JP2007114156A - Measuring method and device utilizing attenuated total reflection - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To effectively utilize a dynamic range of a photodetector. <P>SOLUTION: In the measuring method utilizing attenuated total reflection, a reaction of a sample on a sensor plane is measured by acquiring the light intensity distribution of the reflected light reflected on the rear face of sensor plane with a photodetector. Although attenuation arises in the light intensity distribution by attenuated total reflection, the reaction of the sample can be caught by detecting variation of the dark line position. The quantity of light of a light source is adjusted and measured so that a part of the reflected light exceeds the dynamic range and is saturated, using the level where an effective part required for specifying the dark line position of the light intensity distribution is stored in the dynamic range of the photodetector as an upper limit. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

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

  For example, a measuring device using total reflection attenuation is known as a measuring device for measuring the reaction of a sample in order to examine the interaction of biochemical substances such as proteins and DNA, or to perform 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 photodetector that receives reflected light reflected from the interface and detects the intensity of the light. A sensor unit using a membrane is used. The SPR measurement device generates SPR on the sensor surface, and measures the reaction state of a substance generated there by detecting the SPR by a measurement unit (see, for example, Patent Document 1 below).

  When light is incident on the interface so as to satisfy the total reflection condition (at an incident angle greater than the critical angle), total reflection occurs, but a small amount of incident light does not reflect and travels within the metal film. Passes 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 satisfying the total reflection condition is incident on the interface, and the incident light with various angles is reflected by the interface, and the reflected light is output to the light receiving surface of the photodetector. The reflected light of light incident at an incident angle (resonance angle) at which SPR occurs is attenuated in light intensity and 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 capturing the change in the position of the dark line as the attenuation angle of the reflected light corresponding to 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.

In the SPR measurement device described in Patent Document 1, a CCD sensor is used as a photodetector, and a light shielding mask is disposed in front of the light receiving surface. The light shielding mask has a slit that transmits the attenuation portion corresponding to the dark line position in the light intensity distribution of the reflected light, and blocks other portions having a light amount larger than the attenuation portion from entering the light receiving surface. If such a light-shielding mask is used, only the attenuating portion is incident on the light receiving surface, so that a limited dynamic range can be used effectively.
JP-A-10-170430

  However, when the reflected light passes through the slit and is incident on the light receiving surface as in the method of Patent Document 1, there is a concern that the light is diffracted and the measurement accuracy is lowered. Further, when the light shielding mask is used, there is a problem that the structure becomes complicated, for example, a moving mechanism for moving the light shielding mask is required in accordance with the change of the dark line position.

  In the CCD sensor, noise is generated in the light intensity signal due to its electrical characteristics. Since this noise affects the calculation accuracy of the dark line position, a countermeasure against this is desired.

  An object of the present invention is to effectively use the dynamic range of a photodetector without causing a complicated structure and a decrease in measurement accuracy due to light diffraction.

  Another object of the present invention is to prevent a reduction in dark line position calculation accuracy due to noise in the light intensity signal.

  The measurement method using total reflection attenuation according to the present invention uses a sensor comprising a thin film whose surface is a sensor surface for fixing a ligand and a transparent dielectric on which the thin film is formed. The solution is fed into contact with the ligand, and light is incident on the interface between the back surface of the thin film and the dielectric so as to satisfy the total reflection condition from the light source, and the light intensity distribution of the reflected light at the interface is changed to light. A total reflection attenuation obtained by a detector and measuring a binding reaction between the analyte and the ligand by detecting a change in a dark line position where attenuation of the reflected light is caused by attenuation of total reflection in the light intensity distribution. In the measurement method using the above, the upper limit is a level at which an effective portion necessary for specifying the dark line position in the light intensity distribution falls within the dynamic range of the photodetector. As the light intensity of the periphery of the effective portion is saturated beyond the dynamic range, and measuring by increasing the quantity of the reflected light.

  As the photodetector, a CCD sensor having a plurality of light receiving elements arranged in a matrix and vertical and horizontal transfer paths for taking out charges from each of the light receiving elements is used, and the analyte, the ligand, The CCD sensor is arranged and measured so that the moving direction in which the dark line position moves on the light receiving surface of the CCD sensor and the transfer direction of the horizontal transfer path of the CCD sensor coincide with each other. It is preferable.

  Further, as the photodetector, a plurality of light receiving elements arranged in a matrix, vertical and horizontal transfer paths for taking out charges from each of the light receiving elements, and a saturated charge connected to the horizontal transfer path It is preferable to use a CCD sensor with an overflow drain to be discarded.

  As the photodetector, a CCD sensor having a plurality of light receiving elements arranged in a matrix and vertical and horizontal transfer paths for taking out charges from each of the light receiving elements is used, and around the effective portion. The maximum light intensity point is equal to or higher than the saturation level that is the upper limit of the dynamic range, and the upper limit value of the allowable light receiving range that does not cause the charge spilling phenomenon to the adjacent pixels that occurs during the transfer of the charge through the transfer path. Thus, it is preferable to measure the binding reaction after adjusting the light emission amount of the light source.

  The measuring apparatus using total reflection attenuation according to the present invention uses a sensor composed of a thin film whose surface is a sensor surface for fixing a ligand and a transparent dielectric on which the thin film is formed. A liquid-feeding means for feeding the liquid and bringing it into contact with the ligand; a light source for making light incident on the interface between the back surface of the thin film and the dielectric so as to satisfy a total reflection condition; and receiving reflected light at the interface A light detector that obtains the light intensity distribution and an analysis unit that analyzes the light intensity distribution, and detects a change in a dark line position where the reflected light is attenuated by total reflection attenuation in the light intensity distribution. Thus, in the measuring apparatus using total reflection attenuation for measuring the binding reaction between the analyte and the ligand, an effective part necessary for specifying the dark line position in the light intensity distribution is a duplication of the photodetector. Levels that fall Namikkurenji as the upper limit, so that the light intensity near the effective portion is saturated beyond the dynamic range, characterized in that a light amount adjusting means for adjusting the light quantity of the light source.

  In the present invention, when the binding reaction between the ligand contacting the sensor surface and the analyte is measured using the total reflection attenuation of the reflected light reflected from the back surface of the sensor surface, the detection is performed by the photodetector. Of the light intensity distribution of the reflected light, the effective portion necessary for specifying the position of the dark line caused by the attenuation is limited to the level at which the effective portion falls within the dynamic range of the photodetector, and the periphery of the effective portion exceeds the dynamic range. Since the measurement is performed by increasing the amount of reflected light so as to be saturated, the dynamic range of the photodetector can be used effectively. In addition, since no light shielding mask is used, there is no complication of the structure and no decrease in measurement accuracy due to light diffraction.

  In addition, by increasing the amount of reflected light, the fluctuation range of the dark line position with respect to the noise level of the light intensity signal is reduced, so that it is possible to prevent the dark line position calculation accuracy from being reduced due to the noise.

  As shown in FIG. 1, a sensor unit 12 for detecting a binding reaction or a dissociation 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 reaction and a dispensing head 28 provided with a pair of pipettes 28 a and 28 b for injecting and discharging the sample solution to and from the channel 16. The measurement unit receives the illumination unit 26 that irradiates the sensor unit 12 with light beams having various incident angles that satisfy the total reflection condition, and the reflected light reflected by the sensor unit 12, and calculates the light intensity distribution. It comprises a CCD sensor 27 for detection. The CCD sensor 27 outputs the light intensity distribution of the reflected light on the light receiving surface as a measurement signal. By analyzing this measurement signal, the reaction of the sample is measured by detecting the change in the resonance angle.

  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. One sensor cell 17 is constituted by one sensor surface 13 a including the linker film 23 and one 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. After the ligand is fixed, an analyte solution containing an analyte is injected into the channel 16 and is sent to the linker film 23 to come into contact with the ligand. By analyzing the measurement signal detected at this time, the binding reaction between the ligand and the analyte is measured.

  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 typified 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 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 35 and an optical system. 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 optical system includes a collimator lens 36, a condenser lens 37, and the like. The divergent light emitted from the light source 35 is collimated by the collimator lens 36 and enters the condenser lens 37. The condensing lens 37 condenses the parallel light and converges it to a specific incident position of the interface 33. 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 a light beam. The light beam to each region is generated by, for example, spectrally dividing light from one light source.

  The CCD sensor 27 receives the reflected light reflected at the detection position P on the interface 33 and outputs a light intensity signal of a level corresponding to the light intensity of the reflected light as a measurement signal. At the detection position P, light beams incident at various incident angles are reflected. Reflected light of various reflection angles corresponding to them enters the light receiving surface 27a. The CCD sensor 27 outputs a light intensity signal of reflected light for each pixel. Thereby, the light intensity distribution of reflected light is obtained.

  Of the light beams incident at various incident angles, the light intensity of the reflected light Rsp (shown by hatching in FIG. 3) incident at the resonance angle is greatly attenuated, so that the reflected light is reflected on the light receiving surface 27a of the CCD sensor 27. An Rsp light receiving position (hereinafter referred to as a dark line position) D is detected as a dark line. An angle formed by the reflected light Rsp and the normal line LN with respect to the interface 33 is referred to as an attenuation angle θ of the reflected light corresponding to the resonance angle.

  The CCD sensor 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, and the detector 27. The head driving mechanism 41 drives the dispensing head 28. The controller 39 controls the movement of the dispensing head 28 and the suction and discharge operations through the head drive mechanism 41.

  The data analysis unit 42 analyzes the measurement data and obtains a temporal change in the resonance signal (SPR signal) representing the attenuation angle θ. The light source driving unit 43 drives the light source 35 to light it. The light source drive unit 43 adjusts the light emission amount of the light source 35 by changing the current value for driving the light source 35 based on the control signal from the controller 39.

  As shown in the graph of FIG. 4, the attenuation angle θ of the reflected light increases in proportion to the refractive index n of the medium on the sensor surface 13a. The refractive index n increases as the amount of binding between the ligand and the analyte increases. Accordingly, when the binding reaction between the ligand and the analyte proceeds, as shown in FIG. 5, the attenuation angle θ increases (θ0 → θ1), and the dark line position D on the light receiving surface 27a moves accordingly (D0 → D1). ) In the SPR measurement, the binding reaction between the ligand and the analyte is analyzed by capturing the change in the dark line position D based on the measurement data.

  FIG. 6 is a graph showing the light intensity distribution of the reflected light acquired by the detector 27. An approximately chevron-shaped curve BO indicated by a thin line is a light intensity distribution of reflected light when no SPR occurs, and shows a beam profile representing an initial shape of a light beam emitted from the illumination unit 26 and reflected by the interface 33. A curve BA indicated by a bold line shows a light intensity distribution when the reflected light is attenuated by the occurrence of SPR. In the curve BA of this example, the substantially central portion is greatly depressed, and this portion is an attenuation portion including the dark line position D. The dark line position D is obtained by calculating the light intensity centroid corresponding to the vicinity of the minimum value of the attenuation portion based on the measurement data of the attenuation portion. As data used for specifying the dark line position D, an effective part E (area surrounded by a two-dot chain line square) located in a lower part of the attenuated part is used.

  As shown in FIG. 7 in which the effective portion E of the curve BA is enlarged, noise is generated in the light intensity signal acquired by the detector 27. This noise is noise caused by the electrical characteristics of the detector 27, and appears as blurring of the measured light intensity value. When such noise is large, the calculation accuracy is lowered, and the dark line position D cannot be accurately specified. Therefore, when calculating the dark line position D, the difference between the curve BO and the curve BA is obtained in order to cancel such noise, and as shown in FIG. 6, the curve CD (indicated by a dotted line) as the difference data is obtained. Is calculated. And the dark line position D is calculated | required by analyzing the attenuation | damping part of this curve CD. Since this curve CD is calculated to facilitate the analysis of the dark line position D, it is called dark line data.

  However, even if the dark line data is obtained in this way, noise cannot be completely removed. Therefore, in this embodiment, in order to increase the calculation accuracy of the dark line position D, the amount of reflected light received by the detector 27 is increased and measured.

  Curves BAa to BAc shown in the graph of FIG. 8 show the light intensity distribution of the reflected light when the light emission amount of the light source 35 is changed in three stages under the same attenuation angle θ. As can be seen from this graph, as the amount of reflected light is increased, the curve of the attenuation part becomes tighter (the rise and fall slopes become tighter). As described above, when the curve of the attenuation portion is tightened, the fluctuation range of the dark line position D due to noise can be reduced.

  FIG. 9 is an enlarged graph of the attenuation portion of the curve BAa having a low light amount and the curve BAc having a high light amount. As shown in this graph, when noise is generated, the curves BAa and BAc are blurred along the horizontal axis as indicated by dotted lines. When the amount of blur in the horizontal axis direction is the fluctuation width ΔX of the dark line position D and the magnitude of noise is ΔN, even if ΔN is the same, the fluctuation width ΔX at that time is greater than the fluctuation width ΔXa of the curve BAa. The fluctuation range ΔXc is smaller. The calculation accuracy of the dark line position D is preferably such that the fluctuation range ΔX is small, so that the amount of reflected light is large.

  In this embodiment, as shown by a curve BAc in FIG. 8, measurement is performed by increasing the amount of reflected light so that a part of the reflected light saturates beyond the accumulated charge capacity of the detector 27. As described above, the measurement data used to calculate the dark line position D is a part of the attenuation portion (effective portion E) of the curve BA. The upper limit is a level where the effective portion E falls within the dynamic range (DR) of the detector 27, and the periphery of the effective portion E (the peak portions generated on both sides when the attenuation portion is a valley) The light emission amount of the light source 35 is adjusted so as to exceed the saturation level TH.

  As a result, the curve of the attenuation portion becomes tight, and the deterioration of the calculation accuracy of the dark line position D caused by the noise of the light intensity signal is prevented. Further, by saturating the periphery of the effective portion E in this way, most of the dynamic range of the CCD sensor 27 is allocated to the effective portion E, so that the dynamic range can be used effectively. In addition, since the light shielding mask is not used as in the prior art, the structure is simplified and there is no fear of light diffraction due to the slit.

  As described above, in order to reduce the fluctuation range ΔX of the dark line position D, it is necessary to increase the amount of light to such an extent that the detector 27 is saturated and tighten the attenuation portion. In such a case, there is a concern that abnormal data resulting from the charge transfer of the CCD sensor 27 may occur.

  As shown in FIG. 10, the CCD sensor 27 is an area sensor having a light receiving surface 27a in which pixels 52 formed of light receiving elements (photodiodes) are arranged in a matrix. Then, the electric charge accumulated according to the amount of light received by each light receiving element is taken out to the amplifier through the vertical transfer path 53 and the horizontal transfer path 54. The vertical transfer path 53 is arranged for each column, and the charge of each light receiving element 52 is taken out and stored in a potential well, and the well in which the charge is stored is connected to the horizontal transfer path 54 like a bucket relay. Forward sequentially. The horizontal transfer path 54 transfers the wells transferred from the vertical transfer paths 53 one by one in the horizontal direction toward the amplifier like a bucket relay. Such a procedure is repeated, and the charge for one screen is taken out.

  When a certain pixel 52 is saturated during such charge transfer, the charge exceeding the accumulated charge amount of the well overflows, and the overflowed charge spills into the adjacent well transferred from the upstream side. This causes a phenomenon that the electric charge of the battery rises exceeding the original accumulated charge amount.

  As shown in FIG. 11, such a phenomenon appears on the read data in the form of an abnormal increase in the amount of light at the upstream pixel in the transfer direction, which becomes abnormal data. A curve B indicated by a solid line indicates a light intensity distribution when a part of a light beam having a substantially mountain beam profile indicated by a dotted line is saturated. The area A1, which is the peak portion of the beam profile, is a saturated light amount portion. When the charge in the area A1 spills upstream in the transfer direction at the time of charge transfer, the light amount in that portion becomes the light amount of the original beam profile (dotted line). The area A2 that becomes abnormal data occurs. When such abnormal data is generated in the attenuation portion, the calculation accuracy of the dark line position D is significantly reduced.

  Therefore, in this embodiment, as shown in FIG. 12, the transfer direction H of the horizontal transfer path 54 of the CCD sensor 27 is arranged so as to coincide with the moving direction M in which the dark line position D moves as the refractive index n increases. ing. As described above, when the binding reaction between the ligand and the analyte occurs and the refractive index n increases, the attenuation angle θ increases (θ0 → θ1), and the dark line position D moves (D0 → D1).

  As shown in FIG. 13, the curve BA indicating the light intensity distribution when attenuation occurs has an asymmetric shape of the attenuation portion. The amount of light around the attenuation portion is larger on the upstream side than on the downstream side in the movement direction M, so that the saturation area Au on the upstream side is larger than the saturation area Ad on the downstream side.

  Since the saturation light amount spills upstream in the transfer direction H of the horizontal transfer path 54, if the CCD sensor 27 is arranged so that the movement direction M and the transfer direction H coincide with each other, the saturation area Ad with less saturation light amount is attenuated. Since it is located downstream of the portion, the probability that the saturated light amount spills into the attenuation portion is reduced, and even if it spills down, the amount is small, so the occurrence of abnormal data can be suppressed to a small amount.

  Hereinafter, the operation of the above configuration will be described. When measuring the reaction of the sample, the controller 39 increases the light amount of the light source 35 through the light source driving unit 43, and a part of the reflected light incident on the CCD sensor 27 is saturated as shown by a curve BAc in FIG. Adjust as follows. After adjusting the amount of light in this way, the analyte solution is injected into the flow path 16 to contact the analyte and the ligand, and the binding reaction between them is measured. When the analyte and the ligand are combined, the refractive index increases and the dark line position D moves. The data analysis unit 42 analyzes the attenuation portion based on the measurement data acquired by the CCD sensor 27 and examines the change in the dark line position D.

  Since the measurement is performed by increasing the amount of light, measurement data can be obtained with a tight curve in the attenuation portion. Since the fluctuation range ΔX of the dark line position D is suppressed, the calculation accuracy of the dark line position D is ensured at a high level. Further, since the upstream side of the horizontal transfer path 54 of the CCD sensor 27 coincides with the upstream side of the moving direction M of the dark line position D when the refractive index n increases, abnormal data is also suppressed.

  In the above-described embodiment, the example in which the direction in which the CCD sensor is arranged is devised in order to suppress the abnormal data. However, as in the CCD sensor 71 shown in FIG. The abnormal data may be suppressed by using a CCD sensor provided with a drain 72. The overflow drain 72 is connected to the horizontal transfer path 54, and when a well having a potential where charges are accumulated is transferred from each vertical transfer path 53, the saturated charge exceeding the accumulated charge amount of the well (two in the figure). (Shown by a dashed line) is passed through the substrate of the CCD sensor 71 and discarded. The horizontal transfer path 54 sends out charges within the range of the accumulated charge amount to the amplifier as signal charges. When the overflow drain 72 is provided in this way, saturated charges are discarded, and it is difficult for charge spillage during horizontal transfer to occur. For this reason, even if the amount of reflected light incident on the CCD sensor 71 is further increased in order to make the attenuation portion curve tighter, abnormal data is less likely to occur, so that the calculation accuracy of the dark line position D can be further improved. .

  In the above embodiment, any of the methods for suppressing the abnormal data (1. The direction in which the CCD sensor is arranged is a specific direction. 2. A CCD sensor with an overflow drain is used.) Assuming that spillage occurs, we explained how to prevent it from appearing as abnormal data. In addition to these methods, the abnormal data may be suppressed by adjusting the amount of light received by the CCD sensor to a level at which charge spillage does not occur during horizontal transfer using the characteristics of the CCD sensor.

  Even if the amount of received light exceeds the saturation level TH, which is the upper limit of the dynamic range, the CCD sensor does not cause a charge spill phenomenon when transferring charges through a horizontal transfer path if it is below a predetermined light receiving level. It has characteristics. Here, a range that is equal to or higher than the saturation level, which is the upper limit of the dynamic range, and in which the charge spill phenomenon does not occur is called an allowable light receiving range. Therefore, if the light emission amount of the light source 35 is adjusted to an appropriate light emission amount so that the maximum intensity point of the reflected light around the effective portion E becomes the upper limit value of the allowable light receiving range, a spillage of charge during transfer occurs. It is possible to make the curve of the attenuation part as tight as possible.

The upper limit value of the allowable light receiving range varies depending on the type of the detector 27. For example, in a CCD sensor having a ¹⁶ -bit dynamic range (2 ¹⁶ = 65536 digit), even if the reflected light intensity is equal to or higher than a saturation level exceeding 65536 digits, charge spillage does not occur up to 100,000 digits. In such a CCD sensor, 100,000 digits is the upper limit of the allowable light receiving range.

  Therefore, if the amount of light emitted from the light source 35 is adjusted by the light source driving unit 43 so that the reflected light intensity around the effective portion E becomes 100000 digits, it becomes difficult for the charge to be spilled during transfer, thereby causing abnormalities. Data can be suppressed.

  Even in the same CCD sensor, since the reflectance at the interface 33 varies depending on the incident position (sensing point) of light within the interface 33 of the sensor unit 12 and the individual difference of the sensor unit 12, the incident from the light source 35 The intensity of the reflected light is not uniformly determined by simply adjusting the amount of light to a specific value. Therefore, in order to adjust the reflected light intensity to the upper limit value of the allowable light receiving range, it is preferable to adjust the light emission amount of the light source 35 by the following procedure.

  As shown in the flowchart of FIG. 15, the light emission amount adjustment of the light source 35 is performed before the main measurement for measuring the binding reaction between the ligand and the analyte. First, the sensor unit 12 in which the ligand is not fixed to the linker film 23 is set in the SPR measurement device, light is incident from the light source 35 to the interface 33 of the sensor unit 12, and the reflected light is Light is received by the detector 27 to obtain a light intensity distribution of the reflected light.

  Next, as shown in FIG. 16, the light emission amount of the light source 35 is adjusted so that the maximum intensity point M of the obtained reflected light intensity distribution BA1 reaches the saturation level TH. Then, the light emission amount of the light source 35 is increased so that the maximum intensity point M further increases from the saturation level TH and reaches the upper limit value Pmax of the allowable light receiving range. As a result, the light intensity distribution BA1 is changed to the light intensity distribution BA2. Since a region exceeding the saturation level TH (indicated by a two-dot chain line) cannot be measured, the light emission amount of the light source 35 when the maximum intensity point M reaches the saturation level TH is expressed as (the upper limit value Pmax / saturation of the permissible light reception range). The light intensity distribution BA2 is obtained by multiplying the level TH). For example, when the saturation level TH is 65536 digits and the upper limit value Pmax level P of the allowable light receiving range is 100,000 digits, it is multiplied by 100,000 digits / 65536 digits.

  After adjusting the light emission amount of the light source 35 in this way, a ligand fixing process is performed and a measurement process is executed. Since the light emission amount of the light source 35 is adjusted to an appropriate light emission amount that does not cause charge spilling during transfer, abnormal data does not occur, and high-precision measurement is possible. Such light emission amount adjustment is preferably performed every time the sensor unit 12 is replaced.

  The above-described three methods for suppressing abnormal data may be applied one by one, or two or more methods may be combined.

  In the above embodiment, the example in which the CCD sensor is used as the photodetector has been described. However, for example, a photosensor other than the CCD sensor such as a CMOS sensor may be used.

  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.

  In the above 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, and 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 which shows the relationship between a refractive index and an attenuation angle. It is explanatory drawing which shows the relationship between attenuation angle change and the movement of a dark-line position. It is a graph which shows light intensity distribution. It is explanatory drawing of the noise of a light intensity signal. It is a graph which shows the relationship between the light quantity level of reflected light, and the curve of an attenuation part. It is a graph which shows the relationship between noise and a fluctuation range. It is explanatory drawing of the charge transfer method of a CCD sensor. It is explanatory drawing of the abnormal data resulting from electric charge transfer. It is explanatory drawing of the arrangement direction of a CCD sensor. It is a graph explaining the asymmetry of an attenuation part. It is explanatory drawing of a CCD sensor with an overflow drain. It is a flowchart which shows the procedure which performs light quantity adjustment and measures. It is a graph explaining the light quantity adjustment method.

Explanation of symbols

12 Sensor unit 13 Metal film 13a Sensor surface
14 Prism 26 Illumination unit 27 CCD sensor 28 Dispensing head 39 Controller 42 Data analysis unit 43 Light source drive unit

Claims (5)

Using a thin film whose surface is a sensor surface for fixing a ligand and a transparent dielectric formed with the thin film, an analyte is fed to the sensor surface to contact the ligand, and the thin film The light is incident from the light source toward the interface between the back surface of the dielectric and the dielectric so as to satisfy the total reflection condition, and the light intensity distribution of the reflected light at the interface is acquired by a photodetector, In a measurement method using total reflection attenuation for measuring a binding reaction between the analyte and the ligand by detecting a change in a dark line position where attenuation of the reflected light is caused by total reflection attenuation,
The light intensity around the effective portion saturates beyond the dynamic range, with the upper limit being a level at which the effective portion necessary for specifying the dark line position in the light intensity distribution falls within the dynamic range of the photodetector. Thus, the measurement method using the total reflection attenuation, wherein the measurement is performed by increasing the amount of the reflected light.   As the photodetector, a CCD sensor having a plurality of light receiving elements arranged in a matrix and vertical and horizontal transfer paths for taking out charges from each of the light receiving elements is used, and the analyte, the ligand, The CCD sensor is arranged and measured so that the moving direction in which the dark line position moves on the light receiving surface of the CCD sensor and the transfer direction of the horizontal transfer path of the CCD sensor coincide with each other. The measurement method using total reflection attenuation according to claim 1.   As the photodetector, a plurality of light receiving elements arranged in a matrix, vertical and horizontal transfer paths for taking out charges from each of the light receiving elements, and connected to the horizontal transfer path, the saturated charges are discarded. 2. The measuring method using total reflection attenuation according to claim 1, wherein a CCD sensor having an overflow drain is used.   As the photodetector, a CCD sensor having a plurality of light receiving elements arranged in a matrix and vertical and horizontal transfer paths for taking out charges from each of the light receiving elements is used, and around the effective portion. The maximum light intensity point is equal to or higher than the saturation level that is the upper limit of the dynamic range, and the upper limit value of the allowable light receiving range that does not cause the charge spilling phenomenon to the adjacent pixels that occurs during the transfer of the charge through the transfer path. The measurement method using total reflection attenuation according to claim 1, wherein the binding reaction is measured after adjusting the light emission amount of the light source. A liquid supply means for supplying an analyte to the sensor surface and bringing it into contact with the ligand, using a thin film whose surface is a sensor surface for fixing the ligand and a transparent dielectric formed with the thin film; A light source that makes light incident so as to satisfy a total reflection condition toward the interface between the back surface of the thin film and the dielectric, a photodetector that receives the reflected light at the interface and acquires its light intensity distribution; An analysis means for analyzing the light intensity distribution, and detecting a change in a dark line position where attenuation of the reflected light due to total reflection attenuation is detected in the light intensity distribution, and performing a binding reaction between the analyte and the ligand. In a measuring device using total reflection attenuation to be measured,
The light intensity around the effective portion saturates beyond the dynamic range, with the upper limit being the level at which the effective portion necessary for specifying the dark line position in the light intensity distribution falls within the dynamic range of the photodetector. Thus, a measuring device using total reflection attenuation, characterized in that a light amount adjusting means for adjusting the light amount of the light source is provided.

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