Patent Publication Number: US-2019187125-A1

Title: Measurement method

Description:
TECHNICAL FIELD 
     The present invention relates to a measurement method of measuring a substance to be measured in a specimen containing blood. 
     BACKGROUND ART 
     In clinical examinations, if it is possible to quantitatively detect a fine amount of substance to be measured in a specimen such as protein or DNA at high sensitivity, it becomes possible to rapidly grasp a condition of a patient and treat. For example, in a case of measuring an antigen (substance to be measured) in blood, whole blood collected from the patient or plasma or serum acquired by separating blood cell components from the whole blood may be used as the specimen. In addition, in order to grasp the condition of the patient, it is necessary to measure an amount (concentration) of the substance to be measured with respect to the plasma or serum. However, since a proportion of the plasma or serum to the whole blood varies from patient to patient, in a case where a measurement value indicating the amount of the substance to be measured is acquired by using the whole blood as the specimen, it is necessary to correct the measurement value according to the proportion of the plasma or serum to the whole blood. At that time, a hematocrit value may be used for correcting the measurement value. A method of determining the amount of the substance to be measured on the basis of the hematocrit value and the above-described measurement value is known (for example, refer to Patent Literature 1). 
     In a measurement method disclosed in Patent Literature 1, blood in a specimen is first hemolyzed by an oxidizing agent and a surfactant, and a specimen is diluted. Next, a measurement value indicating an amount of a substance to be measured in the specimen in a state in which blood is hemolyzed is acquired. Next, an amount of hemoglobin in the specimen in the state in which the blood is hemolyzed is measured and a hematocrit value of the specimen is acquired. Finally, the measurement value is corrected on the basis of the hematocrit value. According to the above-described procedure, in the measurement method disclosed in Patent Literature 1, the amount of the substance to be measured in plasma or serum is measured. 
     CITATION LIST 
     Patent Literature 
     Patent Literature 1: JP 2013-036959 A 
     SUMMARY OF INVENTION 
     Technical Problem 
     In the measurement method disclosed in Patent Literature 1, in both a step of acquiring the hematocrit value and a step of acquiring the measurement value, the specimen in the state in which the blood is hemolyzed is used. The inventors of the present invention confirmed by experiments that it is sometimes impossible to accurately measure depending on measurement items when the substance to be measured is measured as for the specimen in the state in which the blood is hemolyzed. This is probably because protease (proteolytic enzyme) in a red blood cell flows out of the red blood cell by hemolysis of the blood and decomposes the substance to be measured. As described above, according to the measurement method disclosed in Patent Literature 1, there is a case in which the amount of the substance to be measured in the specimen cannot be measured with a high degree of accuracy. 
     An object of the present invention is to provide the measurement method capable of measuring the measurement value indicating the amount of the substance to be measured in the specimen and the hematocrit value with a high degree of accuracy and measuring the amount of the substance to be measured in the specimen containing blood with a high degree of accuracy. 
     Solution to Problem 
     In order to solve the above-described problem, a measurement method according to one embodiment of the present invention is a measurement method for measuring an amount of a substance to be measured in a specimen containing blood using a measurement chip including an accommodating unit for accommodating liquid provided with a step of dividing the specimen into a first specimen and a second specimen, a step of hemolyzing the blood in the second specimen, a step of introducing the first specimen containing the substance to be measured into the accommodating unit to acquire a measurement value indicating the amount of the substance to be measured in the first specimen in a state in which the blood is not hemolyzed, a step of detecting second light acquired when first light including light of a wavelength absorbed by a red blood cell passes through the second specimen in the accommodating unit in a state in which the second specimen in a state in which the blood is hemolyzed is present in the accommodating unit of the measurement chip, a step of determining a hematocrit value of the specimen on the basis of a detection result of the second light, and a step of correcting the measurement value on the basis of the hematocrit value. 
     Advantageous Effects of Invention 
     According to the present invention, it is possible to measure the amount of the substance to be measured in the specimen containing blood with a high degree of accuracy. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG. 1  is a flowchart illustrating an example of steps included in a measurement method according to one embodiment of the present invention. 
         FIG. 2  is a flowchart illustrating steps in a measuring step of an optical blank value illustrated in  FIG. 1 . 
         FIG. 3  is a view illustrating an example of configurations of a measurement chip and an SPFS device. 
         FIG. 4A  is a graph illustrating accuracy when determining an amount of a substance to be measured in plasma by an absorbance method, and  FIG. 4B  is a graph illustrating accuracy when determining the amount of the substance to be measured in plasma by an absorbance method. 
     
    
    
     DESCRIPTION OF EMBODIMENTS 
     One embodiment of the present invention is hereinafter described in detail with reference to the drawings. Here, as a representative example of a measurement method according to the present invention, a measurement method utilizing surface plasmon-field enhanced fluorescence spectroscopy (hereinafter abbreviated as “SPFS”) is described. 
       FIG. 1  is a flowchart illustrating an example of steps included in the measurement method according to one embodiment of the present invention.  FIG. 2  is a flowchart illustrating steps in a measuring step of an optical blank value (step S 113 ) illustrated in  FIG. 1 .  FIG. 3  is a view illustrating an example of configurations of a measurement chip  10  and a measurement device (SPFS device)  100  which might be used for implementing the measurement method according to this embodiment. The measurement chip  10  and the SPFS device  100  are described later in detail. 
     The measurement method according to this embodiment is a measurement method in which an amount of a substance to be measured in a specimen containing blood is measured by using the measurement chip  10  including an accommodating unit (flow path  41  in this embodiment) for accommodating liquid. In this embodiment, as a measurement value indicating the amount of the substance to be measured, a fluorescence value which is a light amount of fluorescence β (signal) emitted from a fluorescent substance labeling the substance to be measured is measured. 
     The measurement method according to this embodiment includes a step of preparing for measurement (step S 110 ), a step of dispensing and diluting the specimen (step S 111 ), a step of setting an incident angle to an enhancement angle (step S 112 ), a step of measuring the optical blank value (step S 113 ), a step of performing a primary reaction (step S 114 ), a step of performing a secondary reaction (step S 115 ), a step of measuring the fluorescence value (step S 116 ), a step of performing hemolysis (step S 117 ), a step of acquiring a hematocrit related value (step S 118 ), a step of determining a hematocrit value (step S 119 ), and a step of correcting the measurement value (step S 120 ). 
     1) Preparation for Measurement 
     First, the measurement is prepared (step S 110 ). Specifically, the measurement chip  10  is installed in a chip holder  142  arranged in an installation position of the SPFS device  100 . Here, the “installation position” is a position for installing the measurement chip  10  in the SPFS device  100 . 
     (Measurement Chip) 
     Here, the measurement chip  10  used in the SPFS device  100  is described.  FIG. 3  is a view for illustrating an example of the configurations of the measurement chip  10  and the SPFS device  100 . The measurement chip  10  includes a prism  20 , a metal film  30 , and a flow path lid  40 . In this embodiment, the flow path lid  40  of the measurement chip  10  is integrated with a liquid chip  50  for accommodating the liquid. 
     The prism  20  includes an incident surface  21 , a film depositing surface  22 , and an emission surface  23 . The incident surface  21  allows excitation light α (referred to as “fourth light” in claims) from an excitation light emitting unit  110  to be described later to enter the prism  20 . Here, the “excitation light” is light which directly or indirectly excites the fluorescent substance. The metal film  30  is arranged on the film depositing surface  22 . The excitation light α entering the prism  20  is reflected by an interface (film depositing surface  22 ) between the prism  20  and the metal film  30  to be reflected light. The emission surface  23  emits the reflected light out of the prism  20 . 
     A shape of the prism  20  is not especially limited. In this embodiment, the shape of the prism  20  is a columnar body having a trapezoid as a bottom surface. A surface corresponding to one bottom side of the trapezoid is the film depositing surface  22 , a surface corresponding to one leg is the incident surface  21 , and a surface corresponding to the other leg is the emission surface  23 . The trapezoid as the bottom surface is preferably an isosceles trapezoid. As a result, the incident surface  21  and the emission surface  23  become symmetrical, and an S wave component of the excitation light α is less likely to stay in the prism  20 . 
     The incident surface  21  is formed such that the excitation light α from the excitation light emitting unit  110  is not reflected by the incident surface  21  to return to a light source of the SPFS device  100 . In a case where a light source of the excitation light α is a laser diode (hereinafter also referred to as “LD”), when the excitation light α returns to the LD, an excited state of the LD is disturbed and a wavelength and an output of the excitation light α fluctuate. Therefore, in a scanning range centered on an ideal resonance angle or enhancement angle, an angle of the incident surface  21  is set so that the excitation light α does not enter the incident surface  21  perpendicularly. 
     Here, the “resonance angle” means the incident angle when a light amount of the reflected light of the excitation light α emitted from the emission surface  23  becomes the minimum in a case of scanning the incident angle of the excitation light α with respect to the metal film  30 . In addition, the “enhancement angle” means the incident angle when a light amount of scattered light having the same wavelength as that of the excitation light α emitted above the measurement chip  10  (hereinafter referred to as “plasmon scattered light γ”) becomes the maximum in a case where the incident angle of the excitation light α with respect to the metal film  30  is scanned. In this embodiment, an angle between the incident surface  21  and the film depositing surface  22  and an angle between the film depositing surface  22  and the emission surface  23  are both approximately 80°. 
     Note that the resonance angle (and the enhancement angle in the close vicinity thereof) is roughly determined by a design of the measurement chip  10 . Design elements are a refractive index of the prism  20 , a refractive index of the metal film  30 , a thickness of the metal film  30 , an extinction coefficient of the metal film  30 , the wavelength of the excitation light α and the like. The resonance angle and the enhancement angle are shifted by the substance to be measured captured on the metal film  30 , but the amount is less than several degrees. 
     The prism  20  is made of a dielectric body transparent to the excitation light α. The prism  20  has a birefringence characteristic more than little. Examples of materials of the prism  20  include resin and glass. Examples of the resin forming the prism  20  include polymethylmethacrylate (PMMA), polycarbonate (PC), and cycloolefin polymer. The material of the prism  20  is preferably resin having a refractive index of 1.4 to 1.6 and small birefringence. 
     The metal film  30  is arranged on the film depositing surface  22  of the prism  20 . As a result, surface plasmon resonance (hereinafter abbreviated as “SPR”) occurs between photons of the excitation light α incident on the film depositing surface  22  under a total reflection condition and free electrons in the metal film  30 , and it is possible to generate localized field light (generally also referred to as “evanescent light” or “near field light”) on a surface of the metal film  30 . The localized field light reaches a distance approximately the wavelength of the excitation light α from the surface of the metal film  30 . The metal film  30  may be formed on an entire surface on the film depositing surface  22  or on a part of the film depositing surface  22 . In this embodiment, the metal film  30  is formed on the entire surface of the film depositing surface  22 . 
     In addition, in this embodiment, the metal film  30  also serves as a reflecting unit which specularly reflects the light which passes through the flow path  41  so as to pass through the flow path  41  again. In this embodiment, the metal film  30  specularly reflects first light δ 1  including light of a wavelength absorbed by a red blood cell entering the measurement chip  10  at the flow path lid  40  toward the flow path lid  40 . 
     On the metal film  30 , a capturing body for capturing the substance to be measured is immobilized. On the metal film  30 , a region in which the capturing body is immobilized is especially referred to as a “reaction field”. The capturing body may be immobilized on an entire surface of the metal film  30  or may be immobilized on a part of the surface. From a viewpoint of suppressing the light which passes through the flow path  41  from being scattered by the capturing body, it is preferable that the capturing body is not immobilized in a region serving as the reflecting unit (a region where light specularly reflects) of the metal film  30 . Also, the capturing body specifically binds to the substance to be measured. Therefore, the substance to be measured might be immobilized on the metal film  30  via the capturing body. 
     A type of the capturing body is not especially limited as long as this may capture the substance to be measured. For example, the capturing body is an antibody (primary antibody) capable of specifically binding to the substance to be measured, a fragment thereof, an enzyme capable of specifically binding to the substance to be measured or the like. 
     The material of the metal film  30  is not especially limited as long as this is metal capable of causing the surface plasmon resonance. Examples of the material of the metal film  30  include gold, silver, copper, aluminum, and alloys thereof. In this embodiment, the metal film  30  is a gold thin film. Although the thickness of the metal film  30  is not especially limited, this is preferably in a range from 20 to 60 nm from a viewpoint of efficiently causing the SPR. A method of forming the metal film  30  is not especially limited. Examples of the method of forming the metal film  30  include sputtering, vapor deposition, and plating. 
     The flow path lid  40  is arranged on the metal film  30 . In a case where the metal film  30  is formed only on a part of the film depositing surface  22  of the prism  20 , the flow path lid  40  may also be arranged on the film depositing surface  22 . In this embodiment, the flow path lid  40  is arranged on the metal film  30 . By arranging the flow path lid  40  on the metal film  30 , the accommodating unit for accommodating liquid is formed on the metal film  30 . In this embodiment, the accommodating unit is the flow path  41  through which the liquid flows. The flow path  41  includes a bottom surface, a top surface, and a pair of side surfaces connecting the bottom surface and the top surface. In this specification, a surface of the flow path  41  on the prism  20  side is referred to as the “bottom surface of the flow path”, and a surface of the flow path  41  opposed to the bottom surface of the flow path  41  is referred to as the “top surface of the flow path”. Also, an interval between the bottom surface of the flow path  41  and the top surface of the flow path  41  is set as a height of the flow path  41 . Since the height of the flow path  41  might be precisely managed, it is preferable that the accommodating unit is the flow path  41  from a viewpoint of precisely managing an optical path length of the flow path  41  related to absorption of the light by the specimen. 
     A recess (flow path groove) is formed on a rear surface of the flow path lid  40 . The flow path lid  40  is arranged on the metal film  30  (and the prism  20 ), and an opening of the recess is closed by the metal film  30 , so that the flow path  41  is formed. From a viewpoint of sufficiently securing a region where the localized field light reaches, it is preferable that the height of the flow path  41  (a depth of the flow path groove) is large to some extent. From a viewpoint of reducing an amount of impurities mixed in the flow path  41 , the height of the flow path  41  (the depth of the flow path groove) is preferably small. From such a viewpoint, the height of the flow path  41  is preferably in a range from 0.05 to 0.15 mm. Both ends of the flow path  41  are connected to an injection port and a discharge port not illustrated formed on the flow path lid  40  so as to allow the inside and the outside of the flow path  41  to communicate with each other. 
     The flow path lid  40  is preferably formed of a material transparent to the light (fluorescence β and plasmon scattered light γ) emitted from an upper side of the metal film  30  and the first light δ 1  (and the light having the same wavelength as that of the first light δ 1 ) including the light of the wavelength absorbed by the red blood cell emitted toward the metal film  30 . The fact that the material of the flow path lid  40  is transparent to the first light δ 1  is preferable from a viewpoint of suppressing scattering of light which becomes noise and measuring the hematocrit value with a high degree of accuracy. Examples of the material of the flow path lid  40  include glass and resin. Examples of the resin include polymethylmethacrylate resin (PMMA). Also, the other part of the flow path lid  40  may be formed of an opaque material as long as this is transparent to the above-described light. The flow path lid  40  is joined to the metal film  30  or the prism  20  by, for example, bonding with a double-sided tape, an adhesive and the like, laser welding, ultrasonic welding, crimping using a clamp member and the like. 
     The measurement chip  10  is usually exchanged for each measurement is made. Also, the measurement chip  10  is preferably a structure a length of each piece of which is several millimeters to several centimeters, but this may also be a smaller structure or a larger structure not included in a category of “chip”. 
     Note that, in a case where a stored reagent is present on the metal film  30  of the measurement chip  10 , the stored reagent is removed by washing the metal film  30  so that the capturing body may appropriately capture the substance to be measured. 
     2) Dispensing and Dilution of Specimen 
     Then, the specimen is dispensed and diluted (step S 111 ). Specifically, the specimen is divided into a first specimen for measurement value (fluorescence value) measurement and a second specimen for hematocrit value measurement. At step S 111 , the first specimen is further diluted from a viewpoint of measurement accuracy and measurement sensitivity. Unless the first specimen is diluted, an amount of absorption (nonspecific adsorption) of impurities in the specimen to the capturing body increases and noise increases, and as a result, the measurement accuracy might be deteriorated. In addition, in a case where the amount of the substance to be measured is too large as compared with an amount of the capturing body, the amount of the substance to be measured which may be captured by the capturing body is saturated, and it becomes impossible to specify concentration of a highly concentrated specimen. As a diluent, for example, physiological saline may be used. The first specimen is diluted 1 to 500 times, for example. 
     It is preferable that the dispensing of the specimen is performed before a blood cell component in the specimen settles out. By dividing the specimen into the first specimen and the second specimen before the specimen is separated into a blood cell component and a supernatant component, a blood cell amount contained in the first specimen and a blood cell amount contained in the second specimen may be made equivalent to each other. From such a viewpoint, it is preferable that the dispensing of the specimen is performed after sufficiently mixing the specimen in a container in which blood is stored, for example, within a blood collecting tube. In addition, it is preferable that the specimen is dispensed in the SPFS device  100  before the primary reaction (step S 114 ); for example, this is preferably performed within 10 minutes before the step of performing the primary reaction. 
     3) Set Incident Angle to Enhancement Angle 
     Next, the incident angle of the excitation light α with respect to the metal film  30  (film depositing surface  22 ) is set to the enhancement angle (step S 112 ). Specifically, first, reference liquid transparent to the excitation light α is provided in the flow path  41 . Next, in a state in which the reference liquid is present in the flow path  41 , the excitation light α is applied to a rear surface of the metal film  30  corresponding to the region where the capturing body is immobilized via the prism  20  while scanning the incident angle of the excitation light α with respect to the metal film  30  and the plasmon scattered light γ generated in the measurement chip  10  is detected. As a result, data including a relationship between the incident angle of the excitation light α and the light amount of the plasmon scattered light γ is acquired. By analyzing the acquired data, the enhancement angle which is the incident angle at which the light amount of the plasmon scattered light γ becomes the maximum might be determined. Finally, the incident angle of the excitation light α with respect to the metal film  30  (film depositing surface  22 ) is set to the determined enhancement angle. 
     Note that the enhancement angle is determined by the material and shape of the prism  20 , the thickness of the metal film  30 , the refractive index of the liquid in the flow path  41  and the like, but this slightly fluctuates by various factors such as the type and amount of the capturing body in the flow path  41  and an error in shape of the prism  20 . Therefore, it is preferable to determine the enhancement angle each time the measurement is performed. The enhancement angle is determined on the order of approximately 0.1°. 
     4) Measurement of Optical Blank Value 
     Next, the optical blank value is measured (step S 113 ). In this embodiment, the optical blank value includes a first blank value used to determine the fluorescence value (measurement value) and a second blank value used to determine the hematocrit value. Here, the “first blank value” means the amount of light of the same wavelength as that of the fluorescence β emitted above the measurement chip  10  in a state in which the reference liquid transparent to the first light δ 1  is present in the flow path  41 . Also, the “second blank value” means an amount of third light δ 3  acquired when the first light δ 1  passes through the reference liquid in the flow path  41  when the first light δ 1  is applied to the metal film  30  in a state in which the reference liquid is present in the flow path  41 . 
     In this embodiment, the reference liquid is transparent to the excitation light α and the first light δ 1 . Note that a refractive index of the reference liquid is preferably the same as or equivalent to a refractive index of the specimen. As a result, it becomes possible to make reflectance of the first light δ 1  on the bottom surface of the flow path groove (recess) of the flow path lid  40  (top surface of the flow path) the same or equivalent and to make reflectance of the light on the surface of the metal film  30  (reflecting unit) the same or equivalent between a case in which the reference liquid is present in the flow path  41  and a case in which the specimen is present in the flow path  41 . 
     As illustrated in  FIG. 2 , at step S 113 , the first blank value is first measured (step S 1131 ). At the same time as the metal film  30  (film depositing surface  22 ) is irradiated with the excitation light α, the light amount of the light of substantially the same wavelength as the fluorescence β is detected. As a result, it is possible to measure the light amount (first blank value) of light which becomes noise in the measurement of the fluorescence value (step S 116 ). 
     Next, the second blank value is measured (step S 1132 ). In a state in which the reference liquid is present in the flow path  41 , the first light δ 1  is applied to the surface of the metal film  30  corresponding to the region where the capturing body is immobilized via the flow path lid  40 , and at the same time, the third light δ 3  reflected by the metal film  30  is detected. As a result, it is possible to measure the light amount (second blank value) of light which becomes noise in the measurement of the hematocrit value. The second blank value may be absorbance OD 1  of the reference liquid represented by following equation (1). 
     
       
         
           
             
               
                 
                   
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     [In equation (1) above, OD 1  represents the absorbance of the reference liquid, I 0  represents the light amount of the first light δ 1 , and I 1  represents the light amount of the third light δ 3 .] 
     5) Primary Reaction 
     Next, the substance to be measured in the specimen and the capturing body on the metal film  30  are allowed to react (primary reaction; step S 114 ). Specifically, the reference liquid in the flow path  41  is discharged and the first specimen diluted at step S 111  is provided in the flow path  41 . As a result, in a case where the substance to be measured is present in the specimen, at least a part of the substance to be measured may be captured by the capturing body on the metal film  30 . Note that the first specimen is not hemolyzed. Thereafter, the interior of flow path  41  is washed with a buffer solution and the like to remove substances not captured by the capturing body. Note that examples of the substance to be measured include troponin, myoglobin, and creatine kinase-MB (CK-MB). 
     6) Secondary Reaction 
     Subsequently, the substance to be measured captured by the capturing body on the metal film  30  is labeled with the fluorescent substance (secondary reaction; step S 115 ). Specifically, a fluorescent labeling solution is provided in the flow path  41 . As a result, the substance to be measured may be labeled with the fluorescent substance. The fluorescent labeling solution is, for example, a buffer solution containing an antibody (secondary antibody) labeled with the fluorescent substance. Thereafter, the interior of the flow path  41  is washed with the buffer solution and the like to remove free fluorescent substances and the like. 
     7) Measurement of Fluorescence Value 
     Next, the fluorescence emitted from the fluorescent substance labeling the substance to be measured in the reaction field is detected and the fluorescence value is measured (step S 116 ). First, the buffer solution for measurement is provided in the flow path  41 . In a state in which the substance to be measured contained in the first specimen is immobilized and the first specimen and the second specimen are not present, the excitation light α is applied to the rear surface of the metal film  30  corresponding to the region where the capturing body is immobilized via the prism  20  at the incident angle at which the surface plasmon resonance occurs. At the same time, the fluorescence β (signal) generated in the measurement chip  10  is detected. As a result, it is possible to acquire the fluorescence value (measurement value) which is the light amount of the fluorescence β which indicates the amount of the substance to be measured in the first specimen in a state in which blood is not hemolyzed. Note that, in this specification, “a state in which no specimen is present” means a state in which operation of removing the specimen from the flow path  41  is performed. That is, it suffices that there is substantially no specimen in the flow path  41 , and a small amount of specimen which cannot be removed may be left in the flow path  41 . 
     8) Hemolysis and Dilution 
     Next, blood in the second specimen dispensed at step S 111  is hemolyzed and diluted (step S 117 ). 
     Specifically, a hemolytic agent is provided in the second specimen. As a result, the blood in the second specimen may be hemolyzed and diluted. At that time, the second specimen is diluted, for example, 1 to 20 times. At that time, in a case where a dilution ratio of the second specimen is one, this means that the second specimen is not diluted. The larger the dilution ratio, the smaller an amount of light absorbed by the specimen. Therefore, when the dilution ratio is too large, sufficient measurement resolution cannot be acquired. 
     In addition, a type of the hemolytic agent is not especially limited as long as this may hemolyze blood, and this may be appropriately selected from known hemolytic agents. The hemolytic agent is, for example, a surfactant. Examples of types of surfactant include an anionic surfactant, a cationic surfactant, and a nonionic surfactant. Among them, the surfactant is preferably the anionic surfactant. 
     9) Acquisition of Hematocrit Related Value 
     Next, the hematocrit related value is acquired (step S 118 ). Specifically, the specimen in the state in which blood is hemolyzed is provided in the flow path  41 . Next, in a state in which the second specimen in a state in which the blood is hemolyzed is present in the flow path  41 , at the same time as the first light δ 1  is applied to the metal film  30 , second light δ 2  acquired when this passes through the second specimen in the flow path  41 , reflected by the metal film  30 , and passes through again the second specimen in the flow path  41  is detected. As a result, the light amount of the second light δ 2  may be measured. Note that the light amount of the second light δ 2  may be absorbance OD 2  of the second specimen represented by following equation (2). 
     
       
         
           
             
               
                 
                   
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                       2 
                     
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                     2 
                   
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     [In equation (2) above, OD 2  represents the absorbance of the second specimen, I 0  represents the light amount of the first light δ 3 , and I 2  represents the light amount of the second light δ 2 .] 
     Next, the hematocrit value determined on the basis of a detection result of the second light δ 2  is corrected on the basis of a detection result of the third light δ 3 . The absorbance OD 2  of the second specimen includes a signal component resulting from the absorption of the light by the second specimen and a noise component (second blank value) caused by other factors. Therefore, by subtracting the noise component (second blank value) acquired at step S 1132  from the absorbance OD 2  of the specimen acquired at step S 118 , the signal component may be calculated. On the basis of the measurement value (the light amount of the second light δ 2  or the absorbance OD 2  of the specimen) acquired at step S 118  and the measurement value acquired at the step S 1132  (the light amount of the third light δ 3  or the absorbance OD 1  of the reference liquid), a hematocrit related value Hct′ represented by following equation (3) may be calculated. 
     
       
         
           
             
               
                 
                   
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     [In equation (3) above, Hct′ represents the hematocrit related value, I 1  represents the light amount of the third light δ 3 , and I 2  represents the light amount of the second light δ 2 .] 
     Note that the absorbance of the specimen (hemoglobin) changes according to the wavelength of the first light δ 1 . It is preferable to adjust temperature of the light source of the first light δ 1  to be kept constant from a viewpoint of stabilizing the wavelength of the first light δ 1  and measuring the absorbance with a high degree of accuracy. 
     10) Determination of Hematocrit Value 
     Next, on the basis of the detection result of the second light δ 2 , the hematocrit value is determined (step S 119 ). In this embodiment, a hematocrit value Hct is calculated by multiplying the hematocrit related value acquired at step S 118  by a predetermined correction coefficient. 
     Note that, as described above, the absorbance changes according to the wavelength of the first light δ 1 . Therefore, from a viewpoint of acquiring a more correct hematocrit value, it is preferable to correct the hematocrit value on the basis of the wavelength of the first light δ 1 . Consider, for example, a case where the hematocrit value is calculated with a reference value of the wavelength of the first light δ 1  set to 520 nm. In this case, when the wavelength of the second light δ 2  detected is 530 nm, the hematocrit value Hct may be corrected so as to be the value when the wavelength of the first light δ 1  is 520 nm in consideration of a shift amount of an absorption rate (absorption amount) of the red blood cell corresponding to a shift amount (10 nm) between the measurement value and the reference value. 
     In addition, the hematocrit value may also be corrected on the basis of the height of the flow path  41  or the optical path length in the flow path  41  of the light which becomes the second light δ 2 . Consider, for example, a case where the hematocrit value is calculated with the reference value of the height of the flow path  41  set to 100 μm. In this case, when the measurement value of the height of the flow path  41  is 110 μm, the hematocrit value may be corrected so as to be the value when the height of the flow path  41  is 100 μm in consideration of a change amount of the absorption rate (absorption amount) of the red blood cell corresponding to the shift amount (10 μm) between the measurement value and the reference value, that is, the shift amount of the height of the flow path  41 . 
     11) Correction of Measurement Value 
     Finally, the measurement value (fluorescence value) is corrected on the basis of the hematocrit value (step S 120 ). The fluorescence value includes the fluorescent component (signal component) derived from the fluorescent substance which labels the substance to be measured and the noise component (first blank value) caused by the factors other than the fluorescent substance. Therefore, by subtracting the first blank value acquired at step S 1131  from the fluorescence value acquired at step S 116 , it is possible to calculate the measurement value (signal component) indicating the amount of the substance to be measured. Furthermore, by multiplying the calculated measurement value by a conversion coefficient c expressed by following equation (4), the calculated measurement value may be converted into the amount of the substance to be measured in plasma. 
     
       
         
           
             
               
                 
                   
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     [In equation (4) above, Hct represents the hematocrit value (0 to 100%) and df represents the dilution ratio of the diluent.] 
     By the above-described procedure, the amount (concentration) of the substance to be measured in the plasma may be determined. 
     Note that, in this embodiment, a mode in which the step of detecting the second light δ 2  (acquisition of the hematocrit related value; step S 118 ) is performed after the step of acquiring the measurement value (measurement of the fluorescence value; step S 116 ) is described. In a case where the detection of the second light δ 2  and the detection of the fluorescence β are performed in the same measurement chip  10  (flow path  41 ), it is preferable to perform the step of detecting the second light δ 2  after the step of acquiring the measurement value from a viewpoint of acquiring measurement values with a high degree of accuracy. This is because it is possible to suppress the reaction between the substance to be measured in the second specimen to which the hemolytic agent is added and the capturing body on the metal  30  film by performing the step of detecting the second light δ 2  after the step of acquiring the measurement value. 
     Also, in this embodiment, a mode in which the step of hemolyzing the blood in the second specimen (step S 117 ) is performed after the step of acquiring the measurement value (measurement of the fluorescence value; step S 116 ) is described. In a case where a pipette handling the hemolytic agent and a pipette handling the specimen are the same, it is preferable that the step of hemolyzing the blood in the second specimen (step S 117 ) is performed after the step of acquiring the measurement value (measurement of the fluorescence value; step S 116 ) from a viewpoint of acquiring the measurement value with a high degree of accuracy. This is because, since the step of hemolyzing the blood in the second specimen is performed after the step of acquiring the measurement value, it is possible to prevent the hemolytic agent from remaining in the pipette and prevent the remaining hemolytic agent from mixing into the first specimen. 
     Also, in this embodiment, a mode in which the step of dispensing the specimen (step S 111 ) is performed before the step of the primary reaction (step S 114 ) is described. The fact that the step of dispensing the specimen is performed within 10 minutes before the step (primary reaction step) of providing the first specimen in the flow path  41  is preferable from a viewpoint of determining the amount of the substance to be measured in plasma or serum with a high degree of accuracy. This is because, since the step of dispensing the specimen is performed within 10 minutes before the step of the primary reaction, it is possible to dispense the specimen before the blood cell component settles out, and make the hematocrit value of the first specimen and the hematocrit value of the second specimen the same or equivalent. As a result, it is possible to accurately correct the measurement value acquired using the first specimen on the basis of the hematocrit value acquired using the second specimen. 
     Also, from a viewpoint of measuring the hematocrit value with a high degree of accuracy, the measurement of the second blank value may be performed after the measurement of the fluorescence value (step S 116 ). This may shorten a time interval between the measurement of the second blank value (step S 1132 ) and the acquisition of the hematocrit related value (step S 118 ), thereby making an effect of fluctuation in power of the light source or fluctuation in wavelength of the first light δ 1  caused by the change in temperature of the light source. Furthermore, it is conceivable that a scattering state of the first light δ 1  on the surface of the metal film  30  changes due to the primary reaction and the secondary reaction, but by measuring the second blank value (step S 1132 ) and acquiring the hematocrit related value (step S 118 ) after the first reaction and the second reaction, there is no effect of the change in the scattering state described above. From such a viewpoint, it is preferable to measure the second blank value (step S 1132 ) after the measurement of the fluorescence value (step S 116 ). 
     Also, in the above-described embodiment, a mode in which the step of setting the incident angle to the enhancement angle (step S 112 ), the step of measuring the optical blank value (step S 113 ), and the step of performing the primary reaction (step S 114 ) are performed in this order is described. However, in the measurement method according to the present invention, the order is not limited to this. For example, the incident angle may be set to the enhancement angle after the primary reaction is performed, or the primary reaction may be performed after measuring the optical blank value. 
     Also, in the description above, after the step of performing the primary reaction (step S 114 ), the step of performing the secondary reaction (step S 115 ) is performed (two-step method). However, a timing of labeling the substance to be measured with the fluorescent substance is not especially limited. For example, before introducing a specimen solution into the flow path  41  of the measurement chip  10 , the labeling solution may be added to the specimen solution to label the substance to be measured with the fluorescent substance in advance. Alternatively, the specimen solution and the labeling solution may also be simultaneously injected into the flow path  41  of the measurement chip  10 . In the former case, the substance to be measured labeled with the fluorescent substance is captured by the capturing body by injecting the specimen solution into the flow path  41  of the measurement chip  10 . In the latter case, the substance to be measured is labeled with the fluorescent substance and the substance to be measured is captured by the capturing body. In either case, both the primary reaction and the secondary reaction may be completed by introducing the specimen solution into the flow path  41  of the measurement chip  10  (one-step method). 
     (SPFS Device) 
     Next, an example of the SPFS device which operates in accordance with the measurement method according to this embodiment and may measure the substance to be measured in the specimen is described.  FIG. 3  is a configuration diagram illustrating an example of the configuration of the SPFS device  100 . The SPFS device  100  includes the excitation light emitting unit  110 , a signal detecting unit  120 , a liquid sending unit  130 , a transporting unit  140 , a light emitting unit  150 , a light detecting unit  160 , and a control processing unit (processing unit)  170 . The excitation light emitting unit  110  and the signal detecting unit  120  form a measurement value acquiring unit for acquiring the measurement value indicating the amount of the substance to be measured in the specimen. The light emitting unit  150  and the light detecting unit  160  form a hematocrit value acquiring unit for acquiring the hematocrit value of the specimen. 
     In  FIG. 3 , the light emitting unit  150  and the light detecting unit  160  are arranged along a paper surface for the sake of convenience, but the light emitting unit  150  and the light detecting unit  160  are arranged in a direction perpendicular to the paper surface of  FIG. 3 . Note that, in  FIG. 3 , an optical axis of light δ′ is indicated by a dotted line for illustrating that an optical axis of the first light δ 1  and the optical axis of the light δ′ are included within a plane perpendicular to the paper surface of  FIG. 3 . 
     The SPFS device  100  is used in a state in which the above-described measurement chip  10  is mounted on the chip holder (holder)  142  of the transporting unit  140 . As described above, the measurement chip  10  includes the prism  20 , the metal film  30 , and the flow path lid  40 . The flow path lid  40  of the measurement chip  10  is integrated with the liquid chip  50 . The prism  20  includes an incident surface  21 , a film depositing surface  22 , and an emission surface  23 . The flow path  41  is formed between the prism  20  and the flow path lid  40 . 
     The excitation light emitting unit  110  emits the excitation light α (fourth light). When the fluorescence β is detected, the excitation light emitting unit  110  emits a P wave to the metal film  30  toward the incident surface  21  such that the surface plasmon resonance occurs on the metal film  30 . The excitation light α is light which generates the localized field light exciting the fluorescent substance on the surface of the metal film  30  when this is applied to the metal film  30  via the prism  20  at the angle at which the surface plasmon resonance occurs. The excitation light emitting unit  110  includes a first light source unit  111 , an angle adjusting mechanism  112 , and a first light source control unit  113 . 
     The first light source unit  111  emits light collimated and having constant wavelength and light amount so that a shape of an irradiation spot on the rear surface of the metal film  30  is substantially circular. The first light source unit  111  includes, for example, a light source, a beam shaping optical system, an APC mechanism, and a temperature adjusting mechanism (none of them is illustrated). 
     A type of the light source is not especially limited, and is, for example, the laser diode (LD). Other examples of the light source include laser light sources such as light emitting diodes and mercury lamps. The wavelength of the excitation light α emitted from the light source is, for example, in a range of 400 nm to 1000 nm. In a case where the excitation light α emitted from the light source is not a beam, the excitation light α is converted into the beam by a lens, a mirror, a slit and the like. Also, in a case where the excitation light α emitted from the light source is not monochromatic light, the excitation light α is converted into the monochromatic light by a diffraction grating and the like. Furthermore, in a case where the excitation light α emitted from the light source is not linear polarization, the excitation light α is converted into linear polarization light by a polarizer and the like. 
     The beam shaping optical system includes, for example, a collimator, a band pass filter, a linear polarization filter, a half wavelength plate, a slit, a zoom means and the like. The beam shaping optical system may include all of them or a part of them. 
     The collimator collimates the excitation light α emitted from the light source. 
     The band pass filter converts the excitation light α emitted from the light source into narrow band light having only a central wavelength. This is because the excitation light α emitted from the light source has a slight wavelength distribution width. 
     The linear polarization filter makes the excitation light α emitted from the light source the linear polarization light. 
     The half wavelength plate adjusts a polarization direction of the light so that the P wave component is incident on the metal film  30 . 
     The slit and the zoom means adjust a beam diameter, a contour shape and the like of the excitation light α emitted from the light source so that the shape of the irradiation spot on the rear surface of the metal film  30  becomes a circle of a predetermined size. 
     The APC mechanism controls the light source so that an output of the light source is constant. More specifically, the APC mechanism detects an amount of light branched from the excitation light α with a photodiode not illustrated and the like. Then, the APC mechanism controls input energy by a recurrent circuit, thereby controlling the output of the light source to be constant. 
     The temperature adjusting mechanism is, for example, a heater, a Peltier element and the like. The wavelength and energy of the excitation light α emitted from the light source might fluctuate depending on the temperature. Therefore, by keeping the temperature of the light source constant by the temperature adjusting mechanism, the wavelength and energy of the excitation light α emitted from the light source are controlled to be constant. 
     The angle adjusting mechanism  112  adjusts the incident angle of the excitation light α with respect to the metal film  30  (interface (film depositing surface  22 ) between the prism  20  and the metal film  30 ). In order to apply the light at a predetermined incident angle to a predetermined position of the metal film  30  via the prism  20 , the angle adjusting mechanism  112  relatively rotates the optical axis of the excitation light α emitted from the light source and the chip holder  142 . For example, the angle adjusting mechanism  112  rotates the first light source unit  111  around an axis orthogonal to the optical axis of the excitation light α on the metal film  30  (axis perpendicular to the paper surface of  FIG. 3 ). At that time, a position of a rotational axis is set such that a position of the irradiation spot on the metal film  30  scarcely changes even if the incident angle is scanned. Especially, displacement of the irradiation position may be minimized by setting a position of a rotational center in the vicinity of an intersection (between the irradiation position on the film depositing surface  22  and the incident surface  21 ) of the optical axes of the excitation light α emitted from two light sources at both ends of a scanning range of the incident angle. 
     As described above, the angle at which the light amount of the plasmon scattered light γ becomes maximum out of the incident angle of the excitation light α emitted from the light source to the metal film  30  is the enhancement angle. By setting the incident angle of the excitation light α emitted from the light source to the enhancement angle or the angle in the vicinity thereof, high-intensity fluorescence β and plasmon scattered light γ may be detected. Although a basic incident condition of the excitation light α emitted from the light source is determined by the material and shape of the prism  20 , the thickness of the metal film  30 , the refractive index of the liquid in the flow path  41  and the like, an optimal incident condition slightly fluctuates depending on the type and amount of the capturing body in the flow path  41 , the error in shape of the prism  20  and the like. Therefore, it is preferable to acquire an optimum enhancement angle for each measurement. 
     The first light source control unit  113  controls various devices included in the first light source unit  111  to control the emission of the excitation light α from the first light source unit  111 . The first light source control unit  113  is formed of, for example, a well-known computer or microcomputer including an arithmetic device, a control device, a storage device, an input device, and an output device. 
     The signal detecting unit  120  detects a signal (for example, fluorescence β, reflected light, or plasmon scattered light γ) generated in the measurement chip  10  when the excitation light emitting unit  110  applies the excitation light α to the metal film  30  at the incident angle at which the surface plasmon resonance occurs via the prism  20  in a state in which the substance to be measured in the specimen is present on the metal film  30 . The substance to be measured may be immobilized in the flow path  41  or not. In this embodiment, the signal detecting unit  120  detects the above-described signal in a state in which the substance to be measured contained in the specimen is immobilized on the metal film  30  and the specimen is not present in the flow path  41 . The signal detecting unit  120  outputs a signal indicating a detected signal amount (for example, the light amount of the fluorescence β, the light amount of the reflected light δ′ or the light amount of the plasmon scattered light γ) to the control processing unit  170 . The signal detecting unit  120  includes a light receiving optical system unit  121 , a position switching mechanism  122 , and a first sensor control unit  127 . 
     The light receiving optical system unit  121  is arranged on a normal to the metal film  30  of the measurement chip  10 . The light receiving optical system unit  121  includes a first lens  123 , an optical filter  124 , a second lens  125 , and a first light receiving sensor  126 . 
     The position switching mechanism  122  switches a position of the optical filter  124  on the optical path or out of the optical path in the light receiving optical system unit  121 . Specifically, when the first light receiving sensor  126  detects the fluorescence β, the optical filter  124  is arranged on the optical path of the light receiving optical system unit  121 , and when the first light receiving sensor  126  detects the plasmon scattered light γ, the optical filter  124  is arranged outside the optical path of the light receiving optical system unit  121 . 
     The first lens  123  is, for example, a condensing lens, and condenses light (signal) emitted from the upper side of the metal film  30 . The second lens  125  is, for example, an image forming lens, and forms an image of the light condensed by the first lens  123  on a light receiving surface of the first light receiving sensor  126 . Between the two lenses, the light is a substantially parallel light flux. 
     The optical filter  124  is arranged between the first lens  123  and the second lens  125 . When detecting fluorescence, the optical filter  124  transmits only the fluorescent component out of the light incident on the optical filter  124  and removes an excitation light component (plasmon scattered light γ). As a result, it is possible to guide only the fluorescent component to the first light receiving sensor  126  and detect the fluorescence β with a high S/N ratio. Examples of types of the optical filter  124  include an excitation light reflecting filter, a short wavelength cutting filter, and a band pass filter. Examples of the optical filter  124  include a filter including a multilayer film which reflects a predetermined light component and a color glass filter which absorbs a predetermined light component. 
     The first light receiving sensor  126  detects the fluorescence β and the plasmon scattered light γ. The first light receiving sensor  126  has high sensitivity capable of detecting weak fluorescence β from a minute amount of substance to be measured. The first light receiving sensor  126  is, for example, a photomultiplier tube (PMT), an avalanche photodiode (APD), a silicon photodiode (SiPD) and the like. 
     The first sensor control unit  127  controls detection of an output value of the first light receiving sensor  126 , management of sensitivity of the first light receiving sensor  126  by the output value, change in the sensitivity of the first light receiving sensor  126  for acquiring an appropriate output value and the like. The first sensor control unit  127  is formed of, for example, a well-known computer or microcomputer including an arithmetic device, a control device, a storage device, an input device, and an output device. 
     The liquid sending unit  130  supplies the liquid in the liquid chip  50  into the flow path  41  of the measurement chip  10  held by the chip holder  142 . Also, the liquid sending unit  130  removes liquid from the flow path  41  of the measurement chip  10 . Furthermore, the liquid sending unit  130  dispenses and dilutes the liquid in the liquid chip  50 . The liquid sending unit  130  includes a pipette  131  and a pipette control unit  135 . 
     The pipette  131  includes a syringe pump  132 , a nozzle unit  133  connected to the syringe pump  132 , and a pipette chip  134  attached to a tip end of the nozzle unit  133 . Reciprocating motion of a plunger in the syringe pump  132  quantitatively sucks and discharges the liquid in the pipette chip  134 . 
     The pipette control unit  135  includes a driving device of the syringe pump  132  and a moving device of the nozzle unit  133 . The driving device of the syringe pump  132  is a device for reciprocating the plunger of the syringe pump  132  and includes, for example, a stepping motor. For example, the moving device of the nozzle unit  133  freely moves the nozzle unit  133  in a vertical direction. The moving device of the nozzle unit  133  is formed of, for example, a robot arm, a two-axis stage, or a vertically movable turntable. 
     The pipette control unit  135  drives the syringe pump  132  to suck various types of liquid from the liquid chip  50  into the pipette chip  134 . Then, the pipette control unit  135  moves the nozzle unit  133  to insert the pipette chip  134  into the flow path  41  of the measurement chip  10 , and drives the syringe pump  132  to inject the liquid in the pipette chip  134  into the flow path  41 . Also, after introducing the liquid, the pipette control unit  135  drives the syringe pump  132  to suck the liquid in the flow path  41  into the pipette chip  134 . By sequentially exchanging the liquid in the flow path  41  in this manner, the capturing body and the substance to be measured are allowed to react in the reaction field (primary reaction) and the substance to be measured and the capturing body labeled with the fluorescent substance are allowed to react (secondary reaction). Also, the liquid sending unit  130  sucks or discharges the liquid in the liquid chip  50  in the above-described manner, thereby dispensing or diluting the specimen. 
     The transporting unit  140  transports the measurement chip  10  to fix. The transporting unit  140  includes a transporting stage  141  and the chip holder  142 . 
     The transporting stage  141  moves the chip holder  142  in one direction and in the opposite direction. The transporting stage  141  also has a shape which does not interfere with the optical paths of the light such as the excitation light α, the reflected light of the excitation light α, the fluorescence β, the plasmon scattered light γ, the first light δ 1 , and the reflected light δ′ of the first light δ 1  (second light δ 2  and third light δ 3 ). The transporting stage  141  is driven by, for example, a stepping motor and the like. 
     The chip holder  142  is fixed to the transporting stage  141  and detachably holds the measurement chip  10 . The chip holder  142  has a shape capable of holding the measurement chip  10  which does not interfere with the optical paths of the light such as the excitation light α, the reflected light of the excitation light α, the fluorescence β, the plasmon scattered light γ, the first light δ 1 , and the reflected light δ′ of the first light δ 1 . For example, the chip holder  142  is provided with an opening through which the above-described light passes. 
     The light emitting unit  150  emits the first light δ 1  including light of a wavelength absorbed by the red blood cell. In this embodiment, the light emitting unit  150  emits the first light δ 1  from the flow path  41  side toward the metal film  30 . It is preferable that the first light δ 1  contains light of a wavelength absorbed by hemoglobin contained in the red blood cell. The light emitting unit  150  includes a second light source unit  151  and a second light source control unit  152 . 
     The second light source unit  151  emits the first light δ 1  which is collimated and has constant wavelength and light amount toward the metal film  30 . The second light source unit  151  includes, for example, a light source, a collimator, an APC mechanism, and a temperature adjusting mechanism (none of them is illustrated). The collimator, the APC mechanism, and the temperature adjusting mechanism are similar to the collimator, the APC mechanism, and the temperature adjusting mechanism of the first light source unit  111 , so that the description thereof is omitted. From a viewpoint of suppressing variation in wavelength of the first light δ 1  due to temperature change of the light source, the second light source unit  151  preferably includes the temperature adjusting mechanism. 
     From a viewpoint of applying the first light δ 1  to an inside of an outer edge of the metal film  30  and suppressing reduction in energy efficiency due to irradiation of the first light δ 1  in a region other than the metal film  30 , the light source is preferably the laser light source. The laser light source may irradiate the metal film  30  with the first light δ 1  in a smaller irradiation spot as compared with that of a light source having low directivity such as an LED. The light source is, for example, a laser diode (LD). Other examples of the light source include laser light sources such as light emitting diodes and mercury lamps. A central wavelength of the first light δ 1  from the light emitting unit  150  is, for example, 500 to 650 nm. In a case where the first light δ 1  emitted from the light source is not a beam, the first light δ 1  is converted into a beam by a lens, a mirror, a slit and the like. 
     The second light source control unit  152  controls various devices included in the second light source unit  151  and controls emission of the first light δ 1  from the second light source unit  151 . The second light source control unit  152  is formed of, for example, a well-known computer or microcomputer including an arithmetic device, a control device, a storage device, an input device, and an output device. 
     The light detecting unit  160  detects the light δ′ acquired by the reflection of the first light δ 1  in the measurement chip  10 . For example, in a state in which the specimen is present in the flow path  41 , the light detecting unit  160  detects the second light δ 2  acquired when the first light δ 1  passes through the specimen in the flow path  41 , reflected by the metal film  30 , and passes through again the specimen in the flow path  41  when the light emitting unit  150  emits the first light δ 1  toward the metal film  30 . The light detecting unit  160  outputs a signal indicating a light amount of the detected light δ′. The light detecting unit  160  includes a second light receiving sensor  161  and a second sensor control unit  162 . 
     The second light receiving sensor  161  detects the light δ′ which is the first light δ 1  reflected within the measurement chip  10 . The second light receiving sensor  161  is, for example, a photomultiplier tube (PMT), an avalanche photodiode (APD), a silicon photodiode (SiPD) and the like. 
     The second sensor control unit  162  controls detection of an output value of the second light receiving sensor  161 , management of sensitivity of the second light receiving sensor  161  by the output value, change in the sensitivity of the second light receiving sensor  161  for acquiring an appropriate output value and the like. The second sensor control unit  162  is formed of, for example, a well-known computer or microcomputer including an arithmetic device, a control device, a storage device, an input device, and an output device. 
     The light emitting unit  150  (second light source unit  151 ) and the light detecting unit  160  (second light receiving sensor  161 ) are preferably arranged such that a plane including the optical axis of the first light δ 1  and the optical axis of the reflected light δ′ of the first light δ 1  is in a longitudinal direction of the flow path  41 . As a result, even if the position of the irradiation spot of the first light δ 1  is displaced in the longitudinal direction of the flow path  41 , it is possible to suppress the irradiation of the region other than the flow path  41  with the first light δ 1 . Also, as compared with a case where the first light δ 1  is perpendicularly incident on the metal film  30 , in a case where the first light δ 1  is obliquely incident on the metal film  30 , the shape of the irradiation spot of the first light δ 1  on the metal film  30  extends in one direction. The direction in which the irradiation spot extends depending on the incident angle of the first light δ 1  with respect to the metal film  30  is preferably in the longitudinal direction of the flow path  41 . As a result, as compared with a case where the direction in which the irradiation spot extends is in a lateral direction of the flow path  41 , even if the position of the irradiation spot is displaced, the first light δ 1  may be suppressed from being applied to the region other than the flow path  41  (metal film  30 ). Note that a cause of the positional displacement of the irradiation spot includes a positioning error of the chip holder  142  on the transporting stage  141 , an installation error of the measurement chip  10  with respect to the chip holder  142  and the like. 
     The control processing unit  170  controls the angle adjusting mechanism  112 , the first light source control unit  113 , the position switching mechanism  122 , the first sensor control unit  127 , the pipette control unit  135 , the transporting stage  141 , the second light source control unit  152 , and the second sensor control unit  162 . The control processing unit  170  also serves as a processing unit which processes detection results of the signal detecting unit  120  (first light receiving sensor  126 ) and the light detecting unit  160  (second light receiving sensor  161 ). In this embodiment, the control processing unit  170  determines the measurement value indicating the amount of the substance to be measured in the specimen on the basis of the detection result of the fluorescence β by the signal detecting unit  120 . In addition, the control processing unit  170  determines the hematocrit value of the specimen on the basis of the detection result of the second light δ 2  by the light detecting unit  160 . Along with this, the control processing unit  170  corrects the above-described measurement value on the basis of the hematocrit value. As a result, the control processing unit  170  determines the amount (concentration) of the substance to be measured in plasma or serum. In addition, predetermined information (for example, various conversion coefficients and data regarding calibration curve) and the like used when processing the above-described detection results may also be recorded in the control processing unit  170  in advance. In this embodiment, a coefficient for converting the hematocrit related value to the hematocrit value is recorded in the control processing unit  170  in advance. The control processing unit  170  is formed of, for example, a well-known computer or microcomputer including an arithmetic device, a control device, a storage device, an input device, and an output device. 
     (Optical Path in SPFS Device) 
     As illustrated in  FIG. 3 , the excitation light α enters the prism  20  from the incident surface  21 . The excitation light α entering the prism  20  is incident on the metal film  30  at a total reflection angle (angle at which SPR occurs). In this manner, the localized field light may be generated on the metal film  30  by irradiating the metal film  30  with the excitation light α at an angle at which the SPR occurs. By this localized field light, the fluorescent substance which labels the substance to be measured present on the metal film  30  is excited and the fluorescence β is released. The SPFS device  100  detects the light amount (intensity) of the fluorescence β emitted from the fluorescent substance. Note that, although not especially illustrated, the reflected light of the excitation light α on the metal film  30  is emitted out of the prism  20  from the emission surface  23 . 
     Also, as illustrated in  FIG. 3 , in this embodiment, the first light δ 1  enters the flow path  41  (measurement chip  10 ) via the flow path lid  40 . The first light δ 1  is reflected within the measurement chip  10 . The light reflected in the measurement chip  10  is emitted out of the flow path  41  (measurement chip  10 ) via the flow path lid  40 . The SPFS device  100  detects the light emitted from the measurement chip  10 . 
     (Effect) 
     In this embodiment, the measurement value (fluorescence value) indicating the amount of the substance to be measured is acquired by the first specimen in a state in which the blood is not hemolyzed, and the hematocrit related value is acquired by the specimen of the second specimen in a state in which the blood is hemolyzed to determine the hematocrit value. Since the blood in the first specimen is not hemolyzed, it is possible to suppress protease (proteolytic enzyme) in the red blood cell from flowing out of the red blood cell to decompose the substance to be measured. As a result, the measurement value may be acquired with a high degree of accuracy. In addition, since the blood in the second specimen is hemolyzed, the effect of scattering of light by the red blood cell may be reduced. As a result, the hematocrit related value may be acquired with a high degree of accuracy, and the hematocrit value may be determined with a high degree of accuracy. Therefore, in the measurement method according to this embodiment, the measurement value indicating the amount of the substance to be measured in the specimen and the hematocrit value may be measured with a high degree of accuracy and the amount of the substance to be measured in the specimen containing blood may be measured with a high degree of accuracy. 
     Note that, in the measurement method according to this embodiment, the light which becomes the second light δ 2  is allowed to reciprocate in the flow path  41  by the metal film  30 . This makes it possible to lengthen the optical path length of the light which becomes the second light δ 2  in the flow path  41  as compared with a case where reflection by the metal film  30  is not utilized. As a result, it is possible to measure the hematocrit value with a high degree of accuracy by increasing the amount of light absorbed by the specimen. As a result, the amount of the substance to be measured may be determined with a high degree of accuracy. 
     Also, in the measurement method according to this embodiment, it is possible to measure the second blank value at step S 1132  and remove the effect of the noise component (step S 118 ). Therefore, it is possible to measure the hematocrit value with a higher degree of accuracy. 
     Also, in this embodiment, the second blank value is measured (step S 1132 ) before the primary reaction (step S 114 ). Therefore, even if the washing in the flow path  41  after the primary reaction is not sufficient and the blood remains in the flow path  41 , the measurement of the second blank value is not affected by residual blood. Therefore, the hematocrit value may be measured with a high degree of accuracy. From such a point of view, it is preferable to measure the second blank value (step S 1132 ) before the first reaction (step S 114 ). 
     Furthermore, although a mode in which the SPFS method is used and the fluorescence value of the fluorescence β from the fluorescent substance is measured as the measurement value is described in this embodiment, the present invention is not limited to this mode. For example, it is also possible to measure the light amount of the reflected light of the excitation light α as the measurement value by utilizing an SPR method. Also, in the present invention, the measurement value may also be acquired by using an ELISA method, an RIfS method, a QCM method and the like. 
     [Reference Experiment] 
     In a reference experiment, a hematocrit value was measured by using an absorbance method, a microhematocrit method, or an electric resistance method, a measurement value indicating an amount of a substance to be measured previously acquired was corrected with the hematocrit value Hct, and results of the measurement were compared. 
     1. Acquisition of Measurement Value 
     As specimens, 24 types of whole blood containing cardiac troponin (cTn)I to which heparin was added were used. When acquiring the measurement value, blood in the specimen is not hemolyzed. Concentration of cTnI in the specimen was measured using a high-sensitivity automatic immunoassay device (SPFS device according to the above-described embodiment) having a function of measuring the hematocrit value by the absorbance method. 
     2. Micro Hematocrit Method 
     The specimen in a state in which blood is not hemolyzed was placed in a capillary made of glass to be centrifuged for five minutes at 12000 rpm by using a well-known hematocrit centrifuge (Centec 3220; manufactured by KUBOTA CORPORATION, “Centec” is the registered trademark of the company). Using a measuring instrument attached to the hematocrit centrifuge, the hematocrit value of the specimen after the centrifugation was measured. Subsequently, on the basis of the measured hematocrit value, the concentration of cTnI in the specimen was corrected to the concentration of cTnI in plasma. 
     3. Absorbance Method 
     First, blood was hemolyzed with a surfactant. Next, the hematocrit value of the specimen in a state in which blood was hemolyzed was measured by an absorbance method using a high-sensitivity automatic immunoassay device. Subsequently, on the basis of the measured hematocrit value, the concentration of cTnI in the specimen was corrected to the concentration of cTnI in plasma. 
     4. Electric Resistance Method 
     Using a well-known hematocrit analyzer (i-STAT; manufactured by Abbott Point of Care Inc., “i-STAT” is the registered trademark of the company), the hematocrit value of the specimen in a state in which blood is not hemolyzed was measured by an electric resistance method. Subsequently, on the basis of the measured hematocrit value, the concentration of cTnI in the specimen was corrected to the concentration of cTnI in plasma. 
     A specimen number, cTnI concentration WB-cTnI in the specimen, the hematocrit value Hct and the cTnI concentration in the plasma (correction value A (reference value)) acquired by the microhematocrit method, the hematocrit value Hct, the cTnI concentration in plasma (correction value B), and a shift amount BiasB of the correction value B with respect to the reference value acquired by the absorbance method, and the hematocrit value Hct, the cTnI concentration (correction value C) in the plasma, and a shift amount BiasC of the correction value C with respect to the reference value acquired by the electric resistance method are illustrated in Table 1. In Table 1, BiasB is a value calculated by following equation (5) and BiasC is a value calculated by following equation (6). 
     
       
         
           
             
               
                 
                   
                     [ 
                     
                       Equation 
                        
                       
                           
                       
                        
                       5 
                     
                     ] 
                   
                    
                   
                       
                   
                 
               
               
                 
                     
                 
               
             
             
               
                 
                   BiasB 
                   = 
                   
                     
                       
                         B 
                         - 
                         A 
                       
                       
                         
                           ( 
                           
                             A 
                             + 
                             B 
                           
                           ) 
                         
                          
                         
                           / 
                         
                          
                         2 
                       
                     
                     × 
                     100 
                   
                 
               
               
                 
                   ( 
                   5 
                   ) 
                 
               
             
             
               
                 
                   
                     [ 
                     
                       Equation 
                        
                       
                           
                       
                        
                       6 
                     
                     ] 
                   
                    
                   
                       
                   
                 
               
               
                 
                     
                 
               
             
             
               
                 
                   BiasC 
                   = 
                   
                     
                       
                         C 
                         - 
                         A 
                       
                       
                         
                           ( 
                           
                             A 
                             + 
                             C 
                           
                           ) 
                         
                          
                         
                           / 
                         
                          
                         2 
                       
                     
                     × 
                     100 
                   
                 
               
               
                 
                   ( 
                   6 
                   ) 
                 
               
             
           
         
       
     
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 1 
               
             
            
               
                   
                   
               
               
                   
                 MICROHEMATOCRIT METHOD 
                 ABSORBANCE METHOD 
                 ELECTRIC RESISTANCE METHOD 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                 CORRECTION 
                   
                 CORRECTION 
                   
                   
                 CORRECTION 
                   
               
               
                 SPECIMEN 
                 WB-cTnI 
                 Hct 
                 VALUE A 
                 Hct 
                 VALUE B 
                 Bias B 
                 Hct 
                 VALUE C 
                 Bias C 
               
               
                 NUMBER 
                 [pg/mL] 
                 [%] 
                 [pg/mL] 
                 [%] 
                 [pg/mL] 
                 [%] 
                 [%] 
                 [pg/mL] 
                 [%] 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 1 
                 50.0 
                 37.0 
                 69.6 
                 39.8 
                 72.1 
                 +3.52 
                 39.0 
                 71.3 
                 +2.5 
               
               
                 2 
                 55.2 
                 30.0 
                 71.0 
                 29.4 
                 70.5 
                 −0.60 
                 34.0 
                 74.2 
                 +4.4 
               
               
                 3 
                 60.4 
                 39.0 
                 86.1 
                 36.1 
                 83.1 
                 −3.57 
                 40.0 
                 87.2 
                 +1.3 
               
               
                 4 
                 61.1 
                 40.0 
                 88.3 
                 38.9 
                 87.1 
                 −1.34 
                 40.0 
                 88.3 
                 0 
               
               
                 5 
                 61.8 
                 38.0 
                 87.1 
                 38.4 
                 87.4 
                 +0.46 
                 39.0 
                 88.1 
                 +1.2 
               
               
                 6 
                 74.5 
                 30.0 
                 95.8 
                 28.0 
                 93.8 
                 −2.07 
                 32.0 
                 97.9 
                 +2.2 
               
               
                 7 
                 76.0 
                 36.0 
                 104.5 
                 38.4 
                 107.6 
                 +2.91 
                 34.0 
                 102.1 
                 −2.3 
               
               
                 8 
                 90.4 
                 39.0 
                 128.9 
                 40.6 
                 131.6 
                 +2.06 
                 38.0 
                 127.3 
                 −1.2 
               
               
                 9 
                 95.1 
                 39.0 
                 135.6 
                 35.8 
                 130.5 
                 −3.88 
                 37.0 
                 132.3 
                 −2.5 
               
               
                 10 
                 123.8 
                 45.0 
                 191.3 
                 47.1 
                 197.1 
                 +2.99 
                 56.0 
                 228.8 
                 +17.9 
               
               
                 11 
                 181.3 
                 37.0 
                 252.3 
                 39.8 
                 261.1 
                 +3.45 
                 38.0 
                 255.4 
                 +1.2 
               
               
                 12 
                 198.0 
                 46.0 
                 310.4 
                 46.2 
                 311.4 
                 +0.32 
                 51.0 
                 335.4 
                 +7.7 
               
               
                 13 
                 198.1 
                 43.5 
                 299.8 
                 44.3 
                 303.2 
                 +1.13 
                 49.0 
                 325.0 
                 +8.1 
               
               
                 14 
                 203.2 
                 41.0 
                 297.3 
                 41.4 
                 298.9 
                 +0.52 
                 44.0 
                 309.6 
                 +4.1 
               
               
                 15 
                 257.1 
                 46.5 
                 406.1 
                 49.0 
                 421.5 
                 +3.74 
                 50.0 
                 428.5 
                 +5.4 
               
               
                 16 
                 593.0 
                 46.0 
                 929.8 
                 44.0 
                 904.1 
                 −2.80 
                 47.0 
                 943.6 
                 +1.5 
               
               
                 17 
                 598.8 
                 37.0 
                 833.3 
                 37.4 
                 837.1 
                 +0.46 
                 37.0 
                 833.3 
                 0 
               
               
                 18 
                 662.1 
                 31.0 
                 860.4 
                 28.9 
                 841.3 
                 −2.25 
                 30.0 
                 851.2 
                 −1.1 
               
               
                 19 
                 748.4 
                 50.0 
                 1247.3 
                 49.3 
                 1233.5 
                 −1.11 
                 49.0 
                 1227.7 
                 −1.6 
               
               
                 20 
                 752.6 
                 38.0 
                 1060.1 
                 37.8 
                 1057.5 
                 −0.25 
                 37.0 
                 1047.3 
                 −1.2 
               
               
                 21 
                 785.9 
                 43.0 
                 1181.2 
                 41.9 
                 1164.4 
                 −1.43 
                 43.0 
                 1181.2 
                 0 
               
               
                 22 
                 814.9 
                 35.0 
                 1107.4 
                 33.1 
                 1083.3 
                 −2.20 
                 34.0 
                 1094.8 
                 −1.2 
               
               
                 23 
                 908.4 
                 43.0 
                 1365.3 
                 42.3 
                 1351.7 
                 −1.00 
                 41.0 
                 1329.3 
                 −2.7 
               
               
                 24 
                 954.6 
                 39.0 
                 1361.5 
                 37.3 
                 1333.2 
                 −2.10 
                 38.0 
                 1344.7 
                 −1.2 
               
               
                   
               
            
           
         
       
     
       FIG. 4A  is a graph illustrating accuracy of the amount of the substance to be measured in the plasma determined by the absorbance method, and  FIG. 4B  is a graph illustrating the accuracy of the amount of the substance to be measured in the plasma determined by the electric resistance method. In  FIG. 4A , mean cTnI concentration is plotted along the abscissa and BiasB which is the shift amount of the correction value B with respect to the correction value A (reference value) is plotted along the ordinate. In  FIG. 4B , mean cTnI concentration is plotted along the abscissa and BiasC which is the shift amount of the correction value C with respect to the correction value A (reference value) is plotted along the ordinate 
     As illustrated in  FIG. 4A  and Table 1, BiasB was within 5% in any of the specimens used in this embodiment. On the other hand, as illustrated in  FIG. 4B  and Table 1, BiasC was more than 5% in at least a part of the specimens used in this embodiment, and it is confirmed that BiasC tends to be larger in the specimen having a high hematocrit value of equal to or more than 50%. In this manner, it is understood that the absorbance method using the specimen containing hemolyzed blood may determine the amount of the substance to be measured in the plasma with a higher degree of accuracy as compared with the electric resistance method using the specimen containing blood which is not hemolyzed. In addition, compared to the micro hematocrit method, with the absorbance method, it is unnecessary to separately prepare a device such as a centrifuge, and it is possible to simply and highly accurately measure in a device for measuring the substance to be measured. As described above, according to the present invention, it is not necessary to separately prepare a device for measuring the hematocrit value, and the hematocrit value and the amount of the substance to be measured may be determined with a high degree of accuracy. 
     This application claims priority on the basis of JP 2016-160756 A filed on Aug. 18, 2016. The contents described in the specification and illustrated in the drawings of this application are entirely incorporated herein by reference. 
     INDUSTRIAL APPLICABILITY 
     Since the measurement method of the substance to be measured according to the present invention may highly reliably detect the substance to be measured, this is useful for examining diseases, for example. 
     REFERENCE SIGNS LIST 
     
         
         
           
               10  Measurement chip 
               20  Prism 
               21  Incident surface 
               22  Film-depositing surface 
               23  Emission surface 
               30  Reflecting unit 
               30  Metal film 
               40  Flow path lid 
               41  Flow path 
               50  Liquid chip 
               100  SPFS device 
               110  Excitation light emitting unit 
               111  First light source unit 
               112  Angle adjusting mechanism 
               113  First light source control unit 
               120  Signal detecting unit 
               121  Light receiving optical system unit 
               122  Position switching mechanism 
               123  First lens 
               124  Optical filter 
               125  Second lens 
               126  First light receiving sensor 
               127  First sensor control unit 
               130  Liquid sending unit 
               131  Pipette 
               132  Syringe pump 
               133  Nozzle unit 
               134  Pipette chip 
               135  Pipette control unit 
               140  Transporting unit 
               141  Transporting stage 
               142  Chip holder 
               150  Light emitting unit 
               151  Second light source unit 
               152  Second light source control unit 
               160  Light detecting unit 
               161  Second light receiving sensor 
               162  Second sensor control unit 
               170  Control processing unit 
             α Excitation light (fourth light) 
             β Fluorescence 
             γ Plasmon scattered light 
             δ 1  First light 
             δ′ Reflected light of first light in measurement chip 
             δ 2  Second light 
             δ 3  Third light