1. Field of the Invention
The present invention relates to a detection method and a detection system for detecting a target material (i.e., a material to be detected) in a specimen, and in particular, to a detection method and a detection system for detecting the target material by detection of an optical signal by use of an evanescent field or an enhanced optical field.
2. Description of the Related Art
Conventionally, fluorescence detection is widely used as a simple high-sensitivity measurement technique in the field of biomeasurement and the like. The fluorescence detection is a technique for confirming existence of a target material which emits fluorescence when excited by light at a specific wavelength, by irradiating with excitation light having the specific wavelength a specimen which is expected to contain the target material, and detecting the emitted fluorescence. In addition, according to another technique which is widely used in the case where the target material is not a fluorescent material, a material which is labeled with a fluorescent dye and can be specifically bonded to the target material is brought into contact with the specimen, and the existence of the target material or the specific bonding is confirmed by detecting fluorescence in a similar manner to the case where the target material is a fluorescent material.
In the field of biomeasurement, for example, in order to detect an antigen as a target material contained in a specimen, assays such as sandwich ELISA (Enzyme-linked Immunosorbent Assay) or competitive ELISA are performed. According to sandwich ELISA, a primary antibody which can be specifically bonded to the target material is immobilized on a substrate, and a specimen is supplied onto the substrate, so that the target material is specifically bonded to the primary antibody. Subsequently, a secondary antibody which is fluorescence labeled and can be specifically bonded to the target material is added so as to make the secondary antibody bonded to the target material. Thus, the so-called sandwich of the primary antibody, the target material, and the secondary antibody is formed. Then, fluorescence emitted from the fluorescence label of the secondary antibody is detected. According to competitive ELISA, a fluorescent-labeled secondary antibody which can be specifically bonded to a primary antibody in competition with a target material is put in competition with the target material for specific bonding to the primary antibody, and fluorescence emitted from the secondary antibody bonded to the primary antibody is detected.
In order to detect, in sandwich ELISA or competitive ELISA, the fluorescence which is emitted from the secondary antibody bonded through the target material to the primary antibody immobilized on the substrate, or from only the secondary antibody directly bonded to the primary antibody, the evanescent-excited fluorescence technique, in which fluorescence is excited by evanescent light, has been proposed. According to the evanescent-excited fluorescence technique, excitation light which is totally reflected at the front surface of a substrate is injected from the rear surface of the substrate, and fluorescence emitted by excitation by an evanescent wave leaking from the front surface of the substrate is detected.
In addition, techniques of utilizing an effect of plasmon resonance enhancing an electric field in order to increase the sensitivity in the evanescent-excited fluorescence technique have been proposed in the Patent Literature 1, the Non-patent Literature 1, and the like. In the surface plasmon-enhanced fluorescence technique, a metal layer is arranged on a substrate for causing plasmon resonance, and excitation light is injected from the rear surface of the substrate to the interface between the substrate and the metal layer at an angle equal to or greater than the total reflection angle so as to produce surface plasmons in the metal layer. Thus, the fluorescence signal is enhanced by the field enhancing effect of the surface plasmons, so that the S/N is increased.
Further, a technique of enhancing the electric field in a sensor portion by utilizing the field enhancing effect of the optical waveguide mode has been proposed in the Non-patent Literature 2. In the optical waveguide mode-enhanced fluorescence spectroscopy (OWF) proposed in the Non-patent Literature 2, a metal layer and an optical waveguide layer of a dielectric or the like are formed in this order on a substrate, and excitation light is injected from the rear surface of the substrate at an angle equal to or greater than the total reflection angle so as to cause an optical waveguide mode in the optical waveguide layer. Thus, the fluorescence signal is enhanced by the field enhancing effect of the optical waveguide mode.
Furthermore, according to the techniques proposed in the Patent Literature 2 and the Non-patent Literature 3, the fluorescence emitted from the fluorescent label which is excited by the electric field enhanced by surface plasmons is not detected, and instead the surface plasmon-coupled emission (SPCE) caused by surface plasmons newly induced in the metal film by the fluorescence is extracted from the prism side.
As mentioned above, in the field of biomeasurement, various techniques for detecting the target material have been proposed. According to the proposed techniques, the plasmon resonance or the optical waveguide mode is caused by irradiation with excitation light, the fluorescent label is excited by an electric field enhanced by the plasmon resonance or the optical waveguide mode, and the fluorescence is detected directly or indirectly.
The produced evanescent field and the enhanced electric field in the evanescent-excited fluorescence technique are known to rapidly damp with increase in the distance from the surface at which the electric field is produced. FIG. 12 is a graph indicating the dependence of the effect of surface plasmons enhancing an electric field on the distance from the surface (the metal surface) on which the enhanced electric field is produced. Specifically, FIG. 12 indicates a result of a simulation which has been performed for a system in which a solvent (water) exists on a sensor constituted by a prism (of polymethyl methacrylate (PMMA) resin) and a gold film having the thickness of 50 nanometers and being formed on the prism, under the condition that excitation light (having the laser wavelength of 656 nanometers) is injected onto the interface between the prism and the gold film at the incident angle of 72.5 degrees. It can be confirmed, from the graph of FIG. 12, that the degree of enhancement of the electric field is reduced by half at the distance of approximately 100 nanometers. Therefore, it is preferable that the fluorescence label be located close to the surface at which the enhanced electric field is produced.
On the other hand, in the field of biomeasurement, there is a demand for enabling measurement in a shorter time. Therefore, various techniques for efficiently causing reactions on the sensor portion and reducing the measurement time have been proposed. For example, a process being performed in a DNA chip and including a plurality of reaction stages has been proposed in the Patent Literature 3. In the process, a fluid is controlled so as to flow at a flow rate appropriate for each of stages in which the fluid is brought into contact with a functional substrate. Further, a technique for moving fluid in a microchannel at extremely high speed for measurement has been proposed in the Patent Literature 4.    [Patent Literature 1] Japanese Unexamined Patent Publication No. (1998)-307141    [Patent Literature 2] U.S. Patent Application Publication No. 20050053974    [Patent Literature 3] International Patent Publication No. WO2004/104584    [Patent Literature 4] Japanese Unexamined Patent Publication No. 2007-101221    [Non-patent Literature 1] M. M. L. M. Vareiro et al., “Surface Plasmon Fluorescence Measurements of Human Chorionic Gonadotrophin: Role pf Antibody Orientation in Obtaining Enhanced Sensitivity and Limit of Detection”, Analytical Chemistry, Vol. 77, pp. 2426-2431, 2005    [Non-patent Literature 2] K. Tsuboi et al., “High-sensitive sensing of catechol amines using by optical waveguide mode enhanced fluorescence spectroscopy”, Preprints for the Spring Meeting 2007 of the Japan Society of Applied Physics, No. 3, p. 1378, 28p-SA-4    [Non-patent Literature 3] T. Libermann and W. Knoll, “Surface-plasmon field-enhanced fluorescence spectroscopy”, Colloid and Surfaces, Vol. A171, pp. 115-130, 2000
As explained above, in the case where an optical signal emitted from the vicinity of the sensor portion is detected by the evanescent evanescent-excited fluorescence technique, or the optical signal is detected after enhancing the optical field of the evanescent light by the plasmon resonance or the optical waveguide mode, the effect of enhancement by the plasmon resonance or the optical waveguide mode rapidly damps with increase in the distance from the surface of the metal layer or the optical waveguide layer. That is, even when the distance from the above surface to the fluorescent label increases by a small amount, the optical signal greatly damps. Therefore, the signal detection is required to be performed under the condition that the label is located as close as possible to the surface of the sensor portion.
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