WO2015029352A1 - 検出装置 - Google Patents
検出装置 Download PDFInfo
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- WO2015029352A1 WO2015029352A1 PCT/JP2014/004155 JP2014004155W WO2015029352A1 WO 2015029352 A1 WO2015029352 A1 WO 2015029352A1 JP 2014004155 W JP2014004155 W JP 2014004155W WO 2015029352 A1 WO2015029352 A1 WO 2015029352A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2201/00—Features of devices classified in G01N21/00
- G01N2201/06—Illumination; Optics
- G01N2201/061—Sources
- G01N2201/06113—Coherent sources; lasers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2201/00—Features of devices classified in G01N21/00
- G01N2201/06—Illumination; Optics
- G01N2201/068—Optics, miscellaneous
- G01N2201/0683—Brewster plate; polarisation controlling elements
Definitions
- the present invention relates to a detection apparatus for detecting a detection target substance based on surface plasmon fluorescence spectroscopy (Surface Plasmon Fluorescence Spectroscopy: SPFS method).
- SPFS method Surface plasmon fluorescence spectroscopy
- the SPFS method uses a light source that emits laser light as excitation light.
- the laser light is incident on a metal thin film attached to the surface of the prism under total reflection conditions.
- the incident angle of the laser beam to the metal thin film is adjusted so that surface plasmon resonance occurs near the surface of the metal thin film. As a result of surface plasmon resonance, the electric field of evanescent light is enhanced.
- the substance to be detected is labeled with a fluorescent substance.
- the specimen containing the detection target substance is disposed in the vicinity of the surface of the metal thin film.
- the electric field enhanced under surface plasmon resonance can enhance the fluorescence generated from the substance to be detected.
- the SPFS method is a technique for detecting and / or measuring enhanced fluorescence from a detection target substance.
- the incident angle at which the electric field of evanescent light is most enhanced is called the resonance angle.
- the resonance angle depends on the wavelength of the excitation light, the refractive index of the prism, the refractive index of the metal thin film, the thickness of the metal thin film, and the refractive index of the detection target substance existing in the vicinity of the metal thin film.
- the measurer measures the incident angle dependency on the intensity of the reflected light from the metal thin film, the measurer can observe the rapid attenuation of the intensity of the reflected light near the resonance angle. If the measurer measures the incident angle dependence on the intensity of the fluorescence from the substance to be detected, the measurer observes a sudden increase in fluorescence intensity near the resonance angle (referred to as the resonance curve). can do. These phenomena are well known.
- a detector that detects fluorescence from a detection target substance In the SPFS method, a detector that detects fluorescence from a detection target substance is used. In the actual measurement, the detector detects not only the fluorescence derived from the detection target substance but also light (unnecessary light) derived from other factors. Autofluorescence generated inside the optical component when the excitation light passes through the optical component such as a prism is exemplified as a factor that generates unnecessary light. If the excitation light scattered inside the apparatus used in the SPFS method is irradiated to the detection target substance existing at a position away from the surface of the metal thin film, the detection target substance existing at a position away from the surface of the metal thin film. Fluorescence generated from the light becomes unnecessary light.
- Patent Document 1 proposes a technique for reducing the influence of unnecessary light.
- the polarization direction of excitation light is modulated.
- the fluorescence signal output from the detector is synchronously detected using a modulation signal in the polarization direction.
- the above-described technique cannot sufficiently eliminate the influence of the unnecessary light. In this case, the technique described above cannot accurately measure the intensity of fluorescence.
- An object of the present invention is to provide a detection apparatus that can accurately measure the intensity of fluorescence.
- a detection apparatus includes a light source that emits excitation light, a storage unit that stores a specimen, a metal film that receives the excitation light and generates evanescent light that illuminates the specimen, and the metal
- a modulation unit that adjusts an incident angle of the excitation light to the film, a drive unit that generates a drive signal for driving the modulation unit, and an intensity of fluorescence generated from the specimen upon receiving the evanescent light.
- a detection unit that outputs a fluorescence signal; and an extraction unit that extracts a signal component derived from the specimen from the fluorescence signal. The incident angle changes according to the change of the drive signal.
- the extraction unit extracts a synchronization signal component that changes in synchronization with a change in the drive signal as the signal component.
- the above-described detection device can accurately measure the intensity of fluorescence.
- FIG. 4 is a schematic enlarged view of the detection apparatus shown in FIG. 3 (fourth embodiment).
- FIG. 14A It is the schematic of the detection apparatus of patent document 1.
- FIG. 15 It is a graph showing the characteristic of the detection signal which the signal processing part of the detection apparatus shown by FIG. 15 produces
- FIG. 15 is a schematic diagram of the detection apparatus 900 of Patent Document 1. With reference to FIG. 15, the problem which the detection apparatus 900 has is demonstrated.
- the detection apparatus 900 includes a light source 910, a polarization modulator 920, a prism 930, a metal thin film 940, a sample cell 950, a detector 960, a polarization control unit 970, and a signal processing unit 980.
- the light source 910 emits the excitation light PL to the polarization modulator 920.
- the excitation light PL passes through the polarization modulator 920 and enters the prism 930.
- the prism 930 refracts the excitation light PL.
- the metal thin film 940 is formed on the surface of the prism 930.
- the metal thin film 940 is located between the prism 930 and the specimen cell 950.
- the excitation light PL refracted by the prism 930 propagates toward the metal thin film 940.
- the metal thin film 940 totally reflects the excitation light PL.
- the specimen cell 950 accommodates a specimen (not shown).
- the specimen includes a detection target substance (not shown) labeled with a fluorescent substance (not shown).
- fluorescence FL is generated from the detection target substance existing near the metal thin film 940.
- the detector 960 receives the fluorescent light FL and generates a fluorescent signal FLS representing the intensity of the fluorescent light FL.
- the fluorescence signal FLS is output from the detector 960 to the signal processing unit 980.
- the polarization controller 970 generates a polarization modulation signal PMS.
- the polarization modulation signal PMS is output from the polarization control unit 970 to the polarization modulator 920 and the signal processing unit 980.
- the polarization modulation signal PMS may have a sine wave, a triangular wave, or a square wave signal waveform.
- the polarization modulator 920 modulates the polarization direction of the excitation light PL in accordance with the polarization modulation signal PMS.
- the signal processing unit 980 synchronously detects the fluorescence signal FLS using the polarization modulation signal PMS.
- a lock-in amplifier may be used as the signal processing unit 980.
- the polarization modulation signal PMS is used as a reference signal for the lock-in amplifier.
- the fluorescence signal FLS is used as an input signal for the lock-in amplifier.
- a signal component that is signaled in synchronization with the modulation in the polarization direction is selectively extracted from the fluorescence signal FLS.
- the fluorescence signal FLS is a signal component Lf that varies with the frequency f and a signal that does not vary with the frequency f. And a component Ls.
- the signal processing unit 980 selectively extracts the signal component Lf. Since the signal processing unit 980 can remove the signal component Ls, the detection apparatus 900 can measure the intensity of the fluorescence FL without being affected by unnecessary light that is not related to the polarization direction modulation. The amount of the detection target substance contained in the specimen is calculated from the intensity of the fluorescence FL.
- Unnecessary light may contain a component synchronized with the polarization direction modulation. If the transmittance of the prism 930 depends on the polarization direction of the excitation light PL, the autofluorescence generated in the prism 930 is also synchronized with the modulation of the polarization direction of the excitation light PL. Therefore, the signal processing unit 980 cannot appropriately remove signal components derived from autofluorescence generated in the prism 930.
- FIG. 16 is a graph showing the characteristics of the detection signal generated by the signal processing unit 980. With reference to FIGS. 15 and 16, the problems of the prior art will be further described.
- FIG. 16 represents the incident angle of the excitation light PL to the metal thin film 940.
- the vertical axis of the graph in FIG. 16 represents the magnitude of the detection signal.
- the graph of FIG. 16 represents the resonance angle with the symbol “ ⁇ res ”.
- the detection signal peaks.
- the orientations of the prism 930, the metal thin film 940, the specimen cell 950, and the detector 960 are set so that the incident angle coincides with the resonance angle ⁇ res . As a result, a large detection signal is obtained.
- the detection signal shown in FIG. 16 includes an offset component P off .
- the offset component P off is caused by unnecessary light that is synchronized with the modulation in the polarization direction. Therefore, the detection signal cannot accurately represent the amount of the detection target substance in the specimen.
- the present inventors have developed a technique for generating a detection signal from which an offset component has been removed.
- a detection apparatus capable of generating a detection signal that can accurately represent the amount of a detection target substance in a specimen is described.
- FIG. 1 is a schematic block diagram of the detection apparatus 100 of the first embodiment.
- the detection apparatus 100 will be described with reference to FIG.
- the detection apparatus 100 includes a light source 200, an incident angle modulation unit 300, a metal thin film 410, a storage unit 420, a detection unit 510, an extraction unit 520, and a drive unit 530.
- the light source 200 emits the excitation light PL toward the incident angle modulation unit 300.
- the excitation light PL may be laser light.
- Various types of light capable of generating evanescent light EL from the metal thin film 410 can be used as the excitation light PL.
- the principle of this embodiment is not limited to a specific type of light source 200.
- the drive unit 530 generates a drive signal DRS.
- the drive signal DRS is output from the drive unit 530 to the incident angle modulation unit 300 and the extraction unit 520.
- the drive signal DRS may have a sine wave, triangular wave, or square wave signal waveform.
- the drive signal DRS may be a pulse signal.
- the principle of this embodiment is not limited to a specific signal waveform of the drive signal DRS.
- the incident angle modulation unit 300 is driven by the drive signal DRS and changes the incident angle ⁇ of the excitation light PL to the metal thin film 410. Therefore, the incident angle ⁇ changes according to the change of the drive signal DRS.
- the modulation unit is exemplified by the incident angle modulation unit 300.
- the incident angle modulation unit 300 may include a reflection member (not shown) that reflects the excitation light PL, a lens unit (not shown) that refracts the excitation light PL, and other various optical elements.
- the reflecting member may be one galvanometer mirror. Alternatively, the reflecting member may be a plurality of galvanometer mirrors. Further alternatively, the reflecting member may be a polygon mirror, an acousto-optic modulator, an electro-optic modulator, or another optical element that can change the optical path of the excitation light PL in accordance with an electric signal.
- the lens unit may include one lens element. Alternatively, the lens unit may include a plurality of lens elements. The principle of the present embodiment is not limited to a specific structure of the incident angle modulation unit 300.
- the excitation light PL is incident on the metal thin film 410 at an incident angle ⁇ modulated by the incident angle modulation unit 300.
- the metal thin film 410 generates evanescent light EL under irradiation of the excitation light PL.
- the metal thin film 410 may be formed of various metals (for example, Au, Ag, Ag alloy, and Al) that can generate the evanescent light EL.
- the metal thin film 410 is formed to a thickness capable of generating evanescent light EL.
- the metal thin film 410 may have a laminated structure (for example, a laminated thin film of Au and Cr or a laminated thin film of Au and Ti).
- the principle of the present embodiment is not limited to a specific material, a specific size, or a specific structure of the metal thin film 410. In the present embodiment, the metal film is exemplified by the metal thin film 410.
- the accommodation unit 420 accommodates a specimen (not shown) including a detection target substance (not shown).
- the detection target substance may be fluorescently labeled with a fluorescent substance.
- the container 420 is made of a material that is transparent to fluorescence generated from the detection target substance.
- the storage unit 420 may be a general sample cell used for fluorescence detection by the SPFS method. The principle of the present embodiment is not limited to a specific material or a specific structure of the accommodating portion 420.
- the specimen may be various substances that can emit fluorescence FL under irradiation of evanescent light EL.
- the specimen may be a solution containing DNA that has undergone a fluorescent labeling process.
- the detection target substance may be DNA.
- the detection target substance may be a protein such as a biomarker or a chemical substance.
- Various substances that can be disposed near the surface of the metal thin film 410 and emit fluorescence FL under irradiation of the excitation light PL can be used as the detection target substance.
- the principle of the present embodiment is not limited to a specific type of specimen and detection target substance.
- the user who measures the amount of the detection target substance using the detection apparatus 100 can arrange the detection target substance near the surface of the metal thin film 410 using various techniques. Binding techniques such as binding of biotin and streptobiazine, DNA hybridization, and protein antigen-antibody reaction can be suitably used for the arrangement of the detection target substance in the vicinity of the surface of the metal thin film 410. Alternatively, the detection target substance may be suspended near the surface of the metal thin film 410. The principle of the present embodiment is not limited to a specific technique for disposing the detection target substance near the surface of the metal thin film 410.
- the detection target substance may be present in the liquid.
- the detection target substance may be present in the gas. Therefore, the principle of this embodiment is not limited to a specific presence state of the detection target substance.
- Fluorescence FL generated from the specimen under irradiation with the evanescent light EL propagates to the detection unit 510.
- the detection unit 510 generates a fluorescence signal FLS corresponding to the intensity of the fluorescence FL. If the fluorescence FL is strong, the fluorescence signal FLS may show a large value. If the fluorescence FL is weak, the fluorescence signal FLS may show a low value.
- the fluorescence signal FLS is output from the detection unit 510 to the extraction unit 520.
- the detection unit 510 may be a general photomultiplier used for fluorescence detection by the SPFS method. The principle of the present embodiment is not limited to a specific device used as the detection unit 510.
- the extraction unit 520 receives the fluorescence signal FLS from the detection unit 510 and receives the drive signal DRS from the drive unit 530.
- the extraction unit 520 extracts a synchronization signal component that changes in synchronization with the change of the drive signal DRS from the fluorescence signal FLS as a signal component derived from the specimen.
- the extraction unit 520 extracts a signal component that is synchronized with the modulation of the incident angle, and thus the extracted signal component is an optical characteristic (for example, prism) of an optical element used in the detection apparatus 100. It is difficult to be affected by unnecessary light due to the transmittance. Therefore, the detection apparatus 100 can accurately detect the detection target substance.
- the detection apparatus described in relation to the first embodiment can accurately detect a detection target substance under various controls.
- exemplary control of the detection apparatus will be described.
- FIG. 2A is a schematic graph of the drive signal DRS.
- FIG. 2B is a graph conceptually showing the fluorescence signal FLS processed by the extraction unit 520.
- FIG. 2C is a schematic graph of the detection signal DTS output from the extraction unit 520. An exemplary control technique of the detection device 100 will be described with reference to FIGS. 1 to 2C.
- the drive signal DRS may have a sinusoidal waveform. Since the amplitude of the drive signal DRS is substantially constant, the fluctuation range of the incident angle ⁇ is substantially constant.
- the intensity of the evanescent light EL also changes according to the change in the incident angle ⁇ . Therefore, the intensity of the fluorescent light FL emitted from the accommodating portion 420 also changes according to the change in the incident angle ⁇ .
- the fluorescent light FL emitted from the housing part 420 may include unnecessary light. Therefore, detection unit 510 outputs fluorescent signal FLS including a signal component derived from unnecessary light (hereinafter referred to as a noise component).
- the graph in FIG. 2B represents a signal component that changes due to a change in the incident angle ⁇ with a solid line.
- the noise component is conceptually represented as a point in the graph of FIG. 2B.
- the extraction unit 520 refers to the drive signal DRS received from the drive unit 530, and extracts a signal component (solid line) that changes at a frequency that matches the fluctuation frequency of the drive signal DRS from the fluorescence signal FLS as a synchronization signal component. .
- the extraction unit 520 processes other signal components as noise components.
- the graph of FIG. 2B represents the amplitude of the synchronization signal component by the symbol “ ⁇ F”.
- the extraction unit 520 processes the synchronization signal component and calculates the amplitude ⁇ F of the synchronization signal component.
- the extraction unit 520 multiplies the calculated amplitude ⁇ F by a predetermined amplification coefficient K to generate a detection signal DTS. Therefore, the detection signal DTS can represent a magnitude proportional to the amount of change in the fluorescence signal FLS due to the change in the incident angle ⁇ .
- ⁇ Third Embodiment> The designer can design various detection devices based on the design principle described in relation to the first embodiment.
- an exemplary detection device is described.
- FIG. 3 is a schematic diagram of the detection apparatus 100A of the third embodiment.
- the detection apparatus 100A will be described with reference to FIGS.
- the detection apparatus 100A includes a light source 200A, a modulation mechanism 300A, a metal thin film 410A, a specimen cell 420A, a detector 510A, a signal detector 520A, and a modulation signal generator 530A.
- the light source 200A corresponds to the light source 200 described with reference to FIG.
- the modulation mechanism 300A corresponds to the incident angle modulation unit 300 described with reference to FIG.
- the metal thin film 410A corresponds to the metal thin film 410 described with reference to FIG.
- the sample cell 420A corresponds to the storage unit 420 described with reference to FIG.
- the detector 510A corresponds to the detection unit 510 described with reference to FIG.
- the signal detector 520A corresponds to the extraction unit 520 described with reference to FIG.
- the detection apparatus 100A further includes a prism 430 and a calculator 540.
- the light source 200A emits excitation light PL.
- the prism 430 includes a first surface 431 and a second surface 432.
- the excitation light PL propagates through the modulation mechanism 300A and enters the first surface 431.
- the metal thin film 410 ⁇ / b> A may be formed on the second surface 432.
- the metal thin film 410A may be formed on a transparent member prepared separately from the prism 430. In this case, the transparent member is fixed to the second surface 432.
- the principle of the present embodiment is not limited to a specific forming technique of the metal thin film 410A.
- the specimen cell 420A is fixed on the metal thin film 410A.
- the excitation light PL is refracted by the first surface 431 and travels toward the metal thin film 410A on the second surface 432.
- evanescent light is generated from the metal thin film 410A.
- the detection target substance located in the vicinity of the metal thin film 410A generates fluorescence FL in the presence of evanescent light.
- Detector 510A receives fluorescence FL.
- the detector 510A generates a fluorescence signal FLS that represents the intensity of the fluorescence FL.
- the fluorescence signal FLS is output from the detector 510A to the signal detector 520A.
- the modulation signal generator 530A generates a modulation signal MDS.
- Modulation signal MDS is output from modulation signal generator 530A to signal detector 520A and modulation mechanism 300A.
- the modulation signal MDS corresponds to the drive signal DRS described with reference to FIG.
- the signal detector 520A can extract the synchronization signal component from the fluorescence signal FLS with reference to the modulation signal MDS based on the control principle described in relation to the second embodiment.
- the signal detector 520A generates a detection signal DTS using the synchronization signal component.
- the detection signal DTS is output from the signal detector 520A to the calculator 540.
- the calculator 540 calculates the amount of the detection target substance from the detection signal DTS.
- the calculator 540 may store in advance reference data representing the relationship between the magnitude of the detection signal DTS and the amount of the detection target substance.
- the calculator 540 can output the amount of the detection target substance using the detection signal DTS and the reference data.
- the principle of this embodiment is not limited to the specific arithmetic processing executed by the calculator 540.
- Various calculation techniques using the detection signal DTS may be used to calculate the amount of the detection target substance.
- the calculator 540 may be a general computer device. The principle of this embodiment is not limited to a specific device used as the calculator 540.
- the modulation mechanism 300 ⁇ / b> A includes a galvanometer mirror 310 and a lens 320.
- the light source 200 ⁇ / b> A emits the excitation light PL toward the galvanometer mirror 310.
- the galvanometer mirror 310 reflects the excitation light PL to the lens 320.
- the excitation light PL passes through the lens 320 and enters the first surface 431 of the prism 430.
- the reflection unit is exemplified by the galvanometer mirror 310.
- the lens unit is exemplified by the lens 320.
- the galvanometer mirror 310 includes a reflection mirror 311 and a drive motor 312.
- Modulation signal MDS is output from modulation signal generator 530A to drive motor 312.
- the drive motor 312 rotates and reciprocates the reflecting mirror 311 according to the modulation signal MDS.
- the excitation light PL is incident on the reflection mirror 311. Since the reflection direction of the excitation light PL is changed by the reciprocating motion of the reflection mirror 311, the modulation mechanism 300 ⁇ / b> A can define the first optical path FOP and the second optical path SOP.
- the optical path of the excitation light PL changes between the first optical path FOP and the second optical path SOP.
- the lens 320 refracts the excitation light PL.
- the excitation light PL that changes in position between the first optical path FOP and the second optical path SOP can propagate to a predetermined position on the metal thin film 410A.
- the second optical path SOP moves away from the first optical path FOP, while as the excitation light PL approaches the metal thin film 410A from the lens 320, the second optical path SOP becomes , Approaches the first optical path FOP.
- the second optical path SOP matches the first optical path FOP on the metal thin film 410A.
- the optical parameters such as the rotation angle of the are determined.
- the incident angle of the excitation light PL on the metal thin film 410A also changes.
- the intensity of the fluorescence FL emitted from the specimen stored in the specimen cell 420A changes in synchronization with the change in the incident angle.
- the detector 510A receives the fluorescence FL and generates a fluorescence signal FLS indicating the intensity of the fluorescence FL.
- the modulation signal MDS is output not only to the drive motor 312 but also to the signal detector 520A. Therefore, the signal detector 520A can extract the signal component that changes in synchronization with the change in the incident angle of the excitation light PL from the fluorescence signal FLS output from the detector 510A with reference to the modulation signal MDS.
- the signal detector 520A generates a detection signal DTS that represents the amplitude of the signal component that changes in synchronization with the change in the incident angle of the excitation light PL.
- the detection signal DTS is output to the calculator 540.
- the conversion technique from the fluorescence signal FLS to the detection signal DTS may depend on the principle of the second embodiment.
- the detection device described in connection with the third embodiment may operate under various optical settings.
- the user using the detection device may determine the optical settings of the detection device so that the signal detector outputs a large value detection signal. If the detection signal has a large value, the calculator can accurately calculate the amount of the detection target substance.
- an exemplary optical setting of the detection device is described.
- FIG. 4 is a schematic enlarged view of the detection apparatus 100A around the prism 430.
- FIG. The optical setting of the detection apparatus 100A will be described with reference to FIGS.
- FIG. 4 shows the first incident angle ⁇ 1 ( ⁇ 1 > 0) and the second incident angle ⁇ 2 ( ⁇ 2 > 0).
- Excitation light PL propagating along the first optical path FOP is incident on the metal thin film 410A in the first incidence angle theta 1.
- Excitation light PL propagating along the second optical path SOP is incident on the metal thin film 410A at second incident angle theta 2.
- the second incident angle ⁇ 2 is set to a value different from the first incident angle ⁇ 1 .
- the second incident angle ⁇ 2 is larger than the first incident angle ⁇ 1 .
- the second incident angle ⁇ 2 may be smaller than the first incident angle ⁇ 1 .
- the principle of this embodiment is not limited by the magnitude relationship between the first incident angle ⁇ 1 and the second incident angle ⁇ 2 .
- FIG. 4 shows the modulation amplitude ⁇ of the incident angle.
- the modulation amplitude ⁇ is defined by the rotation angle of the galvanometer mirror 310.
- the modulation amplitude ⁇ may be expressed by the following mathematical formula.
- Figure 4 shows mean incidence angles theta av of the excitation light PL with respect to the metal thin film 410A.
- the average incident angle ⁇ av may be considered as a reference incident angle when the modulation amplitude ⁇ is set to “0”.
- the average incident angle ⁇ av may be expressed by the following mathematical formula.
- the fluorescence FL from the detection target substance is generated by surface plasmon resonance.
- the intensity of the fluorescence FL increases rapidly near the resonance angle. Therefore, if the user sets the average incident angle ⁇ av close to the resonance angle, the intensity of the fluorescence FL from the detection target substance changes sensitively corresponding to the modulation of the incident angle. In this case, the detection signal DTS can have a large value.
- FIG. 5A is a graph schematically showing the relationship between the incident angle and the magnitude of the fluorescence signal FLS.
- the optical setting of the detection apparatus 100A will be further described with reference to FIGS. 3 to 5A.
- the horizontal axis of the graph in FIG. 5A indicates the incident angle.
- the vertical axis of the graph in FIG. 5A indicates the magnitude of the fluorescence signal FLS.
- a user using the detection apparatus 100A may rotate the metal thin film 410A, the specimen cell 420A, the prism 430, and the detector 510A integrally while stopping the modulation mechanism 300A. As a result, since the incident angle to the metal thin film 410A changes, the user can obtain the graph shown in FIG. 5A.
- the user can determine the incident angle at which the fluorescence signal FLS peaks from the graph of FIG. 5A.
- the incident angle at which the fluorescence signal FLS reaches a peak is the resonance angle ⁇ res .
- the fluorescence signal FLS includes a signal component F off that is unrelated to the change in the incident angle.
- the detection apparatus 100A can appropriately remove the signal component F off using a synchronous detection technique between the fluorescence signal FLS and the modulation signal MDS.
- the user may set the variation range of the incident angle in the range of the incident angle where the change rate of the fluorescence signal FLS is high in the graph of FIG. 5A.
- the magnitude of the fluorescence signal FLS changes by “ ⁇ F” as shown in FIG. 5A.
- the signal detector 520A can selectively extract a signal component that changes by “ ⁇ F” from the fluorescence signal FLS using a synchronous detection technique between the fluorescence signal FLS and the modulation signal MDS.
- the user may set the magnitude of the modulation amplitude ⁇ so that a change “ ⁇ F” of the signal component that provides sufficient detection accuracy with respect to the amount of the measurement target substance occurs.
- FIG. 5B is a graph schematically showing a relationship between the average incident angle ⁇ av and the detection signal DTS.
- the optical setting of the detection apparatus 100A will be further described with reference to FIGS. 3 to 5B.
- the horizontal axis of the graph in FIG. 5B indicates the average incident angle ⁇ av .
- the vertical axis of the graph in FIG. 5B indicates the magnitude of the detection signal DTS.
- a user using the detection device 100A may set the modulation amplitude ⁇ to a predetermined value and operate the modulation mechanism 300A. During this time, the user may integrally rotate the metal thin film 410A, the sample cell 420A, the prism 430, and the detector 510A to change the value of the average incident angle ⁇ av . As a result, since the average incident angle ⁇ av on the metal thin film 410A changes, the user can obtain the graph shown in FIG. 5B.
- the magnitude of the detection signal DTS is substantially proportional to “ ⁇ F” described with reference to FIG. 5A.
- the value of the detection signal DTS is “0”. become.
- Figure 5B shows the average angle of incidence values of the detection signal DTS is maximum at the symbol "theta max".
- FIG. 5B shows the average incident angle at which the value of the detection signal DTS is a minimum value by the symbol “ ⁇ min ”. From the graph of FIG. 5B, if the average incident angle ⁇ av is set to “ ⁇ max ” or “ ⁇ min ”, the absolute value of the detection signal DTS is maximized (ie, the incident angle is the average incident angle). As the value approaches ⁇ av , the value of the detection signal approaches the peak value).
- the user may set the average incident angle ⁇ av to “ ⁇ max ” or “ ⁇ min ”.
- FIG. 5B shows the magnitude of the detection signal DTS when the average incident angle ⁇ av is set to “ ⁇ max ” or “ ⁇ min ” as “ ⁇ P n1 ”.
- the calculator 540 converts the amount of the detection target substance into the detection signal DTS. It may be determined as a proportional value.
- the signal component F off is the magnitude of the detection signal DTS. It does not affect “ ⁇ P n1 ”. Therefore, the detection apparatus 100A can accurately determine the amount of the detection target substance.
- FIG. 5C is a graph showing calibration curve data stored in the calculator 540.
- a technique for calculating the amount of the detection target substance will be described with reference to FIGS. 3 to 5C.
- the term “amount of the detection target substance” may mean the concentration of the detection target substance in the sample. Alternatively, the term “amount of the detection target substance” may mean the number of detection target substances in the specimen. The principle of this embodiment is not limited to a specific definition of “amount of detection target substance”.
- the user prepares a specimen containing a known amount of the detection target substance. Thereafter, the user accommodates the prepared specimen in the specimen cell 420A. The user then causes the excitation light PL to be incident on the metal thin film 410A under the optical conditions of the average incident angle ⁇ av and the modulation amplitude ⁇ described with reference to FIGS. 5A and 5B. As a result, the signal detector 520A can generate the detection signal DTS. As a result, the user can find out the magnitude of the detection signal DTS corresponding to the amount of the detection target substance.
- the user prepares a plurality of samples different in the amount of the detection target substance, and investigates the magnitude of the detection signal DTS corresponding to each of these samples.
- the calibration curve shown in FIG. 5C is obtained.
- the calculator 540 may store the obtained calibration curve data as a mathematical function. Alternatively, the calculator 540 may store the obtained calibration curve data as a lookup table.
- the principle of the present embodiment is not limited to a specific storage format of calibration curve data.
- the calculator 540 refers to the calibration curve data, and the amount “D p of the detection target substance corresponding to the magnitude“ P p ”of the detection signal DTS. Can be calculated.
- the calibration curve shown in FIG. 5C shows a proportional relationship between the magnitude of the detection signal DTS and the amount of the detection target substance.
- the magnitude of the detection signal DTS may not be proportional to the amount of the detection target substance.
- the magnitude of the detection signal DTS may not be proportional to the amount of the detection target substance due to the saturation characteristics of the detector 510A.
- the calculator 540 can accurately determine the amount of the detection target substance based on the calibration curve data creation technique described above. Therefore, the principle of the present embodiment is not limited to specific calibration curve data.
- the detection device described in connection with the third embodiment uses a single lens element to adjust the optical path.
- the detection device may have a plurality of lens elements. If a plurality of lens elements are used, each of the plurality of lens elements may not have an excessively high refractive index.
- a detection apparatus including a plurality of lens elements is described.
- FIG. 6 is a schematic diagram of the detection apparatus 100B of the fifth embodiment.
- the detection device 100B will be described with reference to FIGS. 1 and 6.
- symbol used in common between 3rd Embodiment and 5th Embodiment means that the element to which the said common code
- the detection apparatus 100B includes a light source 200A, a metal thin film 410A, a specimen cell 420A, a prism 430, a detector 510A, a signal detector 520A, and a modulation signal generator 530A. 540. The description of the third embodiment is incorporated for these elements.
- the detection apparatus 100B further includes a modulation mechanism 300B.
- the modulation mechanism 300B corresponds to the incident angle modulation unit 300 described with reference to FIG.
- the modulation mechanism 300B includes a galvanometer mirror 310.
- the description of the third embodiment is applied to the galvanometer mirror 310.
- the modulation mechanism 300B further includes a first lens 321 and a second lens 322.
- the first lens 321 is disposed between the galvanometer mirror 310 and the second lens 322.
- the second lens 322 is disposed between the first lens 321 and the prism 430.
- the light source 200A emits excitation light PL to the galvanometer mirror 310.
- the galvanometer mirror 310 reflects the excitation light PL toward the first lens 321.
- the excitation light PL sequentially passes through the first lens 321 and the second lens 322 and enters the first surface 431 of the prism 430.
- the galvanometer mirror 310 changes the reflection direction of the excitation light PL in accordance with the modulation signal MDS. Therefore, the modulation mechanism 300B can define the first optical path FOP and the second optical path SOP. The optical path of the excitation light PL changes in position between the first optical path FOP and the second optical path SOP.
- the second optical path SOP is substantially parallel to the first optical path FOP. Therefore, the angle difference between the first optical path FOP and the second optical path SOP in the propagation section from the first lens 321 to the second lens 322 is the first optical path FOP in the propagation section from the galvanometer mirror 310 to the first lens 321. And the angle difference between the second optical path SOP and the second optical path SOP.
- the angular difference between the first optical path FOP and the second optical path SOP in the propagation section from the first lens 321 to the second lens 322 is the same as the first optical path FOP and the second optical path in the propagation section from the second lens 322 to the prism 430.
- the first lens 321 and the second lens 322 may not have an excessively high refractive index.
- the designer who designs the detection device 100B so that the second optical path SOP matches the first optical path FOP on the metal thin film 410A is refracted by the first lens 321 and the second lens 322.
- the optical parameters such as the ratio, the positional relationship between the first lens 321, the second lens 322, and the galvanometer mirror 310 and the rotation angle of the reflection mirror 311 are determined.
- the incident angle of the excitation light PL on the metal thin film 410A also changes.
- the intensity of the fluorescence FL emitted from the specimen stored in the specimen cell 420A changes in synchronization with the change in the incident angle.
- the detector 510A receives the fluorescence FL and generates a fluorescence signal FLS indicating the intensity of the fluorescence FL.
- the modulation signal MDS is output not only to the drive motor 312 but also to the signal detector 520A. Therefore, the signal detector 520A can extract the signal component that changes in synchronization with the change in the incident angle of the excitation light PL from the fluorescence signal FLS from the detector 510A with reference to the modulation signal MDS.
- the signal detector 520A generates a detection signal DTS that represents the amplitude of the signal component that changes in synchronization with the change in the incident angle of the excitation light PL.
- the detection signal DTS is output to the calculator 540.
- the conversion technique from the fluorescence signal FLS to the detection signal DTS may depend on the principle of the second embodiment and / or the fourth embodiment.
- the technique for determining the amount of the detection target substance from the detection signal DTS may depend on the principle of the fourth embodiment.
- the detection device described in connection with the third embodiment uses a single lens element to adjust the optical path.
- the designer may design the detection device without using lens elements.
- a detection device designed without using a lens element will be described.
- FIG. 7 is a schematic diagram of a detection apparatus 100C according to the sixth embodiment.
- the detection apparatus 100C will be described with reference to FIGS.
- symbol used in common between 3rd Embodiment and 6th Embodiment means that the element to which the said common code
- the detection apparatus 100C includes a light source 200A, a metal thin film 410A, a specimen cell 420A, a prism 430, a detector 510A, a signal detector 520A, and a calculator 540.
- the description of the third embodiment is incorporated for these elements.
- the detection apparatus 100C further includes a modulation mechanism 300C and a modulation signal generator 530C.
- the modulation mechanism 300C corresponds to the incident angle modulation unit 300 described with reference to FIG.
- the modulation signal generator 530C corresponds to the driving unit 530 described with reference to FIG.
- the modulation mechanism 300C includes a first galvanometer mirror 330, a second galvanometer mirror 340, and a signal conditioner 350.
- Modulation signal generator 530C outputs modulation signal MDS to first galvanometer mirror 330, signal conditioner 350, and signal detector 520A.
- the first galvanometer mirror 330 includes a reflection mirror 331 and a drive motor 332.
- Second galvanometer mirror 340 includes a reflection mirror 341 and a drive motor 342.
- the signal conditioner 350 adjusts the amplitude and / or phase of the modulation signal MDS to generate an adjustment signal AJS.
- the modulation signal MDS is output from the modulation signal generator 530C to the drive motor 332 of the first galvanometer mirror 330.
- the drive motor 332 rotates and reciprocates the reflecting mirror 331 according to the modulation signal MDS.
- the excitation light PL is incident on the reflection mirror 331 from the light source 200A.
- the reflection mirror 331 reflects the excitation light PL toward the second galvanometer mirror 340.
- the first reflection mirror is exemplified by the first galvanometer mirror 330.
- the adjustment signal AJS is output from the signal adjuster 350 to the drive motor 342 of the second galvanometer mirror 340.
- the drive motor 342 is rotated and reciprocated on the reflection mirror 341 in accordance with the adjustment signal AJS.
- the excitation light PL reflected by the first galvanometer mirror 330 is incident on the reflection mirror 341 of the second galvanometer mirror 340.
- the reflection mirror 341 of the second galvanometer mirror 340 reflects the excitation light PL toward the first surface 431 of the prism 430.
- the second reflecting mirror is exemplified by the second galvanometer mirror 340.
- the modulation mechanism 300C can define the first optical path FOP and the second optical path SOP.
- the incident angle of the excitation light PL to the metal thin film 410A is, when set to a first angle of incidence theta 1, first galvanometer mirror 330 and the second galvanometer mirror 340 cooperate to define a first optical path FOP .
- the incident angle of the excitation light PL to the metal thin film 410A is, when set to the second incident angle theta 2, the first galvanometer mirror 330 and the second galvanometer mirror 340 cooperate to define a second optical path SOP .
- the optical path of the excitation light PL changes in position between the first optical path FOP and the second optical path SOP.
- the second optical path SOP moves away from the first optical path FOP.
- the second optical path SOP approaches the first optical path FOP.
- the second optical path SOP coincides with the first optical path FOP on the metal thin film 410A.
- the signal conditioner 350 adjusts the amplitude and / or phase of the modulation signal MDS so that the irradiation position of the excitation light PL on the metal thin film 410A is stabilized.
- the optical path of the excitation light PL Since the changes between the first optical path FOP and second optical path SOP, the incident angle is also first incident angle theta 1 and the second incident angle theta 2 of the excitation light PL to the metal thin film 410A Vary between.
- the intensity of the fluorescence FL emitted from the specimen stored in the specimen cell 420A changes in synchronization with the change in the incident angle.
- the detector 510A receives the fluorescence FL and generates a fluorescence signal FLS indicating the intensity of the fluorescence FL.
- the modulation signal MDS is output to the signal detector 520A. Therefore, the signal detector 520A can extract the signal component that changes in synchronization with the change in the incident angle of the excitation light PL from the fluorescence signal FLS from the detector 510A with reference to the modulation signal MDS.
- the signal detector 520A generates a detection signal DTS that represents the amplitude of the signal component that changes in synchronization with the change in the incident angle of the excitation light PL.
- the detection signal DTS is output to the calculator 540.
- the conversion technique from the fluorescence signal FLS to the detection signal DTS may depend on the principle of the second embodiment and / or the fourth embodiment.
- the technique for determining the amount of the detection target substance from the detection signal DTS may depend on the principle of the fourth embodiment.
- the detection apparatus may include an element for adjusting the center value of the incident angle variation range.
- a detection device capable of adjusting the center value of the fluctuation range of the incident angle will be described.
- FIG. 8 is a schematic block diagram of the detection apparatus 100D of the seventh embodiment.
- the detection apparatus 100D will be described with reference to FIGS. 3, 5A, 5B, and 8.
- FIG. The reference numerals used in common between the first embodiment, the second embodiment, and the seventh embodiment are the same as those in the first embodiment and / or the second embodiment. It means having a function. Therefore, description of 1st Embodiment and / or 2nd Embodiment is used for these elements.
- the detection device 100D includes the light source 200, the incident angle modulation unit 300, the metal thin film 410, the storage unit 420, the detection unit 510, the extraction unit 520, and the drive unit 530. Prepare. The description of the first embodiment is incorporated in these elements.
- the detection apparatus 100D further includes a calculation unit 540D and an adjustment unit 600.
- the calculation unit 540D corresponds to the calculator 540 described with reference to FIG.
- the adjustment unit 600 adjusts the average incident angle ⁇ av of the excitation light PL that is incident on the metal thin film 410.
- the center value of the incident angle variation range is exemplified by the average incident angle ⁇ av .
- the adjustment unit 600 may move the metal thin film 410, the storage unit 420, and the detection unit 510 integrally to set the average incident angle ⁇ av to an appropriate value.
- the adjustment unit 600 may set the average incident angle ⁇ av to an appropriate value using the drive signal DRS output from the drive unit 530 to the incident angle modulation unit 300.
- the principle of this embodiment is not limited to a specific technique for adjusting the average incident angle ⁇ av .
- the adjustment unit 600 may determine an appropriate value of the average incident angle ⁇ av with reference to the fluorescence signal FLS output from the detection unit 510. As described with reference to FIG. 5A, the incident angle at which the rate of change in the magnitude of the fluorescence signal FLS increases may be set as the average incident angle ⁇ av . Alternatively, the adjustment unit 600 may determine an appropriate value of the average incident angle ⁇ av with reference to the detection signal DTS output from the extraction unit 520. As described with reference to FIG. 5B, “ ⁇ max ” or “ ⁇ min ” may be set as the average incident angle ⁇ av . The principle of the present embodiment is not limited to specific feedback control for the adjustment unit 600.
- the average incident angle may be adjusted mechanically.
- a detection device capable of mechanically adjusting the average incident angle will be described.
- FIGS. 9A and 9B are schematic views of the detection apparatus 100E according to the eighth embodiment.
- the detection apparatus 100E will be described with reference to FIGS. 5B and 8 to 9B.
- the code used in common between the third embodiment and the eighth embodiment means that the element to which the common code is attached has the same function as that of the third embodiment. Therefore, description of 3rd Embodiment is used for these elements.
- the detection apparatus 100E includes a light source 200A, a modulation mechanism 300A, a metal thin film 410A, a specimen cell 420A, a prism 430, a detector 510A, a signal detector 520A, and a modulation signal generation.
- a calculator 530A and a calculator 540 The description of the third embodiment is incorporated in these elements.
- the detection device 100E further includes an adjustment mechanism 600E.
- the adjustment mechanism 600E corresponds to the adjustment unit 600 described with reference to FIG.
- the adjustment mechanism 600E includes a holding plate 610 and a drive motor 620.
- the metal thin film 410A, the specimen cell 420A, the prism 430, and the detector 510A are fixed to the holding plate 610.
- the drive motor 620 rotates the holding plate 610.
- the rotation center of the holding plate 610 may coincide with the intersection of the first optical path FOP and the second optical path SOP on the metal thin film 410A.
- the holding unit is exemplified by the holding plate 610.
- the rotating unit is exemplified by a drive motor 620.
- the average incident angle ⁇ av is set to the incident angle ⁇ a .
- the mean incidence angle theta av is set to a small angle of incidence theta b than the incident angle theta a.
- the detection apparatus 100E can easily change the value of the average incident angle ⁇ av using the adjustment mechanism 600E.
- the resonance angle ⁇ res may change. If the user sets the average incident angle ⁇ av to the incident angle ⁇ a and the resonance angle ⁇ res changes while measuring the amount of the object to be detected, the user operates the adjustment mechanism 600E. The data shown in FIG. 5B may be acquired. If the incident angle ⁇ b corresponds to “ ⁇ max ” or “ ⁇ min ” shown in FIG. 5B, the user may reset the incident angle ⁇ b as the average incident angle ⁇ av . Thereafter, the user can continue to measure the amount of the detection target substance.
- 10A to 10D are graphs showing detection signals calculated based on the experimental results. An experiment conducted by the present inventors will be described with reference to FIGS. 9A to 10D.
- the inventors used a He—Ne laser as the light source 200A.
- the output of the He—Ne laser was 0.1 mW.
- the He—Ne laser emitted laser light having a wavelength of 633 nm.
- the laser beam was used as the excitation light PL.
- the laser light has p-polarized light.
- the present inventors used a prism element made of SF11 glass material as the prism 430.
- the prism element has a regular triangular prism shape.
- the present inventors used an Au thin film formed by sputtering on an SF11 glass substrate as the metal thin film 410A.
- the present inventors fixed the SF11 glass substrate to the prism element using a refractive index matching liquid after forming the Au thin film.
- the thickness of the Au thin film was 45 nm.
- the present inventors spin-coated a polystyrene film on the Au thin film in order to prevent quenching of the fluorescent FL.
- the thickness of the polystyrene film was 20 nm.
- the present inventors used a small container made of quartz glass as the specimen cell 420A.
- the present inventors sealed the gap between the container and the Au thin film using a rubber ring. As a result, liquid leakage from between the container and the Au thin film was appropriately prevented.
- Two holes were formed in the container.
- the inventors of the present invention supplied liquid to the container and discharged liquid from the container through these holes.
- the inventors used a peristaltic pump (not shown) for supplying and discharging liquid.
- the present inventors used a photomultiplier as the detector 510A.
- the inventors of the present invention arranged a band pass filter between the above-described container and the photomultiplier.
- the inventors blocked the laser beam propagating from the container to the photomultiplier using a bandpass filter. Therefore, the fluorescence FL emitted from the detection target substance in the container selectively entered the photomultiplier.
- the inventors measured the pulse generation frequency of photon pulses detected by the photomultiplier using a frequency counter.
- the present inventors supplied a carbonate buffer solution of biotinylated BSA (bovine serum albumin) to the aforementioned container. As a result, biotinylated BSA was fixed on the above polystyrene film.
- biotinylated BSA bovine serum albumin
- the inventors then supplied a PBS buffer (phosphate buffered saline) solution of streptavidin to the above-described container. As a result, streptavidin bound to biotinylated BSA.
- PBS buffer phosphate buffered saline
- the present inventors used biotinylated DNA labeled with Dy647 fluorescent agent as a substance to be detected.
- the concentration of biotinylated DNA was 10 nM.
- the inventors of the present invention operated the adjustment mechanism 600E with the galvanometer mirror 310 stopped, and measured the magnitude of the fluorescence signal FLS.
- the holding plate 610 is 0.5 deg.
- the inventors plotted the measured fluorescence intensity on a graph and created a resonance curve.
- the incident angle is 58 deg.
- the resonance curve represented the maximum value.
- the full width at half maximum w of the resonance curve is 3.3 deg. Met.
- the resonance curve approximated the Gaussian curve in shape.
- the present inventors calculated the waveform of the detection signal DTS under the modulation of the incident angle based on the result obtained from the measurement of the incident angle dependency.
- the present inventors approximated the Gaussian curve to the resonance curve.
- the relationship between the full width at half maximum of the Gaussian curve and the standard deviation ⁇ of the Gaussian curve is expressed by the following equation.
- a resonance curve I ( ⁇ ) approximated by a Gaussian curve is represented by the following mathematical formula.
- the symbol “ ⁇ ” represents an incident angle.
- the symbol “ ⁇ res ” represents the resonance angle.
- a time-varying waveform m (t, ⁇ av ) of the incident angle is expressed by the following mathematical formula.
- the symbol “ ⁇ ” represents the modulation amplitude of the incident angle.
- the symbol “ ⁇ av ” represents the average incident angle.
- the symbol “f” represents the modulation frequency.
- the symbol “t” represents time.
- the present inventors assign the time-varying waveform m (t, ⁇ av ) of the incident angle to the incident angle ⁇ of the resonance curve I ( ⁇ ), and the time-varying waveform s (of the fluorescence signal FLS expressed by the following equation: t, ⁇ av ) was obtained.
- the magnitude P n ( ⁇ av ) of the detection signal corresponds to a value obtained by integrating “s (t, ⁇ av ) ⁇ m (t, ⁇ av )” over one modulation period ( reference).
- 10A to 10D are graphs showing the magnitude P n ( ⁇ av ) of the detection signal obtained when the resonance angle ⁇ av is “58 deg.”.
- the full width at half maximum w of the resonance curve is obtained under the condition of “1 deg.”.
- the full width at half maximum w of the resonance curve is obtained under the condition of “2 deg.”.
- the full width at half maximum w of the resonance curve is obtained under the condition of “4 deg.”.
- the full width at half maximum w of the resonance curve is obtained under the condition of “8 deg.”.
- Each of the graphs in FIGS. 10A to 10D represents a plurality of curves.
- subjected on each of several curves represents the value of modulation
- the actual incident angle may deviate from the set value determined for the incident angle.
- An error between the actual incident angle and the set value causes an error in the calculation of the amount of the detection target substance.
- a technique for reducing the influence of the error between the actual incident angle and the set value on the calculation of the amount of the detection target substance will be described.
- FIG. 11A and FIG. 11B are schematic views of the detection apparatus 100F of the tenth embodiment.
- the detection apparatus 100F will be described with reference to FIGS. 11A and 11B.
- a symbol used in common between the eighth embodiment and the tenth embodiment means that an element to which the common symbol is attached has the same function as that of the eighth embodiment. Therefore, description of 8th Embodiment is used for these elements.
- the detection apparatus 100F includes a light source 200A, a modulation mechanism 300A, a metal thin film 410A, a specimen cell 420A, a prism 430, a detector 510A, a signal detector 520A, and a modulation signal generation. 530A and adjustment mechanism 600E. The description of the eighth embodiment is incorporated in these elements.
- the detection apparatus 100F further includes a calculator 540F.
- Calculator 540F includes a calculation unit 541 and a determination unit 542.
- the detection signal DTS is output from the signal detector 520A to the calculation unit 541.
- the calculation unit 541 performs an integration calculation using the detection signal DTS.
- the result of the integral calculation is output from the calculation unit 541 to the determination unit 542.
- the determination unit 542 determines the amount of the detection target substance based on the result of the integration calculation.
- the user can operate both the drive motors 312 and 620 to measure the amount of the detection target substance.
- the user can operate the drive motor 620 to change the average incident angle from the average incident angle ⁇ 0 sufficiently away from the resonance angle ⁇ res to a predetermined average incident angle ⁇ av .
- the value of the detection signal DTS is substantially “0”. If the value of the detection signal DTS at the average incident angle ⁇ av is represented by the symbol “P n ”, the integral value P i calculated by the calculation unit 541 is represented by the following mathematical formula.
- the determination unit 542 may store in advance calibration curve data representing the relationship between the integral value Pi and the amount of the detection target substance.
- the determination unit 542 can determine the amount of the detection target substance by comparing the integral value Pi with the calibration curve data.
- the technique for creating calibration curve data may conform to the method described in relation to the fourth embodiment. The principle of this embodiment is not limited to a specific technique for creating calibration curve data.
- FIG. 12 is a graph obtained from the above formula. The advantageous features of the principle of this embodiment over the prior art will be described with reference to FIGS.
- the horizontal axis of the graph in FIG. 12 represents the average incident angle.
- the vertical axis of the graph in FIG. 12 indicates the integral value represented by the above mathematical formula. Similar to the graph of FIG. 16, the peak of the integral value appears at the resonance angle ⁇ res .
- the full width at half maximum w of the curve in the graph of FIG. 16 is the magnitude of the detection signal having a value obtained by adding the offset component P off to the half value of the difference between the maximum value P p2 of the detection signal and the offset component P off .
- Position ie, ((P p2 ⁇ P off ) / 2 + P off )).
- 13A to 13D are graphs showing detection signals calculated based on experimental results. With reference to FIGS. 11A, 11B, and 13A to 13D, a technique for appropriately setting the modulation range of the incident angle will be described.
- the following formula represents the integrated value P i ( ⁇ av ) of the magnitude P n ( ⁇ av ) of the detection signal described in the context of the seventh embodiment.
- 13A to 13D are graphs showing the integrated value P i ( ⁇ av ) obtained when the resonance angle ⁇ av is “58 deg.”.
- the full width at half maximum w of the fluorescence signal FLS is obtained under the condition of “1 deg.”.
- the full width at half maximum w of the fluorescence signal FLS is obtained under the condition of “2 deg.”.
- the full width at half maximum w of the fluorescence signal FLS is obtained under the condition of “4 deg.”.
- the full width at half maximum w of the fluorescence signal FLS is obtained under the condition of “8 deg.”.
- Each of the graphs in FIGS. 13A to 13D represents a plurality of curves.
- subjected on each of several curves represents the value of modulation
- a large integrated value P i ( ⁇ av ) means that the fluorescence intensity is measured with high sensitivity. From the graphs of FIGS. 13A to 13D, it can be seen that there is a preferable range of the modulation amplitude ⁇ for obtaining a large detection signal.
- ⁇ Twelfth embodiment> The detection device described in connection with the eighth embodiment mechanically adjusts the average incident angle. Alternatively, the average incident angle may be adjusted electrically. In this case, the mechanical structure of the detection device is simpler than the detection device described in connection with the eighth embodiment. In the twelfth embodiment, a detection apparatus capable of electrically adjusting the average incident angle will be described.
- FIGS. 8, 14A, and 14B are schematic views of a detection device 100G according to the twelfth embodiment.
- the detection apparatus 100G will be described with reference to FIGS. 8, 14A, and 14B.
- symbol used in common between 3rd Embodiment and 12th Embodiment means that the element to which the said common code
- the detection apparatus 100G includes a light source 200A, a modulation mechanism 300A, a metal thin film 410A, a specimen cell 420A, a prism 430, a detector 510A, a signal detector 520A, and a calculator 540. And comprising. The description of the third embodiment is incorporated in these elements.
- the detection device 100G further includes a modulation signal generator 530G and an adjustment unit 600G.
- the adjustment unit 600G corresponds to the adjustment unit 600 described with reference to FIG.
- Adjustment unit 600G includes an offset signal generator 630 and an adder 640.
- the offset signal generator 630 generates an offset signal OSS.
- the offset signal OSS is output from the offset signal generator 630 to the adder 640.
- the modulation signal generator 530G outputs the modulation signal MDS to the signal detector 520A. Unlike the third embodiment, the modulation signal generator 530G outputs the modulation signal MDS to the adder 640.
- the adder 640 adds the offset signal OSS to the modulation signal MDS to generate a drive signal DRS.
- the average incident angle represented by the drive signal DRS is a value that is increased or decreased by an angle represented by the offset signal OSS from the average incident angle defined by the modulation signal MDS.
- the drive signal DRS is output from the adder 640 to the drive motor 312 of the galvanometer mirror 310.
- the drive motor 312 rotates and reciprocates the reflection mirror 311 according to the drive signal DRS.
- the detection apparatus 100G can adjust the average incident angle of the excitation light PL on the metal thin film 410A using the offset signal OSS.
- the offset signal generator 630 shown in FIG. 14A generates the offset signal OSS so that the average incident angle of the excitation light PL to the galvanometer mirror 310 is set to “ ⁇ a ”. Therefore, the offset signal OSS is modulated signals MDS and cooperate, it is possible to define the variation range of incident angles around the mean incidence angle theta a to the metal thin film 410A.
- the adjustment signal is exemplified by the offset signal OSS.
- the adjustment signal generator is exemplified by an offset signal generator 630.
- the offset signal generator 630 shown in FIG. 14B generates the offset signal OSS so that the average incident angle of the excitation light PL to the galvanometer mirror 310 is set to “ ⁇ b ”. Therefore, the offset signal OSS is modulated signals MDS and cooperate, it is possible to define the variation range of incident angles around the mean incidence angle theta b to the metal thin film 410A.
- the technology relating to the exemplary detection device described in connection with the various embodiments described above mainly comprises the following features.
- the detection apparatus includes a light source that emits excitation light, a storage unit that stores a specimen, a metal film that receives the excitation light and generates evanescent light that illuminates the specimen, A modulation unit that modulates the incident angle of the excitation light to the metal film, a drive unit that generates a drive signal that drives the modulation unit, and the intensity of fluorescence generated from the specimen upon irradiation with the evanescent light A detection unit that outputs a corresponding fluorescence signal, and an extraction unit that extracts a signal component derived from the specimen from the fluorescence signal.
- the incident angle changes according to the change of the drive signal.
- the extraction unit extracts a synchronization signal component that changes in synchronization with the change in the drive signal as the signal component.
- the extraction unit extracts, as a signal component, the synchronization signal component that changes in synchronization with the change of the drive signal, so that the light component that is not affected by the variation in incident angle is the extracted signal component It becomes easy to be excluded from. Therefore, the detection apparatus can accurately measure the intensity of fluorescence from the specimen illuminated by the evanescent light.
- the detection device may further include a prism including a first surface on which the excitation light is incident and a second surface to which the metal film is attached.
- the metal film may be disposed between the housing portion and the second surface.
- the excitation light is appropriately guided to the metal film by the prism. Therefore, the metal film can generate evanescent light. Since the metal film is disposed between the accommodating portion and the second surface, the reflecting portion includes a first optical path through which the excitation light propagates when the incident angle is set to the first incident angle. A second optical path through which the excitation light propagates when the incident angle is set to a second incident angle different from the first incident angle, and the evanescent light appropriately illuminates the specimen Can do.
- the modulation unit may include a reflection unit that reflects the excitation light, and a lens unit that is disposed between the reflection unit and the prism.
- the reflection unit may change a reflection direction of the excitation light according to the drive signal.
- the excitation light may be refracted by the lens unit and propagate toward a predetermined position on the metal film.
- the excitation light is refracted by the lens unit and propagates toward a predetermined position on the metal film while the reflection unit changes the reflection direction of the excitation light according to the drive signal. . Therefore, the detection apparatus can accurately measure the intensity of fluorescence from the specimen illuminated by the evanescent light.
- the reflection unit includes a first optical path through which the excitation light propagates when the incident angle is set to the first incident angle, and the incident angle is different from the first incident angle.
- the second optical path moves away from the first optical path as the excitation light approaches the lens part from the reflection part, and enters the first optical path as the excitation light approaches the metal film from the lens part. You may approach.
- the detection apparatus can accurately measure the intensity of fluorescence from the specimen illuminated by the evanescent light.
- the lens unit may include a first lens on which the excitation light reflected by the reflecting unit is incident, and a second lens disposed between the first lens and the prism.
- the reflection unit has a first optical path through which the excitation light propagates when the incident angle is set to a first incident angle, and a second incident angle that is different from the first incident angle.
- a second optical path through which the excitation light propagates may be defined.
- the angular difference between the first optical path and the second optical path in the propagation section from the first lens to the second lens is the same as the first optical path in the propagation section from the reflecting section to the first lens.
- the angle difference from the second optical path may be smaller.
- the designer can design the detection device without using a lens element having an excessively high refractive index.
- alteration part may include the 1st reflective mirror which reflects the said excitation light radiate
- the first reflecting mirror and the second reflecting mirror may cooperate to define the first optical path.
- the incident angle is set to a second incident angle different from the first incident angle
- the second optical path moves away from the first optical path as the excitation light approaches the second reflection mirror from the first reflection mirror, and as the excitation light approaches the metal film from the second reflection mirror.
- the first optical path may be approached.
- the detection apparatus can accurately measure the intensity of fluorescence from the specimen illuminated by the evanescent light.
- an adjustment unit that adjusts the center value of the fluctuation range of the incident angle may be further provided.
- the adjustment unit adjusts the central value of the fluctuation range of the incident angle, so that the detection apparatus can accurately measure the intensity of the fluorescence from the specimen illuminated by the evanescent light.
- the adjustment unit may include an adjustment signal generation unit that generates an adjustment signal for adjusting the center value.
- the adjustment signal may define the variation range in cooperation with the drive signal.
- the adjustment unit electrically adjusts the center value of the variation range of the incident angle, so that the detection device can accurately measure the intensity of the fluorescence from the specimen illuminated by the evanescent light. .
- the adjustment unit may include a holding unit that holds the prism, the metal film, the housing unit, and the detection unit, and a rotation unit that rotates the holding unit.
- the rotating unit may rotate the holding unit and change the center value.
- the adjustment unit mechanically adjusts the center value of the fluctuation range of the incident angle, so that the detection device can accurately measure the intensity of the fluorescence from the specimen illuminated by the evanescent light. .
- the modulation unit varies the incident angle with a variation width that is greater than or equal to the full width at half maximum of the fluorescence signal when the rotation unit rotates the holding unit with the modulation unit stopped. You may let them.
- the detection apparatus can accurately measure the intensity of fluorescence from the specimen illuminated by the evanescent light.
- the fluctuation range may be three times or less the full width at half maximum.
- the detection apparatus can accurately measure the intensity of fluorescence from the specimen illuminated by the evanescent light.
- the detection apparatus may further include a calculation unit that calculates the amount of the detection target substance in the sample from the signal component.
- the amount of the detection target substance in the specimen is appropriately calculated from the signal component.
- the calculation unit may include a calculation unit that performs an integration operation on the signal component, and a determination unit that determines the amount of the detection target substance based on a result of the integration calculation.
- the amount of the detection target substance in the specimen is appropriately determined by the integral calculation with respect to the signal component.
- the modulation unit is configured so that the modulation unit has the incident width with a fluctuation width of at least twice the full width at half maximum of the fluorescence signal when the rotation unit rotates the holding unit with the modulation unit stopped
- the angle may be varied.
- the detection apparatus can accurately measure the intensity of fluorescence from the specimen illuminated by the evanescent light.
- the signal component may approach a peak value as the incident angle approaches the center value.
- the detection apparatus can accurately measure the intensity of the fluorescence from the specimen illuminated by the evanescent light. .
- the principle of the above-described embodiment can be applied to various technical fields (for example, biotechnology) that are required to detect a minute amount of a substance.
- the above technical principle may be applied not only to the field of biotechnology but also to measurement technology used in the environmental field.
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Abstract
Description
本発明者等は、従来の検出技術を研究し、様々な課題を見出した。後述の様々な実施形態によって説明される検出装置は、これらの課題を解消すべく開発されている。
本発明者等は、オフセット成分が除去された検波信号を生成する技術を開発した。第1実施形態において、検体中の検出対象物質の量を正確に表すことができる検波信号を生成することができる検出装置が説明される。
第1実施形態に関連して説明された検出装置は、様々な制御の下で、検出対象物質を正確に検出することができる。第2実施形態において、検出装置の例示的な制御が説明される。
設計者は、第1実施形態に関連して説明された設計原理に基づいて、様々な検出装置を設計することができる。第3実施形態において、例示的な検出装置が説明される。
第3実施形態に関連して説明された検出装置は、様々な光学的設定の下で動作してもよい。信号検波器が大きな値の検波信号を出力するように、検出装置を使用する使用者は、検出装置の光学的設定を決定してもよい。検波信号が大きな値を有するならば、算出器は、検出対象物質の量を正確に算出することができる。第4実施形態において、検出装置の例示的な光学的設定が説明される。
第3実施形態に関連して説明された検出装置は、単一のレンズ素子を利用し、光路を調整する。代替的に、検出装置は、複数のレンズ素子を有してもよい。複数のレンズ素子が利用されるならば、複数のレンズ素子それぞれは、過度に高い屈折率を有さなくてもよい。第5実施形態において、複数のレンズ素子を備える検出装置が説明される。
第3実施形態に関連して説明された検出装置は、単一のレンズ素子を利用し、光路を調整する。代替的に、設計者は、レンズ素子を用いることなく、検出装置を設計してもよい。第6実施形態において、レンズ素子を用いることなく設計された検出装置が説明される。
第4実施形態に関連して説明された如く、入射角の変動範囲の中心値(すなわち、平均入射角)の設定は、蛍光信号から検波信号への変換処理における感度に大きく影響する。したがって、検出装置は、入射角の変動範囲の中心値を調整するための要素を有してもよい。第7実施形態において、入射角の変動範囲の中心値を調整することができる検出装置が説明される。
平均入射角は、機械的に調整されてもよい。第8実施形態において、平均入射角を機械的に調整することができる検出装置が説明される。
本発明者等は、第8実施形態に関連して説明された設計原理に基づいて構築された検出装置を用いて、様々な実験を行い、変調振幅の適切な範囲を見出した。第9実施形態において、本発明者等が行った実験が説明される。
本発明者等は、光源200Aとして、He-Neレーザを用いた。He-Neレーザの出力は、0.1mWであった。He-Neレーザは、633nmの波長を有するレーザ光を出射した。レーザ光は、励起光PLとして利用された。レーザ光は、p偏光を有する。
本発明者等は、ビオチン化BSA(ウシ血清アルブミン)の炭酸バッファ溶液を、上述の容器へ供給した。この結果、ビオチン化BSAは、上述のポリスチレン膜上に固着された。
本発明者等は、ガルバノミラー310を停止させたまま、調整機構600Eを作動し、蛍光信号FLSの大きさを測定した。保持板610が0.5deg.回転するごとに、本発明者等は、蛍光強度を測定した。本発明者等は、測定された蛍光強度を、グラフ上にプロットし、共鳴曲線を作成した。入射角が、58deg.であるとき、共鳴曲線は、最大値を表した。共鳴曲線の半値全幅wは、3.3deg.であった。共鳴曲線は、形状において、ガウシアンカーブに近似していた。
本発明者等は、入射角依存性の測定から得られた結果に基づいて、入射角の変調下における検波信号DTSの波形を算出した。
検出装置の機械的な誤差に起因して、実際の入射角が入射角に対して定められた設定値からずれることもある。実際の入射角と設定値との間の誤差は、検出対象物質の量の算出に誤差を生じさせる。第10実施形態において、実際の入射角と設定値との間の誤差が、検出対象物質の量の算出に与える影響を低減する技術が説明される。
本発明者等は、第9実施形態に関連して説明された知見を用いて、積分機能を有する検出装置にとって、適切な変調振幅範囲を見出した。第11実施形態において、入射角の変調範囲の適切な設定技術が説明される。
第8実施形態に関連して説明された検出装置は、平均入射角を機械的に調整する。代替的に、平均入射角は、電気的に調整されてもよい。この場合、検出装置の機械的構造は、第8実施形態に関連して説明された検出装置よりも簡素になる。第12実施形態において、平均入射角を電気的に調整することができる検出装置が説明される。
Claims (15)
- 励起光を出射する光源と、
検体が収容された収容部と、
前記励起光を受け、前記検体を照らすエバネセント光を生じさせる金属膜と、
前記金属膜への前記励起光の入射角を変調する変調部と、
前記変調部を駆動する駆動信号を生成する駆動部と、
前記エバネセント光の照射を受けて前記検体から生じた蛍光の強度に応じた蛍光信号を出力する検出部と、
前記蛍光信号から前記検体由来の信号成分を抽出する抽出部と、を備え、
前記入射角は、前記駆動信号の変化に応じて変化し、
前記抽出部は、前記駆動信号の前記変化に同期して変化する同期信号成分を、前記信号成分として抽出することを特徴とする検出装置。 - 前記励起光が入射する第1面と、前記金属膜が取り付けられた第2面と、を含むプリズムを更に備え、
前記金属膜は、前記収容部と前記第2面との間に配置されることを特徴とする請求項1に記載の検出装置。 - 前記変調部は、前記励起光を反射する反射部と、前記反射部と前記プリズムとの間に配置されたレンズ部と、を含み、
前記反射部は、前記駆動信号に応じて、前記励起光の反射方向を変え、
前記励起光は、前記レンズ部によって屈折され、前記金属膜上の所定の位置へ向けて伝搬することを特徴とする請求項2に記載の検出装置。 - 前記反射部は、前記入射角が、第1入射角に設定されたときに、前記励起光が伝搬する第1光路と、前記入射角が、前記第1入射角とは異なる第2入射角に設定されたときに、前記励起光が伝搬する第2光路と、を規定し、
前記第2光路は、前記励起光が前記反射部から前記レンズ部に近づくにつれて、前記第1光路から離れ、且つ、前記励起光が前記レンズ部から前記金属膜に近づくにつれて、前記第1光路に近づくことを特徴とする請求項3に記載の検出装置。 - 前記レンズ部は、前記反射部によって反射された前記励起光が入射する第1レンズと、前記第1レンズと前記プリズムとの間に配置された第2レンズと、を含み、
前記反射部は、前記入射角が、第1入射角に設定されたときに、前記励起光が伝搬する第1光路と、前記入射角が、前記第1入射角とは異なる第2入射角に設定されたときに、前記励起光が伝搬する第2光路と、を規定し、
前記第1レンズから前記第2レンズまでの伝搬区間における前記第1光路と前記第2光路との間の角度差は、前記反射部から前記第1レンズまでの伝搬区間における前記第1光路と前記第2光路との間の角度差よりも小さいことを特徴とする請求項3に記載の検出装置。 - 前記変調部は、前記光源から出射された前記励起光を反射する第1反射ミラーと、前記第1反射ミラーの後に前記励起光を反射する第2反射ミラーと、を含み、
前記入射角が、第1入射角に設定されたときに、前記第1反射ミラー及び前記第2反射ミラーは、協働して、第1光路を規定し、
前記入射角が、前記第1入射角とは異なる第2入射角に設定されたときに、前記第1反射ミラー及び前記第2反射ミラーは、協働して、第2光路を規定し、
前記第2光路は、前記励起光が前記第1反射ミラーから前記第2反射ミラーに近づくにつれて、前記第1光路から離れ、且つ、前記第2反射ミラーから前記金属膜に近づくにつれて、前記第1光路に近づくことを特徴とする請求項2に記載の検出装置。 - 前記入射角の変動範囲の中心値を調整する調整部を更に備えることを特徴とする請求項2乃至6のいずれか1項に記載の検出装置。
- 前記調整部は、前記中心値を調整する調整信号を生成する調整信号生成部を含み、
前記調整信号は、前記駆動信号と協働して、前記変動範囲を規定することを特徴とする請求項7に記載の検出装置。 - 前記調整部は、前記プリズム、前記金属膜、前記収容部及び前記検出部を保持する保持部と、前記保持部を回転させる駆動部と、を含み、
前記駆動部は、前記保持部を回転し、前記中心値を変更することを特徴とする請求項7に記載の検出装置。 - 前記変調部の停止下で、前記駆動部が前記保持部を回転させたときの前記入射角の半値全幅以上の大きさの変動幅で、前記変調部は、前記入射角を変動させることを特徴とする請求項9に記載の検出装置。
- 前記変動幅は、前記半値全幅の3倍以下であることを特徴とする請求項10に記載の検出装置。
- 前記信号成分から前記検体中の検出対象物質の量を算出する算出部を更に備えることを特徴とする請求項9に記載の検出装置。
- 前記算出部は、前記信号成分に対して積分演算を行う演算部と、前記積分演算の結果に基づき、前記検出対象物質の前記量を決定する決定部と、を含むことを特徴とする請求項12に記載の検出装置。
- 前記変調部の停止下で、前記駆動部が前記保持部を回転させたときの前記入射角の半値全幅の2倍以上の大きさの変動幅で、前記変調部は、前記入射角を変動させることを特徴とする請求項13に記載の検出装置。
- 前記入射角が、前記中心値に近づくにつれて、前記信号成分は、ピーク値に近づくことを特徴とする請求項7乃至14のいずれか1項に記載の検出装置。
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