JP2007155403A - Measuring instrument using total reflection return loss and measuring method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To measure the bonding reaction of a sample at a proper detecting position. <P>SOLUTION: A metal film 13 becoming a sensor surface is formed on the prism 14 of a sensor unit 12 and a linker film 23 for fixing a ligand is formed on the metal film 13 at the position opposed to a flow channel 16. The light from an illumination part 26 is thrown on the interface 33 of the prism 14 and the metal film 13 and the luminous intensity of the reflected light from the interface is detected by a detector 27 to perform the measurement of reaction. The sensor unit 12 is mounted on a table 31 freely movable in an X-Y direction. The detecting position of the reflected light is changed while moving the table 31 fore the measurement of reaction to perform preparatory measurement. On the basis of a preparatory measurement result, the optimum detecting position is judged and the measurement of reaction is performed at the optimum detecting position. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a measuring apparatus and method using total reflection attenuation for measuring a reaction state of a sample using total reflection attenuation of light.

  For example, a measurement device using total reflection attenuation is known as a measurement device for measuring the reaction of a sample when investigating the interaction of biochemical substances such as proteins and DNA, or when performing drug screening.

  A measuring device using total reflection attenuation causes a sample reaction on the surface of a thin film formed on one surface of a transparent dielectric block, and causes a total reflection condition at the interface between the thin film and the dielectric block. The reaction is measured by making light incident so as to satisfy the above condition and detecting the attenuation of the reflected light. One of the measuring devices using such total reflection attenuation is a measuring device using a surface plasmon resonance phenomenon (hereinafter referred to as an SPR measuring device). The surface plasmon is a density wave of free electrons generated by collective vibration of free electrons in a metal and traveling along the surface of the metal.

  The SPR measurement device includes a measurement unit including a light source that makes light incident on the interface and a light detection unit that receives reflected light reflected from the interface and detects the light intensity thereof. A sensor unit using a membrane is used. The SPR measurement device generates SPR on the sensor surface, and measures the reaction state of the substance generated there by detecting the SPR by the measurement unit.

  When light is incident toward the interface so as to satisfy the total reflection condition (at an incident angle greater than the critical angle), total reflection occurs at the interface, but only a small amount of incident light is not reflected. It passes through the metal film and oozes out to the sensor surface. This light wave that oozes out is called an evanescent wave. When the frequencies of the evanescent wave and the surface plasmon coincide and resonate (when SPR occurs), the intensity of the reflected light is greatly attenuated. Incident light with various angles that satisfy the total reflection condition is incident on the interface, and the incident light with various angles is reflected by the interface and output to the light receiving surface of the light detection unit. For this reason, the incident angle (resonance angle) at which SPR occurs is regarded as a dark line on the light receiving surface.

  The resonance angle depends on the refractive index of the medium through which the evanescent wave and the surface plasmon propagate. In other words, if the refractive index of the medium changes, the resonance angle changes. The substance in contact with the sensor surface becomes a medium for propagating evanescent waves and surface plasmons. For example, when a chemical reaction such as bonding or dissociation between two types of molecules occurs on the sensor surface, it is determined by the refractive index of the medium. It appears as a change, and the resonance angle changes. The SPR measurement device measures the interaction between molecules by detecting the change of the dark line and the change of the resonance angle.

  In experiments and research in the field of biochemistry, proteins, DNA, drugs and the like are used as ligands and analytes. For example, when screening a drug, a biological substance such as a protein is used as a ligand, and a plurality of kinds of drugs serving as analytes are brought into contact with the ligand, and their interaction is examined.

  A fixed film for fixing the ligand is formed on the sensor surface, and the ligand is fixed to the fixed film. Thereafter, an analyte solution containing the analyte is fed to the sensor surface and brought into contact with the ligand, and the change in the dark line position of the reflected light at that time is detected.

However, since the ligand used as the sample is expensive, it is difficult in terms of cost to prepare the sample to the extent that a sufficient amount of fixation is secured on the entire surface of the fixed membrane, and therefore the amount of fixation is not uniform within the surface of the fixed membrane. It tends to be. In a region where the amount of fixation is small, the amount of change in the dark line position is too small, and a satisfactory measurement signal cannot be obtained and the binding reaction cannot often be measured. In addition, if there is a flaw in the fixed film, the measurement signal at that portion becomes an error. As described above, when there is a scratch on the fixed film or the amount of ligand immobilized is not uniform within the film surface of the fixed film, the measurement accuracy varies depending on the detection position of the reflected light. Therefore, in the SPR measurement device disclosed in Patent Document 1 below, reflected light is detected at a plurality of predetermined positions in the region corresponding to the fixed film in the interface, and the detected measurement data is statistically calculated. Thus, the variation in measurement accuracy is eliminated.
JP 2003-270130 A

  However, in the above-described conventional method, since the detection position is determined in advance, the detection position is not necessarily set in an area having a large fixed amount. Since the measurement accuracy increases as the amount of change in the dark line position increases, there is a demand for reliable detection in a region with a large fixed amount.

  In addition, there is a demand to measure the binding reaction of multiple types of samples at a desired fixed amount, for example, by examining the reaction with different types of analytes with the same fixed amount of ligand. Measurement was impossible.

  An object of the present invention is to measure the binding reaction of a sample at an appropriate detection position.

  The measurement apparatus using total reflection attenuation of the present invention includes a transparent dielectric block, a thin film formed on one surface of the dielectric block, and a surface serving as a sensor surface for detecting a binding reaction between a ligand and an analyte, A light source that uses a sensor unit that is formed on the sensor surface and has a ligand fixing film that fixes the ligand, and that makes light incident on the interface between the thin film and the dielectric block so as to satisfy a total reflection condition; A light detection unit that receives reflected light reflected from the interface and detects the intensity of the reflected light, and detects the coupling angle by examining a change in resonance angle at which total reflection attenuation occurs based on the light intensity of the reflected light. In a measurement apparatus using total reflection attenuation for measuring a reaction, detection for moving a detection position for detecting the light intensity of the reflected light within a region of the interface corresponding to the ligand-fixed film After the ligand is immobilized on the ligand-immobilizing film and before the binding reaction is measured, preliminary measurement is performed by the light source and the light detection unit while moving the detection position, based on the result. And an optimum detection position determining means for determining an optimum detection position for the measurement of the binding reaction.

  Here, the detection position moving means moves the detection position by relative movement of the light source and the light detection unit and the sensor unit. The relative movement includes a case where any one of the light source and the light detection unit and the sensor unit is moved, and a case where both are moved.

  The optimum detection position determination means determines, for example, a position where the change in the resonance angle before and after the ligand fixation becomes maximum as the optimum detection position as a result of the preliminary measurement.

  The detection position moving means is, for example, a sensor unit moving mechanism that moves the sensor unit in a plane parallel to the interface in a measurement stage where the sensor unit is arranged in the measurement.

  The sensor unit moving mechanism is disposed in the measurement stage, and includes a table on which the sensor unit is mounted and provided so as to be movable in a plane parallel to the interface, and a drive unit that drives the table. Is preferred.

  The detection position moving means is preferably incident position changing means for changing an incident position of incident light incident on the interface by displacing at least a part of an illumination optical system including the light source.

  The incident position changing means is, for example, a movable mirror that is arranged in the incident optical path and changes the incident position of the incident light by a change in posture.

  An illuminating unit that causes the spot light emitted from the light source to enter a predetermined area in the interface, and the detection position moving unit causes only the reflected light reflected at a specific position out of the reflected light reflected at the predetermined area to be the light. You may comprise from the light reception optical system output toward a detection part, and the moving mechanism which changes the said detection position by moving the said specific position by displacing this light reception optical system.

  The sensor unit preferably includes a flow path member in which a plurality of flow paths for supplying liquid to the sensor surface are formed.

  The measurement method using total reflection attenuation according to the present invention includes a transparent dielectric block, a thin film formed on one surface of the dielectric block, and a surface serving as a sensor surface for detecting a binding reaction between a ligand and an analyte, Using a sensor unit having a ligand fixing film formed on the sensor surface and fixing the ligand, light is incident toward the interface between the thin film and the dielectric block so as to satisfy a total reflection condition, and the interface In the measuring method using total reflection attenuation, the reflected light reflected by the light is received and the light intensity is detected, and the change in the resonance angle at which total reflection attenuation occurs based on the light intensity is measured. A detection position for detecting the light intensity of the reflected light in a region corresponding to the ligand-fixed film in the interface after fixing the ligand and before measuring the binding reaction While moving to perform the preliminary measurement, the results are based on determining an optimal detection position for the measurement of the binding reaction, and wherein at the determined optimal detection position to perform the measurement of the binding reaction.

  In the present invention, a ligand-fixed film is formed on a thin film serving as a sensor surface for detecting a reaction of a sample, light is incident on an interface between the thin film and a dielectric block, and total reflection is performed based on the light intensity of the reflected light. In a measurement method using total reflection attenuation that measures a binding reaction of a sample by examining a change in a resonance angle at which attenuation occurs, after fixing a ligand and before measuring the binding reaction, the ligand is included in the interface. Preliminary measurement was performed while moving the detection position for detecting the light intensity of the reflected light within the region corresponding to the fixed film, and the optimal detection position for the measurement of the binding reaction was determined based on the result. Since the binding reaction is measured at the optimal detection position, the binding reaction of the sample can be measured at an appropriate detection position.

  As shown in FIG. 1, a sensor unit 12 for detecting a binding reaction between a ligand and an analyte is detachably set in the SPR measurement device. The SPR measuring device is provided with a measuring unit for measuring a binding reaction, and a dispensing head 28 having a pair of pipettes 28a and 28b for injecting and discharging the sample solution to and from the channel 16. . The measurement unit includes an illumination unit 26 that irradiates light to the sensor unit 12 and a detector 27 that receives reflected light reflected by the sensor unit 12 and outputs a measurement signal.

  As shown in FIG. 2, the sensor unit 12 includes a prism 14 that is a transparent dielectric, a flow path member 18 in which a flow path 16 for sending a liquid is formed, and the flow path member 18 that is connected to the prism 14. The holding member 19 is configured to be pressed against the upper surface and integrally hold the flow path member 18 and the prism 14, and the lid member 22 is attached to the upper surface of the holding member 19 with a double-sided tape 21.

  The flow path member 18 has a long column shape with a square cross section, and is formed of an elastic member. The lower surface of the flow path member 18 is pressed against the upper surface of the prism 14. The flow path 16 is a substantially U-shaped liquid feeding pipe, and a facing portion 16c that flows the liquid injected facing the upper surface of the prism 14 along the upper surface of the prism 14 and flows from both ends of the facing portion 16c. A through portion 16d that penetrates the flow path member 18 in the vertical direction toward the upper surface 18a of the path member 18 is formed. At the upper end of each penetrating portion 16d, the ends of pipettes 28a and 28b are inserted, and inlets 16a and 16b serving as sample solution inlets and outlets are formed.

  The tube diameter of the flow path 16 is, for example, about 1 mm, and the interval between the entrances 16a, 16b is, for example, about 10 mm. The facing portion 16 c is a groove formed on the bottom surface of the flow path member 18, and the open portion is covered and sealed by the top surface of the prism 14 pressed against the bottom surface. The flow path member 18 is provided with, for example, three such flow paths 16, and the flow paths 16 are arranged side by side along the longitudinal direction of the flow path member 18.

  A metal film 13 whose surface is the sensor surface 13a is formed on the upper surface of the prism 14 by vapor deposition. The metal film 13 is formed in a strip shape so as to face the plurality of channels 16 formed in the channel member 18. Further, on the upper surface of the metal film 13, a plurality of linker films 23 serving as a ligand fixing film for fixing a ligand are formed at portions corresponding to the respective flow paths 16. The linker film 23 is formed when the sensor unit 12 is manufactured. A sensor cell 17 is constituted by the sensor surface 13 a including the linker film 23 and each flow path 16.

  As shown in FIG. 1, on the linker film 23, a measurement region (act region) 23a in which a ligand is fixed and a binding reaction between an analyte and a ligand occurs, and a ligand is not fixed. The reference region (ref region) 23b for obtaining the reference signal is formed. The ref region 23b is formed when the linker film 23 is formed. As a formation method, for example, the linker film 23 is subjected to a surface treatment to deactivate a binding group that binds to a ligand in a region about half of the linker film 23. As a result, half of the linker film 23 becomes the act region 23a and the other half becomes the ref region 23b.

  First, a ligand solution containing a ligand is fed to the linker film 23 through the flow path 16 so that the ligand is fixed to the act region 23a. In the binding reaction between the analyte and the ligand, after the ligand is immobilized, the analyte solution containing the analyte is sent to the linker film 23 through the channel 16 and the analyte comes into contact with the immobilized ligand. The binding reaction between the ligand and the analyte is measured by detecting the SPR signal at that time.

  The prism 14 has, for example, a bar shape with a trapezoidal cross section. Examples of the material of the prism 14 include optical glass represented by borosilicate crown (BK7) and barium crown (Bak4), polymethyl methacrylate (PMMA), polycarbonate (PC), and amorphous polyolefin (APO). Representative optical plastics are used.

  The prism 14 and the flow path member 18 are integrally held by a holding member 19. Engaging claws 14 a that engage with the engaging portions 19 a of the holding member 19 are provided on both side surfaces of the prism 14 in the longitudinal direction. With these engagements, the flow path member 18 is sandwiched between the holding member 19 and the prism 14. In addition, protrusions 14 b are provided at both ends in the short side direction of the prism 14. The protrusion 14b is a positioning member that engages with the inner wall of the sensor unit 12 to position the storage position when the sensor unit 12 is stored in a holder (not shown).

  In the upper part of the holding member 19, a receiving port 19b for guiding the tips of the pipettes 28a, 28b is formed at positions corresponding to the inlets 16a, 16b of the respective channels 16. When the holding member 19 sandwiches the flow path member 18 and engages with the prism 14, the receiving ports 19 b are connected to the doorways 16 a and 16 b. Also, cylindrical bosses 19c are provided on both sides of each receiving port 19b. These bosses 19 c are for fitting the holes 22 a formed in the lid member 22 to position the lid member 22.

  The lid member 22 covers the receiving port 19 b that communicates with the flow path 16, thereby preventing the liquid in the flow path 16 from evaporating. The lid member 22 is formed of an elastic member, for example, rubber or plastic, and a cross-shaped slit 22b is formed at a position corresponding to each receiving port 19b. The pipettes 28a and 28b are inserted while expanding the slit 22b. When the pipettes 28a and 28b are pulled out, the slit 22b returns to the initial state by the elastic force and closes the receiving port 19b.

  Although not shown, a bar code including information such as an identification ID for each unit is recorded in the sensor unit 12 so that each unit can be identified. If an identification ID is recorded in each sensor unit 12, for example, it becomes possible to manage data by associating the measurement result for each sensor unit 12 with the type of sample solution injected. Instead of providing a barcode, for example, an IC tag such as an RFID tag may be provided.

  The measurement stage on which the sensor unit 12 is set is provided with a table 31 serving as a mounting portion that detachably holds the sensor unit 12. The sensor unit 12 is carried from the standby position to the measurement stage by a handling mechanism (not shown) and set on the table 31. The table 31 is provided with a guide rail 31 a that fits with the bottom of the sensor unit 12. The sensor unit 12 is slidably provided along the guide rail 31a, and each sensor cell 17 is selectively inserted into the measurement position by the slide. The sliding of the sensor unit 12 is performed by the handling mechanism.

  As will be described later, the table 31 is movable in the X direction and the Y direction. By this movement, the incident position of the light beam on the interface 33 is changed, and the detection position of the measurement signal is adjusted. The The illumination unit 26 and the detector 27 are arranged to face each other with the table 31 interposed therebetween.

  As shown in FIG. 3, the illumination unit 26 irradiates light toward the interface 33 between the prism 14 and the metal film 13. As described above, since the reaction state between the ligand and the analyte appears as a change in the resonance angle, the illumination unit 26 causes the light beam having various incident angles that satisfy the total reflection condition to enter the interface 33. The illumination unit 26 includes a light source and an optical system. As the light source, 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 optical system includes a collimator lens, an optical fiber 36, a condensing lens 37, and the like. The divergent light emitted from the optical fiber 36 is converged to a specific incident position on the interface 33 by the condenser lens 37. As a result, light beams with various incident angles are irradiated onto the interface 33. The incident position of the light beam is a detection position P for detecting the light intensity of the reflected light and obtaining a measurement signal. The act region 23a and the ref region 23b are irradiated with light beams, and each light beam is generated, for example, by splitting light from one light source.

  The detector 27 is composed of, for example, a CCD area sensor or a photodiode array, receives reflected light reflected at the detection position P on the interface 33, and uses an electric signal at a level corresponding to the light intensity of the reflected light as a measurement signal. Output. At the detection position P, the light beams incident at various incident angles are reflected, so that the detector 33 receives the light beams with these various reflection angles.

  The resonance angle at which SPR occurs is determined according to the refractive index of the medium on the surface of the metal film 13. As shown in the graph of FIG. 4, the light intensity of the reflected light Rsp (shown by hatching in FIG. 3) incident at the resonance angle θsp among the light beams incident at various incident angles θ is greatly attenuated. On the light receiving surface 27a of the device 27, a light receiving position (hereinafter referred to as a dark line position) D of the reflected light Rsp is detected as a dark line. As shown in the graph of FIG. 4, when the reaction between the ligand and the analyte occurs and the refractive index changes, the resonance angle θsp changes (θsp0 → θsp1), and the dark line position D moves within the light receiving surface 27a. Based on the measurement signal, the change in the dark line position D is captured to measure the binding reaction between the ligand and the analyte.

  The detector 27 outputs a measurement signal corresponding to the act region 23a as an act signal, and outputs a measurement signal corresponding to the ref region 23b as a ref signal. The signal processing unit 38 generates measurement data based on the difference or ratio between the act signal and the ref signal. By generating measurement data based on the act signal and the ref signal, it is possible to cancel noise caused by disturbances such as individual differences of sensor units and sensor cells, mechanical fluctuations of the apparatus, and temperature changes of the liquid. Therefore, it is possible to measure with high accuracy.

  The controller 39 comprehensively controls each part of the SPR measurement device such as the dispensing head 28, the illumination unit 26, the detector 27, and the table 31. The table driving unit 44 includes a motor and a table moving mechanism, and moves the table 31 in the XY direction. The SPR measurement device performs a main measurement for measuring a reaction between an analyte and a ligand, and a preliminary measurement for determining a detection position for executing the main measurement. The main measurement data output from the detector 27 during the main measurement is input to the data analysis unit 41 through the controller 39. The data analysis unit 41 performs data analysis based on the measurement data, and analyzes the binding reaction between the analyte and the ligand. The preliminary measurement data output from the detector 27 during the preliminary measurement is output to the optimum detection position determination unit 42 through the controller 39. The optimum detection position determination unit 42 determines the optimum position of the detection position P of the main measurement signal based on the preliminary measurement data.

  As shown in FIG. 5, since it is difficult to make the amount of ligand 46 fixed to the linker film 23 uniform within the film surface, a portion where the amount of ligand 46 is fixed (high density) in the linker film 23 , A portion where the amount of ligand 46 immobilized is small (density is low). Since the amount of the fixed amount affects the amount of binding between the analyte and the ligand, the resonance signal (RU) representing the amount of change in the resonance angle differs depending on the detection position P in the interface 33 corresponding to the linker film 23. . That is, the detection signal P2 with a larger fixed amount has a larger resonance signal than the detection position P1 with a smaller fixed amount. In order to improve the measurement accuracy, it is preferable to detect in a portion having as much fixed amount as possible. The optimal detection position determination unit 42 determines the optimal detection position based on preliminary measurement data obtained by preliminary measurement after fixing the ligand to the linker film 23.

  As shown in FIG. 6, in the preliminary measurement, while moving the table 31 in the XY direction, the region corresponding to the linker film 23 in the interface 33 is scanned, and the optimum detection is performed based on the scanning result. Search for position P. When the table 31 is moved, the sensor unit 12 is moved accordingly. Therefore, the incident position of the light beam of the illumination unit 26, that is, the detection position P at the interface 33 is moved. Along with this, a preliminary measurement signal at each detection position P is acquired by the detector 27, and the preliminary measurement data is sequentially input to the optimum detection position determination unit 42. Based on the preliminary measurement data, the optimum detection position determination unit 42 examines the detection position where the change in the resonance angle θsp before and after the ligand fixation is maximum, and determines that position as the optimum detection position.

  Hereinafter, the operation of the above configuration will be described with reference to the flowchart shown in FIG. After setting one sensor cell 17 of the sensor unit 12 to the measurement position, a preliminary measurement is performed before performing the main measurement. In the preliminary measurement, first, before the ligand is fixed, the measurement signal is detected by the detector 27, and the resonance angle θsp before the ligand is fixed is measured. Thereafter, the ligand solution is injected into the flow path 16 by the dispensing head 28, and the ligand is fed to the linker film 23 to fix the ligand. After the ligand is fixed, the detection position P is changed in the region corresponding to the linker film 23 in the interface 33 while moving the table 31, and preliminary measurement data at each detection position P is acquired. Based on the preliminary measurement data of each detection position P, the optimal detection position determination unit 42 determines the optimal detection position where the change in the resonance angle after the ligand fixation becomes the maximum with the resonance angle θsp before the ligand fixation as a reference.

  This measurement is performed with the detection position P set as the optimum detection position. In this measurement, the analyte solution is injected into the flow path 16 by the dispensing head 28, and a measurement signal is acquired. Since the detection position P is set to the optimum detection position, highly accurate main measurement data can be obtained. Thus, after the main measurement of one sensor cell 17 is completed, the next sensor cell 17 is inserted into the measurement position, and the preliminary measurement and the main measurement are performed in the above-described procedure. After measurement of all sensor cells 17 in one sensor unit 12 is completed, the next sensor unit 12 is set on the measurement stage and measurement is performed.

  In the above-described embodiment, the detection position is changed by moving the sensor unit in the preliminary measurement. However, as shown in FIG. 8, the illumination unit 26 is translated in a plane parallel to the interface 33. Thus, the detection position P may be changed.

  However, in this case, since the relative position between the illumination unit 26 and the detector 27 changes when the illumination unit 26 is moved, there is no change in the resonance angle θsp between the detection position P1 and the detection position P2. However, the dark line position D moves (D1 → D2) on the light receiving surface 27a. Therefore, the movement amount of the dark line position D based on the change in the resonance angle θsp cannot be specified only by the apparent movement amount of the dark line position D on the light receiving surface 27a. Therefore, as shown in the graph of FIG. 9, a memory is provided in which table data indicating the correspondence between the amount of movement of the illumination unit 26 and the amount of movement of the light receiving position of the reflected light on the light receiving surface 27a is stored in advance. It is necessary to keep. The optimum detection position determination unit 42 corrects the preliminary measurement data acquired by the detector 27 based on the table data, thereby calculating the movement amount of the dark line position D based on the change of the resonance angle θsp, and thereby calculating the resonance angle. The change amount of θsp is specified.

  In the above-described embodiment, the example in which the plurality of sensor cells 17 in one sensor unit 12 are measured one by one in order has been described. For example, as illustrated in FIG. You may perform what is called multichannel measurement which performs the measurement process with respect to the sensor cell 17 simultaneously. When performing such multi-channel measurement, the light beam from one light source may be dispersed according to the number of each channel, or a plurality of light sources corresponding to the number of channels may be used. Good.

  In the example shown in FIG. 10, the reflection mirror plate 51 that constitutes the illumination optical system together with the light source is disposed on the incident optical path of the light beam from the light source to the interface 33, and the detection position is detected by the reflection mirror plate 51. Change P. The reflection mirror plate 51 includes a long reflection surface 51 a that reflects each light beam directed to each sensor cell 17. A movable mirror unit 52 is disposed on the reflection mirror plate 51 on the incident optical path of the light beam toward the act region 23 b of each linker film 23.

  As shown in FIG. 11, the movable mirror unit 52 includes a mirror portion 53 that has a reflecting surface 53a and is rotatable around the X axis and the Y axis. By changing the orientation of the mirror 53, the direction of the light beam B is changed, and the incident position at the interface 33 is changed. Each movable mirror unit 52 is driven by a mirror driving unit 54 and controlled independently for each channel. Thereby, the optimum detection position is set for each channel.

  As described above, when the direction of the light beam is changed by the movable mirror unit 52, the relative position of the mirror unit 53 and the detector 27 changes as in the case of parallel movement of the illumination unit. Even if there is no change, the dark line position D of the light receiving surface 27a changes due to the change in the attitude of the mirror 53. For this reason, the amount of change in the dark line position D detected by the detector 27 is calculated based on the correspondence between the amount of change in the attitude of the mirror 53 and the amount of movement of the light receiving position of the reflected light on the light receiving surface 27a. It is necessary to correct and specify the amount of change in the resonance angle θsp.

  Further, the detection position P may be changed by moving the light receiving optical system 61 as in the SPR measurement apparatus shown in FIG. 13A is a side view of the optical path from the interface 33 to the light receiving optical system 61 as seen from the side, and FIG. 13B is a top view of the optical path as seen from the top. In this case, the illumination unit 62 is not a light beam that causes light to enter a specific point in the interface 33 corresponding to the act region 23a, but light in a region A having a predetermined area as shown in FIG. A light source that emits spot light is used. As a light source that emits such spot light, for example, an LED or the like is preferable. The area A is set to an area that covers most of the entire area of the act area 23a.

  The light receiving optical system 61 receives the reflected light reflected at a certain point among the reflected light reflected by the region A. When the light receiving optical system 61 is moved, the reflection position of the reflected light, that is, the detection position P changes. Thus, the optimum detection position is specified by scanning the area A.

  The light receiving optical system 61 includes a collimator lens 62, a condenser lens 63, a diaphragm plate 66 in which a pinhole 64 is formed, and a detector 27. The reflected light reflected from the region A is collimated by the collimator lens 62 and then condensed toward the pinhole 64 by the condenser lens 63. One point in the region A and the pinhole 64 are both arranged to be confocal, that is, to be in an optically beneficial position relative to the objective lens including the collimator lens 62 and the condenser lens 63. Is done.

  As a result, the reflected light emitted from one point of the confocal region A is collected toward the pinhole 64 serving as the other confocal point, so that the reflected light passing through the pinhole 64 is detected. The reflected light from one point in the region A is detected by receiving the light at the light receiving surface 27a of the device 27. By displacing the light receiving optical system 61 in the D1 direction and the D2 direction, the detection position P can be changed in the region A. The light receiving optical system 61 is moved by a displacement mechanism 67. The displacement mechanism 67 is controlled by a controller.

  In the above embodiment, in the optimum detection position determination, an example is described in which the position of the linker film with the largest amount of ligand fixation is determined as the optimum detection position in the linker film. May be. For example, when it is desired to examine the reactions of a plurality of types of analytes at a specific ligand fixation amount, the position of the specific fixation amount may be set as the optimum detection position.

  In the above embodiment, an example is described in which a sensor unit in which three sensor cells are arranged in one row is used. However, the number of sensor cells included in one unit is not limited to three, and may be one or three. You may provide above.

  Moreover, although the example which integrated the metal film, the flow path, and the prism was demonstrated as a sensor unit, among these, you may remove a prism and a flow path member from the component of a sensor unit, and may provide in an apparatus side.

  Further, in the present embodiment, the SPR sensor that generates SPR on the sensor surface and detects the attenuation of the reflected light at that time has been described as an example. However, the present invention is not limited to the SPR sensor, but other total reflections. It can also be applied to measurements using attenuation. As a sensor using total reflection attenuation, for example, a leakage mode sensor is known in addition to the SPR sensor. 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 reflected light, the reaction on the sensor surface is measured.

It is a schematic explanatory drawing of a SPR measuring method. It is a block diagram of a sensor unit. It is the schematic of an SPR measuring device. It is a graph explaining reflected light intensity and resonance angle change. It is explanatory drawing which shows the relationship between the fixed amount of a ligand, and a resonance signal. It is explanatory drawing of the scanning method of a detection position. It is a flowchart which shows a measurement procedure. It is explanatory drawing which shows the relationship between the movement of an illumination part, and the movement of the light reception position of reflected light. It is a graph which shows the relationship of FIG. It is explanatory drawing in the case of performing multichannel measurement. It is explanatory drawing of a movable mirror unit. It is explanatory drawing in the case of changing a detection position by moving a light-receiving optical system.

Explanation of symbols

12 Sensor unit 13 Metal film 13a Sensor surface
14 prism 16 flow path 17 sensor cell 18 flow path member 23 linker film 23a act area 23b ref area 26 illuminating section 27 detector 27a light receiving surface 31 table

Claims (9)

A transparent dielectric block, a thin film formed on one surface of the dielectric block, the surface serving as a sensor surface for detecting a binding reaction between the ligand and the analyte, and a ligand formed on the sensor surface and fixing the ligand Using a sensor unit having a fixed film, a light source that makes light incident toward the interface between the thin film and the dielectric block so as to satisfy the total reflection condition, and the reflected light reflected by the interface are received and A measuring device using a total reflection attenuation that measures the coupling reaction by examining a change in a resonance angle at which total reflection attenuation occurs based on the light intensity of the reflected light. ,
A detection position moving means for moving a detection position for detecting the light intensity of the reflected light within a region corresponding to the ligand-fixing film in the interface;
After the ligand is immobilized on the ligand-immobilized membrane, before the binding reaction is measured, a preliminary measurement is performed by the light source and the light detection unit while moving the detection position, and the binding reaction is measured based on the result. A measuring apparatus using total reflection attenuation, comprising: an optimum detection position determination means for determining an optimum detection position.   2. The total reflection attenuation according to claim 1, wherein the optimum detection position determination unit determines, as a result of the preliminary measurement, a position where a change in resonance angle before and after ligand fixation is maximum as an optimum detection position. Measuring equipment used.   The detection position moving means is a sensor unit moving mechanism that moves the sensor unit in a plane parallel to the interface in a measurement stage on which the sensor unit is arranged in the measurement. Item 3. A measuring apparatus using total reflection attenuation according to item 1 or 2.   The sensor unit moving mechanism is disposed in the measurement stage, and includes a table on which the sensor unit is mounted and provided so as to be movable in a plane parallel to the interface, and a drive unit that drives the table. The measuring apparatus using total reflection attenuation according to claim 3.   The detection position moving means is incident position changing means for changing an incident position of incident light incident on the interface by displacing at least a part of an illumination optical system including the light source. A measuring apparatus using total reflection attenuation according to 1.   6. The measuring apparatus using total reflection attenuation according to claim 5, wherein the incident position changing means is a movable mirror that is disposed in the incident optical path and changes the incident position of the incident light by a change in posture. .   An illumination unit is provided for causing the spot light emitted from the light source to enter a predetermined area in the interface, and the detection position moving means only reflects the reflected light reflected at a specific position out of the reflected light reflected at the predetermined area. 2. A light receiving optical system that outputs the light toward the light detection unit, and a moving mechanism that changes the detection position by moving the specific position by displacing the light receiving optical system. A measuring device using the total reflection attenuation described.   The measurement using the total reflection attenuation according to claim 1, wherein the sensor unit includes a flow path member in which a plurality of flow paths for supplying a liquid to the sensor surface are formed. apparatus. A transparent dielectric block, a thin film formed on one surface of the dielectric block, the surface serving as a sensor surface for detecting a binding reaction between the ligand and the analyte, and a ligand formed on the sensor surface and fixing the ligand Using a sensor unit having a fixed film, light is incident toward the interface between the thin film and the dielectric block so as to satisfy the total reflection condition, and the reflected light reflected at the interface is received and the light intensity is measured. In a measurement method using total reflection attenuation, which detects and measures the binding reaction by examining a change in resonance angle at which total reflection attenuation occurs based on the light intensity,
After immobilizing the ligand and before measuring the binding reaction, perform a preliminary measurement while moving the detection position for detecting the light intensity of the reflected light within the region corresponding to the ligand-immobilized film in the interface. And determining the optimum detection position for the measurement of the binding reaction based on the result,
A measurement method using total reflection attenuation, wherein the binding reaction is measured at the determined optimum detection position.

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