JP2010223802A - Light signal detection method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To perform high quantitativeness measurement by simply reducing dispersion in an electric field enhancing effect received by an optical responsiveness labeling substance in a light signal detection method. <P>SOLUTION: A sensor chip 10 having a sensor section 14 including a flat metal layer 12 is used, a binding substance B<SB>2</SB>labeled by the optical responsiveness labeling substance F is bound to the sensor section 14, an enhancing electric field D is generated by irradiating the sensor section 14 by epi-illumination with measuring light L<SB>0</SB>having a wavelength that induces plasmon in the metal layer 12 and is optically responded by the optical responsiveness labeling substance F, a light signal Lf<SB>1</SB>generated from the optical responsiveness labeling substance F is enhanced by the enhancing electric field D, and the amount of a substance A to be detected is detected based on the amount of the light signal Lf<SB>1</SB>. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to an optical signal detection method for detecting an amount of a specific substance in a sample by irradiating the sample with light and detecting an optical signal from the photoresponsive labeling substance.

  In bio-measurement and the like, the fluorescence method is widely used as a highly sensitive and easy measurement method. The fluorescence method is qualitative or qualitative by irradiating a sample considered to contain a substance to be detected that emits fluorescence when excited by light of a specific wavelength, by irradiating the excitation light of the specific wavelength and detecting the fluorescence emitted at this time. This is a method for quantitatively confirming the presence of a substance to be detected. In addition, when the substance to be detected is not a fluorescent material, the substance to be detected is labeled with a fluorescent label such as an organic fluorescent dye, and then the fluorescence is detected in the same manner. It is a method to confirm.

  In the above-described fluorescence method, there is a technique in which only a specific target substance can be efficiently detected while flowing a sample, and the target substance is fixed to the surface of the sensor unit by the following two methods and then fluorescence detection is performed. It is common. One such technique is, for example, when the substance to be detected is an antigen, the antigen is specifically bound to the primary antibody immobilized on the surface of the sensor unit, and then a fluorescent label is attached. A secondary antibody that specifically binds to the above antigen is bound to the above antigen to form a primary antibody-antigen-secondary antibody binding state, and fluorescence from the fluorescent label attached to the bound secondary antibody This is a so-called sandwich method. The other is, for example, when the substance to be detected is an antigen, a secondary antibody in which an antigen and a fluorescent label are attached to the primary antibody immobilized on the surface of the sensor unit (unlike the above-described secondary antibody, Is a so-called competitive method in which the primary antibody is bound to the primary antibody in a competitive manner, and the fluorescence from the fluorescent label attached to the bound secondary antibody is detected.

  Further, for example, as described in Patent Document 1, for the reason that the S / N ratio can be improved in fluorescence detection, the fluorescent label fixed to the sensor unit by the above-described method is enhanced by local plasmons. A method of exciting by an electric field has been proposed. Localized plasmons are plasmons that are locally generated in a fine structure having the same size as the wavelength of irradiation light. When this localized plasmon is excited, a significantly enhanced electric field is formed around the microstructure. For example, when this fine structure is a gold fine particle, localized plasmon is induced for light in the vicinity of 520 nm.

JP 2007-240361 A

  However, in patent document 1, since the fine structure for producing the said localized plasmon is formed on the optical base material using the imprint technique, it takes time to manufacture the sensor chip. In addition, there is a problem of yield, for example, the fine structure is broken during the manufacture of the sensor chip.

  Furthermore, when fixing a labeling substance such as a fluorescent label on the surface of the sensor unit having a large number of convex portions having a height of 100 to 1000 nm, a width of 20 to 1000 nm, and an aspect ratio of 2 to 10 as described in Patent Document 1, The influence of the labeling substance by the enhanced electric field varies depending on whether the labeling substance is fixed on the top of the convex part or between the convex parts. For example, when the above-described sandwich assay is performed by antigen-antibody reaction, the labeling substance is separated from the surface of the fixed sensor part by more than 50 nm. Therefore, the labeling substance immobilized on the top of the convex part is enhanced. You may not be able to benefit from signal enhancement by electric fields. As described above, the difference in the fixing position of the labeling substance results in a difference in signal enhancement, which may cause a decrease in the quantitativeness of the measurement.

  The present invention has been made in view of the above problems, and in the optical signal detection method, it is possible to perform measurement with higher quantitativeness by simply reducing variation in the electric field enhancement effect received by the photoresponsive labeling substance. An object of the present invention is to provide an optical signal detection method.

  The applicant of the present invention pays attention to the fact that plasmons are induced in the metal layer when the measurement light is irradiated with epi-illumination on the photoresponsive labeling substance present on the sensor unit having a flat metal layer, It came.

That is, in order to solve the above problems, an optical signal detection method according to the present invention includes:
Using a sensor chip having a dielectric plate and a sensor unit including a flat metal layer provided in a predetermined region on one surface of the dielectric plate,
By bringing the sample into contact with the sensor unit, the binding substance labeled with the photoresponsive labeling substance in an amount corresponding to the amount of the substance to be detected contained in the sample is bound to the sensor part,
By irradiating the sensor part with measuring light having a wavelength capable of inducing plasmon in the metal layer and having a photoresponsive labeling substance capable of photoresponsiveness, an enhanced electric field is generated on the sensor part,
Enhance the optical signal from the photoresponsive labeling substance by the enhanced electric field, detect this optical signal,
An optical signal detection method for detecting the amount of a substance to be detected based on the amount of the optical signal,
The photoresponsive labeling substance comprises a plurality of photoresponsive substances that generate an optical signal in response to measurement light, and a light transmitting material that includes the photoresponsive substance and transmits the optical signal. is there.

  Here, the “flat metal layer” means a metal film formed on one surface of a dielectric plate having no fine structure such as metal fine particles. That is, the metal film does not have a fine structure that generates localized plasmons when irradiated with measurement light.

  The “binding substance” means a substance that specifically binds to a specific target substance. For example, when an antigen is considered as a specific target substance, an antibody that specifically binds to the antigen is exemplified as a binding substance.

  “Photo-responsive labeling substance” means a labeling substance that generates an optical signal due to irradiation of measurement light. Here, for example, fluorescence can be cited as an optical signal. The photoresponsive labeling substance is a substance in which a plurality of photoresponsive substances are present in a particulate light transmitting material. A part of the plurality of photoresponsive substances may be exposed to the outside of the light transmitting material. Further, the distribution state of the photoresponsive substance in the light transmitting material may be any, may be uniformly distributed, or may not be uniformly distributed. Furthermore, in the central part of the particulate photoresponsive labeling substance, there may be a region where no photoresponsive substance is present.

  In the assay, a binding substance labeled with a photoresponsive labeling substance is added in an amount corresponding to the quantity of the substance to be detected via a first binding substance that is immobilized on the sensor unit and specifically binds to the substance to be detected. It is coupled on the sensor unit. There are the following two methods for “binding the sensor” to the binding substance labeled with the photoresponsive labeling substance. For example, when an assay by the sandwich method is performed, a second binding substance that specifically binds to the substance to be detected is used as the binding substance. As a result, a sandwich structure of the first binding substance-the substance to be detected-the second binding substance is created. Here, the binding site for the first binding substance of the substance to be detected is different from the binding site for the second binding substance. On the other hand, when the assay by the competition method is performed, a third binding substance that competes with the substance to be detected and specifically binds to the first binding substance is used as the binding substance. This creates a first binding substance-third binding substance bond.

  “Detecting the amount of a substance to be detected” includes detecting the presence or absence of the substance to be detected, and means detecting not only a qualitative amount but also a quantitative amount and a degree of activity. .

  Furthermore, in the optical signal detection method according to the present invention, the metal layer is mainly composed of at least one metal selected from the group consisting of Au, Ag, Cu, Al, Pt, Ni, Ti, and alloys thereof. It is preferable that the main component is Au or Ag.

  The photoresponsive substance is preferably one substance selected from the group consisting of fluorescent dye molecules, fluorescent fine particles, and quantum dots. In other words, the “photoresponsive substance” only needs to have photoresponsiveness to the measurement light, and may be a fluorescent dye molecule, a fluorescent fine particle, a quantum dot (semiconductor fine particle) or the like that generates fluorescence when irradiated with the measurement light. Good. Therefore, “light from the photoresponsive labeling substance” means light (fluorescence, phosphorescence, etc.) generated from the photoresponsive substance when irradiated with the measurement light.

  The optical signal detection method according to the present invention induces plasmons on a metal layer by irradiating measurement light with epi-illumination on a photoresponsive labeling substance present on a sensor unit having a flat metal layer. Yes. This is considered that plasmon is induced in the metal layer by the optical signal from the photoresponsive labeling substance. Therefore, the electric field on the sensor unit can be enhanced by the electric field enhancing effect of the plasmon. This indicates that an enhanced electric field can be easily formed by simply performing epi-illumination on the sensor unit without forming a fine structure or adjusting a total reflection illumination optical system. In addition, since the metal layer is flat, the positional relationship between each photoresponsive labeling substance and the metal layer is almost constant, and the electric field enhancement effect is achieved so that each photoresponsive labeling substance has a substantially uniform enhancement. Obtainable. As a result, in the optical signal detection method, variation in the electric field enhancement effect received by the photoresponsive labeling substance can be easily reduced, and measurement with higher quantitativeness is possible.

Schematic sectional view showing an apparatus used in the optical signal detection method according to the first embodiment Schematic plan view showing a chip used in the optical signal detection method according to the second embodiment Schematic sectional view showing a chip used in the optical signal detection method according to the second embodiment FIG. 6 is a schematic cross-sectional view showing an assay process of the optical signal detection method according to the second embodiment. Schematic cross section explaining the principle of the competitive assay Schematic sectional view showing the steps of the assay of the optical signal detection method according to the third embodiment Schematic cross-sectional view showing an example of design change of photoresponsive labeling substance The graph which shows the measurement result of an Example and a comparative example

  Hereinafter, although an embodiment of the present invention is described using a drawing, the present invention is not limited to this.

<First Embodiment of Optical Signal Detection Method>
First, the configuration of the optical signal detection apparatus 1 used in the optical signal detection method according to the present embodiment will be described. FIG. 1 is a schematic cross-sectional view showing an overall configuration of an optical signal detection device 1 according to the present embodiment. In each figure, for convenience of explanation, the dimensions of each part are different from the actual ones.

Optical signal detecting apparatus as shown in FIG. 1 1, the sensor chip 10 having a sensor unit 14 having the metal layer 12 provided in a predetermined area of the one surface of the dielectric plate 11, on the measurement light L ₀ sensor unit When the epi-illumination optical system 20 that irradiates by epi-illumination and the binding substance B ₂ labeled with the photo-responsive labeling substance F are present in the sample in contact with the metal layer 12, the photo-responsive labeling substance F And a photodetector 30 for detecting the generated light.

Epi-illumination optical system 20 includes a light source 21 composed of a semiconductor laser (LD) for outputting the measurement light L _0, and a half mirror 23 for guiding to the sensor chip 10 to reflect the measuring light L _0. The half mirror 23 reflects the measurement light L ₀ and transmits light generated from the photoresponsive labeling substance.

  The sensor chip 10 is obtained by forming a metal film as a metal layer 12 in a predetermined region in a flow path of a dielectric plate 11 having a flow path.

  The dielectric plate 11 is made of a transparent material such as transparent resin or glass. The dielectric plate 11 is preferably made of a resin. In this case, it is more preferable to use a resin such as polymethyl methacrylate (PMMA), polycarbonate (PC), or amorphous polyolefin (APO) containing cycloolefin. .

The metal layer 12 can be formed by a known vapor deposition method by forming a mask having an opening in a predetermined region on one surface of the dielectric plate 11. The metal layer 12 does not have a fine structure that can induce localized plasmons by light irradiation. The thickness of the metal layer 12 is preferably determined as appropriate so that the material of the metal film, the surface plasmon by the wavelength of the measuring light L ₀ is strongly excited. For example, when laser light having a center wavelength of 780 nm is used as the measurement light L ₀ and an Au film is used as the metal layer 12, the thickness of the metal layer 12 is preferably 50 nm ± 20 nm. More preferably, it is 47 nm ± 10 nm. The metal layer 12 is preferably composed mainly of at least one metal selected from the group consisting of Au, Ag, Cu, Al, Pt, Ni, Ti, and alloys thereof, and particularly Au or Ag. Is preferred. Here, the “main component” is defined as a component having a content of 90% by mass or more. When Ag is used as the metal layer, it is preferable to form an antioxidant film on the surface because Ag is easily oxidized. As the antioxidant film, an oxide film such as a SiO ₂ film or another metal film that is difficult to oxidize such as an Au film can be used.

  In the following, the optical signal detection device of the embodiment shown in FIG. 1 is used as a fluorescence detection device, and a fluorescence detection method will be described as an optical signal detection method. In addition, a fluorescent labeling substance including a fluorescent dye molecule is used as the photoresponsive labeling substance F, and fluorescence is detected as an optical signal.

The principle of fluorescence detection using the fluorescence detection apparatus 1 will be described.
The measurement light L ₀ is irradiated onto the sensor unit 14 by epi-illumination by the epi-illumination optical system 20, whereby fluorescence Lf is generated from the fluorescent labeling substance F. At this time, the fluorescence Lf ₁ from the fluorescent labeling substance F ₁ fixed to the sensor unit is enhanced by the following two effects. One is a “mirror effect” by the metal layer 12. Due to this mirror effect, the amount of the measurement light L ₀ irradiated to the fluorescent labeling substance F ₁ increases, so that the amount of fluorescence generated from the fluorescent labeling substance F ₁ is increased. The other is the electric field enhancement effect by surface plasmons. This surface plasmon is considered to be induced in the metal layer 12 when a part of the fluorescence Lf generated from the fluorescent labeling substance F is absorbed by the metal layer 12. As a result, an electric field distribution is generated on the surface of the metal layer 12 by the surface plasmons, and an electric field enhancement region D is formed. In this case, if the fluorescent labeling substance F (F ₁ in FIG. ₁₎ is present in the electric field enhancing region D, the fluorescence Lf ₁ occurs the fluorescent labels F ₁ is further excited. The photodetector 30 condenses the fluorescence with a condenser lens (not shown) and detects the fluorescence Lf. Here, when the fluorescent labeling substance F (F _{2 in} FIG. 1) exists outside the electric field enhancement region D, the fluorescence Lf ₂ is not enhanced. In this manner, the optical detector 30 detects the fluorescence Lf ₁ augmented. Here, it is preferable to carry out the fluorescence measurements from wash away fluorescent labels F ₂ floating in the sample.

In the present embodiment, the fluorescent labeling substance F includes a plurality of fluorescent dye molecules 15 that are photoresponsive substances, and a light transmissive material 16 that includes the plurality of fluorescent dye molecules 15 and transmits fluorescence. Therefore, since the fluorescent labeling substance F contains a plurality of fluorescent dye molecules 15, the amount of emitted fluorescence can be greatly increased as compared with the case where the conventional fluorescent dye molecules 15 themselves are used as labels. The fluorescent labeling substance F preferably has a particle size of 5300 nm or less from the viewpoint of diffusion time. Further, from the viewpoint of the amount of fluorescence and the closest packing density on the sensor part and from the viewpoint of disturbance of the surface plasmon, those of 100 nm to 700 nm are more preferred, and those of 130 nm to 500 nm are particularly preferred. Here, the particle diameter of the fluorescent labeling substance (photoresponsive labeling substance) F is the diameter in the case of a substantially spherical particle, and the average length of the maximum width and the minimum width in the case of a non-spherical particle. Let us define it. Specific examples of the light transmissive material 16 include dielectrics such as polystyrene and SiO _2, but the fluorescent dye molecule 15 can be included, and the fluorescence from the fluorescent dye molecule 15 is transmitted and emitted to the outside. There is no particular limitation as long as it is possible.

  In addition, instead of the fluorescent dye molecule, fluorescent fine particles and quantum dots (semiconductor fine particles) may be provided as the photoresponsive substance in the fluorescent labeling substance.

The fluorescent labeling substance F used in the present embodiment can be produced as follows.
First, polystyrene particles (Estapoor, φ500 nm, 10% solid, carboxyl group, product number K1-050) are prepared to prepare 0.1% solid inphosphate (polystyrene solution: pH 7.0).
Next, a 0.3 mg ethyl acetate solution (1 mL) of a fluorescent dye (Molecular Probes, BODIPY-FL-SE, product number D2184) is prepared.
The polystyrene solution and the fluorescent dye solution are mixed and impregnated while evaporating, and then centrifuged (15000 rpm, 4 ° C., 20 minutes twice) to remove the supernatant. Through the above steps, a fluorescent labeling substance F formed by encapsulating fluorescent dye molecules with polystyrene can be obtained. In such a procedure, the particle diameter of the fluorescent labeling substance F produced by impregnating the fluorescent particles with polystyrene particles is the same as the particle diameter of the polystyrene particles (in the above example, φ500 nm).

Sensing using the fluorescence detection method using the fluorescence detection apparatus 1 having the above-described configuration will be described.
First, the sample S to be inspected is brought into contact with the sensor unit 14 including the metal layer 12 of the sensor chip 10. Here, as an example, a case where the antigen A is detected as a detection target substance contained in the sample S will be described. On the metal layer 12 is the primary antibody B ₁ is being modified as a first binding substance that specifically binds an antigen A. Sample S is passed through the flow path of the sensor chip 10, and then flows fluorescent labeling substance F which is a second binding substance secondary antibody B ₂ is modified on the surface to bind to antigen A specifically similar . In this case, the primary antibody B ₁ that is surface-modified to the metal layer 12 and the secondary antibody B ₂ that is surface-modified to the fluorescent labeling substance F are those that bind to the antibody A at different sites. . Thereafter, the measurement light L ₀ is emitted from the epi-illumination optical system 20 toward the sensor unit 14, and fluorescence detection is performed by the photodetector 30. At this time, if a predetermined fluorescence Lf by the light detector 30 is detected, the bond between the secondary antibody B ₂ and the antigens A, that is, being able to confirm the presence of the antigen A in the sample.

In addition, the timing of labeling of the substance to be detected (antigen A) is not particularly limited, and before the substance to be detected (antigen A) is bound to the first binding substance (primary antibody B ₁ ), the sample is fluorescently labeled in advance. Substances may be added.

As described above, in the optical signal detection method according to the present embodiment, the metal layer is formed by irradiating the photoresponsive labeling substance existing on the sensor unit including the flat metal layer with the epi-illumination. Inducing plasmons in the. This is considered that plasmon is induced in the metal layer by the optical signal from the photoresponsive labeling substance. Therefore, the electric field on the sensor unit can be enhanced by the electric field enhancing effect of the plasmon. This indicates that an enhanced electric field can be easily formed by simply performing epi-illumination on the sensor unit without forming a fine structure or adjusting a total reflection illumination optical system. In addition, since the metal layer is flat, the positional relationship between each photoresponsive labeling substance and the metal layer is almost constant, and the electric field enhancement effect is achieved so that each photoresponsive labeling substance has a substantially uniform enhancement. Obtainable. As a result, in the optical signal detection method, variation in the electric field enhancement effect received by the photoresponsive labeling substance can be easily reduced, and measurement with higher quantitativeness is possible.
(Design changes)
In the first embodiment, the description has been given using the coaxial epi-illumination type optical system as the epi-illumination optical system. However, the present invention is not particularly limited to this. Further, the epi-illumination does not necessarily have to be irradiated so as to be perpendicular to the sensor unit as shown in FIG.

<Second Embodiment of Optical Signal Detection Method>
The optical signal detection method according to the present embodiment is a method of performing sensing using a sample cell 50 as shown in FIGS. 2A and 2B. FIG. 2A is a plan view showing the configuration of the sample cell 50, and FIG. 2B is a side sectional view of the sample cell 50. Also in this embodiment, a fluorescent labeling substance that includes fluorescent dye molecules as a photoresponsive labeling substance will be described as an example.

  The sample cell 50 includes a base 51 made of a dielectric plate, a spacer 53 that holds the liquid sample S on the base 51 and forms a flow path 52 of the liquid sample S, an injection port 54a for injecting the sample S, and A sample contact surface between the inlet 54a and the air hole 54b of the flow channel 52 is configured by an upper plate 54 made of a glass plate provided with an air hole 54b serving as an outlet for discharging the sample flowing down the channel 52. Sensor portions 58a and 59a made of a metal layer provided on a predetermined region of the base 51 to be provided. Further, a membrane filter 55 is provided at a location from the inlet 54 a to the flow path 52, and a waste liquid reservoir 56 is formed at a portion connected to the air hole 54 b downstream of the flow path 52.

  In this embodiment, the base 51 is composed of a dielectric plate and also serves as the dielectric plate of the sensor unit. However, as the base, only the part where the sensor unit is composed is composed of a dielectric plate. May be used.

On the base 51 of the sample cell 50 from the flow passage 52 upstream, fluorescent labels F which second antibody specifically binds to an antigen to be detected substance (second binding substance) B ₂ is surface-modified The first antibody (first binding substance) B ₁ that specifically binds to the labeled secondary antibody adsorption area 57 and the antigen to be detected is fixed on the metal layer 58a. A measurement area 58 and a second measurement area 59 in which a primary antibody B ₀ that specifically binds to the labeled secondary antibody BF without binding to the antigen to be detected is fixed on the metal layer 59a are sequentially provided. ing. That is, the regions where the sensor parts 58a and 59a are formed correspond to the first and second measurement areas 58 and 59, respectively. Here, the labeled secondary antibody BF means a secondary antibody (second binding substance) labeled with the fluorescent labeling substance F. Further, in this example, an example in which two measurement areas are provided in the flow path 52 is described, but only one measurement area may be provided.

Au films 58a and 59a are formed as metal layers on the base 51 in the first measurement area 58 and the second measurement area 59, respectively. A primary antibody B ₁ is further immobilized on the Au film 58a in the first measurement area 58, and a primary antibody B ₀ different from the primary antibody B ₁ is further deposited on the Au film 59a in the second measurement area 59. Is fixed. The first measurement area 58 and the second measurement area 59 have the same configuration except that different primary antibodies are provided. Second measuring primary antibody is fixed in the area 59 B ₀ do not bind to the antigen A, it is one which binds directly with secondary antibody B _2. As a result, fluctuation factors relating to the reaction such as the amount and activity of the labeled secondary antibody BF flowing through the flow path 52 and fluctuation factors relating to the surface plasmon enhancement such as the epi-illumination optical system 20, the Au films 58a and 59a, and the liquid sample S are detected. Can be used for calibration.

Note that the second measurement area primary antibody B ₀ instead, a known amount of the labeling substance may be fixed in advance. The labeling substance may be the same type as the fluorescent labeling substance surface-modified with the secondary antibody, or may be a fluorescent labeling substance having a different wavelength and size. Furthermore, other photoresponsive substances such as metal fine particles may be used. In this case, only the fluctuation factors relating to the surface plasmon enhancement such as the epi-illumination optical system 20, the Au films 58a and 59a, and the liquid sample S can be detected and used for calibration. Which of the primary antibody B ₀ and the known amount of the labeling substance is fixed in the second measurement area 59 may be appropriately determined depending on the calibration purpose and method.

  By using the sample cell 50, after the fluorescence detection measurement from the first measurement area 58, the second measurement area 59 is moved to the fluorescence detection position to detect the fluorescence from the second measurement area 59. it can.

  For the epi-illumination optical system, the same optical system as in the first embodiment can be used.

Furthermore, FIG. 3 shows a procedure for performing an assay by the sandwich method to determine whether or not an antigen as a substance to be detected is contained in blood (whole blood) using the detection sample cell 50 and the fluorescence detection method of the present embodiment. The description will be given with reference.
Step 1: Blood (whole blood) So to be examined is injected from the injection port 54a. Here, the case where the antigen A which is a to-be-detected substance is contained in this blood So is demonstrated. In FIG. 3, the whole blood So is indicated by a shaded area.
step 2: The whole blood So is filtered by the membrane filter 55, and large molecules such as red blood cells and white blood cells become residues.
Step 3: The blood (plasma) S separated by the membrane filter 55 oozes out into the flow path 52 by capillary action. In order to speed up the reaction and shorten the detection time, a pump may be connected to the air hole 54b, and the blood cells separated by the membrane filter 55 may be caused to flow down by suction and push-out operations of the pump. In FIG. 3, the plasma S is indicated by a hatched area.
step4: is a fluorescent plasma S and the secondary antibody _{B 2} exuded into the flow path 52 is modified labeling substance F (primary labeled 2 antibody BF) is mixed, combined with antigen A secondary antibody _{B 2} in plasma To do.
step5: Plasma S gradually flows into the air hole 54b side along the flow path 52, the secondary antibody _{B 2} antigen A bound to the first measurement area 58 primary antibody is fixed on _{B 1} bound, so-called sandwich antigen a is sandwiched with primary antibody B ₁ and the secondary antibody B ₂ is formed with.
Step 6: A part of the labeled secondary antibody BF that has not bound to the antigen A binds to the primary antibody B ₀ fixed on the second measurement area 59. Even if further antigens A or the primary antibody B ₀ and unbound labeled secondary antibody BF remains on the measurement area, a subsequent plasma functions as a cleansing, floating and non-on plate Wash away adsorbed material.

In this way, blood is injected from the injection port, and after Step 1 to Step 6 until a sandwich is formed in which the antigen A is sandwiched between the primary antibody B ₁ and the secondary antibody B ₂ on the first measurement area 58. By detecting the fluorescence intensity from the first measurement area 58, the presence and / or concentration of the antigen can be detected. Thereafter, the sample cell 50 is moved so that the fluorescence signal from the second measurement area 59 can be detected, and the fluorescence signal from the second measurement area 59 is detected. Fluorescence signal flow down the amount of labeled secondary antibody BF from the second measurement area 59 is the primary antibody B ₀ that bind with the labeling secondary antibodies BF fixed, it is a signal that reflects the reaction conditions, such as active By using this signal as a reference and correcting the signal from the first measurement area 58, a more accurate detection result can be obtained. Further, as described above, even when a known amount of a labeling substance (fluorescent substance or the like) is fixed in advance in the second measurement area 59, similarly, the fluorescence signal from the second measurement area 59 is used as a reference. The signal from the first measurement area 58 can be corrected.

Here, in FIG. 3, the primary antibody B ₁ fixed to the first measurement area 58 is two-dimensionally provided on the surface of the metal layer 59a, but in addition to this, 3 on the metal layer 58a. place the membrane spread dimensionally, it may be fixed to the primary antibody B ₁ to the membrane.

  As described above, also in the optical signal detection method according to the present embodiment, the metal layer is obtained by irradiating the photoresponsive labeling substance existing on the sensor unit including the flat metal layer with the epi-illumination. Inducing plasmons in the. That is, the principle of the optical signal detection method (fluorescence detection method) using the sample cell 50 is the same as that of the first embodiment, and the same effect as that of the first embodiment can be obtained in this embodiment. It is possible to perform measurement with higher quantitativeness by a simple method.

<Third Embodiment of Optical Signal Detection Method>
The optical signal detection method according to the present embodiment is a method of performing sensing using the same sample cell as that of the second embodiment. This embodiment is different from the second embodiment in that an assay by a competition method is performed. Accordingly, the same elements as those in the second embodiment are denoted by the same reference numerals, and detailed description thereof is omitted unless particularly necessary. Also in this embodiment, a fluorescent labeling substance that includes fluorescent dye molecules as a photoresponsive labeling substance will be described as an example.

The competition method will be briefly described with reference to FIG.
As shown in FIG. 4 (A), a third binding substance (for example, competitive antigen) C ₃ showing the same immune reaction as the substance to be detected (for example, antigen) A is modified with a fluorescent labeling substance F. On the metal layer 12, a first binding substance C ₁ (for example, a primary antibody) that specifically binds to both the antigen A and the competitive antigen C ₃ is immobilized. Competing antigen C ₃ is modified fluorescent labels F (labeling competitor CF) at a predetermined concentration, was mixed with antigen A, thereby competitively react the primary antibody C ₁ immobilized on the metal layer 12 ( Antigen-antibody reaction). The concentration of the labeled competitor CF at the time of mixing the antigen A and the labeled competitor CF is known.

In the competition method, as shown in FIG. 4B, when the concentration of the substance A to be detected is high, the amount of the labeled competitor CF that binds to the _first binding substance C1 decreases, that is, on the metal layer 12. Since the number of fluorescent labeling substances F decreases, the fluorescence intensity decreases. On the other hand, as shown in FIG. 4C, when the concentration of the substance A to be detected is low, the amount of the label competitor CF that binds to the _first binding substance C ₁ increases, that is, the fluorescent label on the metal layer 12. Since the number of substances F increases, the fluorescence intensity increases. The competitive method is suitable for detection of a low molecular weight substance because it can be measured if the substance to be detected has one epitope.

Reference numeral 50 ′ in FIG. 5 shows a sample cell of the third embodiment suitable for the assay by the competition method. 'In the first measurement area 58' sample cell 50 of this embodiment material further primary antibody C ₁ on the Au film 58a of fixed, will be described later on the second Au film 59a of the measurement area 59 ' D ₁ is fixed. The first measurement area 58 ′ and the second measurement area 59 ′ have the same configuration except that different substances are provided. Antigen A and competing antigen C ₃ are those that competitively bind to the primary antibody C ₁ which is fixed to the first measurement area 58 '. The substance D ₁ fixed in the second measurement area 59 ′ does not bind to the antigen A and the competitive antigen C _3, and the substance D ₂ modified on the surface of the fluorescent labeling substance F together with the competitive antigen C ₃ It binds specifically. As a result, fluctuation factors relating to the reaction such as the amount and activity of the labeled competitor CF flowing through the flow path and fluctuation factors relating to the surface plasmon enhancement such as the epi-illumination optical system, the Au films 58a and 59a, and the liquid sample S are detected for calibration. Can be used.

Incidentally, instead of substance D ₁ in the second measurement area, a known amount of the labeling substance may be fixed in advance. The labeling substance may be the same type as the fluorescent labeling substance whose surface has been modified with a competitive antigen, or may be a fluorescent labeling substance having a different wavelength and size. In this case, only the fluctuation factors relating to the surface plasmon enhancement such as the epi-illumination optical system, the Au films 58a and 59a, and the liquid sample S can be detected and used for calibration. Whether to fix the substance D ₁ or the known amount of the labeled substance in the second measurement area 59 ′ can be appropriately selected depending on the calibration purpose and method.

In the detection method of the present invention, FIG. 5 shows a procedure for performing an assay according to the competitive method to determine whether or not an antigen as a substance to be detected is contained in blood (whole blood) using the detection sample cell 50 ′ of the present embodiment. The description will be given with reference.
Step 1: Blood (whole blood) So to be examined is injected from the injection port 54a. Here, a case will be described in which an antigen that is a substance to be detected is contained in the blood So. In FIG. 5, whole blood So is indicated by a shaded area.
step 2: The whole blood So is filtered by the membrane filter 55, and large molecules such as red blood cells and white blood cells become residues.
Step 3: The blood (plasma) S separated by the membrane filter 55 oozes out into the flow path 52 by capillary action. Alternatively, in order to speed up the reaction and shorten the detection time, a pump may be connected to the air hole 54b, and the blood cells separated by the membrane filter 55 may be caused to flow down by suction and push-out operations of the pump. In FIG. 5, the plasma S is indicated by a hatched area.
step4: fluorescent labels plasma S competing antigen _{C 3} exuded is applied to the flow passage 52 F and (labeled competitor CF) is mixed.
Step 5: The plasma S gradually flows along the flow path 52 toward the air hole 54b, and the antigen A and the labeled competitor CF compete to be immobilized on the first measurement area 58 ′. It binds to C _1.
step6: 'Some on the primary antibody _{C 1} and unbound labeled competitor CF, the label competitor CF substance _{D 2} is the second measurement area 59' first measurement area 58 fixed on combined with substances D ₁ being, it is fixed on the second measurement area 59 '. In addition, even if the labeled competitor CF that is not bound to the primary antibody C ₁ and the substance D ₁ may remain on the measurement area, the subsequent plasma plays a role of washing, floating and non-specific on the plate Wash away adsorbed material.

In this way, blood is injected from the injection port, and after step 1 to step 6 until antigen A and labeled competitor CF are competitively bound to the primary antibody C ₁ on the first measurement area 58 ′, the first measurement is performed. By detecting the detection signal from the area 58 ′, the presence and / or concentration of the antigen can be detected. Thereafter, the detection sample cell 50 ′ is moved so that the detection signal from the second measurement area 59 ′ can be detected, the detection signal from the second measurement area 59 ′ is detected, and this detection signal is used as a reference. By correcting the detection signal from the first measurement area 58 ′, a more accurate detection result can be obtained.

  As described above, also in the optical signal detection method according to the present embodiment, the metal layer is obtained by irradiating the photoresponsive labeling substance existing on the sensor unit including the flat metal layer with the epi-illumination. Inducing plasmons in the. That is, the principle of the optical signal detection method (fluorescence detection method) using the sample cell 50 ′ is the same as that of the first embodiment, and the same effect as that of the first embodiment is obtained in this embodiment. Therefore, it is possible to perform measurement with higher quantitativeness by a simple method.

<Design changes for the first to third embodiments>
In the first to third embodiments, as shown in FIG. 6, the metal coating film 19 having a thickness that transmits light generated from the photoresponsive material to the surface of the light transmitting material 16 as the photoresponsive labeling material. Those provided with may be used. The metal coating 19 may cover the entire surface of the light transmitting material 16, or may be provided so that a part of the surface is exposed instead of covering the entire surface. As a material of the metal coating 19, the same metal material as that of the above-described metal layer can be used.

  When the metal coating 19 is provided on the surface of the photoresponsive labeling substance, the surface plasmon or local plasmon generated on the metal layer 12 of the sensor chip 10 is coupled to the whispering gallery mode of the metal coating 19, and the optical response The photoresponsive substance 15 in the sex labeling substance F can be excited with higher efficiency. The whispering gallery mode is an electromagnetic wave mode that localizes and circulates on the surface of a microsphere having a diameter of about 5300 nm or less used here.

An example of a metal coating method on a photoresponsive labeling substance will be given.
First, a photoresponsive labeling substance is prepared by the above-described procedure, and polyethyleneimine (PEI) (for example, trade name: Epomin (registered trademark), Nippon Shokubai Co., Ltd.) is modified on the surface.
Next, Pd nanoparticles having a particle size of about 15 nm (for example, an average particle size of 19 nm, Tokuru Honten Co., Ltd.) are adsorbed on the PEI on the surface of the photoresponsive labeling substance.
Surface of photoresponsive labeling substance using electroless plating with Pd nanoparticles as catalyst by immersing polystyrene particles adsorbed with Pd nanoparticles in electroless gold plating solution (HAuCl ₄ , Kojima Chemical Co., Ltd.) An Au film is prepared.

<Example>
Examples of the optical signal detection method according to the present invention will be described below.

  The sensor chip 10 was manufactured by the following method. First, a 50 nm Au film was formed on a ZEONEX (registered trademark) substrate. Then, the fluorescent labeling substance described in the first embodiment was fixed to the sensor part on which the Au film was formed, and the amount of fluorescence was measured by irradiating a semiconductor laser having a wavelength of 660 nm.

  And the fixing method of a fluorescent labeling substance is as follows. First, UV treatment was performed on the Au film-coated ZEONEX substrate for 30 seconds. Then, a Teflon (registered trademark) cuvette was placed on the ZEONEX substrate and fixed with a jig. Next, 20 uL of an aqueous solution adjusted so that the concentration of the fluorescent labeling substance was 0.002% was dispensed into a cuvette and allowed to stand for 1 hour. Here, fluorescent particles (trade name: FC02F, average particle size 0.21 um, excitation wavelength 660 nm, fluorescence wavelength 690 nm) manufactured by Bangs Laboratories were used as the fluorescent labeling substance.

  In addition, the amount of scattered light when particles that do not contain fluorescent dye molecules (photoresponsive substances) were fixed on the sensor portion in the same manner as described above was also measured.

<Comparative example>
As a comparative example with respect to Example 1, using a ZEONEX substrate on which no Au film is formed, a fluorescent labeling substance is fixed to a predetermined region in the same manner as in Example 1, and fluorescence is applied to this sensor chip as in Example 1. Quantity measurement was performed.

  Similarly to Example 1, the amount of scattered light was measured when particles that did not contain fluorescent dye molecules (photoresponsive substances) were immobilized on the sensor portion in the same manner as described above. Here, the measured light amount is adjusted so that the scattered light amount of the comparative example and the scattered light amount of the example are equal.

<Result>
FIG. 7 is a graph showing the measurement results of the above examples and comparative examples. In this result, when a value obtained by subtracting the signal amount without the photoresponsive substance (scattering light amount) from the signal amount with the photoresponsive substance (fluorescence amount) is about 2 as compared with the case without the Au film by providing the Au film. The signal amount (fluorescence amount) increased by 9 times. Thus, it has been demonstrated that the optical signal detection method according to the present invention can obtain an optical signal increase rate that is equal to or greater than the specular effect in which the measurement light is reflected by the Au film and the measurement light quantity increases.

DESCRIPTION OF SYMBOLS 1, 2 Optical signal detection apparatus 10 Sensor chip 11 Dielectric plate 12 Metal film 14 Sensor part 15 Photoresponsive substance 16 Light transmission material 20 Epi-illumination optical system 21 Light source 23 Half mirror 30 Photo detector 50, 50 'Sample cell A Cover Detection substance D Electric field enhancement region F Photoresponsive labeling substance Lf Fluorescence L ₀ Measurement light Ls Scattered light S Sample

Claims (4)

Using a sensor chip having a dielectric plate and a sensor unit including a flat metal layer provided in a predetermined region on one surface of the dielectric plate,
By bringing the sample into contact with the sensor unit, the binding substance labeled with the photoresponsive labeling substance in an amount corresponding to the amount of the substance to be detected contained in the sample is bound to the sensor part,
Increasing the electric field on the sensor unit by irradiating the sensor unit with incident light having a wavelength capable of inducing plasmon in the metal layer and having a wavelength with which the photoresponsive labeling substance can respond to light. Generated
Enhance the optical signal from the photoresponsive labeling substance by the enhanced electric field, detect the optical signal,
An optical signal detection method for detecting the amount of the substance to be detected based on the amount of the optical signal,
The photoresponsive labeling substance includes a plurality of photoresponsive substances that generate an optical signal in response to the measurement light, and a light transmitting material that includes the photoresponsive substance and transmits the optical signal. Optical signal detection method.   The metal layer is mainly composed of at least one metal selected from the group consisting of Au, Ag, Cu, Al, Pt, Ni, Ti, and alloys thereof. 2. The optical signal detection method according to 1.   The optical signal detection method according to claim 2, wherein the metal layer contains Au or Ag as a main component.   4. The optical signal detection method according to claim 1, wherein the photoresponsive substance is one substance selected from the group consisting of fluorescent dye molecules, fluorescent fine particles, and quantum dots.

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