Patent Publication Number: US-2019187058-A1

Title: Calibration reference body for fluorescence measurement device

Description:
TECHNICAL FIELD 
     The present disclosure relates to a calibration reference body for a fluorescence measurement device. 
     BACKGROUND ART 
     Immunochromatography assay (lateral flow immunoassay) is known as a method of a sample test such as a blood test. In recent, in a quantitative diagnosis including a low color development region which is difficult to visually determine, a method using a fluorescent label has been used. For example, Patent Literature 1 discloses a technique related to an immunochromatography assay using a fluorescent label. 
     CITATION LIST 
     Patent Literature 
     Patent Literature 1: Japanese Unexamined Patent Publication No. 2006-208386 
     SUMMARY OF INVENTION 
     Technical Problem 
     In the method using a fluorescent label as described above, a fluorescence measurement device is used. However, in order to obtain a highly reliable test result, fluorescence measurement by the fluorescence measurement device needs to be performed with a constant measurement sensitivity. For this reason, a reference body for calibrating the fluorescence measurement device (for example, identifying a difference between machines, adjusting the measurement sensitivity, performing failure diagnosis, or the like) is required. 
     The present disclosure has been made in view of the above problems, and an object of the present disclosure is to provide a calibration reference body for a fluorescence measurement device that can stably identify a measurement sensitivity of the fluorescence measurement device in a wide dynamic range. 
     Solution to Problem 
     According to an embodiment of the present disclosure, there is provided a calibration reference body for a fluorescence measurement device, including: a support provided with a first light passing portion, a second light passing portion, a first accommodating space facing the first light passing portion, and a second accommodating space facing the second light passing portion; a first fluorescent body accommodated in the first accommodating space and configured to emit first fluorescence in a second wavelength band when irradiated with first excitation light in a first wavelength band through the first light passing portion; a second fluorescent body accommodated in the second accommodating space and configured to emit second fluorescence in the second wavelength band when irradiated with second excitation light in the first wavelength band through the second light passing portion; and a light shielding portion disposed between the first accommodating space and the second accommodating space. In the reference body, in a case where a light amount of the first excitation light incident on the first light passing portion and a light amount of the second excitation light incident on the second light passing portion are equal to each other, a light amount of the first fluorescence emitted from the first light passing portion and a light amount of the second fluorescence emitted from the second light passing portion are different from each other. 
     In this reference body, it is possible to suppress the influence of the excitation light and the fluorescence in one of the fluorescent bodies on the excitation light and the fluorescence in the other fluorescent body by the light shielding portion disposed between the first accommodating space and the second accommodating space, and in a case where the light amount of the first excitation light incident on the first light passing portion and the light amount of the second excitation light incident on the second light passing portion are equal to each other, the light amount of the first fluorescence emitted from the first light passing portion and the light amount of the second fluorescence emitted from the second light passing portion are different from each other, so that it is possible to stably identify the measurement sensitivity in a wide dynamic range by one measurement. 
     In one aspect, the support may include a first holding portion that holds the first fluorescent body accommodated in the first accommodating space and a second holding portion that holds the second fluorescent body accommodated in the second accommodating space. The first fluorescent body is held in the first accommodating space, and the second fluorescent body is held in the second accommodating space, so that variations in measured values are suppressed. Therefore, it is possible to improve the accuracy of calibration. 
     In one aspect, the first holding portion may be a first wall portion that defines the first accommodating space, and the second holding portion may be a second wall portion that defines the second accommodating space. At least one of the first wall portion and the second wall portion may constitute the light shielding portion. In this case, since light shielding by the light shielding portion can be reliably performed, variations in measured values are suppressed. Therefore, it is possible to improve the accuracy of calibration. 
     In one aspect, at least the first region surrounding the first light passing portion and the second region surrounding the second light passing portion of the support may have a light shielding property. In this case, the influence of light (first excitation light) irradiated to the first region and light (second excitation light) irradiated to the second region on the measurement of the first fluorescence and the second fluorescence can be suppressed, so that variations in measured values are suppressed. Therefore, it is possible to improve the accuracy of calibration. 
     In one aspect, the support may include a main body provided with the first accommodating space and the second accommodating space, and a cover provided with the first light passing portion and the second light passing portion. In this case, it is easy to configure the main body and the cover with materials corresponding to respective functions. 
     In one aspect, the reference body may further include a first light transmitting member which is disposed between the first light passing portion and the first fluorescent body and transmits the first excitation light and the first fluorescence and a second light transmitting member which is disposed between the second light passing portion and the second fluorescent body and transmits the second excitation light and the second fluorescence. In this case, deterioration of the fluorescent bodies (the first fluorescent body and the second fluorescent body) caused by physical and chemical factors can be prevented by the first light transmitting member and the second light transmitting member, so that it is possible to perform calibration with a high accuracy over a long period of time. 
     In one aspect, the reference body may further include a first optical bonding material disposed between the first fluorescent body and the first light transmitting member and a second optical bonding material disposed between the second fluorescent body and the second light transmitting member. In this case, since the optical loss at an interface between the fluorescent body and the light transmitting member is suppressed, variations in the measured values are suppressed. Therefore, it is possible to improve the accuracy of calibration. 
     In one aspect, the first light transmitting member may have a function of changing characteristics of the first excitation light, and the second light transmitting member may have a function of changing characteristics of the second excitation light. In this case, by selecting the light transmitting member (the first light transmitting member and the second light transmitting member), for example, the light amount and the wavelength of the transmitted light, that is, the light amount and the wavelength of the excitation light (the first excitation light and the second excitation light) irradiated to the fluorescent body (the first fluorescent body and the second fluorescent body) can be adjusted. 
     In one aspect, at least one of the first fluorescent body and the second fluorescent body may include a semiconductor layer having a light emitting layer containing a semiconductor material as a fluorescent substance. By appropriately selecting the semiconductor material, the measurement sensitivity in a desired fluorescence wavelength band can be identified. 
     In one aspect, the semiconductor material may be a compound semiconductor containing Ga. By appropriately selecting the compound material, the measurement sensitivity in a desired fluorescence wavelength band can be identified. 
     In one aspect, the compound semiconductor may be GaAs (1-x) P x  (0≤x≤1). In this case, good fluorescence is obtained, particularly, in the red range. 
     In one aspect, the semiconductor layer may further include a layer containing AlGaAsP on a side of the light emitting layer on which the excitation light is incident and on the side opposite thereto. In this case, the luminous efficiency of the fluorescent body tends to be excellent. 
     In one aspect, at least one of the first fluorescent body and the second fluorescent body may further include an antioxidation layer on a side of the light emitting layer on which the excitation light is incident. In this case, since deterioration of the fluorescent body caused by oxidation can be prevented, it is possible to perform calibration with a high accuracy over a long period of time. 
     In one aspect, at least one of the first fluorescent body and the second fluorescent body may be made of a fluorescent resin containing a light transmissive resin and a fluorescent substance dispersed in the light transmissive resin. By appropriately selecting the fluorescent substance, the measurement sensitivity in a desired fluorescence wavelength band can be identified. 
     In one aspect, at least one of the first fluorescent body and the second fluorescent body may be made of a fluorescent glass containing a glass and a fluorescent substance dispersed in the glass. By appropriately selecting the fluorescent substance, the measurement sensitivity in a desired fluorescence wavelength band can be identified. 
     Advantageous Effects of Invention 
     According to the present disclosure, it is possible to provide a calibrating reference body for a fluorescence measurement device capable of stably identifying a measurement sensitivity of the fluorescence measurement device in a wide dynamic range. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG. 1  is a perspective view of an optical head and a chromatographic test tool of a fluorescence measurement device. 
         FIG. 2  is a plan view of a reference body according to an embodiment of the present disclosure. 
         FIG. 3  is a cross-sectional view of a reference body taken along line III-III of  FIG. 2 . 
         FIG. 4  is a plan view of a main body of the reference body of  FIG. 2 . 
         FIG. 5  is a cross-sectional view of a semiconductor fluorescent body constituting a fluorescent body of the reference body of  FIG. 2 . 
         FIG. 6( a )  is a perspective view of Modified Example 1 of the fluorescent body of the reference body according to an embodiment of the present disclosure.  FIG. 6( b )  is a perspective view of Modified Example 2 of the fluorescent body of the reference body according to an embodiment of the present disclosure. 
         FIG. 7  is a diagram illustrating a fluorescence excitation spectrum of the fluorescent body according to an embodiment of the present disclosure. 
         FIG. 8  is a diagram illustrating a fluorescence profile obtained by fluorescence measurement of a reference body according to an embodiment of the present disclosure. 
     
    
    
     DESCRIPTION OF EMBODIMENTS 
     Hereinafter, embodiments according to the present disclosure will be described in detail with reference to the drawings. In the following description, the same reference numerals are used for the same or corresponding elements, and redundant description thereof is omitted. 
     First, a fluorescence measurement device using a reference body according to this embodiment will be described. A fluorescence measurement device  100  illustrated in  FIG. 1  is a fluorescence measurement device (fluorescent immunochromato reader) used for an immunochromatography assay. As illustrated in  FIG. 1 , the fluorescence measurement device  100  includes a support base  110 , an optical head  120 , and a scanning mechanism (not illustrated). 
     The support base  110  supports a chromatographic test tool  50 . 
     The chromatographic test tool  50  has a casing  51  and a chromatographic test piece  52 . The casing  51  has a rectangular plate shape having an X axis direction as the longitudinal direction and a Z axis direction as the thickness direction. The casing  51  is foiled with an opening  51   a  which is open to one side in the Z axis direction. The chromatographic test piece  52  is accommodated in the casing  51 . As a result of, for example, antigen-antibody reaction, a plurality of colored lines CL extending along a Y axis direction are formed in the chromatographic test piece  52 . The plurality of colored lines CL are aligned along the X axis direction and are exposed to the outside through the opening  51   a . Since each colored line CL contains a fluorescent substance (and a fluorescent reagent containing the fluorescent substance) bonded to an antigen or an antibody, each colored line CL excited by excitation light in the first wavelength band emits fluorescence in the second wavelength band. 
     The optical head  120  includes an irradiation optical system  130  that irradiates the chromatographic test tool  50  with excitation light in the first wavelength band and a detection optical system  140  that detects fluorescence in the second wavelength band emitted from the chromatographic test tool  50 . The irradiation optical system  130  includes a semiconductor light emitting element  131 , a collimator lens  132 , a light flux shaping member  133 , a short pass filter  134 , and a condenser lens  135 . The detection optical system  140  includes a long pass filter  141  and a semiconductor light receiving element  142 . In addition, the fluorescence measurement device  100  has a mechanism that prevents disturbance light from entering. 
     In the fluorescence measurement device  100 , the optical head  120  is scanned in the X axis direction by a scanning mechanism. At this time, the chromatographic test tool  50  is irradiated with the excitation light of the first wavelength band emitted from the semiconductor light emitting element  131  through the collimator lens  132 , the light flux shaping member  133 , the short pass filter  134 , and the condenser lens  135 . When each colored line CL is irradiated with the excitation light through the opening  51   a , fluorescence in the second wavelength band is emitted from each colored line CL, and the fluorescence is incident on the semiconductor light receiving element  142  through the long pass filter  141 . As a result, the chromaticity of each colored line CL is measured. 
     Herein, the first wavelength band denotes a wavelength of light (excitation light) having energy necessary for exciting electrons in a fluorescent substance emitting fluorescence in the second wavelength band to emit the fluorescence in the second wavelength band. Therefore, usually, the first wavelength band and the second wavelength band do not overlap each other in the wavelength range, and the peak wavelength of the first wavelength band is shorter than the peak wavelength of the second wavelength band. 
     In other words, the first wavelength band, which is the wavelength band of the excitation light, is determined according to excitation characteristics of the fluorescent substance used, and the second wavelength band, which is the wavelength band of fluorescence emitted by the colored line CL, is determined according to fluorescence characteristics of the colored line CL, that is, the fluorescence characteristics of the fluorescent substance contained in the colored line CL. In addition, the first wavelength band is adjusted by the types of the semiconductor light emitting element  131  and the short pass filter  134  and the like. 
     Next, a reference body according to this embodiment will be described. A reference body  1  illustrated in  FIGS. 2 and 3  is used for calibration of the fluorescence measurement device  100  described above. As illustrated in  FIGS. 2 and 3 , the reference body  1  has a rectangular plate shape having the X axis direction as the longitudinal direction and the Z axis direction as the thickness direction. The shape and size of the reference body  1  are the shape and size in accordance with the chromatographic test tool  50 . 
     The reference body  1  includes a support  10 , a first fluorescent body  20 , and a second fluorescent body  30 . The reference body  1  further includes a first light transmitting member  2 , a second light transmitting member  3 , a first optical bonding material  4 , and a second optical bonding material  5 . 
     The support  10  has a main body  11  and a cover  12 . Each of the main body  11  and the cover  12  has a rectangular plate shape having the X axis direction as the longitudinal direction and the Z axis direction as the thickness direction. The cover  12  is disposed on a surface  11   a  (a surface on one side in the Z axis direction) of the main body  11  and is fixed to the main body  11  by bolts  6 . 
     As illustrated in  FIGS. 3 and 4 , a first accommodating space  14  and a second accommodating space  15  that are open to one side in the Z axis direction are provided on the surface  11   a  of the main body  11 . The first accommodating space  14  and the second accommodating space  15  are aligned in the X axis direction. The first accommodating space  14  includes a first recessed portion  14   a  formed in the surface  11   a  of the main body  11  and a first width-expanded portion  14   b  of which the width is expanded on the opening side of the first recessed portion  14   a . The second accommodating space  15  includes a second recessed portion  15   a  formed in the surface  11   a  of the main body  11  and a second width-expanded portion  15   b  of which the width is expanded on the opening side of the second recessed portion  15   a.    
     The first fluorescent body  20  is disposed inside the first recessed portion  14   a . The first fluorescent body  20  is held by a first wall portion (first holding portion)  16  that defines the first recessed portion  14   a . The first fluorescent body  20  emits first fluorescence in the second wavelength band when irradiated with the first excitation light in the first wavelength band. The first light transmitting member  2  is disposed inside the first width-expanded portion  14   b . The first light transmitting member  2  transmits the first excitation light irradiated to the first fluorescent body  20  and the first fluorescence emitted from the first fluorescent body  20 . The first light transmitting member  2  has a function of changing the characteristics of the first excitation light. As an example, the first light transmitting member  2  has a dimming function of decreasing the light amount of the first excitation light that is transmitted, a wavelength selecting function of transmitting only light having a specific wavelength band, and the like. The first optical bonding material  4  is disposed between the first fluorescent body  20  and the first light transmitting member  2 . The first optical bonding material  4  optically bonds the first fluorescent body  20  and the first light transmitting member  2  and suppresses a change in optical properties (wavelength, light amount, and the like) of the light passing between the first fluorescent body  20  and the first light transmitting member  2 . As an example, the first optical bonding material  4  may be a resin having an optical transmissive property and an adhesive property. In addition, in  FIG. 4 , the first light transmitting member  2  and the first optical bonding material  4  are indicated by two-dot dashed lines. 
     The second fluorescent body  30  is disposed in the second recessed portion  15   a . The second fluorescent body  30  is held by a second wall portion (second holding portion)  17  that defines the second recessed portion  15   a . The second fluorescent body  30  emits second fluorescence in the second wavelength band when irradiated with the second excitation light in the first wavelength band. The second light transmitting member  3  is disposed in the second width-expanded portion  15   b . The second light transmitting member  3  transmits the second excitation light irradiated to the second fluorescent body  30  and the second fluorescence emitted from the second fluorescent body  30 . The second light transmitting member  3  has a function of changing the characteristics of the second excitation light. As an example, the second light transmitting member  3  has a dimming function for decreasing the light amount of the second excitation light that is transmitted, a wavelength selecting function of transmitting only light having a specific wavelength band, and the like. The second optical bonding material  5  is disposed between the second fluorescent body  30  and the second light transmitting member  3 . The second optical bonding material  5  optically bonds the second fluorescent body  30  and the second light transmitting member  3  and suppresses a change in optical properties (wavelength, light amount, and the like) of the light passing between the second fluorescent body  30  and the second light transmitting member  3 . As an example, the second optical bonding material  5  may be a resin having an optical transmissive property and an adhesive property. In  FIG. 4 , the second light transmitting member  3  and the second optical bonding material  5  are indicated by two-dot dashed lines. 
     The main body  11  is made of a material having a light shielding property (light absorbing property or light reflecting property), and more preferably made of a material (for example, a black ABS resin) having a light absorbing property. That is, the first wall portion  16  and the second wall portion  17  have a light shielding property. Therefore, the first wall portion  16  and the second wall portion  17  also function as a light shielding portion  13  that shields light being incident between the first accommodating space  14  and the second accommodating space  15 . 
     As illustrated in  FIGS. 2 and 3 , the cover  12  is provided with a first light passing portion  18  and a second light passing portion  19 . Each of the first light passing portion  18  and the second light passing portion  19  is a slit extending along the Y axis direction and has the same size and shape. The first light passing portion  18  faces the first accommodating space  14  in the Z axis direction, and the second light passing portion  19  faces the second accommodating space  15  in the Z axis direction. That is, the first light transmitting member  2  is disposed between the first light passing portion  18  and the first fluorescent body  20 , and the second light transmitting member  3  is disposed between the second light passing portion  19  and the second fluorescent body  30 . The shapes and sizes of the first light passing portion  18  and the second light passing portion  19  may be appropriately adjusted in accordance with the shape and size of the colored line CL of the chromatographic test tool  50 . 
     The cover  12  is made of a material having a light shielding property, and more preferably made of a material having a light absorbing property (for example, black acrylic resin). That is, a first region  18   a  surrounding the first light passing portion  18  and a second region  19   a  surrounding the second light passing portion  19  in the cover  12  have a light shielding property (for example, a light absorbing property). 
     The first fluorescent body  20  is a fluorescent body that emits first fluorescence in the second wavelength band when irradiated with the first excitation light in the first wavelength band, and the second fluorescent body  30  is a fluorescent body that emits second fluorescence in the second wavelength band when irradiated with the second excitation light in the first wavelength band. In this embodiment, the first fluorescent body  20  and the second fluorescent body  30  are configured with a semiconductor fluorescent body  40  illustrated in  FIG. 5 , which includes a substrate  41 , a graded layer  42  disposed on the substrate  41 , a semiconductor layer  43  disposed on the graded layer  42 , and an antioxidation layer  44  disposed on the semiconductor layer  43 . For the convenience of illustration, the thickness of each layer is illustrated to be uniform regardless of the actual thickness. The first fluorescent body  20  and the second fluorescent body  30  are accommodated in the first accommodating space  14  and the second accommodating space  15 , respectively, so that the side of the antioxidation layer  44  with respect to the semiconductor layer  43  becomes the side of the cover  12 . 
     The semiconductor layer  43  includes a barrier layer  43   a , a light emitting layer  43   b , and a window layer  43   c . The barrier layer  43   a  is disposed on the substrate  41  side with respect to the light emitting layer  43   b , and the window layer  43   c  is disposed on the cover  12  side with respect to the light emitting layer  43   b.    
     The light emitting layer  43   b  is made of a semiconductor material that is a fluorescent substance that emits fluorescence in the second wavelength band (first fluorescence or second fluorescence) by irradiation with excitation light (first excitation light or second excitation light) in the first wavelength band. The semiconductor material constituting the light emitting layer  43   b  is a compound semiconductor containing Ga, which is, for example, a mixed crystal of GaAs and GaP and is represented by GaAs (1-x) P x  (0≤x≤1) (hereinafter, also referred to simply as “GaAsP”). As listed in the following Table 1, it is preferable that x is 0.5 or less from the viewpoint that the transition type is a direct transition type and the luminous efficiency is excellent. Such a fluorescent body is preferable as a fluorescent body, particularly, in the red range. The emission wavelength (wavelength of fluorescence) listed in Table 1 is a value calculated from the band gap energy Eg. 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 Compound 
                   
                   
                   
                   
               
               
                 Semiconductor 
                   
                   
                 Emission 
               
               
                 (Compositional 
                   
                 Eg 
                 Wavelength 
               
               
                 Formula) 
                 x 
                 (ev) 
                 (nm) 
                 Type of Transition 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 GaAs 1−x P x   
                 0 
                 1.42 
                 873 
                 Direct Transition 
               
               
                   
                 0.05 
                 1.48 
                 840 
                 Direct Transition 
               
               
                   
                 0.1 
                 1.53 
                 809 
                 Direct Transition 
               
               
                   
                 0.15 
                 1.59 
                 779 
                 Direct Transition 
               
               
                   
                 0.2 
                 1.65 
                 751 
                 Direct Transition 
               
               
                   
                 0.25 
                 1.71 
                 725 
                 Direct Transition 
               
               
                   
                 0.3 
                 1.77 
                 700 
                 Direct Transition 
               
               
                   
                 0.35 
                 1.83 
                 676 
                 Direct Transition 
               
               
                   
                 0.4 
                 1.9 
                 653 
                 Direct Transition 
               
               
                   
                 0.45 
                 1.96 
                 632 
                 Direct Transition 
               
               
                   
                 0.5 
                 2.03 
                 612 
                 Direct Transition 
               
               
                   
                 0.55 
                 2.05 
                 605 
                 Indirect Transition 
               
               
                   
                 0.6 
                 2.07 
                 599 
                 Indirect Transition 
               
               
                   
                 0.65 
                 2.09 
                 593 
                 Indirect Transition 
               
               
                   
                 0.7 
                 2.11 
                 587 
                 Indirect Transition 
               
               
                   
                 0.75 
                 2.13 
                 581 
                 Indirect Transition 
               
               
                   
                 0.8 
                 2.16 
                 575 
                 Indirect Transition 
               
               
                   
                 0.85 
                 2.18 
                 569 
                 Indirect Transition 
               
               
                   
                 0.9 
                 2.21 
                 562 
                 Indirect Transition 
               
               
                   
                 0.95 
                 2.23 
                 555 
                 Indirect Transition 
               
               
                   
                 1 
                 2.26 
                 549 
                 Indirect Transition 
               
               
                   
               
            
           
         
       
     
     As an example, the thickness of the light emitting layer  43   b  is 0.01 to 5 μm. In this embodiment, the thickness of the light emitting layer  43   b  of the first fluorescent body  20  and the thickness of the light emitting layer  43   b  of the second fluorescent body  30  are different from each other. Since the light amount of fluorescence (first fluorescence and second fluorescence) emitted from the semiconductor fluorescent body  40  increases in proportion to the thickness of the light emitting layer  43   b , in this embodiment, in a case where the light amount of the first excitation light incident on the first light passing portion  18  and the light amount of the second excitation light incident on the second light passing portion  19  are equal to each other, the light amount of the first fluorescence emitted from the first light passing portion  18  and the light amount of the first fluorescence emitted from the second light passing portion  19  are different from each other. When the thickness of the light emitting layer  43   b  is equal to or larger than a certain value, the light amount of fluorescence hardly increases. Therefore, in at least one of the first fluorescent body  20  and the second fluorescent body  30 , it is preferable that the thickness of the light emitting layer  43   b  is 3 μm or less. 
     The barrier layer  43   a  and the window layer  43   c  are layers made of a semiconductor material represented by Al y Ga (1-y) As (1-z) Pz (0≤y≤1, 0≤z≤1)) (hereinafter, also referred to simply as “AlGaAsP”). As an example, the thickness of the barrier layer  43   a  is 0.01 to 5 μm, and the thickness of the window layer  43   c  is 0.01 to 5 μm. 
     The antioxidation layer  44  has a function of preventing oxidation of the semiconductor layer  43 . The antioxidation layer  44  has, for example, an antireflection layer  44   a  and a protective layer  44   b  disposed on the side opposite to the semiconductor layer  43  with respect to the antireflection layer  44   a . The antireflection layer  44   a  is a layer having a function of preventing reflection of the excitation light incident on the light emitting layer  43   b  in addition to the antioxidant function. The protective layer  44   b  is a layer that protects the surface exposed to the cover  12  side of the semiconductor fluorescent body  40  from physical and chemical factors. In this embodiment, a layer containing Si 3 N 4  is provided as the antireflection layer  44   a , and a layer containing SiO 2  is provided as the protective layer  44   b . As an example, the thickness of the antireflection layer  44   a  is 0.01 to 0.3 μm and the thickness of the protective layer  44   b  is 0.01 to 0.5 μm. 
     The substrate  41  has a function of fixing the semiconductor layer  43 . In this embodiment, the substrate is a GaAs substrate. As an example, the thickness of the substrate  41  is 100 to 1200 μm. 
     The graded layer  42  is a layer having a function of relaxing the lattice mismatch between the substrate  41  and the semiconductor layer  43 . The graded layer  42  is a layer containing Ga, As, and P (for example, a layer made of GaAsP) similarly to the semiconductor layer  43 , but the graded layer  42  is configured so that the content of P increases as approaching from the substrate  41  side to the semiconductor layer  43  side. Specifically, the graded layer  42  is configured so that the content of P is about the same as that of the semiconductor layer  43  in the vicinity of the interface with the semiconductor layer  43 . The graded layer  42  may be a layer further containing Al (for example, a layer made of AlGaAsP). In a case where the graded layer  42  further includes Al, the graded layer  42  may be configured so that the content of Al increases as approaching from the substrate  41  side to the semiconductor layer  43  side, and may be configured so that the content of Al is about the same as that of the semiconductor layer  43  in the vicinity of the interface with the semiconductor layer  43 . 
     The above-described semiconductor fluorescent body  40  is obtained, for example, by growing the graded layer  42 , the barrier layer  43   a , the light emitting layer  43   b , and the window layer  43   c  in this order on the substrate  41  and forming the antireflection layer  44   a  and the protective layer  44   b  in this order thereon. 
     Next, calibration of the fluorescence measurement device  100  by using the reference body  1  according to this embodiment will be described. In this specification, the calibration of the fluorescence measurement device  100  denotes an operation of identifying the presence or absence of a deviation from a reference value of a fluorescence intensity, profile, and the like measured by the fluorescence measurement device  100 . The reference body  1  according to this embodiment is useful, for example, for shipment adjustment, periodic test, and the like of the fluorescence measurement device  100 . For example, at the time of shipment of the fluorescence measurement device  100 , first, the fluorescence measurement of the reference body  1  according to this embodiment is performed with a reference machine. Subsequently, the fluorescence measurement of the reference body  1  according to this embodiment is performed by the fluorescence measurement device  100  (actual machine) to be tested, and a deviation between the emission intensity, profile, and the like measured by the reference machine and the emission intensity, profile, and the like measured by the actual machine is identified. At this time, if the emission intensity, profile, and the like of the actual machine are not within specified values, machine base adjustment is performed. By such calibration operation, the fluorescence measurement device  100  capable of performing stable fluorescence measurement can be shipped. In addition, at the periodic test of the fluorescence measurement device  100 , the fluorescence measurement of the reference body  1  according to this embodiment is performed, and by comparing with the emission intensity, the profile, and the like measured by the reference machine, it is identified whether or not the measured emission intensity, the profile, and the like are within the specified values. As a result, it is possible to perform the failure diagnosis of the fluorescence measurement device  100 . In addition, by measuring the reference body  1  according to this embodiment with a plurality of fluorescence measurement devices  100  and comparing the measured emission intensities, profiles, and the like, it is also possible to identify a difference between machines. 
     According to the reference body  1  of this embodiment, it is possible to identify the measurement sensitivity in a wide dynamic range by one measurement. 
     In addition, in this embodiment, since the support  10  includes the main body  11  and the cover  12 , the main body  11  and the cover  12  may be made of materials corresponding to the respective functions. In addition, since the cover  12  is fixed to the main body  11  by the bolts  6 , replacement of the first fluorescent body  20  and the second fluorescent body  30  can be easily performed. In addition, since there is a region where the first fluorescent body  20  and the second fluorescent body  30  are not accommodated in the first accommodating space  14  and the second accommodating space  15 , removal of the first fluorescent body  20  and the second fluorescent body  30  can be easily performed. 
     In addition, in this embodiment, since the main body  11  includes the first holding portion and the second holding portion, the first fluorescent body  20  is held in the first accommodating space  14 , and the second fluorescent body  30  is held in the second accommodating space  15 . As a result, since variations in measured values are suppressed, it is possible to improve the accuracy of calibration. 
     In addition, in this embodiment, since the main body  11  is made of a material having a light shielding property, it is possible to shield the light incident between the first accommodating space  14  and the second accommodating space  15 , and it is possible to suppress the incidence of excitation light and the emission of fluorescence from regions other than the first light passing portion  18  and the second light passing portion  19 . Therefore, it is possible to suppress the influence of the excitation light and the fluorescence in one fluorescent body on the excitation light and the fluorescence in the other fluorescent body. In particular, in this embodiment, since the main body  11  is made of a material having a light absorbing property, the influence of scattering of the excitation light and the fluorescence on the fluorescence measurement can be suppressed. In addition, in this embodiment, since the first wall portion  16  and the second wall portion  17  have a light shielding property, the first wall portion  16  and the second wall portion  17  also function as the light shielding portion  13  that shields light being incident between the first accommodating space  14  and the second accommodating space  15 . For this reason, the fluorescent substance accommodated in one of the accommodating spaces is hardly affected by the light (excitation light and fluorescence) leaked from the wall portion of the other accommodating space. As a result, since variations in measured values are suppressed, it is possible to improve the accuracy of calibration. 
     In this embodiment, the cover  12  is made of a material having a light shielding property, and the first region  18   a  surrounding the first light passing portion  18  and the second region  19   a  surrounding the second light passing portion  19  have a light shielding property, so that the influence of the light (first excitation light) irradiated to the first region  18   a  and the light (second excitation light) irradiated to the second region  19   a  on the fluorescence measurement can be suppressed. As a result, since variations in measured values are suppressed, it is possible to improve the accuracy of calibration. In particular, in this embodiment, since the first region  18   a  and the second region  19   a  are made of a material having a light absorbing property, variations in measured values caused by noise components such as scattered light are suppressed. 
     Furthermore, in this embodiment, since the first light transmitting member  2  which is disposed between the first light passing portion  18  and the first fluorescent body  20  and transmits the first excitation light and the first fluorescence and the second light transmitting member  3  which is disposed between the second light passing portion  19  and the second fluorescent body  30  and transmits the second excitation light and the second fluorescence are provided, deterioration of the first fluorescent body  20  and the second fluorescent body  30  caused by physical and chemical factors can be prevented. As a result, it is possible to perform calibration with a high accuracy over a long period of time. 
     In addition, in this embodiment, since the space between the first fluorescent body  20  and the first light transmitting member  2  and the space between the second fluorescent body  30  and the second light transmitting member  3  are filled with the first optical bonding material  4  and the second optical bonding material  5 , respectively, optical loss at the interface between the fluorescent body and the light transmitting member is suppressed. As a result, since variations in measured values are suppressed, it is possible to improve the accuracy of calibration. 
     In addition, in this embodiment, since the fluorescent bodies (the first fluorescent body  20  and the second fluorescent body  30 ) are configured with the semiconductor fluorescent body  40  and the semiconductor material constituting the light emitting layer  43   b  of the semiconductor fluorescent body  40  is a compound semiconductor containing Ga, it is possible to easily change the fluorescence characteristics by adjusting the types and content ratios of the compound materials as described later. In addition, in this embodiment, since the semiconductor material constituting the light emitting layer  43   b  is a compound semiconductor represented by GaAs (1-x) P x  (0≤x≤1), good fluorescence can be obtained, particularly, in the red range. In this embodiment, the barrier layer  43   a  made of AlGaAsP, the light emitting layer  43   b  made of GaAsP, and the window layer  43   c  made of AlGaAsP are stacked in this order, and the semiconductor layer  43  has a so-called well structure, so that luminous efficiency is excellent. In addition, in this embodiment, since the semiconductor fluorescent body  40  includes the antioxidation layer  44 , deterioration of the fluorescent body caused by oxidation can be prevented, and it is possible to perform calibration with a high accuracy over a long period of time. In addition, since the semiconductor fluorescent body  40  described above generally has a fluorescence excitation spectrum approximate to the fluorescence excitation spectrum of a red excitation reagent used for immunochromatography assay, the semiconductor fluorescent body can be appropriately used for a calibration reference body for the fluorescence measurement device  100  (immunochromato reader) used for an immunochromatography assay. 
     Although one embodiment of the reference body according to the present disclosure has been described above, the present disclosure is not limited to the above embodiment. 
     For example, the reference body may not be provided with the bolts  6 , and for example, the main body  11  and the cover  12  may be fixed with a resin having an adhesive property. In addition, the main body  11  and the cover  12  may be integrally molded. 
     In addition, for example, in the main body  11 , the first wall portion  16  and the second wall portion  17  and portions other than the first wall portion  16  and the second wall portion  17  may be made of different materials. For example, in the main body  11 , only the first wall portion  16  and the second wall portion  17  may be made of a material having a light shielding property. In addition, only one of the first wall portion  16  and the second wall portion  17  may be made of a material having a light shielding property. In addition, the first wall portion  16  and the second wall portion  17  do not have a light shielding property, but a light shielding member may be separately provided between the first accommodating space  14  and the second accommodating space  15 . 
     In addition, for example, in the cover  12 , the first region  18   a  and the second region  19   a  and portions other than the first region  18   a  and the second region  19   a  may be made of different materials. For example, in the cover  12 , only the first region  18   a  and the second region  19   a  may be made of a material having a light shielding property. In addition, only one of the first region  18   a  and the second region  19   a  may be made of a material having a light shielding property. In addition, the first region  18   a  and the second region  19   a  do not have a light shielding property, but a light shielding member may be separately provided between the first region  18   a  and the second region  19   a.    
     In addition, the first light passing portion  18  and the second light passing portion  19  may be made of, for example, a light transmissive material. The first light passing portion  18  and the second light passing portion  19  may be light transmitting regions formed in the cover  12 . 
     In addition, for example, by changing the content ratio x of P in GaAs (1-x) P x  constituting the light emitting layer  43   b , the fluorescence characteristics of the fluorescent body may be changed. Since the band gap energy is increased as increasing the content ratio x (bringing x close to 1), the wavelength band (second wavelength band) of fluorescence can be shifted to the shorter wavelength side. When the content ratio x is 0.5 or less, direct transition occurs, so that the fluorescent intensity tends to be excellent. 
     In addition, for example, by changing the content ratio y of Al and the content ratio z of P in Al y Ga (1-y) As (1-z) Pz (0≤y≤1, 0≤z≤1) constituting the window layer  43   c  and the barrier layer  43   a , the fluorescence characteristics of the fluorescent body may be changed. From the viewpoint of further improving the luminous efficiency, the content ratio y in the window layer  43   c  may be 0.05 to 1. Similarly, from the viewpoint of further improving the luminous efficiency, the content ratio y in the barrier layer  43   a  may be 0.05 to 1. 
     In addition, the light emitting layer  43   b  may contain, for example, a material other than a semiconductor material. In addition, the semiconductor material contained in the light emitting layer  43   b  is not limited to GaAsP. The semiconductor material contained in the light emitting layer  43   b  may be, for example, InGaAs, InGaP, InAsP, AlInAs, AlAsP, AlGaAs, AlGaP, InGaN, AlGaN, InNAs, GaNAs, InGaAsP, AlInGaAs, AlInGaP, AlInAsP, AlGaAsP, GaInNAs, AlInGaN, AlInNAs, AlGaNAs, AlInGaAsP, AIGaInNAs, or the like. By changing the semiconductor material, the fluorescence characteristics of the fluorescent body can be changed. That is, by changing the semiconductor material, the wavelength band (second wavelength band) of fluorescence emitted from the fluorescent body can be changed. Table 2 lists the fluorescence wavelength (emission wavelength) assumed when each compound semiconductor is used for a fluorescent body. In addition, the emission wavelength listed in Table 2 is a value calculated from the band gap energy Eg. 
     
       
         
           
               
               
               
             
               
                 TABLE 2 
               
               
                   
               
               
                 Compound Semiconductor 
                   
                 Emission 
               
               
                 (Compositional Formula) 
                 Composition 
                 Wavelength (nm) 
               
               
                   
               
             
            
               
                 In x Ga 1−x As 
                 0 ≤ x ≤ 1 
                 873 to 3444 
               
               
                 In x Ga 1−x P 
                 0 ≤ x ≤ 1 
                 549 to 919 
               
               
                 GaAs 1−x P x   
                 0 ≤ x ≤ 1 
                 549 to 873 
               
               
                 InAs 1−x P x   
                 0 ≤ x ≤ 1 
                 919 to 3444 
               
               
                 Al x In 1−x As 
                 0 ≤ x ≤ 1 
                 574 to 3444 
               
               
                 AlAs 1−x P x   
                 0 ≤ x ≤ 1 
                 500 to 574 
               
               
                 Al x Ga 1−x As 
                 0 ≤ x ≤ 1 
                 574 to 873 
               
               
                 Al x Ga 1−x P 
                 0 ≤ x ≤ 1 
                 500 to 549 
               
               
                 In x Ga 1−x N 
                 0 ≤ x ≤ 1 
                 366 to 1550 
               
               
                 Al x Ga 1−x N 
                 0 ≤ x ≤ 1 
                 200 to 366 
               
               
                 InN 1−x As x   
                 0 ≤ x ≤ 1 
                 1550 to 3444 
               
               
                 GaN 1−x As x   
                 0 ≤ x ≤ 1 
                 366 to 873 
               
               
                 In x Ga 1−x As y P 1−y   
                 0 ≤ x ≤ 1, 0 ≤ y ≤ 1 
                 549 to 3444 
               
               
                 Al x In y Ga 1−x−y As 
                 0 ≤ x ≤ 1, 0 ≤ y ≤ 1 
                 549 to 3444 
               
               
                 Al x In y Ga 1−x−y P 
                 0 ≤ x ≤ 1, 0 ≤ y ≤ 1 
                 500 to 919 
               
               
                 Al x In 1−x As y P 1−y   
                 0 ≤ x ≤ 1, 0 ≤ y ≤ 1 
                 500 to 3444 
               
               
                 Al x Ga 1−x As y P 1−y   
                 0 ≤ x ≤ 1, 0 ≤ y ≤ 1 
                 500 to 873 
               
               
                 Ga x In 1−x N y As 1−y   
                 0 ≤ x ≤ 1, 0 ≤ y ≤ 1 
                 366 to 3444 
               
               
                   
               
            
           
         
       
     
     In addition, the window layer  43   c  and the barrier layer  43   a  may contain, for example, a material other than the semiconductor material. In addition, the semiconductor material contained in the window layer  43   c  and the barrier layer  43   a  is not limited to AlGaAsP. The semiconductor material contained in the window layer  43   c  and the barrier layer  43   a  may be, for example, AlGaAs, AlGaP, AlGaN, InNAs, GaNAs, InGaAsP, AlInGaAs, AlInGaP, AlInAsP, GaInNAs, AlInAs, AlAsP, AlInGaN, AlGaAsP, AlInNAs, AlGaNAs, AlInGaAsP, AlGaInNAs, or the like. In addition, the semiconductor layer  43  may not include the window layer  43   c  and the barrier layer  43   a.    
     In addition, for example, the antioxidation layer  44  may be configured with only one of the antireflection layer  44   a  and the protective layer  44   b , may have layers other than the antireflection layer  44   a  and the protective layer  44   b , and may be configured with only layers other than the antireflection layer  44   a  and the protective layer  44   b . The antireflection layer  44   a  may contain a component other than Si 3 N 4  and may be made of a component other than Si 3 N 4 . The protective layer  44   b  may contain a component other than SiO 2  and may be made of a component other than SiO 2 . The fluorescent body may not have the antioxidation layer  44 . 
     In addition, for example, the fluorescent body may be made of a fluorescent resin  60 . The fluorescent resin  60  contains a light transmissive resin and a fluorescent substance (and a fluorescent reagent containing the fluorescent substance) dispersed in the light transmissive resin. In the case of using the fluorescent resin  60 , by appropriately selecting the fluorescent substance, the measurement sensitivity in a desired fluorescence wavelength band can be identified. The fluorescent reagent may be in a liquid state and may be in a solid state. As the fluorescent reagent, for example, organic dyes such as Alexa Fluor 647 (manufactured by Thermo Fisher Scientific Inc. “Fluor” is a registered trademark), semiconductor crystals such as Q-dot, metal complexes such as DTBTA-Eu 3+ , or the like may be used. In the case of using the fluorescent resin  60  as the fluorescent body, for example, the light amount of the fluorescence can be adjusted by adjusting the concentration of the fluorescent substance in the fluorescent resin  60 . 
     For example, as illustrated in  FIG. 6( a ) , the fluorescent resin  60  is used in the state where the fluorescent resin is fixed to a fixing member  61 . That is, the reference body is manufactured by accommodating the fixing member  61  in the first accommodating space  14  and the second accommodating space  15 . The fixing member  61  illustrated in  FIG. 6( a )  includes a light transmissive substrate  63  such as glass and a flow path  62  manufactured by using a resin having a light shielding property (for example, one formed by mixing carbon, boron, or the like into a resin such as PDMS (polydimethylsiloxane)). The fluorescent resin  60  can be produced by using, for example, the following method. First, a light-transmitting ultraviolet curable resin and a fluorescent reagent are mixed to prepare a resin composition. Next, by using a pipette, the resin composition is injected into the flow path  62  of the fixing member  61  illustrated in  FIG. 6( a ) . Next, the resin composition is cured by irradiating ultraviolet rays from the light transmissive substrate  63  side of the fixing member  61 . Therefore, the fluorescent resin  60  fixed in the fixing member  61  can be obtained. 
     In addition, for example, the fluorescent body may be configured with a fluorescent glass  70  illustrated in  FIG. 6( b ) . The fluorescent glass  70  contains a glass and a fluorescent substance (and a fluorescent reagent containing the fluorescent substance) dispersed in the glass. In the case of using the fluorescent glass  70 , the measurement sensitivity in a desired fluorescence wavelength band can be identified by appropriately selecting the fluorescent substance. In addition, since the fluorescent glass is not easily oxidized, it is possible to perform calibration with a high accuracy over a long period of time. Examples of the fluorescent reagent are the same as those of the fluorescent reagent used for the fluorescent resin  60 . As the fluorescent glass  70 , for example, commercially available products such as “Lumilass-R7”, “Lumilass-G9”, and “Lumilass-B” manufactured by SUMITA OPTICAL GLASS, Inc. may be used. The above-described fluorescent resin  60  may be a fluorescent body having a shape as illustrated in  FIG. 6( b ) . 
     In addition, for example, the same fluorescent body may be used as the first fluorescent body  20  and the second fluorescent body  30 . In this case, the light amount of the first fluorescence emitted from the first light passing portion  18  and the light amount of the second fluorescence emitted from the second light passing portion  19  are adjusted by a light amount adjusting means other than the means for adjusting the light amount of the fluorescence emitted from the first fluorescent body  20  and the second fluorescent body  30 . Adjustment of the light amount may be performed, for example, by changing the shapes and sizes of the first light passing portion  18  and the second light passing portion  19 . In this case, the shapes of the first light passing portion  18  and the second light passing portion  19  may be slits having a mesh structure. In a case where the first light passing portion  18  and the second light passing portion  19  are light transmitting regions formed in the cover  12 , the adjustment of the light amount may be performed by forming the regions with materials having different light transmissivities. In addition, the adjustment of the light amount may be performed by using the first light transmitting member  2  and the second light transmitting member  3  which are made of materials having different light transmissivities. As the member, an ND filter (neutral density filter) may be exemplified. In addition, the above-described light amount adjusting means may be combined. 
     EXAMPLE 
     Hereinafter, the contents of the present disclosure will be described in more detail by using examples, but the present disclosure is not limited to the following examples. 
     Example 1 
     A GaAs substrate (thickness: 350 μm) was prepared, and a graded layer (thickness: 10 μm), a barrier layer (thickness: 0.1 μm), a light emitting layer (thickness: 0.7 μm), and a window layer (thickness: 0.035 μm) were grown in this order on the substrate. The graded layer was configured as a layer containing Ga, As, Al, and P. The contents of Al and P were adjusted to be increased as approaching from the substrate side toward the bather layer side so as to have the same configuration as the barrier layer in the vicinity of the interface with the barrier layer. In addition, the barrier layer was made of Al y Ga (1-y) As (1-z) Pz (y=0.65, z=0.23), and the light emitting layer was made of GaAs (1-x) P x  (x=0.23), and the window layer was made of Al y Ga (1-y) As (1-z) Pz (y=0.65, z=0.23). Next, as an antioxidation layer, an antireflection layer (thickness: 0.095 μm) and a protective layer (thickness: 0.3 μm) were sequentially formed on the window layer. The antireflection layer was made of Si 3 N 4 , and the protective layer was made of SiO 2 . By the above operation, a semiconductor fluorescent body was obtained. 
     Subsequently, the reference body illustrated in  FIGS. 2 to 4  was produced using the semiconductor fluorescent body obtained in Example 1 as the first fluorescent body and the second fluorescent body. The main body of the reference body was made of a black ABS resin so that the first wall portion and the second wall portion had a light shielding property. An acrylic plate made of a black acrylic resin was used for the cover. An ND filter (extinction ratio: 80%) was used as the first light transmitting member, and an ND filter (extinction ratio: 90%) was used as the second light transmitting member. 
     (Fluorescence Measurement) 
     Fluorescence measurement of the obtained reference body was performed by using the fluorescent immunochromato reader illustrated in  FIG. 1 . For excitation, a 655-nm semiconductor laser was used as the semiconductor light emitting element, and a 670-nm short pass filter was used as the short pass filter. In addition, for the fluorescence measurement, a 690-nm long pass filter was used as a long pass filter, and a Si photodiode was used as a semiconductor light receiving element. Irradiation of excitation light was performed at 1 mW. The results are illustrated in  FIGS. 7 and 8 . 
       FIG. 7  is a diagram illustrating a fluorescence excitation spectrum of the semiconductor fluorescent body itself. Spectrum A illustrates a spectrum of the excitation light irradiated to the semiconductor fluorescent body and spectrum B illustrates a spectrum of fluorescence emitted from the semiconductor fluorescent body by irradiation with the excitation light. In  FIG. 7 , the horizontal axis represents the wavelength, and the vertical axis represents the intensity. In addition, the vertical axis on the left side represents the intensity of the excitation spectrum, and the vertical axis on the right side represents the intensity of the fluorescence spectrum. As illustrated in  FIG. 7 , it was identified that the semiconductor fluorescent body of Example 1 emits fluorescence having a peak at 730 nm by excitation light having a wavelength band of 450 nm to 700 nm. 
       FIG. 8  is a diagram illustrating a fluorescence profile obtained by fluorescence measurement of a reference body using the fluorescent immunochromato reader illustrated in  FIG. 1 . In  FIG. 8 , the vertical axis represents the intensities of the first fluorescence (a in  FIG. 8 ) and the second fluorescence (b in  FIG. 8 ). As illustrated in  FIG. 8 , according to the reference body of Example 1, it is possible to stably identify the measurement sensitivity in a wide dynamic range by one measurement. 
     REFERENCE SIGNS LIST 
       1 : reference body,  2 : first light transmitting member,  3 : second light transmitting member,  4 : first optical bonding material,  5 : second optical bonding material,  10 : support,  11 : main body,  12 : cover,  13 : light shielding portion,  14 : first accommodating space,  15 : second accommodating space,  16 : first wall portion,  17 : second wall portion,  18 : first light passing portion,  18   a : first region,  19 : second light passing portion,  19   a : second region,  20 : first fluorescent body,  30 : second fluorescent body,  40 : semiconductor fluorescent body,  43 : semiconductor layer,  43   b : light emitting layer,  44 : antioxidation layer,  60 : fluorescent resin,  70 : fluorescent glass.