JP2008249361A - Surface plasmon sensor and immunological measuring method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To detect fluorescence of such a measuring material as antibody at extremely high sensitivity without immobilizing it on a sensor. <P>SOLUTION: This sensor comprises: a metal film formed on one surface of a light guide; a non-flexible film formed on the metal film; a light source generating light beam; an optical system passing the light beam through a prism to make it incident into the interface between the light guide and the metal film at an incident angle to generate surface plasmon; and a detecting means detecting fluorescence generated with being excited by an evanescent wave which is reinforced with the surface plasmon. A unit capable of attracting magnetic material on the opposite prism face with respect to the non-flexible film is arranged. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

The present invention relates to a surface plasmon sensor and an immunological measurement method for analyzing a substance in a sample using generation of surface plasmons.

  In biomeasurement and the like, the fluorescence method is widely used as a highly sensitive and simple measurement method. This fluorescence method irradiates a sample that is considered to contain a detection target substance that emits fluorescence when excited by light of a specific wavelength, and detects the presence of the detection target substance by detecting the fluorescence at that time. It is a method to confirm. When the detection target substance is not a fluorescent substance, this binding, that is, detection is performed by contacting the sample with a substance that is labeled with the fluorescent substance and specifically binds to the detection target substance, and then detecting fluorescence in the same manner as described above. The existence of the target substance is also widely confirmed.

  FIG. 2 schematically shows an example of a sensor that performs the fluorescence method using the labeled substance. As an example, this fluorescent sensor is for detecting the measurement target substance 2 contained in the sample 1, and an antibody 4 that specifically binds to the measurement target substance 2 is applied to the substrate 3. Then, the sample 1 is flowed in the sample holder 5 provided on the substrate 3, and then the antibody 6 that specifically binds to the measurement target substance 2 labeled with the phosphor 10 is flowed. Thereafter, excitation light 8 is emitted from the light source 7 toward the surface portion of the substrate 3, and fluorescence is detected by the photodetector 9. At this time, if a predetermined fluorescence is detected by the photodetector 9, it is possible to confirm the binding between the above 6 and the measurement target substance 2, that is, the presence of the measurement target substance 2 in the sample 1.

  In the above example, the antibody 6 that specifically binds to the antigen 2 labeled with the phosphor 10 is actually confirmed by fluorescence detection, but this antibody 6 must bind to the antigen 2. Since it has flowed away and cannot exist on the substrate 3, the presence of the detection target substance 2 is indirectly confirmed by confirming the presence of the antibody 6.

  However, in the conventional fluorescent sensor as shown in FIG. 2, reflected light / scattered light of excitation light at the interface between the substrate and the sample, or scattered light due to impurities other than the detection target substance / floating matter M or the like becomes noise. Therefore, the actual situation is that the S / N in fluorescence detection is not improved even if the performance of the photodetector is improved.

  As a solution to this, for example, an evanescent fluorescence method as shown in Non-Patent Document 1, that is, a fluorescence method using an evanescent wave has been proposed. An example of a fluorescence sensor that implements this fluorescence method is schematically shown in FIG. In FIG. 3, the same components as those in FIG. 2 are denoted by the same reference numerals, and description thereof will be omitted unless necessary (the same applies hereinafter).

  In this fluorescent sensor, a prism (dielectric block) 13 is used as an alternative to the substrate 3 described above, and the excitation light 8 from the light source 7 is totally reflected at the interface between the prism 13 and the sample 1. Irradiated through the prism 13. In this configuration, when the excitation light 8 is totally reflected at the interface, the phosphor 10 labeled with the antibody 6 that specifically binds to the measurement target substance 2 is excited by the evanescent wave 11 that leaks out in the vicinity of the interface. Fluorescence detection is performed by a photodetector 9 arranged on the opposite side of the sample 1 from the prism 13 (upward in the drawing).

  In this fluorescence sensor, when a prism is used as the substrate, the excitation light 8 is totally reflected downward in the figure, so that the excitation light becomes a background for the fluorescence detection signal in fluorescence detection from above. There is no. Further, since the evanescent wave 11 reaches only a region of several hundred nm from the interface, scattering from the impurities / floating matter M in the sample can be almost eliminated. Therefore, this evanescent fluorescence method is attracting attention as a method that can significantly reduce (light) noise as compared with the conventional fluorescence method and can measure the fluorescence of the detection target substance in units of one molecule.

  In addition, in the fluorescence method using the above evanescent wave, in Patent Document 2, an optical waveguide is used as a substrate that transmits light instead of a prism. In this case, since the excitation light component travels while totally reflecting the optical waveguide, the excitation light component does not become a background for the fluorescence detection signal in fluorescence detection from above and / or below.

  A surface plasmon enhanced fluorescence sensor as shown in FIG. 4 is also known as a sensor capable of measuring fluorescence with higher sensitivity. This surface plasmon enhanced fluorescence sensor is described in, for example, Patent Document 1, and differs from the fluorescence sensor of FIG. 3 in that a metal film 20 is basically formed on the prism 13. That is, by forming such a metal film 20, surface plasmons are generated in the metal film 20 when the excitation light 8 is irradiated, and fluorescence is amplified by the electric field amplification action. According to a simulation, it has been found that the fluorescence intensity in that case is amplified to about 1000 times.

  However, in the surface plasmon enhanced fluorescence sensor as described above, as shown in Non-Patent Document 2, if the phosphor in the sample and the metal film are too close, the energy excited in the phosphor Transition to the metal film before generating fluorescence, and a phenomenon that fluorescence does not occur (so-called metal quenching) may occur.

  In order to cope with this metal quenching, Non-Patent Document 2 forms a SAM (self-assembled film) on the metal film, thereby separating the phosphor and the metal film in the sample by more than the thickness of the SAM. It has been proposed to let In FIG. 4 also, this SAM is indicated by number 21. Further, in Non-Patent Document 3, in relation to the metal quenching, the dependence of the fluorescence intensity enhanced by the surface plasmon on the distance from the metal film is examined.

  By the way, in the measurement methods described so far, in any case, it is necessary to immobilize a substance that specifically recognizes the measurement target substance on the surface of the sensor substrate. As an example of a measurement method using a substance that specifically recognizes a measurement target substance, an immunological measurement method using an antibody is known. For example, when the immunological measurement method is performed using a surface plasmon sensor, it is necessary to immobilize the antibody on the sensor surface in advance. As a method for immobilizing an antibody, an immobilization method by physical adsorption described in Non-Patent Document 4, an immobilization method by forming a covalent bond, and the like are used.

In general, when a substance that specifically recognizes a substance to be measured, such as an antibody, is immobilized, it becomes a solid phase-liquid phase reaction format, so that the reaction efficiency is remarkably lowered or the binding constant is lowered. It is known that For this reason, there are problems such as a decrease in detection sensitivity and a long reaction time. Furthermore, it is necessary to immobilize specific antibodies for different substances to be measured, which is a factor that hinders the improvement of the versatility of the sensor.
Japanese Patent No. 3562912 Japanese Patent No. 3321469 “Understanding this with bioimaging” p.104-113 Akihiro Shiomi et al. Yodosha W. Knoll et al., Analytical Chemistry (Anal. Chem.) 75 (2003) p.2610 W. Knoll et al., Colloids and Surfaces. A. (Colloids Surf. A), 171 (2000) p.115 "Immunoassay" p.129-163 JP Gosling et al. Oxford University Press

  When a substance that specifically recognizes a substance to be measured such as an antibody is immobilized, there is a concern that the reaction efficiency may be significantly lowered or the binding constant may be lowered due to a solid-liquid phase reaction. For this reason, there are problems such as a decrease in detection sensitivity and a long reaction time. Furthermore, it is necessary to immobilize specific antibodies for different substances to be measured, which is a factor that hinders the improvement of the versatility of the sensor.

The present invention has been made in view of the above circumstances, and a surface plasmon sensor that can be detected with extremely high sensitivity without immobilizing a substance that specifically recognizes a measurement target substance such as an antibody on the sensor. It is intended to provide.

  The surface plasmon sensor of the present invention includes an optical waveguide, a metal film formed on one surface of the optical waveguide, an inflexible film having a thickness of 10 to 100 nm formed on the metal film, A light source that generates a beam; an optical system that causes the light beam to pass through a prism and is incident at an incident angle that generates a surface plasmon to an interface between the prism and the metal film; and an evanescent wave that is enhanced by the surface plasmon A surface plasmon sensor comprising fluorescence detecting means for detecting fluorescence generated by being excited, and having a unit capable of attracting a magnetic substance to an optical waveguide surface opposite to an inflexible film. It is a feature.

  Here, the above-mentioned “inflexibility” means that it has such a rigidity that it does not deform so as to change the film thickness while performing fluorescence detection enhanced by surface plasmon. means. The film thickness of the inflexible film is preferably in the range of 10 to 100 nm. As the inflexible film, a polymer film is preferably used.

  The surface plasmon sensor according to the present invention holds a sample with a metal film formed on the surface of an optical waveguide made of a material that transmits excitation light, forms an inflexible film on the surface of the metal film, and transmits excitation light from the optical waveguide. In the surface plasmon fluorescence method, the material contained in the sample is excited by an evanescent wave that exudes to the surface of the metal film enhanced by the surface plasmon, and the fluorescence emitted by the material is detected by this excitation. By installing a unit capable of attracting a magnetic substance on the opposite prism surface with respect to the conductive film, the measurement target substance labeled with the magnetic substance and the fluorescent substance detectable by the surface plasmon sensor is measured in advance. Substances that specifically recognize substances can be collected on the sensor surface without immobilization.

  As a result, the concentration of the measurement target substance labeled with the magnetic substance and the fluorescent substance that can be detected by the surface plasmon sensor or the complex thereof is increased in the portion of the sample solution that is in contact with the metal film. It changes greatly, and it becomes possible to analyze the measurement target substance with high sensitivity in a short time. In particular, when detecting substances in a sample using the antigen-antibody reaction, it is not necessary to immobilize the antibody (or antigen) on the sensor, improving the reaction efficiency and enabling detection with extremely high sensitivity. And the reaction time can be shortened. Further, since it is not necessary to immobilize the antibody (or antigen) in advance, the versatility of the sensor is improved.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

    The surface plasmon sensor of the present invention has an optical waveguide formed from a material that transmits excitation light of a predetermined wavelength, a metal film formed on one surface of the optical waveguide, and a film thickness formed on the metal film. An inflexible film in the range of 10 to 100 nm, a light source that generates a light beam, and the light beam is passed through an optical waveguide and incident at an incident angle that generates a surface plasmon with respect to the interface between the optical waveguide and the metal film. A surface plasmon sensor comprising: an optical system for detecting light; and fluorescence detection means for detecting fluorescence generated by excitation by an evanescent wave enhanced by the surface plasmon. It is characterized by having a unit capable of attracting a magnetic substance on the surface.

  FIG. 1 is a schematic side view showing a surface plasmon sensor (hereinafter simply referred to as a sensor) used for carrying out the surface plasmon fluorescence detection method of the present invention. As shown in the figure, this sensor includes a light source 7 such as a semiconductor laser that emits excitation light 8 and an optical waveguide 12 that is made of a material that transmits the excitation light 8 and that is disposed at a position where the excitation light 8 enters from one end face. The metal film 20 made of, for example, gold or silver formed on the one surface 12a of the optical waveguide 12, the inflexible film 31 made of a polymer formed on the metal film 20, and the one surface of the optical waveguide 12. A magnet 41 installed in 12 b, a sample holder 5 that holds the liquid sample 1 so that the inflexible film 31 contacts the inflexible film 31 from the side opposite to the optical waveguide 12, and a sample holder 5 disposed above the sample holder 5. The optical detector (fluorescence detection means) 9 is provided.

  In FIG. 1, the light source 7 is arranged so that the excitation light 8 is incident through the optical waveguide 12 so as to satisfy the total reflection condition toward the interface between the optical waveguide 12 and the metal film 20. That is, the light source 7 itself constitutes an incident optical system that makes the excitation light 8 incident on the optical waveguide 12 as described above. However, the present invention is not limited to this configuration, and there is no problem even if an incident optical system including a lens, a mirror, or the like that makes the excitation light 8 incident as described above is provided separately from the light source 7.

  As the magnet 41, in addition to permanent magnets such as alnico magnets, ferrite magnets, MK steel, KS steel, samarium cobalt magnets and neodymium magnets, electromagnets can also be used.

  Note that phosphor molecules existing in the vicinity of the metal are quenched by energy transfer to the metal. The degree of energy transfer is inversely proportional to the third power of the distance if the metal has a semi-infinite thickness, inversely proportional to the fourth power of the distance if the metal is infinitely thin, and It becomes smaller in inverse proportion to the sixth power. As described in Non-Patent Document 3 described above, in the case of a metal film, it is desirable to secure a distance between the metal and the fluorescent molecule of at least several nm or more, more preferably 10 nm or more. Therefore, the lower limit of the film thickness of the inflexible film 31 is preferably 10 nm.

  On the other hand, the phosphor molecules are excited by evanescent waves that have been enhanced by surface plasmons and ooze out on the surface of the metal film. It is known that the reach of the evanescent wave (distance from the surface of the metal film) is at most about a wavelength, and the electric field strength attenuates exponentially and exponentially according to the distance from the surface of the metal film. Actually, in the near-infrared light having a wavelength of 808 nm, the evanescent wave oozes out at about the wavelength (808 nm), and the electric field strength rapidly attenuates when the wavelength exceeds 100 nm. Since it is desirable that the electric field intensity for exciting the phosphor molecules is larger, it is desirable to make the distance between the metal film surface and the phosphor molecules smaller than 100 nm for effective excitation. Accordingly, the upper limit value of the film thickness of the inflexible film 31 is preferably 100 nm.

  Hereinafter, sample analysis by the surface plasmon sensor having the above-described configuration will be described. The sample liquid 1 containing the analysis target substance 2, the substance 4 that specifically recognizes the analysis target substance 2, and the substance 6 that specifically recognizes the analysis target substance 2 labeled with the phosphor 10 that can be detected by the sensor, Are arranged in the sample holder 5 in contact with the inflexible film. Here, the substance 4 that specifically recognizes the analysis target substance 2 is labeled with a magnetic body 51, and the analysis target substance 2 and the substances 4 and 6 that specifically recognize the analysis target substance 2 by magnetic interaction are included. The complex is concentrated on the sensor surface.

  In the sample analysis, the light beam 8 focused as described above by the action of the cylindrical lens 15 is irradiated toward the metal film 20. The phosphor 10 emits fluorescence when excited by an evanescent wave that has been enhanced by surface plasmons and oozes out on the surface of the metal film. Based on this fluorescence intensity, the substance 2 in the sample liquid 1 can be quantitatively analyzed.

  In addition, as described above, the substance 4 that specifically recognizes the analyte 2 is labeled with the magnetic substance 51, and examples of the magnetic substance used here include magnetic fine particles. From the reach of such an evanescent wave, it is preferably 1 nm to 100 nm.

  As the substance 4 that specifically recognizes the analyte 2, for example, when the analyte 2 is a single-stranded nucleic acid, the single-stranded nucleic acid that complementarily binds to the single-stranded nucleic acid is the analyte 2 In the case of protein or the like, an antibody against the analyte 2 is mentioned.

  As described above, since the substance 4 that specifically recognizes the substance 2 to be analyzed is labeled with the magnetic substance 51 and is not fixed to the sensor, the reaction with the substance to be measured proceeds in the liquid phase. In addition, due to magnetic interaction, the concentration of the analyte 2 is high in the portion of the sample liquid 1 that is in contact with the metal film 20, so that the analyte 2 can be analyzed with high sensitivity within a short time.

  Examples of the present invention and comparative examples will be described below, but the present invention is not limited to these examples.

(Example 1) <Preparation of biotinylated antibody>
Biotinylation of the anti-CRP antibody (Fitzgerald clone No. M701289) was performed using a commercially available kit (Biotin Labeling kit-SH, manufactured by Dojindo Laboratories).

(Example 2) <Preparation of magnetic bead-labeled antibody>
Magnetic beads coated with streptavidin (Ademtech SA, particle size 0.2 μm) and the previously prepared biotinylated anti-CRP antibody were added to a binding buffer (Tris-Cl 5 mM, EDTA 0.1 mM, NaCl 0.1%). The magnetic bead-labeled anti-CRP antibody was prepared by reacting for 2 hours in 5 mM). Recovery and washing with a magnet were repeated to remove unbound antibody, and then blocking was performed using BSA to prevent non-specific adsorption.

(Example 3) <Preparation of fluorescently labeled antibody>
Dissolve in carbonate buffer (pH 9.2) to a final concentration of 0.6 mg / mL IRDye800CW-NHS (LI-COR) and 0.5 mg / mL anti-CRP antibody (Fitzgerald, # 701289) and react at room temperature for 30 minutes I let you. Next, the reaction solution is transferred to a desalting column (PIERCE) equilibrated with PBS (pH7.4), and unreacted IRDye800CW-NHS is removed with PBS (pH7.4), so that IRDye800CW labeled anti-CRP antibody Got.
Absorbance at 280 nm and 780 nm was measured using a spectrophotometer (Ultraspec, GE Healthcare), and the labeling ratio (D / P ratio) of the fluorescent dye per antibody molecule was determined to be 2.4.

(Example 4) <Sensor>
A gold film of 50 nm was formed on the surface of a substrate made of ZEONEX (optical waveguide, refractive index 1.50) by sputtering. Further, a 20 nm of polystyrene-based polymer film (refractive index 1.59) was formed on the gold film by spin coating. A well-like reaction vessel (φ5 mm × 11 mm (H)) was placed on the polymer film of the sensor stick so that the sensor polymer film would be on top, and used for the reaction.

(Example 5) <Detection of CRP>
In the reaction well on the sensor prepared in Example 4, 50 μL of 0.1 μg / mL (1 nM) magnetic bead-labeled antibody solution, 50 μL of 0.5 μg / mL (1 nM) fluorescent dye-labeled antibody solution and 50 μL of a sample containing CRP at the concentration shown in Table 1 was added and incubated at room temperature for 15 minutes to form an antigen-antibody complex. A magnet was contacted from the bottom of the sensor stick, and the antigen-antibody complex contained in the reaction solution was collected on the sensor surface. Fluorescence generated when excitation light is incident at an incident angle that generates surface plasmons with respect to the interface between the optical waveguide and the metal film is measured using an LAS-1000 manufactured by Fujifilm. did. The results are shown in Table 1.

(Comparative Example 1)
50 μL of 10 μg / mL (1 nM) anti-CRP antibody (Fitzgerald clone No. M701289) was added to the reaction well on the sensor prepared in (Example 4), and incubated at room temperature for 2 hours. The antibody solution was removed, washed with a PBS solution containing 0.05% Tween-20, and blocked with PBS containing 1% BSA to prepare a sensor stick on which an anti-CRP antibody was immobilized.

(Comparative Example 2)
To the reaction well on the sensor stick prepared in (Comparative Example 1), 50 μL of a 0.5 μg / mL (1 nM) fluorescent dye-labeled antibody solution and 50 μL of a sample containing CRP at the concentrations shown in Table 1 were added. Incubated for minutes. This was passed through a prism, and excitation light was incident on the interface between the prism and the metal film at an incident angle that generates surface plasmons, and the generated fluorescence was measured using LAS-1000 manufactured by Fujifilm. The results are shown in Table 1.

As shown in Table 1, according to the present invention, since the antigen-antibody reaction can be performed in a liquid layer, the reaction efficiency is improved as compared with the reaction in a solid phase. As a result, the measurement sensitivity can be improved and the reaction time can be shortened. Further, since it is not necessary to immobilize the antibody in advance, versatility can be improved.

The schematic side view which shows the surface plasmon sensor which can implement the fluorescence detection method of this invention Schematic side view showing an example of a conventional fluorescent sensor Schematic side view showing another example of a conventional fluorescent sensor Schematic side view showing still another example of a conventional fluorescent sensor

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Sample 2 Antigen 4 The 1st substance which recognizes specifically a to-be-analyzed substance 5 The sample holding part 6 The 2nd substance to specifically recognize an to-be-analyzed substance 7 Light source 8 Light beam 9 Photodetector 10 Phosphor 12 Light Waveguide 13 Prism 20 Metal film 31 Inflexible film 41 Magnet 51 Magnetic body

Claims (6)

  An optical waveguide; a metal film formed on the optical waveguide; an inflexible film formed on the metal film; a light source that generates a light beam; and passing the light beam through the optical waveguide. An optical system that makes incident light at an incident angle that generates surface plasmon to the interface with the film, and fluorescence detection means that detects fluorescence generated by excitation by an evanescent wave enhanced by the surface plasmon. A surface plasmon sensor comprising a unit capable of attracting a magnetic substance to an optical waveguide surface opposite to an inflexible film.   The surface plasmon fluorescence sensor according to claim 2, wherein the inflexible film has a thickness in a range of 10 to 100 nm. A sample containing a specimen, a first protein (X) that specifically recognizes a measurement target substance (A) labeled with magnetic particles, and a measurement target substance modified with a fluorescent dye that can be detected by the surface plasmon sensor A process of forming a complex of X, A, and Y by simultaneously or stepwise acting a second protein (Y) to be recognized, and collecting the X, A, and Y complex generated in the above process on the sensor surface by magnetic force A method comprising the steps of: measuring the X, A and Y complex using the surface plasmon sensor according to claim 1. 3. The analysis method according to claim 2, wherein the particle size of the magnetic particles is in the range of 10 to 100 nm. 4. The measurement method according to claim 2-3, wherein the protein that specifically recognizes the analyte is an antibody. 5. The measuring method according to claim 2, wherein both or one of the first protein and the second protein that specifically recognizes the analyte is a monoclonal antibody.

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