may be.

  As another preferred aspect of the sensor of the present invention, the metal part is a metal layer whose surface is roughened, or the base of the metal part is a dielectric, and the metal part is the dielectric. And a metal particle layer composed of a plurality of metal particles fixed to the surface. In addition, another preferred embodiment of the sensor of the present invention includes a sensor in which the base of the metal part is a dielectric, and the metal part is a metal pattern layer patterned on the surface of the dielectric.

  In the sensor of the present invention, the localized plasmon resonance wavelength of the metal part and the absorption wavelength of the fluorescent label that emits two-photon excitation fluorescence or multiphoton excitation fluorescence substantially coincide, and the measurement light has the wavelength of It is preferable that light is used.

In the sensor of the present invention, a transparent insulator layer may be formed on the surface of the metal part. The “transparent insulator layer” is specifically a layer composed of an inorganic substance such as SiO ₂ or an organic substance such as a polymer, and may be an insulator layer that substantially transmits light irradiated to the sensor surface. That's fine.

  The surface of the metal part may be subjected to surface modification that selectively binds to the substance to be detected directly or via the transparent insulator layer.

  In the sensor of the present invention, the sensing surface may have one sensing area, or may be divided into a plurality of sensing areas in which the sensing is performed independently. When divided into a plurality of sensing regions, different types of the detected substances can be sensed by applying different types of surface modification according to the detected substances in units of sensing areas.

  In the sensor with a sample cell of the present invention, at least a portion through which the sensor of the present invention passes the measurement light and the light emitted from the sensing surface including the two-photon excitation fluorescence or multiphoton excitation fluorescence of the fluorescent label passes. A sensor with a sample cell attached to a sample cell having optical properties, wherein the sensor is attached to the sample cell so that the metal part of the sensor contacts the sample in the sample cell. It is characterized by.

  The sensing device of the present invention includes the sensor of the present invention, light irradiation means for irradiating the sensor with the measurement light, and two-photon excitation fluorescence or multiphoton excitation fluorescence of the fluorescent label included in the light emitted from the sensor. And a detecting means for detecting.

  In the sensing device according to the aspect of the invention, it is preferable that the optical path between the sensor and the detection unit further includes a wavelength selection unit that removes light having the wavelength of the measurement light.

  In the sensing device of the present invention, the presence and / or amount of the substance to be detected can be analyzed.

  In the sensing method of the present invention, the substance to be detected is labeled with a fluorescent label that selectively binds to the substance to be detected, and the two-photon excited fluorescence or multiphoton excited fluorescence of the fluorescent label is detected. The fluorescent label is excited by a localized plasmon electric field to generate two-photon excitation fluorescence or multiphoton excitation fluorescence, and the two-photon excitation fluorescence or multiphoton excitation fluorescence is detected.

  The sensor of the present invention has a sensing surface to which a specific substance to be detected can bind, and has a metal part that is at least partially exposed on the sensing surface and can excite localized plasmons.

  In the sensor of the present invention, sensing is performed by labeling a substance to be detected with a fluorescent label that selectively binds to the substance to be detected, and detecting two-photon excitation fluorescence or multiphoton excitation fluorescence of the fluorescence label. It is.

  In such a configuration, when measurement light having a wavelength capable of exciting the localized plasmon is incident on the metal portion of the sensing surface, the localized plasmon is excited in the metal portion, and the electric field enhancement effect by the localized plasmon effect causes the vicinity of the metal portion. Therefore, the transition probability of the fluorescent label bonded to the metal part is improved, and the fluorescence efficiency can be effectively increased.

  In addition, since the sensor of the present invention uses two-photon excitation fluorescence or multiphoton excitation fluorescence of a fluorescent label, the wavelength of measurement light can be set to a wavelength twice or more that of one-photon excitation. Therefore, absorption of measurement light and fluorescence due to coexisting substances can be suppressed, and fluorescence can be detected with a higher S / N ratio.

  Furthermore, the sensor of the present invention only needs to have a metal part that is at least partially exposed on the sensing surface and can excite localized plasmons, and the sensor itself has a simple configuration, and is also suitable for a measurement system. Since a complicated optical system is not required, the sensing device can also have a simple configuration.

  When a transparent insulator layer is formed on the surface of the metal part, the surface modification that selectively binds to the substance to be detected is modified to the surface of the metal part via the transparent insulator layer. For this reason, since direct charge transfer from the fluorescent label combined with the surface modification to the surface of the metal part is prevented, there is no possibility that the fluorescence emitted from the fluorescent label is weakened by this direct charge transfer.

"Sensor"
A structure of a sensor according to an embodiment of the present invention will be described with reference to the drawings. 1A is an overall perspective view, and FIG. 1B is a sectional view in the thickness direction during sensing. FIG. 1B also shows a diagram schematically showing the state of coupling on the sensing surface during sensing. FIG. 2 is a diagram showing a manufacturing process of the sensor of this embodiment.

  The sensor 1 of the present embodiment is used for sensing in which the detection target substance R is labeled with a fluorescent label Lu that selectively binds to the detection target substance R, and two-photon excitation fluorescence or multiphoton excitation fluorescence of the fluorescence label Lu is detected. It is used and has a sensing surface 1s to which only a specific target substance R can be bound.

In the present embodiment, the sensor 1 has a metal portion 20 that is at least partially exposed on the sensing surface 1 s and can excite the localized plasmon, and the localized plasmon is detected in the metal portion 20 with respect to the sensing surface 1 s. The measurement light L1 having an excitation wavelength and an absorption wavelength that emits the two-photon excitation fluorescence or multiphoton excitation fluorescence of the fluorescent label is irradiated.
The measurement light L1 is single wavelength light emitted from a light source such as a laser.

  Since the localized plasmon effect is large, it is preferable that the metal part 20 forming the sensing surface 1s has an uneven structure smaller than the wavelength of the measurement light L1.

  As shown in FIG. 1 (a), the sensor 1 includes a dielectric substrate 11 on a conductor 13 on which a large number of micropores 12 are open and substantially regularly arranged. Filled portion 21 that is filled and a protruding portion that is formed so as to protrude from the substrate surface 11 s on the micropore 12 and has a diameter that is larger than the diameter of the filled portion 21 and that can excite localized plasmons. Further, a metal part 20 composed of 22 is provided.

  In the sensor 1, the surface of the metal portion 20 on the protruding portion 22 side is a sensing surface 1s. That is, in the present embodiment, the sensing surface 1 s is configured by the base material surface 11 s and the surface 22 s of the protruding portion 22.

  In the sensor 1, the fine hole 12 is a through-hole that is opened by reaching the substrate back surface 11 r substantially straight from the surface of the dielectric base material 11 in the thickness direction.

As shown in FIG. 2, the dielectric substrate 11 is made of alumina (Al) obtained by anodizing a part of the anodized metal body 10 that contains aluminum (Al) as a main component and may contain minute impurities. Al ₂ O ₃ ) layer (metal oxide layer). The conductor 13 is composed of a non-anodized portion of the anodized metal body 10 that remains without being anodized.

  The shape of the anodized metal body 10 is not limited, and examples thereof include a plate shape. Further, it may be used in a form with a support such as a layered metal object 10 to be anodized on a support.

  In anodization, for example, the metal body 10 to be anodized is used as an anode, carbon or aluminum is used as a cathode (counter electrode), these are immersed in an anodizing electrolyte, and a voltage is applied between the anode and the cathode. Can be implemented. The electrolytic solution is not limited, and an acidic electrolytic solution containing one or more acids such as sulfuric acid, phosphoric acid, chromic acid, oxalic acid, sulfamic acid, benzenesulfonic acid, and amidosulfonic acid is preferably used.

  When the anodized metal body 10 shown in FIG. 2 (a) is anodized, as shown in FIG. 2 (b), an oxidation reaction proceeds from the surface 10s (upper surface in the drawing) in a direction substantially perpendicular to the surface, An alumina layer 11 is produced.

  The alumina layer 11 produced by anodic oxidation has a structure in which fine columnar bodies 14 having a substantially regular hexagonal shape in plan view are arranged adjacent to each other. A minute hole 12 is opened in a depth direction from the surface 10 s at a substantially central portion of each minute columnar body 14. Further, the bottom surfaces of the fine holes 12 and the fine columnar bodies 14 have rounded shapes as shown in the figure. The structure of the alumina layer produced by anodization is described in Hideki Masuda, “Preparation of mesoporous alumina by anodization and its application as a functional material”, Material Technology Vol.15, No.10, 1997, p.34, etc. Are listed.

  As an example of suitable anodic oxidation conditions when the alumina layer 11 having a regular arrangement structure is produced, when oxalic acid is used as the electrolytic solution, the preferred example of the conditions is an electrolyte concentration of 0.5 M, a liquid temperature of 15 ° C., and application. An example of the voltage is 40V. By changing the electrolysis time, the alumina layer 11 having an arbitrary layer thickness can be generated.

  Usually, the pitch between adjacent fine holes 12 can be controlled in the range of 10 to 500 nm, and the diameter of the fine holes can be controlled in the range of 5 to 400 nm. Japanese Patent Application Laid-Open Nos. 2001-9800 and 2001-138300 disclose methods for finely controlling the formation position and the hole diameter of fine holes. By using these methods, the micropores having an arbitrary pore diameter and depth within the above range can be arranged almost regularly.

The metal part 20 composed of the filling part 21 and the protruding part 22 is formed by subjecting the fine holes 12 of the dielectric substrate 11 to an electroplating process or the like.
When electroplating is performed, the conductor 13 functions as an electrode, and the metal is preferentially deposited from the bottom of the fine hole 12 where the electric field is strong. By continuing this electroplating process, the fine holes 12 are filled with metal, and the filling portion 21 of the metal portion 20 is formed. If the electroplating process is continued after the filling portion 21 is formed, the filling metal overflows from the fine holes 12, but since the electric field in the vicinity of the fine holes 12 is strong, the metal is continuously deposited around the fine holes 12. As a result, a protruding portion 22 that protrudes from the base material surface 11 s and has a diameter larger than the diameter of the filling portion 21 is formed on the filling portion 21.

  When the metal part 20 is grown by electroplating, depending on conditions, a thin layer between the bottom surface of the fine hole 12 and the conductor 13 formed of the non-anodized part of the metal body 10 to be anodized is broken, and the metal part The 20 filling portions 21 reach the substrate back surface 11r to obtain the configuration of the present embodiment (FIG. 2C).

  In this embodiment, the protrusion part 22 of the metal part 20 is a particle form, and if it sees from the surface of the sensor 1, it has the structure where the metal particle layer was formed in the base material surface 11s. In such a configuration, since the protruding portion 22 is a convex portion of the metal portion 20, it is preferable that the average diameter and pitch are designed to be smaller than the wavelength of the measuring light L1. Although the metal part 20 should just be the magnitude | size which can excite a localized plasmon, the magnitude | size of the protrusion part 22 is a range whose diameter of the protrusion part 22 is 10 nm or more and 300 nm or less considering the wavelength of the measurement light to be used. It is preferable that

  The adjacent protrusions 22 are preferably separated from each other, and the average separation distance w is more preferably in the range of several nm to 10 nm. When the average separation distance is within the above range, the electric field enhancement effect by the localized plasmons can be effectively obtained.

  The localized plasmon phenomenon is a phenomenon in which a free electric field in a convex portion resonates with an electric field of light and vibrates to generate a strong electric field around the convex portion. Therefore, the metal portion 20 may be any metal having free electrons. . The sensor 1 has a wavelength that can excite localized plasmons in the metal part 20 on the sensing surface 1s, that is, the protrusion 22, with respect to the sensing surface 1s, and the fluorescent label Lu is two-photon excitation fluorescence or multiphoton. The measurement light L1 having an absorption wavelength that emits excitation fluorescence is irradiated. Therefore, it is preferable to determine the combination of the fluorescent label Lu and the metal part 20 so that the fluorescent label Lu emits fluorescence with high efficiency. The metal part 20 is preferably a metal that generates localized plasmons at a wavelength that approximately matches the absorption wavelength at which the fluorescence label Lu emits two-photon excitation fluorescence or multiphoton excitation fluorescence. The wavelength of the excitation light of the fluorescence label Lu described later is taken into consideration. In this case, Au, Ag, Cu, Pt, Ni, Ti and the like are preferable.

  Fluorescent label Lu is a fluorescent label that absorbs excitation light due to coexisting substances in the sample and noise due to fluorescence and absorbs light in the near-infrared region or longer and has two-photon excitation or multiphoton excitation. preferable. Examples of such fluorescent labels include R6G and Cye5.

  As shown in FIG. 1B, the surface modification A2 that can selectively bind to the detection target substance R is applied to the surface 22s of the protrusion 22 on the sensing surface 1s. In the sensor 1 of the present embodiment, sensing is performed by labeling with a fluorescent label Lu previously bound to the substance R to be detected. The fluorescent label Lu is subjected to surface modification A1 that selectively binds to the substance R to be detected.

  The binding reaction between the substance to be detected R and the fluorescent label Lu and the reaction between the protrusion 22 and the substance to be detected R are not particularly limited, and specific binding reactions such as an antigen-antibody reaction can be mentioned.

  For example, when the substance to be detected R is an antigen, the fluorescent label Lu is surface-modified with a first antibody that specifically binds to the substance to be detected R (surface modification A1), and the protrusion 22 specifically binds to the substance to be detected R. May be modified with a second antibody (surface modification A2). As shown in FIG. 1 (b), the first antibody surface-modified to the fluorescent label Lu and the second antibody surface-modified to the protrusion 22 are mutually opposite to the detection target substance R that is an antigen. Those that bind to another site are used. That is, as shown in FIG. 1B, the protrusion 22 and the fluorescent label Lu are modified so that the surface modification A2 of the protrusion 22, the substance R to be detected, and the surface modification A1 of the fluorescent label Lu are bound to each other. Is given.

Hereinafter, a sensing procedure using the sensor 1 of the present embodiment will be described.
First, the sample is flowed on the sensing surface 1s. When the detected substance R is contained in the sample, the detected substance R is bound to the surface modification A2 of the protrusion 22. Next, a liquid containing a fluorescent label Lu that has been previously surface-modified A1 is allowed to flow on the sensing surface 1s. If the substance R to be detected is bound to the detection unit 31, the fluorescent label Lu is bound to the substance R to be detected.

  Instead of sequentially flowing the sample and the liquid containing the fluorescent label Lu, when the fluorescent substance Lu is previously added to the sample and the target substance R is contained in the sample, the target substance R and the fluorescent label Lu are included. Can be combined in advance, and sensing can be performed by flowing only this sample on the sensing surface 1s.

  When the measurement light L1 is incident on the sensing surface 1s in which the fluorescent label Lu is coupled to the protruding portion 22, the fluorescent label Lu is two-photon excited or multiphoton excited with a certain transition probability to emit fluorescence. The substance R to be detected can be sensed by detecting this fluorescence. In the sensor 1, the fluorescent light Lu is excited by the measurement light L <b> 1 to emit fluorescence, and at the same time, localized plasmons are excited in the constituent metal of the protrusion 22. When localized plasmons are excited by a metal, the electric field near the surface of the metal is enhanced. Therefore, the power of the measurement light L1 is increased in the vicinity of the protrusion 22 and the transition probability of the fluorescent label Lu is increased. When the transition probability is increased, the fluorescence intensity is enhanced, so that sensing with higher sensitivity can be performed.

  For example, the transition probability due to normal one-photon absorption is proportional to the power of the excitation light, but in two-photon absorption, it is proportional to the square of the power of the excitation light. Therefore, the square probability of the electric field enhancement effect by the localized plasmon is obtained in the transition probability.

The electric field enhancement effect by localized plasmons is said to be 100 times or more at the localized plasmon resonance wavelength. Therefore, it is preferable to use light having a wavelength that causes localized plasmon resonance in the protrusion 22 as the measurement light L1, and in this case, the transition probability can be improved by four digits or more.
As described above, sensing can be performed by the sensor 1 of the present embodiment.

  The sensor 1 of the present embodiment has a sensing surface 1s to which only a specific substance to be detected R can bind, and has a metal part 20 that is at least partially exposed on the sensing surface 1s and can excite localized plasmons. Is. The sensor 1 of the present embodiment detects the detection target substance R with a fluorescent label Lu that selectively binds to the detection target substance R, and detects the two-photon excitation fluorescence or multiphoton excitation fluorescence of the fluorescence label Lu. Sensing is performed by.

  In such a configuration, when the measurement light L1 having a wavelength capable of exciting the localized plasmon is incident on the metal portion 20 of the sensing surface 1s, the localized plasmon is excited in the metal portion 20, and the electric field enhancement effect due to the localized plasmon effect. As a result, the power of the measurement light L1 in the vicinity of the metal part 20 is enhanced, so that the transition probability of the fluorescent label Lu coupled to the metal part 20 is improved, and the fluorescence efficiency can be effectively increased.

  In addition, since the sensor 1 of the present embodiment performs sensing using the two-photon excitation fluorescence or multiphoton excitation fluorescence of the fluorescent label Lu, the wavelength of the measurement light L1 is at least twice that of the one-photon excitation. It can be. Therefore, absorption and fluorescence of the measurement light L1 due to coexisting substances can be suppressed, and fluorescence can be detected with a higher S / N ratio.

  Furthermore, the sensor 1 of the present embodiment only needs to have a metal part 20 that is at least partially exposed on the sensing surface 1s and can excite localized plasmons, and the configuration of the sensor itself is simple.

  In the sensor 1 of the present embodiment, the sensing surface 1s may have one sensing area, or may be divided into a plurality of sensing areas in which sensing is performed independently. In the case of being divided into a plurality of sensing regions, different types of detected substances R can be sensed by applying different types of surface modification A2 corresponding to the detected substances R in units of sensing areas.

  As described above, according to the present embodiment, the fluorescence efficiency of the fluorescence emitted from the fluorescent label Lu is high, the fluorescence emitted from the fluorescent label Lu can be detected with a high S / N ratio, and the configuration of the sensor itself is provided. It is possible to provide a sensor 1 that is simple and does not require a complicated optical system for irradiation of measurement light and detection of detection light.

<Design changes>
In the above embodiment, the alumina layer obtained by anodizing a part of the anodized metal body 10 is the dielectric base material 11, and the non-anodized portion is the conductor 13. The method for forming the metal portion 20 by depositing metal in the holes 12 by electroplating has been described. However, all of the anodized metal body 10 is anodized or part of the anodized metal body 10 is anodized. After that, the non-anodized portion and the vicinity thereof may be removed to obtain the dielectric base material 11 having the fine holes 12 made of through holes, and the conductor 13 may be separately formed by vapor deposition or the like. In this case, the material of the conductor 13 is not limited, and may be any metal or conductive material such as ITO (indium tin oxide).

  Further, although the case where the conductor 13 is provided on the back surface 11r of the base material has been described, as a method of filling the metal portion 20 into the fine hole 12, when a method that requires an electrode such as electroplating is not used, the conductor 13 may not be provided. Alternatively, the conductor 13 may be removed after the metal portion 20 is formed.

  In the above embodiment, the case where the microhole 12 is a through hole has been described, but the microhole 12 may be a non-through hole.

  In the above embodiment, only Al is cited as the main component of the anodized metal body 10 used for the production of the dielectric substrate 11, but any metal can be used as long as it can be anodized. Other than Al, Ti, Ta, Hf, Zr, Si, In, Zn, etc. can be used. The anodized metal body 10 may contain two or more types of metals that can be anodized.

  Depending on the type of anodized metal to be used, the planar pattern of the fine holes 12 to be formed varies, but the dielectric substrate 11 having a structure in which the fine holes 12 having substantially the same shape in plan view are arranged adjacent to each other is formed. Will not change.

  Moreover, although the case where the micropores 12 are regularly arranged using anodization has been described, the method of forming the micropores 12 is not limited to anodic oxidation. The above-described embodiment using anodization is preferable because the entire surface can be collectively processed, can cope with an increase in area, and does not require an expensive apparatus, but other than using anodization, the surface of a substrate such as a resin is used. Forming a plurality of recesses regularly arranged by nanoimprint technology, drawing a plurality of recesses regularly arranged by electron drawing technology such as focused ion beam (FIB) and electron beam (EB) on the surface of a substrate such as metal, etc. These microfabrication technologies can be mentioned.

  Other preferred embodiments of the sensor 1 include the sensors 1A to 1F shown in FIGS. Other preferred aspects of the sensor 1 will be described with reference to FIGS. 3 is a perspective view, and FIGS. 4 and 5 are cross-sectional views.

  A sensor 1A shown in FIG. 3A is a device in which a plurality of metal particles 20a are fixed on a flat dielectric 11 in an array. In this example, the metal part 20 is a metal particle layer composed of a plurality of metal particles 20a. The arrangement pattern of the metal particles 20a can be designed as appropriate and is preferably substantially regular. In such a configuration, the individual metal particles 20a are convex, and the average diameter and pitch of the metal particles 20a are designed to be smaller than the wavelength of the measurement light L1.

  A sensor 1B shown in FIG. 3B is a device in which a metal part 20 made of a metal pattern layer in which fine metal wires 20b are formed in a lattice pattern on a flat dielectric 11 is formed. The pattern of the metal pattern layer can be designed as appropriate and is preferably substantially regular. In such a configuration, the average line width and pitch of the fine metal wires 20b are designed to be smaller than the wavelength of the measurement light L1.

The sensor 1 may be configured by only the metal part 20 whose sensing surface 1s can excite localized plasmons.
A sensor 1C shown in FIG. 4 (a) performs anodization as shown in FIGS. 2 (a) and 2 (b), removes the alumina layer 11 formed by anodization, and forms an anodized metal body. This is a device in which only the non-anodized portion is left (see JP-A-2006-250924). In such a device, the metal part 20 is constituted by the conductor 13 composed of a non-anodized part having a plurality of dimple-like recesses 20c on the surface.

  A sensor 1D shown in FIG. 4B is obtained by forming a metal layer 20d on the surface of the sensor 1C along the uneven shape (see JP-A-2006-250924).

  In the sensor 1E shown in FIG. 4C, the metal layer 20d of the sensor 1D is formed into particles by annealing, and metal particles 20e are formed on the non-anodized portion 13 of the metal to be anodized (special feature). Application No. 2006-198009 (see unpublished at the time of filing this patent).

  In the sensors 1A to 1E shown in FIG. 3 and FIG. 4, since the metal portion 20 having a substantially regular concavo-convex structure is obtained, the localized plasmon effect can be obtained without variation over the entire surface of the device, which is preferable.

  The metal part 20 may be composed of a metal layer whose surface is roughened. Examples of the roughening method include an electrochemical method using oxidation reduction and the like.

A sensor 1F shown in FIG. 5 has a transparent insulator layer 24 formed on the surface of the sensor 1 shown in FIG. In this case, the surface modification A2 modifies the protruding portion 22 of the metal portion 20 via the transparent insulator layer 24. The transparent insulator layer 24 is a SiO ₂ film having a thickness of 50 nm or less, and substantially transmits the measurement light L1 irradiated on the surface of the sensor 1. The main function of the transparent insulator layer 24 is to prevent direct charge transfer from the fluorescent label Lu bound to the surface modification A1 bound to the detected substance R bound to the surface modification A2 to the protrusion 22. is there. In general, the degree of energy transfer from a substance in the vicinity of a metal to the metal is inversely proportional to the third power of the distance if the metal has a semi-infinite thickness, and the fourth power of the distance if the metal is an infinitely thin plate. If the metal is a fine particle, it decreases in inverse proportion to the sixth power of the distance. In the sensor 1, the thickness of the transparent insulator layer 24 that can prevent direct charge transfer from the fluorescent label Lu to the projecting portion 22 depends on the material, size, and shape of the projecting portion 22, and is several nm to several It is about 10 nm. As the film thickness of the transparent insulator layer 24, it is preferable to ensure a film thickness equal to or greater than a thickness capable of preventing direct charge transfer. On the other hand, it is known that the electric field enhancement effect by the localized plasmon effect attenuates exponentially according to the distance from the metal surface. In addition, the strength of the electric field enhancement effect also depends on the shape of the metal surface. Therefore, in consideration of the diameter and shape of the protrusion 22 in the present embodiment, the film thickness of the transparent insulator layer 24 is preferably 50 nm or less so that an effective electric field enhancement effect can be obtained.

Furthermore, the material of the transparent insulator layer 24 is not limited to the SiO ₂ film. Other specific examples of preferred materials include polymers. More specifically, examples of preferred materials for the transparent insulator layer include hydrophobic polymers and inorganic oxides.

  The hydrophobic polymer preferably contains 50% by weight or more of a monomer having a water solubility of 20% by weight or less. Specific examples of monomers having a water solubility of 20% by weight or less include vinyl esters, acrylic acid esters, methacrylic acid esters, olefins, styrenes, crotonic acid esters, itaconic acid diesters, maleic acid diesters. , Fumaric acid diesters, allyl compounds, vinyl ethers, vinyl ketones and the like, and styrene, methyl methacrylate, hexafluoropropane methacrylate, vinyl acetate, acrylonitrile and the like are preferably used. The hydrophobic polymer compound may be a homopolymer composed of one type of monomer or a copolymer composed of two or more types of monomers.

  Furthermore, you may use together the high molecular compound which copolymerized the monomer whose solubility with respect to water is 20 weight% or more. Specific examples of the monomer having a solubility in water of 20% by weight or more include 2-hydroxyethyl methacrylate, methacrylic acid, acrylic acid, and allyl alcohol. As the hydrophobic polymer, polyacrylic acid ester, polymethacrylic acid ester, polyester, and polystyrene are more preferable. By doing so, film formation becomes easy and it becomes easy to expose a functional group for immobilizing a physiologically active substance on the surface. For example, a film made of polyacrylic acid ester, polymethacrylic acid ester, or polyester can easily expose the carboxyl group and hydroxyl group on the surface by hydrolyzing the surface with acid or base, and it is made of polystyrene. The film can be easily exposed to a carboxyl group by performing an oxidation treatment such as UV / ozone treatment.

  As an inorganic oxide that can be used as a material for the transparent insulator layer, silica, alumina, titania, zirconia, ferrite, and composite materials and derivatives thereof can be selected.

  The transparent insulator layer 24 can be formed by a conventional method, for example, a sol-gel method, a sputtering method, a vapor deposition method, a plating method, or the like can be employed.

"Sensor with sample cell"
With reference to FIG. 6, the structure of the sensor with a sample cell of embodiment which concerns on this invention is demonstrated. FIG. 6 is a cross-sectional view corresponding to FIG.

  The sensor 2 with a sample cell of the present embodiment is one in which the sensor 1 of the above embodiment is fixed to the sample cell 30 so that the metal part 20 of the sensor 1 contacts the sample in the sample cell 30 (fixed structure). (Not shown).

  The sample cell 30 mainly includes a cell main body 31 made of a non-translucent material such as a metal that can be filled with the sample X, and a translucent window 32 is fitted into the cell main body 31. The window 32 is fitted on the opposite side of the sensing surface 1 s of the sensor 1.

  The sample cell 30 may be a flow cell in which the sample X can be injected and discharged, and the sample X is continuously injected and discharged.

  In the sensor 2 with the sample cell, the sensor 1 is only required to be fixed so that the sensing surface 1s is in contact with the sample X filled in the sample cell 30, and the entire sensor is placed inside the sample cell 30 as shown in the figure. It may be accommodated, or may be fitted into a wall on the opposite side of the window 32 of the sample cell 30. The sensor 1 may be completely fixed to the sample cell 30 or may be detachable.

  In the sensor 2 with the sample cell, the measurement light L1 is incident on the sensing surface 1s of the sensor 1 from the outside of the sample cell 30 through the translucent window 32, and the two-photon excitation fluorescence or the multiphoton excitation fluorescence of the fluorescent label Lu. The emitted light L2 from the sensing surface 1s including is emitted to the outside of the sample cell 30 through the window 32 and detected.

  The specular reflection component of the measuring light L1 in the translucent window 32 has a strong power, and if it is detected mixed with the outgoing light L2, it causes a decrease in the S / N ratio. Therefore, it is preferable that the sensor cell-equipped sensor 2 can adjust the angle formed by the window 32 and the sensing surface 1s so that the specular reflection component and the emitted light L2 are separated and detected.

"Sensing device"
Based on FIG. 7, the structure of the sensing apparatus of one Embodiment which concerns on this invention is demonstrated. The sensing device 3 shown in FIG. 7 includes the sensor 1 of the above embodiment, the light irradiation means 40 for irradiating the sensor 1 with the measurement light L1, and the two-photon excitation fluorescence of the fluorescent label Lu contained in the emitted light L2 from the sensor 1. Or the detection means 50 which detects multiphoton excitation fluorescence is comprised.

  The sensor 1 irradiates the sensing surface 1s with measurement light L1 having a wavelength that can excite localized plasmons in the metal portion 20 and that emits two-photon excitation fluorescence or multiphoton excitation fluorescence of the fluorescent label Lu. It is what is done. Therefore, the light irradiation means 40 has a wavelength that can excite localized plasmons in the metal part 20 and can emit light having an absorption wavelength that emits two-photon excitation fluorescence or multiphoton excitation fluorescence of the fluorescent label Lu. A single wavelength light source 41 such as a light emitting diode is provided. The light irradiation means 40 is provided with a light guide optical system including a collimator lens and / or a condensing lens that makes the emitted light from the single-wavelength light source 41 a parallel light flux as necessary.

  The detection means 50 includes a light intensity detector 51 such as a photodiode and a data processing unit 52. The light intensity detector 51 detects two-photon excitation fluorescence or multiphoton excitation fluorescence in the outgoing light L2. In order to detect the emitted fluorescence with higher sensitivity, it is preferable that light having the same wavelength as the measurement light L1 such as a specular reflection component of the measurement light L1 that becomes strong background light is not detected. In order to prevent detection of light having the same wavelength as the measurement light L 1, a light intensity detector 51 that does not detect light having the same wavelength as the measurement light L 1 is used, or between the detection means 50 and the sensor 1. The wavelength selection means 53 for removing the light having the wavelength of the measurement light L1 may be further provided on the optical path.

  Since the sensing device 3 performs sensing using the sensor 1 of the above embodiment, the detection target substance R is labeled with a fluorescent label Lu that selectively binds to the detection target substance R, and the fluorescent label Lu Two-photon excitation fluorescence or multiphoton excitation fluorescence has high fluorescence efficiency, and fluorescence emitted from the fluorescent label Lu can be detected at a high S / N ratio for sensing. Further, as shown in the drawing, the sensing device 3 has a simple configuration because the measurement system does not require a complicated optical system. The sensing device 3 can analyze the presence and / or amount of the substance R to be detected.

  The sensor of the present invention can be preferably used for fluorescence analysis performed in immunoassay or the like in the biochemical field.

(A) is the whole perspective view which shows the structure of the sensor of one Embodiment which concerns on this invention, (b) is thickness direction sectional drawing at the time of sensing The figure which shows the manufacturing process of the sensor of FIG. (A) And (b) is a design change example of the sensor of FIG. (A)-(c) is a design change example of the sensor of FIG. Example of changing the sensor in FIG. 1 is a schematic cross-sectional view showing a configuration of a sensor with a sample cell according to an embodiment of the present invention. The figure which shows the structure of the sensing apparatus of one Embodiment which concerns on this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Sensor 1s Sensing surface 10 Metal object to be anodized 11 Dielectric substrate (metal oxide layer, dielectric)
11s substrate surface 11r substrate back surface 12 micropore 13 conductor (non-anodized portion)
DESCRIPTION OF SYMBOLS 20 Metal part 20a Metal particle 21 Filling part 22 Projection part 24 Transparent dielectric layer 2 Sensor with sample cell 30 Sample cell 3 Sensing apparatus 40 Light irradiation means 50 Detection means 53 Wavelength selection means
L1 Measurement light Lu Fluorescent label L2 Emission light w Separation distance between protrusions R Detected substance A1, A2 Surface modification X Sample

Claims (17)

It has a sensing surface that can bind to a specific substance to be detected,
In the sensor used for sensing, wherein the substance to be detected is labeled with a fluorescent label that selectively binds to the substance to be detected, and two-photon excitation fluorescence or multiphoton excitation fluorescence of the fluorescence label is detected.
At least a portion of which is exposed on the sensing surface and has a metal part that can excite localized plasmons;
The sensing surface is irradiated with measurement light having a wavelength capable of exciting localized plasmons in the metal part and emitting two-photon excitation fluorescence or multiphoton excitation fluorescence of the fluorescent label. A sensor characterized by that.   The sensor according to claim 1, wherein the metal part has a concavo-convex structure smaller than a wavelength of the measurement light. The base of the metal part is a dielectric having a plurality of micropores therein, and at least the plurality of micropores opened on the surface on the sensing surface side,
A plurality of the metal part is formed of a filling part filled in the micropores of the dielectric, and a protruding part formed on the filling part so as to protrude from the surface of the dielectric and larger than the diameter of the filling part. The sensor according to claim 2, comprising: a metal part.   The dielectric is made of a metal oxide obtained by anodizing at least a part of a metal to be anodized, and the plurality of micropores are formed in the metal oxide during the anodization process. The sensor according to claim 3, wherein   The sensor according to claim 2, wherein the metal part is a metal layer having a roughened surface. The base of the metal part is a dielectric,
3. The sensor according to claim 2, wherein the metal part is a metal particle layer composed of a plurality of metal particles fixed to the surface of the dielectric. The base of the metal part is a dielectric,
The sensor according to claim 2, wherein the metal part is a metal pattern layer patterned on the surface of the dielectric.   The localized plasmon resonance wavelength of the metal part and the absorption wavelength that emits the two-photon excitation fluorescence or multiphoton excitation fluorescence of the fluorescent label substantially coincide with each other, and light having this wavelength is used as the measurement light. The sensor according to claim 1, wherein the sensor is provided.   The sensor according to claim 1, wherein a transparent insulator layer is formed on a surface of the metal part.   The sensor according to claim 1, wherein the surface of the metal part is subjected to surface modification that selectively binds to the substance to be detected.   The sensor according to claim 1, wherein the sensing surface is divided into a plurality of sensing areas in which the sensing is performed independently. The surface of the metal part is subjected to surface modification that selectively binds to the substance to be detected,
In addition, the sensing surface has a plurality of sensing regions in which the different types of the surface modification for the different types of the substances to be detected are applied and the sensing is performed independently. The sensor according to claim 11. The sensor according to any one of claims 1 to 10,
At least a part through which the measurement light and the light emitted from the sensing surface including the two-photon excitation fluorescence or the multiphoton excitation fluorescence of the fluorescent label pass is a sensor with a sample cell attached to a sample cell having translucency. And
A sensor with a sample cell, wherein the sensor is attached to the sample cell so that the metal part of the sensor contacts a sample in the sample cell. A sensor according to any one of claims 1 to 13,
Light irradiation means for irradiating the sensor with the measurement light;
A sensing device comprising: detecting means for detecting two-photon excitation fluorescence or multi-photon excitation fluorescence of the fluorescent label contained in light emitted from the sensor.   15. The sensing device according to claim 14, further comprising wavelength selection means for removing light having the wavelength of the measurement light in an optical path between the sensor and the detection means.   16. The sensing device according to claim 14, wherein the presence / absence and / or amount of the substance to be detected is analyzed. In a sensing method in which a substance to be detected is labeled with a fluorescent label that selectively binds to the substance to be detected, and two-photon excitation fluorescence or multiphoton excitation fluorescence of the fluorescence label is detected.
A sensing method, wherein the fluorescent label is excited by a localized plasmon electric field to generate two-photon excited fluorescence or multiphoton excited fluorescence, and the two-photon excited fluorescence or multiphoton excited fluorescence is detected.

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