Surface plasmon-field enhanced fluorescence spectroscopic measurement method and surface plasmon-field enhanced fluorescence spectroscopic measurement device
To provide a surface plasmon-field enhanced fluorescence spectroscopic measurement method and a surface plasmon-field enhanced fluorescence spectroscopic measurement device which are capable of accurately measuring a fluorescent signal regardless of the type of a light detection means even when the concentration of an analyte is high by adjusting the dynamic range of the SPFS device. A surface plasmon-field enhanced fluorescence stereoscopic measurement method wherein an analyte labeled with a fluorescent substance is excited by surface plasmon light generated by applying excitation light to a metallic thin film, and generated fluorescence is received by a light detection means to thereby detect the analyte. The dynamic range is expanded by adjusting the amount of the fluorescence received by the light detection means.
1. A surface plasmon-field enhanced fluorescence stereoscopic measurement method comprising:
exciting a fluorescence substance that has labeled an analyte by surface plasmon light that has been generated by applying an excitation light to a metallic thin film and receiving the generated fluorescence by a light detection means to thereby detect the analyte;
by comparing a first fluorescence signal that is output in the case in which the light detection means receives a fluorescence under a first condition and a second fluorescence signal that is output in the case in which the light detection means receives a fluorescence in which a light amount has been adjusted under a second condition in which a light amount of a fluorescence that is received by the light detection means is adjusted so as to be smaller than that in the first condition, judging whether or not the first fluorescence signal is abnormal; and
obtaining a normal fluorescence signal by correcting the second fluorescence signal in the case in which it is decided that the first fluorescence signal is abnormal.
2. The surface plasmon-field enhanced fluorescence stereoscopic measurement method as defined in claim 1 , further comprising the step of adjusting a light amount of a fluorescence that is received by the light detection means by adjusting a light amount of the fluorescence that has been generated.
3. The surface plasmon-field enhanced fluorescence stereoscopic measurement method as defined in claim 1 , further comprising the step of adjusting a light amount of a fluorescence that is received by the light detection means by adjusting a light amount of the excitation light.
4. The surface plasmon-field enhanced fluorescence stereoscopic measurement method as defined in claim 1 , further comprising the step of:
receiving a fluorescence by the light detection means while changing an incidence angle of the excitation light to the metallic thin film in a predetermined range; and
judging an abnormal fluorescence signal based on the relationship between an incidence angle of the excitation light and a fluorescence signal that is output in the case in which the light detection means receives a fluorescence.
5. A surface plasmon-field enhanced fluorescence spectroscopic measurement apparatus that is configured to carry out a detection of a specimen material by applying an excitation light, in which a metallic thin film that is formed on a dielectric member, a fine flow passage that is formed on an upper surface of the metallic thin film, and a sensor chip that is provided with a sensor part that is formed in the fine flow passage are mounted, comprising:
a light source that is configured to apply an excitation light to the metallic thin film via the dielectric member; and
a light detection means that is disposed over the sensor chip,
wherein the light detection means is configured to receive a fluorescence that is generated by exciting a fluorescence substance that labels an analyte that is fixed to the sensor part by a surface plasmon light that is generated in the case in which the excitation light is applied to the metallic thin film;
a fluorescence amount adjusting means is configured to be able to adjust a light amount of a fluorescence that is received by the light detection means; and
the fluorescence amount adjusting means is disposed between the sensor chip and the light detection means.
6. The surface plasmon-field enhanced fluorescence spectroscopic measurement apparatus as defined in claim 5 , wherein:
by comparing a first fluorescence signal that is output in the case in which the light detection means receives a fluorescence under a first condition and a second fluorescence signal that is output in the case in which the light detection means receives a fluorescence in which a light amount has been adjusted by the fluorescence amount adjusting means under a second condition in which a light amount of a fluorescence that is received by the light detection means is adjusted so as to be smaller than that in the first condition, it is judged whether or not the first fluorescence signal is abnormal; and
a normal fluorescence signal is obtained by correcting the second fluorescence signal in the case in which it is decided that the first fluorescence signal is abnormal.
7. The surface plasmon-field enhanced fluorescence spectroscopic measurement apparatus as defined in claim 5 , further comprising an application angle adjusting means that is configured to adjust an incidence angle of the excitation light to the metallic thin film.
8. The surface plasmon-field enhanced fluorescence spectroscopic measurement apparatus as defined in claim 5 , wherein:
a fluorescence is received by the light detection means while changing an incidence angle of the excitation light to the metallic thin film in a predetermined range by using the application angle adjusting means; and
an abnormal fluorescence signal is judged based on the relationship between an incidence angle of the excitation light and a fluorescence signal that is output in the case in which the light detection means receives a fluorescence.
9. A surface plasmon-field enhanced fluorescence spectroscopic measurement apparatus that is configured to carry out a detection of a specimen material by applying an excitation light, in which a metallic thin film that is formed on a dielectric member, a fine flow passage that is formed on an upper surface of the metallic thin film, and a sensor chip that is provided with a sensor part that is formed in the fine flow passage are mounted, comprising:
a light source that is configured to apply an excitation light to the metallic thin film via the dielectric member; and
a light detection means that is disposed over the sensor chip,
wherein the light detection means is configured to receive a fluorescence that is generated by exciting a fluorescence substance that labels an analyte that is fixed to the sensor part by a surface plasmon light that is generated in the case in which the excitation light is applied to the metallic thin film;
a fluorescence amount adjusting means is configured to be able to adjust a light amount of a fluorescence that is received by the light detection means; and
the fluorescence amount adjusting means is disposed between the light source and the dielectric member.
10. The surface plasmon-field enhanced fluorescence spectroscopic measurement apparatus as defined in claim 9 , wherein:
by comparing a first fluorescence signal that is output in the case in which the light detection means receives a fluorescence under a first condition and a second fluorescence signal that is output in the case in which the light detection means receives a fluorescence in which a light amount has been adjusted by the fluorescence amount adjusting means under a second condition in which a light amount of a fluorescence that is received by the light detection means is adjusted so as to be smaller than that in the first condition, it is judged whether or not the first fluorescence signal is abnormal; and
a normal fluorescence signal is obtained by correcting the second fluorescence signal in the case in which it is decided that the first fluorescence signal is abnormal.