In metal, free electrons vibrate in a group to generate compression waves called plasma waves. The compression waves generated in a metal surface are quantized into surface plasmon.
There have been proposed various surface plasmon resonance sensors for quantitatively analyzing a material in a sample utilizing a phenomenon that such surface plasmon is excited by light waves. Among those, one employing a system called “Kretschmann configuration” is best known. See, for instance, Japanese Unexamined Patent Publication No. 6(1994)-167443.
The surface plasmon resonance sensor using the Kretschmann configuration basically comprises a dielectric block shaped, for instance, like a prism, a metal film which is formed on one surface of the dielectric block and is brought into contact with a sample, a light source emitting a light beam, an optical system which causes the light beam to enter the dielectric block so that the total internal reflection condition is satisfied at the interface of the dielectric block and the metal film and various angles of incidence of the light beam to the interface of the dielectric block and the metal film including an angle of incidence at which surface plasmon is generated can be obtained, and a photodetector which detects the intensity of the light beam reflected in total internal reflection at the interface and detects the state of surface plasmon resonance.
Various angles of incidence of the light beam to the interface can be obtained in the following two ways.                (1) A relatively thin light beam is deflected to impinge upon the interface at various angles.        (2) A relatively thick light beam is caused to impinge upon the interface in the form of convergent light so that components of the light beam impinge upon the interface at various angles.        
In the former case, the light beam which is reflected from the interface at an angle which varies as the incident light beam is deflected may be detected by a photodetector which is moved in synchronization with deflection of the incident light beam or by an area sensor extending in the direction in which reflected light beam is moved as a result of deflection. In the latter case, an area sensor which extends in a direction in which all the components of light reflected from the interface at various angles can be detected may be used. In Japanese Unexamined Patent Publication No. 1(1989)-138443, there is disclosed an apparatus using the latter way in obtaining various angles of incidence of the light beam to the interface.
In such a surface plasmon resonance sensor, when a light beam impinges upon the interface at a particular angle of incidence θsp not smaller than the angle of total internal reflection, evanescent waves having an electric field distribution in the sample in contact with the metal film are generated and surface plasmon is excited in the interface between the metal film and the sample. When the wave vector of the evanescent waves is equal to the wave number of the surface plasmon and wave number matching is established, the evanescent waves and the surface plasmon resonate and light energy is transferred to the surface plasmon, whereby the intensity of light reflected in total internal reflection at the interface of the dielectric block and the metal film sharply drops. The sharp intensity drop is generally detected as a dark line by the photodetector.
The aforesaid resonance occurs only when the incident light beam is p-polarized. Accordingly, it is necessary to set the light beam to impinge upon the interface in the form of p-polarized light.
When the wave number of the surface plasmon can be known from the angle of incidence θsp at which the phenomenon of attenuation in total internal reflection (ATR) takes place, the dielectric constant of the sample can be obtained. That is,             K      sp        ⁡          (      ω      )        =            ω      c        ⁢                                                      ɛ              m                        ⁡                          (              ω              )                                ⁢                      ɛ            s                                                              ɛ              m                        ⁡                          (              ω              )                                +                      ɛ            s                              wherein Ksp represents the wave number of the surface plasmon, ω represents the angular frequency of the surface plasmon, c represents the speed of light in a vacuum, and εm and εs respectively represent the dielectric constants of the metal and the sample.
When the dielectric constant εs of the sample is known, the concentration of a specific material in the sample can be determined on the basis of a predetermined calibration curve or the like. Accordingly, a specific component in the sample can be quantitatively analyzed by detecting the angle of incidence θsp at which the intensity of light reflected in total internal reflection from the interface of the prism and the metal film sharply drops.
As a similar apparatus utilizing the phenomenon of attenuation in total internal reflection (ATR), there has been known a leaky mode sensor described in, for instance, “Spectrum Researches” Vol.47, No.1 (1998), pp21 to 23 & pp26 and 27. The leaky mode sensor basically comprises a dielectric block shaped, for instance, like a prism, a clad layer which is formed on one face of the dielectric block, an optical waveguide layer which is formed on the clad layer and is brought into contact with a sample, a light source emitting a light beam, an optical system which causes the light beam to enter the dielectric block at various angles of incidence so that total internal reflection conditions are satisfied at the interface of the dielectric block and the clad layer and various angles of incidence of the light beam to the interface of the dielectric block and the clad layer including an angle of incidence at which attenuation in total internal reflection is caused by excitation of an optical waveguide mode at the optical waveguide layer can be obtained, and a photodetector means which detects the intensity of the light beam reflected in total internal reflection at the interface thereby detecting an excited state of the waveguide mode, i.e., attenuation in total internal reflection.
In the leaky mode sensor with this arrangement, when the light beam is caused to impinge upon the clad layer through the dielectric block at an angle not smaller than an angle of total internal reflection, only light having a particular wave number and impinging upon the optical waveguide layer at a particular angle of incidence comes to propagate through the optical waveguide layer in a waveguide mode after passing through the clad layer. When the waveguide mode is thus excited, almost all the incident light is taken in the optical waveguide layer and accordingly, the intensity of light reflected in total internal reflection at the interface of the dielectric block and the clad layer sharply drops. That is, attenuation in total internal reflection occurs. Since the wave number of light to be propagated through the optical waveguide layer in a waveguide mode depends upon the refractive index of the sample on the optical waveguide layer, the refractive index and/or the properties of the sample related to the refractive index can be detected on the basis of the angle of incidence at which the attenuation in total internal reflection occurs.
Also in the leaky mode sensor, various angles of incidence of the light beam to the interface can be obtained in the aforesaid two ways.
The surface plasmon resonance sensor and the leaky mode sensor are sometimes used in random screening for finding a specific material combined with a predetermined sensing material in the field of medicine creation. In this case, a sensing material is fixed on the film layer (the metal film layer in the case of the surface plasmon resonance sensor and the clad layer and the optical waveguide layer in the case of the leaky mode sensor), and a sample liquid containing a material to be analyzed is spotted on the sensing material. Then the angle of incidence θsp at which attenuation in total internal reflection takes place is repeatedly measured each time a predetermined time lapses.
When the sample material (the material to be analyzed in the sample liquid) is combined with the sensing material, the refractive index changes with time due to combination with the sample material. Accordingly, by measuring the angle of incidence θsp at which attenuation in total internal reflection takes place for every predetermined time, thereby detecting states of combination of the sample material with the sensing material, whether the sample material is a specific material to be combined with the sensing material can be known. As combinations of such a specific material and a sensing material, there has been known an antigen and an antibody. For example, there has been known measurement of detecting combination of a sample material with rabbit antihuman IgG antibody (sensing material).
In order to detect the state of combination of the sample material with the sensing material, the total reflection attenuation angle θsp (the angle of incidence θsp at which attenuation in total internal reflection takes place) itself need not necessarily be detected. For example, change in the total reflection attenuation angle θsp after the sample liquid is spotted onto the sensing material is measured and the state of combination of the sample material with the sensing material may be measured on the basis of the change of the total reflection attenuation angle θsp.
In the apparatuses utilizing the phenomenon of attenuation in total internal reflection such as a surface plasmon resonance sensor or a leaky mode sensor which have been put into practice, there has been a problem that a long time is required to measure lots of samples. For example, when each sample is to be subjected to measurement a plurality of times at predetermined time intervals, measurement of a second sample cannot be started until measurement of a first sample is finished, which results in a very long time required to measure all the samples.
In view of the foregoing observations and description, a first object of the present invention is to provide a measuring apparatus utilizing the phenomenon of attenuation in total internal reflection which can measure lots of samples in a short time.
A second object of the present invention is to provide a measuring method utilizing the phenomenon of attenuation in total internal reflection which makes it feasible to measure lots of samples in a short time in the case where each sample is to be subjected to measurement a plurality of times at time intervals.
Further, in conventional surface plasmon resonance sensors, there has been a problem that measurements can greatly fluctuate when light beam in the form of convergent light is caused to enter the dielectric block in the aforesaid way (1) in order to obtain various angles of incidence of the light beam to the interface of the dielectric block and the metal film. For example, the fluctuation in the measurements is detected as fluctuation in the position of the dark line described above.
The similar problem is recognized also in conventional leaky mode sensors when light beam is caused to enter the dielectric block in the aforesaid way (1) in order to obtain various angles of incidence of the light beam to the interface of the dielectric block and the clad layer.
Thus a third object of the present invention is to prevent generation of great fluctuation in the measurements and improve the measuring accuracy in the apparatuses utilizing the phenomenon of attenuation in total internal reflection where the light beam is caused to enter the dielectric block in the form of convergent light.
A fourth object of the present invention is to improve the measuring accuracy in a measuring method utilizing the phenomenon of attenuation in total internal reflection.