JP2006208035A - Measuring instrument using attenuated total reflection, and tilt correction method therefor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To correct a measuring error resulting from a tilt of a sensor unit, in a measuring instrument using attenuated total reflection. <P>SOLUTION: Five sensor cells 17 are installed adjacently in the sensor unit 12. The sensor cells 17 positioned in both ends are sealed with a lid member 43 while a buffer liquid is filled, to serve as the sensor cells 17b for the correction. A tilt correction part of a data analyzer detects an angle generating the attenuated total reflection, based on respective correction signals obtained by measuring the respective sensor cells 17b for the correction. Then, a difference ▵a between the detected angles is calculated, a correction data for correcting the inclination is calculated as ▵a×(▵t/t), from the ▵a, a distance t between the respective sensor cells 17b for the correction, and a distance ▵t between an act area 22a and a ref area 22b, and the tilt of the sensor unit 12 is corrected by subtracting the correction data from measured data in the respective sensor cells 17b for the correction. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a measuring apparatus using total reflection attenuation such as surface plasmon resonance.

  2. Description of the Related Art There are known measuring apparatuses that measure the reaction of a sample by using total reflection attenuation when measuring an interaction between biochemical substances such as proteins and DNA, screening drugs, and the like.

  One of the measuring devices that use such total reflection attenuation is a measuring device that uses a surface plasmon resonance phenomenon (hereinafter referred to as an SPR measuring device). The surface plasmon is generated by the collective vibration of free electrons in the metal, and is a density wave of free electrons traveling along the surface of the metal.

  For example, in an SPR measurement apparatus that employs a Kretschmann arrangement known from Patent Document 1 or the like, a surface of a metal film formed on a transparent dielectric (hereinafter referred to as a prism) is used as a sensor surface, and a sample is formed on the sensor surface. After reacting, the metal film is irradiated from the back side of the sensor surface through the prism so as to satisfy the total reflection condition, and the reflected light is measured.

  Of the light irradiated to the metal film so as to satisfy the total reflection condition, a small amount of light called an evanescent wave passes through the metal film without being reflected and oozes out to the sensor surface side. At this time, if the frequency of the evanescent wave coincides with the frequency of the surface plasmon, SPR is generated and the intensity of the reflected light is greatly attenuated. The SPR measurement device measures the reaction state of the sample on the sensor surface by capturing the attenuation of the reflected light.

  In order to prevent denaturation and inactivation due to drying, biological samples such as proteins and DNA are often handled as sample solutions dissolved in a physiological saline solution, a pure solvent, or various buffer solutions. The SPR measurement device described in Patent Document 1 examines such interaction between biological samples, and a flow path for feeding a sample solution is provided on the sensor surface. The sensor surface is provided with a linker film for immobilizing the ligand sample. After injecting the ligand solution into the flow path to immobilize the ligand on the linker film (an immobilization process), the analyte solution is By injecting and contacting the ligand and the analyte (measurement process), the interaction is examined.

  In addition, the SPR measurement device described in Patent Document 1 is provided with a measurement stage in which a prism and a channel are arranged in the device body, and a chip-type sensor unit in which a metal film is formed on a glass substrate is used as the measurement stage. The above-mentioned measurement is performed by attaching to the.

  Usually, it takes about 1 hour to complete the fixation of the ligand, which is much longer than the measurement that is completed in about several minutes. In the SPR measurement device described in Patent Document 1, since the fixing process and the measuring process are performed on the same measurement stage, the measuring stage is occupied during the fixing process, and the measuring process of other sensor units is performed. There was a problem that the work tends to be delayed, such as being unable to do so.

  In addition, in order to increase the work efficiency, it may be possible to perform the fixing process of a plurality of sensor units first, and to perform the measuring process together, but the sensor unit exposes the sensor surface, and the fixing process is performed. If you save what you have done, the sensor surface may dry out, causing the sample to denature or deactivate, or impurities may adhere to the sensor surface, making it impossible to perform accurate measurements. there were.

  In order to solve these problems, the applicant of the present invention has joined the flow path member in which the flow path is formed, the prism in which the metal film is formed on the upper surface, and the bottom surface of the flow path member and the upper surface of the prism. An SPR measurement device using a sensor unit that includes a holding member that holds the flow channel and the metal film facing each other has been proposed (see, for example, Japanese Patent Application No. 2004-287615).

  In this SPR measurement device, since the flow path and the prism are provided in the sensor unit itself, it is possible to perform a plurality of sensor unit fixing steps and measurement steps in parallel, thereby improving work efficiency. . In addition, since the flow path is provided, when the material subjected to the fixing process is stored, it can be stored in a liquid-injected state, and drying of the sensor surface can be prevented. Furthermore, this sensor unit has a long external appearance, and by providing a plurality of linker films on a long metal film, a single sensor unit can simultaneously measure a plurality of samples. .

In addition, a measurement region that binds to the ligand and a reference region that does not bind are formed on the linker film. The above-described SPR measurement device irradiates light from a light source to a measurement region and a reference region, and photoelectrically converts reflected light from each region with a detector to obtain a measurement signal and a reference signal. By obtaining the difference or ratio between the two signals thus obtained and analyzing the data, it is possible to perform highly accurate measurement in which noise caused by disturbances such as individual differences in the sensor unit and liquid temperature change is canceled.
Japanese Patent No. 3294605

  For example, a CCD image sensor or the like is used as the detector, and the position where the total reflection attenuation occurs is detected by associating the pixel position of the received light with the total reflection angle. However, if the sensor unit is tilted in the direction in which the height of the measurement area and the reference area as viewed from the light source and the detector changes, the light receiving position of light having the same angle component changes, and it is as if the angle has shifted. There was a problem that such a result would be obtained. In addition, even if data analysis using the reference area is performed on the error due to the inclination, an error due to the inclination occurs in the measurement area and the reference area itself, and thus the error corresponding to the inclination cannot be corrected.

  The present invention has been made in view of the above problems, and provides a measuring apparatus using total reflection attenuation for correcting a measurement error due to the tilt of a sensor unit, and a tilt correction method thereof. .

  In order to solve the above-described problems, a measuring apparatus using total reflection attenuation according to the present invention includes a thin film layer formed on one surface of a translucent dielectric block and a sample formed on the thin film layer. Total reflection from a direction perpendicular to the arrangement direction of the measurement region and the reference region with respect to a sensor unit having a measurement region where the reaction occurs and a polymer film in which the reference region where the reaction of the sample does not occur is arranged side by side A light source that irradiates light on the back surface of the thin film layer so as to satisfy the conditions, and receives light reflected from the back surface of the thin film layer and photoelectrically converts the light irradiated by the light source to the measurement region. Light detection means for acquiring a corresponding measurement signal and a reference signal corresponding to the reference region, and based on the measurement signal and the reference signal, measurement data corresponding to the reaction state of the sample in the measurement region is obtained. Calculation Measurement data calculation means, and two correction regions located in the optical path of the light and provided in the dielectric block along the arrangement direction are irradiated by the light source, and the reflected light is emitted by the light detection means. The sensor in the direction in which the height of the measurement area and the reference area as seen from the light source and the light detection means changes based on the respective correction signals corresponding to the respective correction areas obtained by receiving light. It is characterized by comprising inclination correction means for calculating correction data corresponding to the inclination of the unit and performing correction for the inclination on the measurement data based on the correction data.

  The light source outputs wide light having a spread in the arrangement direction to irradiate the measurement region, the reference region, and the correction regions together, and the reflected light is received by the light detection means. By doing so, it is preferable that the measurement signal, the reference signal, and the correction signals are acquired together.

  Further, the inclination correction means detects the angle of the light at which total reflection attenuation has occurred from each of the correction signals, and calculates the difference between the detected angles as the distance between the correction regions in the arrangement direction. Preferably, the correction data is calculated by proportionally distributing the distance between the measurement area and the reference area.

  Moreover, it is preferable that each correction | amendment area | region is provided so that the said polymer film may be pinched | interposed.

  The sensor unit is provided with a plurality of the polymer films formed on the thin film layer along the arrangement direction and a flow path for supplying a liquid corresponding to each of the polymer films. It is preferable that each correction region be a contact interface between the thin film layer in the portion where the polymer films located at both ends are formed and the dielectric block.

  Furthermore, it is preferable that the sensor unit is provided with a lid member that seals the flow paths corresponding to the polymer films located at both ends in a state where the liquid is injected.

  An optically readable index may be provided at a position corresponding to the correction area of the dielectric block.

  In addition, the tilt correction method of the present invention includes a thin film layer formed on one surface of a light-transmitting dielectric block, a measurement region formed on the thin film layer in which a sample reaction occurs, and the reaction of the sample. For the sensor unit having a polymer film arranged side by side with a reference region where no thin film occurs, the thin film layer is formed so as to satisfy the total reflection condition from a direction orthogonal to the arrangement direction of the measurement region and the reference region. The back surface is irradiated with light by a light source, and the light reflected from the back surface of the thin film layer received by the light source is received and photoelectrically converted by the light detection means, whereby the measurement signal corresponding to the measurement region and the A reference signal corresponding to a reference region is obtained, measurement data corresponding to a reaction state of the sample in the measurement region is calculated based on the measurement signal and the reference signal, and is positioned in the optical path of the light And before Each correction area corresponding to each correction area obtained by irradiating two correction areas provided in the dielectric block along the arrangement direction with the light source and receiving the reflected light with the light detection means Based on the signal, correction data corresponding to the inclination of the sensor unit in the direction in which the height of the measurement area and the reference area as viewed from the light source and the light detection means changes is calculated. Then, correction for the tilt is performed on the measurement data.

  According to the measuring apparatus using the total reflection attenuation and the tilt correction method of the present invention, the two correction regions provided in the dielectric block of the sensor unit are irradiated with the light source, and the reflected light is received by the light detection means. Correction data is calculated on the basis of the respective correction signals obtained in this way, and based on this correction data, the height of the measurement area and the reference area as viewed from the light source and the light detection means with respect to the inclination in the direction in which the height changes. Since the correction is performed on the measurement data from the polymer film, the measurement error caused by the inclination of the sensor unit can be corrected.

  FIG. 1 is a block diagram showing a schematic configuration of an SPR measurement device 2 as a measurement device using total reflection attenuation. The SPR measuring device 2 includes a fixing device 4 for fixing the ligand (fixing step), a measuring device 6 for adding an analyte to the immobilized ligand and measuring the reaction state of both (measurement step), and the measuring device 6 And a data analyzer 8 that performs analysis (data analysis process) of data obtained by the above. Further, the fixing step and the measuring step are performed on the sensor unit 12 which is a separate body, so that a plurality of samples can be measured smoothly.

  FIG. 2 is an exploded perspective view showing a schematic configuration of the sensor unit 12. The sensor unit 12 includes a prism (dielectric block) 14 having a metal film (thin film layer) 13 formed on the upper surface, a channel member 41 having a channel 16 for feeding a liquid to the metal film 13, and a prism. 14 and the bottom surface of the flow path member 41 are held in pressure contact with each other, and a lid member 43 is disposed above the holding member 42.

  The channel member 41 is formed in a long shape, and five channels 16 are provided along the longitudinal direction thereof. The flow path 16 is a liquid feeding pipe bent into a substantially U-shape, and has an inlet 16a for injecting liquid and an outlet 16b for discharging it. The diameter of the channel 16 is about 1 mm, and the interval between the inlet 16a and the outlet 16b is about 10 mm. Further, the bottom of the flow path 16 is open, and this open part is covered and sealed with the metal film 13 when being pressed against the prism 14. Hereinafter, a portion of the metal film 13 surrounded by the open portion (see FIG. 3) is referred to as a sensor cell 17.

  The flow path member 41 is molded from, for example, an elastic material such as rubber or PDMS (polydimethylsiloxane) in order to improve the adhesion with the metal film 13. Thus, when the flow path member 41 is pressed against the upper surface of the prism 14, the flow path member 41 is elastically deformed to fill the gap between the contact surfaces with the metal film 13, and the open bottom portion of each flow path 16 is watertight together with the prism 14. Cover.

  On the upper surface of the prism 14, a strip-shaped metal film 13 facing each flow path 16 is formed by, for example, vapor deposition. For example, gold or silver is used for the metal film 13, and the film thickness is, for example, 50 nm. This film thickness is appropriately selected according to the material of the metal film 13, the wavelength of the irradiated light, and the like.

  A linker film (polymer film) 22 for fixing a ligand (sample) is formed at a position on the metal film 13 facing each flow path 16 (in the sensor cell 17). Hereinafter, of the metal film 13, the surface on which the linker film 22 is formed is referred to as a sensor surface 13a, and the back surface (the surface in contact with the prism 14) is referred to as a light incident surface 13b. The prism 14 condenses incident light toward the light incident surface 13b, and the metal film 13 is formed on the sensor surface 13a when light is incident on the light incident surface 13b so as to satisfy the total reflection condition. Generate SPR.

  Further, as shown in FIG. 3, the linker film 22 formed on the metal film 13 has a measurement region (hereinafter referred to as an act region) 22a in which a ligand is fixed and a reaction between the analyte and the ligand occurs. A ligand is not fixed, and a reference region (hereinafter referred to as a ref region) 22b for obtaining a reference signal when measuring the signal of the act region 22a is provided. The ref region 22b is formed when the linker film 22 is formed. As the formation method, for example, the linker film 22 is subjected to a surface treatment, and about a half of the linker film 22, the bonding group that binds to the ligand is deactivated, so that the half of the linker film 22 becomes the act region. 22a, and the other half becomes the ref region 22b.

  Engaging claws 14 a that engage with the engaging portions 42 a of the holding member 42 are provided on both side surfaces of the long side of the prism 14. By these engagements, the flow path member 41 is sandwiched between the holding member 42 and the prism 14, and is held in a state where the bottom surface and the top surface of the prism 14 are in pressure contact with each other. Further, protrusions 14 b are provided at both ends of the prism 14. As will be described later, the sensor unit 12 is fixed in a state of being housed in the holder 52 (see FIG. 5). The protrusion 14 b engages with the holder 52 to position the sensor unit 12 at a predetermined storage position in the holder 52.

  The prism 14 is made of, for example, optical glass such as borosilicate crown (BK7) or barium crown (Bak4), polymethyl methacrylate (PMMA), polycarbonate (PC), amorphous polyolefin (APO), or the like. Representative optical plastics can be used.

  On the upper surface of the holding member 42, a receiving port 42b through which the tip of a liquid feeding means such as a pipette enters is formed at a position corresponding to the inlet 16a and the outlet 16b of each channel 16. These receiving ports 42b are formed in a funnel shape so that the liquid discharged from the pipette is guided to each inlet 16a. In addition, each receiving port 42 b comes into contact with each inlet 16 a and outlet 16 b and is connected to the channel 16 when the holding member 42 sandwiches the channel member 41 and engages with the prism 14.

  In addition, columnar bosses 42c are provided on both sides of each receiving port 42b. These bosses 42 c are fitted into holes 43 a formed in the lid member 43 to position the lid member 43. The lid member 43 is affixed to the upper surface of the holding member 42 by a double-sided tape 44 having holes at positions corresponding to the receiving ports 42b and the bosses 42c.

  The lid member 43 covers the receiving port 42 b that communicates with the flow path 16, thereby preventing the liquid injected into the flow path 16 from evaporating. The lid member 43 is made of, for example, an elastic material such as rubber or plastic, and a cross-shaped slit 43b is formed at a location corresponding to each receiving port 42b. As a result, the lid member 43 elastically deforms the slit 43b to allow the pipette to be inserted, and when the pipette is removed, the lid member 43 maintains the initial state and closes the receiving port 42b.

  Of the five sensor cells 17 that are located at both ends, the slit 43 b is not formed at a corresponding portion of the lid member 43 and is completely sealed by the lid member 43. When the sensor unit 12 rotates and tilts around the X axis, as shown in FIG. 4, the optical path length of the light irradiated from the bottom surface side of the prism 14 (the distance until the irradiated light is reflected and received). ) Will change. If this optical path length changes, for example, the light intensity and detection angle of the reflected light change, which causes measurement errors. Further, when rotating around the X axis, the heights of the act region 22a and the ref region 22b of the linker films 22 formed side by side in the Y direction change, so data analysis using the ref region 22b was performed. However, this measurement error cannot be corrected. Although details will be described later, the sensor cells 17 located at both ends are used when correcting a measurement error caused by the inclination of the sensor unit 12.

  The sensor cells 17 positioned at both ends are naturally positioned in the optical path of this light when irradiated with light so as to satisfy the total reflection condition. Each sensor cell 17 is formed in a straight line, and is along the direction in which the act region 22a and the ref region 22b are arranged. That is, the portion of the prism 14 in which the two sensor cells 17 located at both ends are formed corresponds to the correction region described in the claims. Hereinafter, separately from these, the sensor cell 17 used for measurement is referred to as a measurement sensor cell 17a, and the sensor cells 17 located at both ends thereof are referred to as correction sensor cells 17b. However, when collectively referring to both, it is also referred to as sensor cell 17 hereinafter. In this example, three measurement sensor cells 17a are shown, but the number is not limited to three, and may be less or more.

  In addition, in the flow path 16 of the correction sensor cell 17b, in the assembly stage of the sensor unit 12, for example, a liquid that does not include a ligand or analyte sample such as a buffer solution is injected. The lid member 43 completely seals each receiving port 42b of the correction sensor cell 17b, thereby preventing measurement errors due to evaporation of the injected liquid.

  FIG. 5 is a perspective view schematically showing the configuration of the fixing machine 4. A mounting space 51 for mounting the plurality of sensor units 12 is secured on the housing base 50 that serves as a base of the fixed machine 4. The sensor unit 12 is subjected to a fixing process while being placed in the placement space 12.

  The sensor unit 12 is placed in the placement space 51 while being housed in the holder 52. The holder 52 can store a plurality of sensor units 12 (eight in this example). The holder 52 is provided with a slit for positioning the sensor unit 12 by fitting with the protrusion 14 b of the sensor unit 12. In addition, an opening is formed in the bottom of the holder 52 except for support portions that support both ends of the sensor unit 12. As will be described later, when the sensor unit 12 is taken out from the holder 52 in the measurement process, a push-up member 81a (see FIG. 6) is inserted from the opening, and the sensor unit 12 is pushed up.

  In the mounting space 51, for example, ten holders 52 can be arranged side by side, and pedestals 53 corresponding to the number of the holders 52 are provided. Each pedestal 53 is formed with a positioning boss for positioning the holder 52.

  Further, the fixing machine 4 is provided with a pipette head 54 in which three pairs of pipettes 19 are connected. The pipette head 54 accesses the sensor units 12 arranged in the placement space 51 via the holder 52 to inject and discharge liquid. Each pipette pair 19 is composed of a pair of pipettes 19a and 19b (see FIG. 9), and each pipette 19a and 19b is inserted into each of the inlet 16a and outlet 16b of each measurement sensor cell 17a. Each pipette 19a, 19b has a function of injecting liquid into the flow path 16 and sucking out from the flow path 16, and when one is performing an injection operation, the other performs a suction operation. So that they work together. Since three pipette pairs 19 are provided in the pipette head 54, liquid is simultaneously injected into or discharged from the flow paths 16 of the three measurement sensor cells 17 a included in one sensor unit 12. Can do. Further, the fixing device 4 is provided with a controller (not shown). The suction and discharge operations of the pipette head 54 are controlled by this controller, and the suction amount, discharge amount, timing, etc. are determined for each pipette pair 19.

  The housing base 50 is provided with a head moving mechanism 56 that moves the pipette head 54 in three directions of X, Y, and Z. The head moving mechanism 56 is a known moving mechanism including, for example, a conveyance belt, a pulley, and a motor. The head moving mechanism 56 is an elevating mechanism that moves the pipette head 54 up and down, and the pipette head 54 can be moved in the Y direction together with the elevating mechanism. A Y-direction moving mechanism including a guide rail 58 to be held and an X-direction moving mechanism that holds the guide rail 58 at both ends and moves the pipette head 54 in the X direction together with the guide rail 58. The head moving mechanism 56 is controlled by a controller, and the controller drives the head moving mechanism 56 to control the vertical and horizontal positions of the pipette head 54.

  In addition to the mounting space 51, a plurality of liquid storage units 61 for storing various liquids to be injected into the flow path 16 and a pipette tip storage unit 63 for storing the pipette tips 62 are provided on the housing base 50. And a well plate 64 in which a plurality of well-shaped ridges are arranged in a matrix.

  The number of the liquid storage units 61 is determined according to the type of liquid to be used, such as a ligand solution, a cleaning liquid, a fixing buffer liquid, a drying prevention liquid, an activation liquid, and a blocking liquid. Each liquid storage unit 61 is provided with six insertion ports in a straight line. The number of the insertion openings and the arrangement pitch are determined according to the number of pipettes of the pipette head 54 and the arrangement pitch. When the pipette head 54 injects the liquid into the flow path 16, the liquid storage unit 61 is accessed to suck in a desired liquid, and then moves to the placement space 51 to be injected into each flow path 16.

  The pipette tips 62 stored in the pipette tip storage section 63 are attached to the tip portions of the pipettes 19a and 19b in a replaceable manner. Since the pipette tip 62 is in direct contact with the liquid, it is exchanged for each liquid to be used so that different kinds of liquids are not mixed through the pipette tip 62. Each pipette 19a, 19b incorporates a mechanism for picking up and releasing the pipette tip 62 so that the pipette tip 62 is automatically replaced. When exchanging the pipette tip 62, the pipette head 54 first releases the used pipette tip 62 at a disposal unit (not shown), and then accesses the pipette tip storage unit 63 to access an unused pipette. The chip 62 is picked up.

  The well plate 64 is used for temporarily storing the liquid sucked up by the pipettes 19a and 19b, or for preparing a mixed liquid by mixing a plurality of types of liquids.

  Further, when the fixing device 4 performs the fixing process, the housing base 50 is covered with the cover, and the inside of the fixing device 4 including the placement space 51 is shielded. The sensor unit 12 is stored in the mounting space 51 for a predetermined time until the ligand immobilization is completed. At this time, the degree of progress of immobilization depends on the temperature. Therefore, the fixing machine 4 keeps the internal temperature at a predetermined temperature by a temperature controller (not shown) while fixing. The set temperature, standing time, and the like are appropriately determined according to the type of ligand to be immobilized.

  FIG. 6 is a perspective view schematically showing the configuration of the measuring instrument 6. The measuring machine 6 includes a holder transport mechanism 71, a pickup mechanism 72, a head moving mechanism 73, and a measurement stage 74, and these parts are accommodated in a housing 75. The holder conveyance mechanism 71 includes a conveyance belt 76, a carriage 77 attached to the conveyance belt 76, and a plate 78 that is attached to the carriage 77 and on which the holder 52 is placed. The holder transport mechanism 71 moves each sensor unit 12 accommodated in the holder 52 to a pickup position where the pickup mechanism 72 picks up by moving the plate 78 on which the holder 52 is placed in the X direction.

  The pickup mechanism 72 is a mechanism for picking up the sensor unit 12 from the holder 52, a push-up mechanism 81 that pushes up the sensor unit 12 accommodated in the holder 52 from below to above, and the push-up mechanism 81 above the holder 52. A handling head 82 sandwiches and holds the pushed sensor unit 12 from both sides. As described above, an opening is provided at the bottom of the holder 52, and the plate 78 is formed on the frame so as to expose the opening. The push-up mechanism 81 enters the opening of the holder 52 via the plate 78, contacts the bottom surface of the sensor unit 12, pushes up the push-up member 81a, and push-up member that drives the push-up member 81a up and down And a drive mechanism 81b.

  The handling head 82 is provided with a pair of claws that sandwich the sensor unit 12, and the sensor unit 12 is gripped by the claws. The handling head 82 has a head body 82 a attached to a ball screw 86 via a nut 84, and is provided so as to be movable between a pickup position above the holder 52 and the measurement stage 74. The handling head 82 grips the sensor unit 12 at the pickup position, moves in the Y direction in that state, and carries the sensor unit 12 to the measurement stage 74. Further, after the measurement is completed, the sensor unit 12 is released by returning to the pickup position, and the measured sensor unit 12 is returned to the holder 52.

  The measurement stage 74 includes a measurement unit 31 including an illumination unit 32 and a detector (light detection means) 33 below a position where the sensor unit 12 is disposed. The measurement unit 31 irradiates a wide light according to the width of the sensor unit 12 from the illumination unit 32 and receives the reflected light by the detector 33 to measure each sensor cell 17 provided in the sensor unit 12. Do it at once. In addition, as shown in this figure and FIG. 3, the direction of the incident light beam irradiated to the sensor unit 12 and the reflected light beam reflected by the light incident surface 13b, and the arrangement direction of the sensor cells 17, that is, the horizontal direction of the flow paths 16 The illumination unit 32 and the detector 33 are arranged so that the flow direction of the part is orthogonal.

  The reaction state between the ligand and the analyte appears as a change in the resonance angle (the incident angle of the light applied to the light incident surface 13b). The illumination unit 32 includes, for example, a light source 34 and an optical system 36 including a lens, a diffuser plate, a polarizing plate, and the like. The arrangement position and the installation angle are set so that the incident angle of illumination light satisfies the total reflection condition. Adjusted.

  As the light source 34, for example, a light emitting element such as an LED (Light Emitting Diode), an LD (Laser Diode), or an SLD (Super Luminescent Diode) is used. The light source 34 uses one such light emitting element, and irradiates light from the single light source toward each light incident surface 13b. The diffusion plate diffuses light from the light source 34 and suppresses unevenness in the amount of light in the light emitting surface. The polarizing plate passes only p-polarized light (linearly polarized light having an oscillating electric field parallel to the incident surface) that causes SPR in the irradiated light. In addition, when using LD, when the direction of polarization of the irradiation light itself emitted from the light source 34 is uniform, the polarizing plate is unnecessary. Further, even when a light source with uniform polarization is used, if the direction of polarization becomes uneven due to passing through the diffusion plate, the direction of polarization is aligned using a polarizing plate. The light thus diffused and polarized is adjusted to a width corresponding to the prism 14 by the lens and is irradiated toward the prism 14. Thereby, the light which does not vary in light intensity and has various incident angles can be incident on the entire light incident surface 13b.

  The detector 33 receives the light reflected by the light incident surface 13b and outputs an electrical signal having a level corresponding to the light intensity. Since light of various angles is incident on the light incident surface 13b, the light is reflected on the light incident surface 13b at a reflection angle corresponding to each incident angle. The detector 33 receives light having these various reflection angles. For example, a CCD area sensor or a photodiode array is used as the detector 33, and the reflected light reflected at various reflection angles on the light incident surface 13b is received and photoelectrically converted, and this is converted into an SPR signal as a data analyzer. 8 is output.

  A well plate 88 for storing the analyte solution is placed beside the measurement stage 74. For example, each well of the well plate 88 contains different types of analyte solutions.

  The measuring device 6 is provided with three pairs of pipettes 26 having the same functions as the pipette pair 19 of the fixing device 4, and various liquids are injected into the flow channel 16 from the inlet 16 a of each measurement sensor cell 17 a. To do. The head moving mechanism 73 moves the pipette head 87 having the pipette pair 26 in the three directions of X, Y, and Z while moving the pipette head 87 to the sensor unit 12 and the well plate 88 at the measurement position. Carry. The head moving mechanism 73 has substantially the same configuration as the head moving mechanism 56 of the fixed machine 4. The pipette head 87 accesses each measurement sensor cell 17a to be measured, and injects and discharges liquid. Although not shown, the measuring machine 6 has a well plate for storing a measurement buffer solution and a cleaning solution at a position accessible by the pipette head 87, a pipette tip storage unit for storing a replacement pipette tip, and the like. Is provided.

  FIG. 7 is a block diagram schematically showing the configuration of the data analyzer 8. The data analyzer 8 includes, for example, an input device 91, a CPU (Central Processing Unit) 92, a ROM (Read Only Memory) 93, a RAM (Random Access Memory) 94, a monitor 95, an HDD (Hard Disk). A drive 96, a communication I / F 97, a signal processing unit (measurement data calculation unit) 98, an inclination correction unit (inclination correction unit) 99, a data analysis unit 100, and the like. The input device 91 is, for example, a keyboard or a mouse, and inputs an instruction from the user to the data analyzer 8. The CPU 92 comprehensively controls each unit of the data analyzer 8. The ROM 93 stores a control program, various setting data, and the like. The RAM 94 is used as a working memory when the CPU 92 or the signal processing unit 98 performs calculations. The HDD 96 is a known data storage device, and various programs such as a control program and a data analysis program are stored in the HDD 96. In addition, various measurement data measured by the measuring device 6 are recorded in the HDD 96. The communication I / F 97 receives the SPR signal output from the detector 33.

  Since the measuring device 6 measures each sensor cell 17 at once, the SPR signal output from the detector 33 includes all signals from each sensor cell 17. For example, the signal processing unit 98 extracts a signal corresponding to each sensor cell 17 from the SPR signal based on the detection position of the detector 33 (pixel position in the case of a CCD). At this time, the signal processing unit 98 outputs the SPR signal corresponding to each measurement sensor cell 17a as a data signal, and outputs the SPR signal corresponding to each correction sensor cell 17b as a correction signal. As shown in FIG. 3, an act region 22a and a ref region 22b are formed on the linker film 22 of each sensor cell 17, and each of the data signal and the correction signal is stored in the act region 22a. A corresponding act signal (measurement signal) and a ref signal (reference signal) corresponding to the ref region 22b are included.

  Further, the signal processing unit 98 subtracts the ref signal from the act signal of the extracted data signal, and calculates measurement data corresponding to each measurement sensor cell 17a. Note that the measurement data is not limited to the difference but may be based on the ratio of the act signal and the ref signal.

  The inclination correction unit 99 corrects the inclination around the X axis of the sensor unit 12 based on each correction signal. FIG. 8 is an explanatory diagram for explaining the concept of inclination correction. The incident rays P1 and P2 have an angle component that causes total reflection attenuation in each correction sensor cell 17b. In each of the correction sensor cells 17b in which the buffer solution is injected into the flow path 16, total reflection attenuation occurs at the same angle, that is, it is understood that the incident light rays P1 and P2 have the same angle. Similarly, the reflected light rays P3 and P4 totally reflected by the sensor unit 12 also have the same angle component. If the sensor unit 12 is not tilted, the reflected rays P3 and P4 should be received at the same position in the Z-axis direction of the detector 33. However, if the sensor unit 12 is tilted around the X-axis as shown in the figure, As shown in FIG. 4, the optical path length changes, and a deviation of Δd occurs between the light receiving position d1 of the reflected light beam P3 and the light receiving position d2 of the reflected light beam P4. Since the detector 33 determines the reflection angle of the reflected light rays P3 and P4 according to the light receiving position in the Z-axis direction (direction along the arrow Δd) of the light receiving surface, the reflection of the reflected light rays P3 and P4 is actually reflected. Despite the same angle, the detector 33 recognizes that the reflection angle of the reflected light ray P4 is shifted by Δd.

  In order to correct this error, the inclination correction unit 99 first detects an angle at which total reflection attenuation occurs from each correction signal. In FIG. 8, the angle is in accordance with the positions of d1 and d2. Here, when the detection angle of d1 is a1, the detection angle of d2 is a2, and the difference obtained by subtracting a1 from a2 is Δa, a2 can be naturally expressed as a1 + Δa, considering a1 as a reference. That is, by subtracting this difference Δa as correction data from a2, it is possible to correct the inclination of a2. The detection of a1 and a2 may be performed between act signals of the respective correction signals or between ref signals.

Similarly, the measurement data of each measurement sensor cell 17a located between d1 and d2 may be obtained by calculating a difference from a1. However, there is a sample reaction at an angle at which the total reflection attenuation of the measurement data occurs. Because of the contribution, the difference as described above cannot be obtained. However, since the difference value varies linearly according to the inclination of the sensor unit 12, the correction data of each measurement sensor cell 17a includes the difference Δa between d1 and d2, and the distance in the Y direction. It can be obtained from the proportional distribution of Further, the inclination correction of each measurement sensor cell 17a may be performed with respect to the inclination between the act region 22a and the ref region 22b of each linker film 22. That is, the inclination correction unit 99 calculates correction data for each measurement sensor cell 17a as shown in the following equation (1). In the equation (1), t represents the distance in the Y direction between d1 and d2, and Δt represents the distance in the Y direction between the act region 22a and the ref region 22b of each linker film 22.
Correction data = Δa × (Δt / t) (1)

  The inclination correction unit 99 subtracts the correction data obtained by the above equation (1) from the measurement data of each measurement sensor cell 17a, and corrects the measurement data with respect to the inclination around the X axis of the sensor unit 12.

  The data analysis unit 100 analyzes each measurement data corrected by the inclination correction unit 99 according to a data analysis program stored in the ROM 93 or the HDD 96. This makes it possible to cancel noise caused by disturbances such as individual differences of the sensor unit 12 and the linker film 22, mechanical fluctuations of the apparatus, changes in the temperature of the liquid, and the like. It is possible to perform highly accurate measurement with a good S / N ratio while suppressing measurement error due to the inclination of the. The data analysis unit 100 displays the analysis result on the monitor 95 and records it in the HDD 96.

  Next, the SPR measurement method of the SPR measurement device 2 having the above configuration will be described using the explanatory diagram shown in FIG.

  The fixing step of fixing the ligand to the linker film 22 is performed in a state where the sensor unit 12 is placed in the placement space 51 of the fixing machine 4 via the holder 52. The fixing device 4 drives the head moving mechanism 56 in response to a fixing start instruction from the user, and uses the pipette pair 19 to convert the ligand solution 21 in which the ligand 21a is dissolved in the solvent into the inlet 16a of each sensor cell 17a for measurement. Inject from.

  As a pretreatment before performing the ligand immobilization process for injecting the ligand solution 21, the fixing machine 4 first feeds a fixing buffer solution to the linker film 22 to moisten the linker film 22, and then An activation process of the linker film 22 is performed to facilitate the binding of the ligand to the film 22. For example, in the amine coupling method, carboxymethyl dextran is used as the linker film 22, and the amino group in the ligand is directly covalently bonded to the dextran. As the activation liquid in this case, a mixed liquid of N ′-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) is used. The fixing device 4 cleans the flow path 16 with the fixing buffer solution after the activation process.

  Examples of the buffer solution for fixation and the solvent (diluent) of the ligand solution 21 include, for example, various buffer solutions (buffer solutions), physiological salt solutions represented by physiological saline, pure water, and the like. Is used. The type of each of these liquids, the pH value, the type of mixture, the concentration thereof, and the like are appropriately determined according to the type of ligand 21a. For example, when a biological sample is used as the ligand 21a, a physiological saline whose pH is adjusted to near neutral is often used. However, in the amine coupling method, since the linker film 22 is negatively (negatively) charged by carboxymethyldextran, the protein is positively (positively) charged so as to easily bind to the linker film 22. In some cases, a phosphate buffer solution (PBS: phosphatic-buffered, saline) having a strong buffering action containing a high concentration of phosphate may be used.

  After performing the activation process and the washing, the fixing machine 4 injects the ligand solution 21 into the flow path 16 to perform the ligand immobilization process. When the ligand solution 21 is injected into the flow path 16, the ligand 21 a diffusing into the solution is precipitated and gradually deposited on the linker film 22. Thereby, the contacted ligand 21 a is bonded to the linker film 22, and the ligand 21 a is fixed on the linker film 22. The immobilization usually takes about one hour. During this time, the sensor unit 12 is stored in a state where environmental conditions such as temperature are set to predetermined conditions. Further, while the immobilization is proceeding, the ligand solution 21 in the flow channel 16 may be allowed to stand, but the ligand solution 21 in the flow channel 16 may be stirred and flowed. By doing so, the binding between the ligand 21a and the linker film 22 is promoted, and the amount of ligand 21a immobilized can be increased.

  When the fixation of the ligand 21a to the linker film 22 is completed, the fixing device 4 sucks out the ligand solution 21 with the pipette 19b and discharges it from the flow path 16, and then the cleaning liquid is injected into the flow path 16 to complete the fixation. The linker film 22 is washed. Moreover, the fixing machine 4 injects a blocking liquid as necessary, and performs a blocking process to deactivate the reactive group of the linker film 22 that has not been bonded to the ligand 21a. As the blocking liquid, for example, ethanolamine-hydrochloride is used. When this blocking process is performed, the flow path 16 is washed again. After the final cleaning, the fixing device 4 injects the drying prevention liquid into the flow path 16. The sensor unit 12 is stored in the mounting space 51 or another storage place until the measurement in a state where the linker film 22 is immersed in the drying prevention liquid.

  The measurement process starts from a state in which the sensor unit 12 is placed on the plate 78 of the measuring device 6 together with the holder 52. The measuring device 6 moves the plate 78 to the pickup position in accordance with a measurement start instruction from the user and drives the pickup mechanism 72 to carry the predetermined sensor unit 12 to the measurement stage 74.

  The measuring device 6 carrying the sensor unit 12 to the measurement stage 74 drives the head moving mechanism 73 to first inject the measurement buffer solution into the flow path 16. Thereafter, an analyte solution 27 in which the analyte is dissolved in a solvent is injected, and then the measurement buffer solution is injected again. Note that the flow path 16 may be washed once before the measurement buffer solution is injected for the first time. Data reading by the detector 33 is started immediately after the measurement buffer solution is first injected in order to detect a reference signal level. After the analyte solution 27 is injected, the measurement buffer solution is injected again. Until it is done. Thereby, it is possible to measure the SPR signal until the detection of the reference level, the reaction state between the analyte and the ligand, and the desorption by the injection of the buffer solution for measurement of the bound analyte and the ligand.

  Examples of the buffer solution for measurement and the solvent (diluent) of the analyte solution 27 include various buffer solutions (buffer solutions), physiological salt solutions typified by physiological saline, and pure water. used. The type of each of these liquids, the pH value, the type of mixture and its concentration, etc. are appropriately determined according to the type of ligand or analyte. For example, DMSO (dimethyl-sulfo-oxide) may be included in physiological saline in order to facilitate the dissolution of the analyte. This DMSO greatly affects the signal level. As described above, the measurement buffer solution is used for detecting the reference level. Therefore, when the analyte solution 27 contains DMSO, the measurement buffer solution having a DMSO concentration comparable to the DMSO concentration should be used. Is preferred.

  The analyte solution 27 is often stored for a long time (for example, one year). In such a case, a difference in concentration occurs between the initial DMSO concentration and the DMSO concentration at the time of measurement due to a change with time. May end up. When it is necessary to perform strict measurement, it is preferable to estimate such a concentration difference from the level of the ref signal when the analyte solution 27 is injected, and to correct the measurement data (DMSO concentration correction).

  The correction data for correcting the DMSO concentration is obtained by injecting a plurality of types of measurement buffer solutions having different DMSO concentrations into the flow path 16 before injecting the analyte solution 27, and ref according to the DMSO concentration change at this time. It is obtained by examining the respective changes in the signal level and the act signal level.

  The reaction state between the ligand and the analyte appears as a transition of the attenuation position of the reflected light within the light receiving surface of the detector 33. For example, before and after the analyte contacts the ligand, the refractive index on the sensor surface 13a is different, and the resonance angle at which SPR occurs is different. When the analyte comes into contact with the ligand and starts to react, the resonance angle of the reflected light starts to change accordingly, and the attenuation position of the reflected light in the light receiving surface starts to move. The measuring device 6 outputs an SPR signal indicating the reaction state of the sample thus obtained to the data analyzer 8.

  In the data analysis process, the SPR signal obtained by the measuring device 6 is analyzed by the data analyzer 8 as described above, and the characteristics of the analyte are quantitatively analyzed.

  Next, the operation of the SPR measurement device 2 of the present invention will be described using the flowchart shown in FIG. The sensor unit 12 is placed in the placement space 51 of the fixing machine 4 while being fitted in the holder 52. The fixing device 4 in which the sensor unit 12 is set performs a fixing process on each measurement sensor cell 17a in accordance with a fixing start instruction from the user.

  The sensor unit 12 in which the ligand is fixed is moved to the measuring machine 6 together with the holder 52 and placed on the plate 78. The measuring machine 6 having the sensor unit 12 mounted on the plate 78 moves the plate 78 to the pickup position in accordance with a measurement start instruction from the user and drives the pickup mechanism 72 to move the sensor unit 12 to the measurement stage 74. Carry to.

  The measuring device 6 that has moved the sensor unit 12 to the measurement stage 74 starts measurement of each sensor cell 17 by the measurement unit 31, and also drives the head moving mechanism 73 to move the pipette head 87, and each measurement sensor cell 17a. The analyte solution is injected into the flow path 16. The measuring device 6 that has performed the measurement from the reaction between the ligand and the analyte to the desorption outputs the SPR signal to the data analyzer 8.

  The data analyzer 8 that has received the SPR signal via the communication I / F 97 sends this SPR signal to the signal processing unit 98. The signal processing unit 98 extracts a signal corresponding to each sensor cell 17 from the SPR signal, and calculates measurement data of each measurement sensor cell 17a from each extracted data signal.

  The correction signal of each correction sensor cell 17 b and the measurement data of each measurement sensor cell 17 a calculated by the signal processing unit 98 are sent to the inclination correction unit 99. The inclination correction unit 99 detects the angle at which total reflection attenuation occurs from each correction signal, calculates a difference Δa between these angles, and calculates correction data for each measurement sensor cell 17a as shown in equation (1). . In addition, the inclination correction unit 99 subtracts the correction data calculated from the measurement data of each measurement sensor cell 17a, and performs inclination correction according to the inclination of the sensor unit 12 on each measurement data. The corrected measurement data is sent to the data analysis unit 100. The data analysis unit 100 analyzes the characteristics of the analyte based on the corrected measurement data.

  As described above, according to the SPR measurement device 2 of the present embodiment, the inclination of the sensor unit 12 is detected by the correction sensor cells 17b located at both ends, and correction is applied based on the SPR signal. Measurement errors caused by the inclination of the sensor unit 12 can be suppressed.

  In the above embodiment, the positions of the prisms 14 where the correction sensor cells 17b located at both ends of the five sensor cells 17 are formed are used as the correction region. However, the metal film 13 formed on the upper surface of the prism 14 You may make it correct | amend the inclination of the sensor unit 12 using the total contact attenuation | damping by the metal film 13 by making the surface which touches into a correction | amendment area | region. According to this, since the sensor cells 17 at both ends can be used for measurement, the measurement can be performed efficiently. Alternatively, the tilt of the sensor unit 12 may be corrected using the light intensity profile of the total reflected light from the prism 14 simply using the upper surface of the prism 14 as a correction region.

  Furthermore, an index such as a white line or a black line that is optically readable by the illumination unit 32 and the detector 33 is provided on the upper surface (total reflection surface) of the prism 14 or the side surface on which light enters or exits. The part provided with this index may be used as a correction area, and the inclination or the inclination of the sensor unit 12 may be corrected using the shape or position of the read symbol.

  In the above-described embodiment, the prism 14 is shown as the dielectric block. However, the dielectric block may be a plate made of optical glass or optical plastic, or these plate-like one and prism. Are integrated with an optical surface smoothing agent (for example, optical matching oil).

  Further, in the above embodiment, each sensor cell 17 is measured at once using a so-called fan beam, in which light irradiated from a single light source such as an LED is expanded by a lens, but is almost the same as the width of the sensor unit 12. A so-called line illumination having a length may be used for the illumination unit 32 to measure each sensor cell 17 at a time, or a single light source may be scanned with a polygon mirror or the like for measurement. .

  In addition, each sensor cell 17 may be individually measured by moving the sensor unit 12 with the handling head 82 and irradiating light from a single light source such as an LED, but while moving the sensor unit 12 If measurement is performed, there is a concern that the sensor unit 12 may be tilted during movement and correct tilt correction cannot be performed, which is not preferable.

  In the above embodiment, an SPR measuring device is shown as an example of a measuring device using total reflection attenuation. However, for example, a leakage mode sensor is known as a measuring device using total reflection attenuation. ing. The leakage mode sensor is composed of a dielectric, and a thin film composed of a clad layer and an optical waveguide layer that are sequentially layered thereon. One surface of the thin film serves as a sensor surface, and the other surface receives light. It becomes a surface. When light is incident on the light incident surface so as to satisfy the total reflection condition, a part of the light is transmitted through the cladding layer and taken into the optical waveguide layer. In this optical waveguide layer, when the waveguide mode is excited, the reflected light at the light incident surface is greatly attenuated. The incident angle at which the waveguide mode is excited changes according to the refractive index of the medium on the sensor surface, similar to the resonance angle of SPR. By detecting the attenuation of the reflection angle, the chemical reaction on the sensor surface is measured.

It is a block diagram which shows schematic structure of a SPR measuring apparatus. It is a disassembled perspective view which shows schematic structure of a sensor unit. It is explanatory drawing which extracts and demonstrates one sensor cell. It is explanatory drawing explaining the inclination of a sensor unit. It is a perspective view which shows the structure of a fixing machine roughly. It is a perspective view which shows the structure of a measuring machine roughly. It is a block diagram which shows the structure of a data analyzer roughly. It is explanatory drawing explaining the concept of inclination correction. It is explanatory drawing of a SPR measuring method. It is a flowchart which shows the procedure of inclination correction.

Explanation of symbols

2 SPR measurement device (measurement device using total reflection attenuation)
4 Fixing machine 6 Measuring machine 8 Data analyzer 12 Sensor unit 13 Metal film (thin film layer)
14 Prism (dielectric block)
16 flow path 17 sensor cell 22 linker film (polymer film)
22a Measurement area 22b Reference area 32 Illumination section 33 Detector (light detection means)
34 Light source 41 Channel member 43 Lid member 98 Signal processing unit (measurement data calculation means)
99 Inclination correction unit (Inclination correction means)

Claims (8)

A thin film layer formed on one surface of a light-transmitting dielectric block, a measurement region formed on the thin film layer where a sample reaction occurs, and a reference region where the sample reaction does not occur are arranged side by side. For a sensor unit having a polymer film, a light source that irradiates light on the back surface of the thin film layer so as to satisfy a total reflection condition from a direction orthogonal to the arrangement direction of the measurement region and the reference region;
Photodetection for obtaining a measurement signal corresponding to the measurement region and a reference signal corresponding to the reference region by receiving and photoelectrically converting reflected light from the back surface of the thin film layer of the light irradiated by the light source Means,
Based on the measurement signal and the reference signal, measurement data calculation means for calculating measurement data according to the reaction state of the sample in the measurement region;
It was obtained by irradiating two correction regions located in the optical path of the light and provided in the dielectric block along the arrangement direction with the light source and receiving the reflected light with the light detecting means. Based on the respective correction signals corresponding to the respective correction areas, correction according to the inclination of the sensor unit in the direction in which the height of the measurement area and the reference area as viewed from the light source and the light detection means changes. An apparatus for measuring the total reflection attenuation, comprising: inclination correction means for calculating data and performing correction for the inclination on the measurement data based on the correction data.   The light source outputs wide light having a spread in the arrangement direction, irradiates the measurement region, the reference region, and the correction regions together, and receives the reflected light by the light detection means. The measurement apparatus using total reflection attenuation according to claim 1, wherein the measurement signal, the reference signal, and the correction signals are acquired together.   The inclination correction unit detects an angle of the light at which total reflection attenuation has occurred from each of the correction signals, and calculates a difference between the detected angles as a distance between the correction regions in the arrangement direction, and 3. The measuring apparatus using total reflection attenuation according to claim 1, wherein the correction data is calculated by proportionally allocating the distance between the measurement area and the reference area.   The measuring apparatus using total reflection attenuation according to any one of claims 1 to 3, wherein each of the correction regions is provided so as to sandwich the polymer film.   In the sensor unit, a plurality of the polymer films formed on the thin film layer along the arrangement direction and a flow path for feeding a liquid are provided corresponding to each of the polymer films. Each of the correction regions is a contact interface between the thin film layer in the portion where the polymer films located at both ends are formed and the dielectric block. The measuring apparatus using the total reflection attenuation according to claim 4.   6. The sensor unit is provided with a lid member that seals the flow paths corresponding to the polymer films positioned at both ends in a state where the liquid is injected. Measuring device using total reflection attenuation.   5. The total reflection attenuation according to claim 1, wherein an optically readable index is provided at a position corresponding to the correction region of the dielectric block. Measuring device. A thin film layer formed on one surface of a light-transmitting dielectric block, a measurement region formed on the thin film layer where a sample reaction occurs, and a reference region where the sample reaction does not occur are arranged side by side. For a sensor unit having a polymer film, irradiate light with a light source on the back surface of the thin film layer so as to satisfy a total reflection condition from a direction orthogonal to the arrangement direction of the measurement region and the reference region,
The reflected light from the back surface of the thin film layer irradiated by the light source is received and photoelectrically converted by a light detection means, whereby a measurement signal corresponding to the measurement region and a reference signal corresponding to the reference region are obtained. Acquired,
Based on the measurement signal and the reference signal, to calculate measurement data according to the reaction state of the sample in the measurement region,
Two correction regions located in the optical path of the light and provided in the dielectric block along the arrangement direction are irradiated with the light source,
Based on the respective correction signals corresponding to the respective correction areas obtained by receiving the reflected light by the light detection means, the measurement area and the reference area viewed from the light source and the light detection means. Calculate correction data according to the inclination of the sensor unit in the direction in which the height changes,
An inclination correction method for a measuring apparatus using total reflection attenuation, wherein the measurement data is corrected based on the correction data.

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