JP2006220605A - Measurement method and measurement device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To more correctly calibrate measurement error caused by individual differences in the measurement sensitivities with the measurement method and measurement device for analysing a sample by generating evanescent waves, by making a light beam reflected totally at the boundary of the thin-film layer that is in contact with the sample etc., and a dielectric body, by measuring the change in the intensity of totally reflected light beam. <P>SOLUTION: The preliminary measurements for the 4 buffers or more of known refractive indexes are performed in the reference area, the 3-D calibration curves are calculated by the signal process part 20, representing the relation between the refractive indexes of the buffers and dark lines, and the calibration curves are stored to a storage means in the signal process part 20. The results of actual measurements are calculated by the calibration means in the signal process part 20, based on the calibration curves stored in the memory means at the time of the actual measurement. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention generates an evanescent wave by totally reflecting a light beam at an interface between a thin film layer in contact with a measurement object such as a sample and a dielectric block, thereby measuring a change in the intensity of the totally reflected light beam. The present invention relates to a measuring method and a measuring apparatus for analyzing a sample.

  Conventionally, a surface plasmon sensor is known as one of measuring devices using an evanescent wave. In the metal, free electrons collectively vibrate to generate a dense wave called a plasma wave. A quantized version of this dense wave generated on the metal surface is called surface plasmon. The surface plasmon sensor analyzes the characteristics of a sample using a phenomenon in which the surface plasmon is excited by a light wave, and various types of sensors have been proposed. Among them, one that uses a system called Kretschmann configuration is well known (for example, see Patent Document 1).

  A surface plasmon sensor using the above system basically includes, for example, a dielectric block formed in a prism shape, a metal film formed on one surface of the dielectric block and brought into contact with a sample, and a light source that generates a light beam. An optical system that causes the light beam to be incident on the dielectric block at various angles so that a total reflection condition is obtained at the interface between the dielectric block and the metal film, and a light beam that is totally reflected at the interface A light detecting means for detecting the intensity of the light and a measuring means for measuring the surface plasmon resonance state based on the detection result of the light detecting means.

  In order to obtain various incident angles as described above, a relatively thin light beam may be incident on the interface by changing the incident angle, or a component incident on the light beam at various angles is included. As described above, a relatively thick light beam may be incident on the interface in a convergent light state or a divergent light state. In the former case, a light beam whose reflection angle changes according to the change in the incident angle of the incident light beam is detected by a small photodetector that moves in synchronization with the change in the reflection angle, or the direction in which the reflection angle changes Can be detected by an area sensor extending along the line. On the other hand, in the latter case, it can be detected by an area sensor extending in a direction in which all light beams reflected at various reflection angles can be received.

In the surface plasmon sensor having the above configuration, when a light beam is incident on the metal film at a specific incident angle θ _SP that is equal to or greater than the total reflection angle, an evanescent wave having an electric field distribution is generated in the sample in contact with the metal film, This evanescent wave excites surface plasmons at the interface between the metal film and the sample. When the wave number vector of the evanescent light is equal to the wave number of the surface plasmon and the wave number matching is established, both are in a resonance state and the energy of the light is transferred to the surface plasmon, so that the entire energy is transferred to the interface between the dielectric block and the metal film. The intensity of the reflected light decreases sharply. This decrease in light intensity is generally detected as a dark line by the light detection means.

  The resonance described above occurs only when the incident beam is p-polarized light. Therefore, it is necessary to set in advance so that the light beam is incident as p-polarized light.

When the wave number of the surface plasmon is found from a specific incident angle θ _SP (hereinafter referred to as total reflection attenuation angle θ _SP ) that is greater than or equal to the total reflection angle at which the light intensity is reduced, the dielectric constant of the sample is obtained. That is, when the wave number of the surface plasmon is K _SP , the angular frequency of the surface plasmon is ω, c is the speed of light in vacuum, ε _m and ε _s are each a metal, and the dielectric constant of the sample is as follows.

If the dielectric constant ε _s of the sample is known, the refractive index of the sample can be known based on a predetermined calibration curve or the like, and therefore, by knowing the total reflection attenuation angle θ _SP , the dielectric constant of the sample, that is, the refractive index is related. The characteristics to be obtained can be obtained.

  Further, a leak mode sensor is also known as a similar sensor using an evanescent wave (see, for example, Non-Patent Document 1). This leakage mode sensor is basically a dielectric block formed in a prism shape, for example, a clad layer formed on one surface of the dielectric block, and formed on the clad layer to be brought into contact with a sample. An optical waveguide layer, a light source that generates a light beam, and the light beam are incident on the dielectric block at various angles so that a total reflection condition is obtained at the interface between the dielectric block and the cladding layer. An optical system, a light detecting means for measuring the intensity of the light beam totally reflected at the interface, and a measuring means for measuring the excited state of the waveguide mode based on the detection result of the light detecting means. is there.

In the leaky mode sensor having the above configuration, when a light beam is incident on the cladding layer through the dielectric block at an incident angle greater than the total reflection angle, the light waveguide layer transmits a specific wave number after passing through the cladding layer. Only light having a specific incident angle is propagated in the guided mode. When the waveguide mode is excited in this way, most of the incident light is taken into the optical waveguide layer, resulting in total reflection attenuation in which the intensity of light totally reflected at the interface is sharply reduced. Since the wave number of the waveguide light depends on the refractive index of the sample on the optical waveguide layer, by knowing the total reflection attenuation angle theta _SP, it can be analyzed refractive index of the sample and the properties of the sample related thereto .

In addition, the surface plasmon sensor and leakage mode sensor described above may be used for random screening to find an analyte that binds to a desired ligand in the field of drug discovery research. In this case, the thin film layer (surface plasmon sensor) is used. In the case of a sensor, it is a metal film, and in the case of a leak mode sensor, a ligand is fixed on a clad layer and an optical waveguide layer), and buffers (liquid samples) containing various analytes are added on the ligand, every time elapses measures the ATR angle theta _SP described above. If the analyte in the buffer is one that binds to the ligand, the binding causes the refractive index of the ligand to change over time. Therefore, the attenuated total reflection angle theta _SP was measured every predetermined time, by a change in the attenuated total reflection angle theta _SP to measure whether occurring, whether binding between the analyte and ligand have been made That is, it can be determined whether or not the analyte is a specific substance that binds to the ligand. Examples of such a combination of an analyte and a ligand include an antigen and an antibody or an antibody and an antibody. As a specific measurement related to such an analyte, for example, a ligand is a rabbit anti-human IgG antibody, Examples include detection of the presence or absence of binding to a human IgG antibody, which is a light, and quantitative analysis thereof.

In order to measure a binding state between the analyte and ligand in buffer, it is not always necessary to detect the angle itself of an attenuated total reflection angle theta _SP. For example after first measuring the baseline as a reference by using the buffer without analyte was measured angle variation of the attenuated total reflection angle theta _SP upon addition of a buffer containing the analyte on the ligand Thus, the coupling state can be measured based on the magnitude of the angle change amount.
JP-A-6-167443 Japanese Patent Laid-Open No. 2003-279478 “Spectroscopy” Vol. 47, No. 1 (1998)

By the way, the amount of change in the total reflection attenuation angle θ _SP detected by the measuring device such as the above-described surface plasmon sensor accurately reflects the change in the refractive index due to the binding between the analyte and the ligand. Instead, some errors may occur due to individual differences in the measurement sensitivity of the measurement apparatus.

  In order to eliminate such errors due to individual differences, a measurement method for performing preliminary measurement on a plurality of buffers (liquid samples) having a known refractive index in advance and calibrating the result at the time of actual measurement based on the result, and Although a measurement device has been proposed by the applicant of the present application (see Patent Document 2), there is a possibility that a measurement error due to individual differences in measurement sensitivity of the measurement device may occur even if such a measurement method and measurement device are used.

  The present invention has been made in view of such problems, and generates an evanescent wave by totally reflecting a light beam at an interface between a thin film layer in contact with a measurement object such as a sample and a dielectric block, thereby A measurement method and measurement device that measures changes in the intensity of the totally reflected light beam and analyzes the sample, allowing measurement errors due to individual differences in measurement sensitivity of the measurement device to be calibrated more accurately It is an object to provide a method and apparatus.

  In order to calibrate the measurement error due to individual differences in the measurement sensitivity of the measurement device, calibration data on the relationship between the refractive index of the buffer and the position of the dark line is necessary, and if this calibration data is not accurate, the measurement device Measurement errors due to individual differences in measurement sensitivity cannot be accurately corrected. Therefore, the present inventor conducted a detailed investigation on the relationship between the refractive index of the buffer and the position of the dark line.

  As a result, it was found that there is a relationship as shown in FIG. FIG. 12 is a graph showing the relationship between the refractive index of the buffer and the position of the dark line. The vertical axis represents the SPR signal (corresponding to the position of the dark line), and the horizontal axis represents the DMSO concentration in the buffer (corresponding to the refractive index).

  Conventionally, the relationship between the refractive index and the position of the dark line is approximated to a first-order or second-order low-order straight line or curve based on the results of preliminary measurements performed on a plurality of buffers having a known refractive index. However, if the relationship between the refractive index and the position of the dark line is approximated to a straight line in the narrow range of the DMSO concentration in the buffer, that is, the refractive index as shown by the dotted line in FIG. 12, an error in the DMSO concentration in the buffer. And the slope of the straight line changes in response to measurement errors and the like, and as shown by the one-dot chain line in FIG. When the relationship with the position is approximated to a straight line, the influence of the DMSO concentration error in the buffer, the measurement error, and the like are reduced, but there may be a large deviation from the actual measurement result. For this reason, it has been found that these relationships can be reflected more accurately by approximating a cubic curve of higher order or higher.

  The present invention has been made on the basis of the above-mentioned new knowledge, and the measurement method according to the present invention uses a light beam at the interface between the dielectric block and the thin film layer with respect to the dielectric block on which the thin film layer is formed. The incident light is incident at various angles so that the total reflection condition is obtained, and the position of the dark line in the light beam totally reflected at the various reflection angles at the interface is detected. Based on the detection result, the substance in contact with the thin film layer is detected. In a measurement method for measuring characteristics depending on the refractive index, preliminary measurement is performed on at least four buffers having known refractive indexes, and the relationship between the refractive index of the buffer and the position of the dark line is determined based on the result of the preliminary measurement. A high-order calibration curve is calculated, and the result of this measurement is calibrated based on this calibration curve.

  A measuring apparatus according to the present invention includes a dielectric block in which a thin film layer is formed, a measurement unit having a sample holding mechanism for holding a sample on the thin film layer, a light source that generates a light beam, and a light block that is a dielectric block. In contrast, an incident optical system that is incident at various angles so that a total reflection condition can be obtained at the interface between the dielectric block and the thin film layer, and a dark line that detects the position of the dark line in the light beam that is totally reflected at the interface A measuring apparatus comprising: a position detecting unit; and a measuring unit that measures characteristics depending on a refractive index of a substance in contact with a thin film layer based on a detection result by a dark line position detecting unit. Calculating means for calculating a higher-order calibration curve representing the relationship between the refractive index of the buffer and the position of the dark line based on the preliminary measurement results for two or more buffers, and a school calculated by the calculating means. Storage means for storing the curve, is characterized in that a calibration means for calibrating the result of the measurement based on the calibration curve.

  In the measurement method and measurement apparatus according to the present invention, the higher-order calibration curve is preferably a third-order calibration curve.

  The “high-order calibration curve representing the relationship between the refractive index and the position of the dark line” is not limited to the refractive index and the position of the dark line itself, and includes a variable corresponding to the refractive index and a variable corresponding to the position of the dark line. It only has to represent a relationship. For example, the variable corresponding to the refractive index includes DMSO concentration in the buffer, and the variable corresponding to the position of the dark line includes an SPR signal indicating the amount of movement of the dark line.

  As will be described later, the measurement apparatus in the measurement method of the present invention and the measurement apparatus of the present invention make light beams of a plurality of wavelengths incident at an incident angle at which the total reflection condition is obtained at the interface, and for each wavelength, By measuring the intensity of the reflected light beam and detecting the degree of total reflection attenuation for each wavelength, the refractive index or refractive index change may be measured. Instead of “position”, a higher-order calibration curve representing the relationship between “the wavelength at which the dark line is generated” and the refractive index may be obtained.

  Further, as a method for calculating a higher-order (third-order or higher) calibration curve, any method such as a least square method may be used.

  In addition, the measuring device in the measuring method of the present invention and the measuring device of the present invention are configured as a so-called surface plasmon sensor in which the thin film layer is made of a metal film, and measurement is performed using the effect of the surface plasmon resonance described above. Can be. In addition, the thin film layer is composed of a clad layer formed on the one surface of the dielectric block and an optical waveguide layer formed on the clad layer, and measurement is performed by using the effect of waveguide mode excitation in the optical waveguide layer. It can be configured as a so-called leakage mode sensor.

  The method for measuring the state on the thin film layer is to detect the reflected light at the interface between the dielectric block and the thin film layer at various incident angles and detect the total reflection attenuation angle. Alternatively, the refractive index or refractive index change may be measured by detecting the angle change, and DVNoort, K. johansen, C.-F. Mandenius, Porous Gold in Surface Plasmon Resonance Measurement, As described in EUROSENSORS XIII, 1999, pp.585-588, a light beam having a plurality of wavelengths is incident at an incident angle at which the total reflection condition is obtained at the interface, and is totally reflected at the interface for each wavelength. The refractive index or refractive index change may be measured by measuring the intensity of the light beam and detecting the degree of total reflection attenuation for each wavelength.

  According to the measurement method and the measurement apparatus of the present invention, preliminary measurement is performed on at least four buffers having a known refractive index, and the relationship between the refractive index of the buffer and the position of the dark line is expressed based on the result of the preliminary measurement. By calculating the next calibration curve and calibrating the result of the main measurement based on this calibration curve, it becomes possible to calibrate the measurement error due to the individual difference in the measurement sensitivity of the measuring apparatus more accurately.

  Further, in the measurement method and the measurement apparatus described above, the calibration curve can be obtained with a small amount of calculation compared to the case of obtaining a fourth-order or higher-order calibration curve by making the calibration curve a cubic curve.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. The measurement apparatus according to the first embodiment of the present invention is a surface plasmon sensor capable of simultaneously analyzing a plurality of samples by allowing light beams to enter the plurality of measurement units of the measurement unit in parallel. 1 is a plan view showing a schematic configuration of a surface plasmon sensor according to the present embodiment, FIG. 2 is a plan view of a measurement system of the surface plasmon sensor, FIG. 3 is a side view of the measurement system of the surface plasmon sensor, and FIG. It is the VIII-VIII sectional view taken on the line in FIG.

  As shown in FIG. 1, the surface plasmon sensor 1 has a surface that can simultaneously analyze a plurality of samples by allowing a light beam to enter in parallel for each of a plurality of measurement units provided in the measurement unit 10. A plasmon sensor, which is composed of a plurality of surface plasmon measurement systems 1A, 1B,. The configuration of each measurement system will be described by omitting the symbols A, B,.

  As shown in FIGS. 2, 3, and 8, each measurement system measures a light source 14 (hereinafter referred to as a laser light source 14) composed of a semiconductor laser or the like that generates one light beam 13, and the light beam 13. Through the unit 10, the light is incident in parallel on the two interfaces 50f and 50g between the dielectric block 50 and the metal film 55 below the flow channel 60 (measurement unit) so that various incident angles can be obtained. An optical system 15, two collimator lenses 16 for collimating the light beams 13 totally reflected at the interfaces 50f and 50g, and two photodiode arrays 17 for detecting the collimated light beams 13, respectively. A differential amplifier array 18 connected to the two photodiode arrays 17, a driver 19, a signal processing unit 20 including a computer system, and a display unit 21 connected to the signal processing unit 20. Yes. The dark line position detection means includes a photodiode array 17, a differential amplifier array 18, a driver 19, and a signal processing unit 20, and the signal processing unit 20 uses a buffer based on the result of preliminary measurement described later. As a calculation means for calculating a calibration curve representing the relationship between the refractive index and the position of the dark line, a storage means for storing the calibration curve calculated by the calculation means, and a calibration means for calibrating the result of this measurement based on the calibration curve Also works. Details of the processing of this computing means will be described later.

  First, the measurement unit 10 will be described. 4 is a perspective view of the measurement unit 10, FIG. 5 is an exploded perspective view of the measurement unit, FIG. 6 is a top view of the measurement unit, and FIG. 7 is a sectional view taken along the line VII-VII in FIG.

  The measurement unit 10 is transparent to the light beam and is in close contact with the dielectric block 50 in which a metal film 55 as a thin film layer is formed on a smooth upper surface 50a, and the metal film 55 of the dielectric block 50. A flow path member (sample supply mechanism) 51 and a holding member 52 that engages with the dielectric block 50 and holds the flow path member 51 on the upper surface 50a of the dielectric block 50 are configured.

  The dielectric block 50 is made of, for example, a transparent resin, and has a trapezoidal body whose section perpendicular to the longitudinal direction is shorter at the lower base than at the upper base, and at both ends in the longitudinal direction of the main body. A holding portion 50b having a width smaller than that of the main body when viewed from the upper surface (or lower surface) direction is formed, and a light beam emitted from a light source of a measuring apparatus described later is applied to the dielectric block 50 and the metal film. A prism portion is formed integrally so as to be incident on the interface with 55 and to emit the light beam totally reflected on the interface toward the light detection means of the measuring apparatus. On both side surfaces in the longitudinal direction of the main body are engaging convex portions 50c for engaging with engaging holes 52c formed in a holding member 52, which will be described later, and vertical convex portions 50d whose side surfaces are formed vertically. Sliding grooves 50e extending in parallel with the longitudinal direction are formed on the bottom surface.

  The flow path member 51 includes a plurality of flow paths 60 including a supply path 62 extending from the inlet 61 to the measurement section 63 and a discharge path 64 extending from the measurement section 63 to the outlet 65 in the longitudinal direction of the flow path member 51. The plurality of flow paths 60 are arranged in a straight line.

  As shown in FIG. 7, the lower portion of the flow path member 51 has the outlet of the supply path 62 and the inlet of the discharge path 64 opened, and a region in contact with the surface of the metal film 55 located on the lower surface of the flow path member 51. Further, a seal part 51a is formed surrounding the outlet of the supply path 62 and the inlet of the discharge path 64, and the inside of the seal part 51a becomes the measurement part 63. For this reason, when the flow path member 51 is brought into close contact with the metal film 55 of the dielectric block 50, the measurement section 63 in the seal section 51a functions as a flow path. The seal portion 51a may be integrally formed with the upper portion of the flow path member 51, or may be formed of a material different from the upper portion and attached later, for example, O A ring or the like attached to the lower portion of the flow path member 51 may be used.

  In a measuring device such as a surface plasmon sensor using the measuring unit of the present invention, it is assumed that a liquid sample containing a protein is used. However, if the protein in the liquid sample is fixed in the flow channel 60, the measurement is performed. Therefore, it is preferable that the material of the flow path member 51 does not have non-specific adsorptivity to proteins, and specifically, silicon, polypropylene, or the like may be used. In addition, since the flow path member 51 is made of such an elastic material, the flow path member 51 can be reliably brought into close contact with the metal film 55, thereby preventing liquid leakage of the liquid sample from the contact surface. can do.

  The holding member 52 is made of an elastic material such as polypropylene, and the cross section in the direction orthogonal to the longitudinal direction has a substantially square shape, and the inlet 61 of the flow path member 51 of the upper plate (holding plate portion) of the holding member 52. Further, a tapered pipette insertion hole 52a that narrows toward the flow path member 51 is formed at a position facing the outlet 65, and pipette insertion at the middle of each pipette insertion hole 52a on the upper surface of the holding member 52 and at both ends A positioning boss 52b is formed on the outer side of the hole 52a.

  Further, an evaporation preventing member 54 is attached to the upper surface of the holding member 52 with a double-sided tape (adhesive member) 53. As shown in FIG. 5, a pipette insertion hole 53a is formed at a position facing the pipette insertion hole 52a of the double-sided tape 53, and a positioning hole 53b is formed at a position facing the boss 52b. Similarly, a slit 54a is formed at a position facing the pipette insertion hole 52a of the evaporation preventing member 54, and a positioning hole 54b is formed at a position facing the boss 52b. In a state where the hole 53b and the hole 54b of the evaporation preventing member 54 are inserted, the evaporation preventing member 54 is attached to the upper surface of the holding member 52, whereby the slit 54a of the evaporation preventing member 54, the inlet 61 and the outlet 65 of the flow path member 51 are obtained. Are configured to face each other. The evaporation preventing member 54 needs to be made of a material having elasticity so that a pipette can be inserted from the slit 54a, and specifically silicon or polypropylene may be used. The holding member 52 and the evaporation preventing member 54 may be integrally formed, and in addition to this, the flow path member 51 may be integrally formed.

  Furthermore, an engagement hole 52c for engaging with an engagement protrusion 50c formed in the dielectric block 50 is formed in the longitudinal side plate of the holding member 52, and the engagement hole 52c is engaged with the engagement protrusion 52c. In a state where the holding member 52 and the dielectric block 50 are engaged with each other by the portion 50c, the flow path member 51 is sandwiched between the holding member 52 and the dielectric block 50, and the flow path member 51 is the dielectric block. 50 is configured to be held on the upper surface 50a.

  As shown in FIG. 7, in a state where the flow path member 51 is sandwiched between the holding member 52 and the dielectric block 50, the inlet 61 and the outlet 65 of the flow path member 51 are separated from the outside air by the slit 54a of the evaporation preventing member 54. The liquid sample that is blocked and injected into the flow path 60 is configured to prevent evaporation.

  The incident optical system 15 includes a collimator lens 15a that collimates the light beam 13 emitted from the laser light source 14 in a divergent light state, a half mirror 15c that divides the collimated light beam 13, and a half mirror 15c. The light beam 13 reflected by the mirror 15d is reflected in the direction of the measurement unit 10, the light beam 13 transmitted through the half mirror 15c, and the light beam 13 reflected by the mirror 15d are converged on the interfaces 50f and 50g, respectively. It is composed of two condenser lenses 15b.

  Since the light beam 13 is condensed as described above, the light beam 13 includes components incident on the interfaces 50f and 50g at various incident angles θ. In addition, this incident angle (theta) shall be an angle more than a total reflection angle. Therefore, the light beam 13 is totally reflected at the interfaces 50f and 50g, and the reflected light beam 13 includes components reflected at various reflection angles. The optical system 15 may be configured to cause the light beam 13 to enter the interfaces 50f and 50g in a defocused state. By doing so, errors in surface plasmon resonance state detection are averaged, and measurement accuracy is improved.

  The light beam 13 is incident on the interfaces 50f and 50g as p-polarized light. In order to do so, the laser light source 14 may be disposed in advance so that the polarization direction thereof is a predetermined direction. In addition, the direction of polarization of the light beam 13 may be controlled with a wave plate.

As shown in FIG. 8, in the present embodiment, the measurement unit 63 of each flow path 60 of the measurement unit 10 is used.
The light beam 13 is incident on the two interfaces 50f and 50g in parallel, of which the metal film 55 on one interface 50f is used as a reference region where nothing is fixed, and the other interface On the metal film 55 above 50 g, a detection region in which the ligand 73 is fixed is used so that the measurement result can be calibrated by the reference method described later.

  Hereinafter, sample analysis by the surface plasmon sensor 1 having the above-described configuration will be described. Prior to the measurement, the measurement unit 10 is moved from the temperature-controlled room 2 toward the measurement position on the chip holder 11. The chip holding portion 11 is formed with a rail 11a that engages with a sliding groove 50e formed in the dielectric block 50, so that high positional accuracy can be ensured when the measuring unit 10 is moved. ing. Further, after the measurement unit 10 is placed on the chip holding part 11, the vertical convex part 50d formed on the dielectric block 50 is clamped by a fixing mechanism (not shown) and fixed at the measurement position on the chip holding part 11. Is done. Thereafter, as shown in FIG. 8, the pipette tip 70 for supplying the liquid sample is inserted into the inlet 61 of the flow path member 51, and the pipette tip 71 for sucking the liquid sample is inserted into the outlet 65. After supplying the buffer 72 containing the analyte as a sample to the measuring unit 63 of the flow channel 60, the measurement is started.

  As shown in FIG. 3, the light beam 13 emitted from the laser light source 14 in a divergent light state is caused on the interfaces 50 f and 50 g between the dielectric block 50 and the metal film 55 below the measuring unit 63 by the action of the optical system 15. Converge. At this time, the light beam 13 includes components incident on the interfaces 50f and 50g at various incident angles θ. In addition, this incident angle (theta) shall be an angle more than a total reflection angle. Therefore, the light beam 13 is totally reflected at the interfaces 50f and 50g, and the reflected light beam 13 includes components reflected at various reflection angles.

  After total reflection at the interfaces 50f and 50g, the two light beams 13 respectively collimated by the two collimator lenses 16 are detected by the two photodiode arrays 17, respectively. In the photodiode array 17 in this example, a plurality of photodiodes 17a, 17b, 17c... Are arranged in a line, and in the traveling direction of the collimated light beam 13 in the plane shown in FIG. On the other hand, the photodiodes are arranged in a direction in which the parallel arrangement direction of the photodiodes is substantially a right angle. Therefore, different photodiodes 17a, 17b, 17c,... Receive the components of the light beam 13 totally reflected at various reflection angles at the interfaces 50f and 50g.

  FIG. 9 is a block diagram showing an electrical configuration of the surface plasmon sensor. As shown in the figure, the driver 19 samples and holds the outputs of the differential amplifiers 18a, 18b, 18c,... Of the differential amplifier array 18, and these sample-hold circuits 22a, 22b. , 22c... Are input to the multiplexer 23, the A / D converter 24 which digitizes the output of the multiplexer 23 and inputs it to the signal processing unit 20, the multiplexer 23 and the sample hold circuits 22a, 22b, 22c. And a controller 26 that controls the operation of the drive circuit 25 based on an instruction from the signal processing unit 20. The differential amplifier array 18, the driver 19, and the signal processing unit 20 are configured to perform the same processing in parallel on the inputs from the two photodiode arrays 17.

  The outputs of the photodiodes 17a, 17b, 17c,... Are input to the differential amplifiers 18a, 18b, 18c,. At this time, the outputs of two photodiodes adjacent to each other are input to a common differential amplifier. Therefore, it can be considered that the outputs of the differential amplifiers 18a, 18b, 18c,... Are obtained by differentiating the photodetection signals output from the plurality of photodiodes 17a, 17b, 17c,.

  The outputs of the differential amplifiers 18a, 18b, 18c... Are sampled and held at predetermined timings by the sample hold circuits 22a, 22b, 22c. The multiplexer 23 inputs the sampled and held outputs of the differential amplifiers 18a, 18b, 18c,... Into the A / D converter 24 in a predetermined order. The A / D converter 24 digitizes these outputs and inputs them to the signal processing unit 20.

  FIG. 10 illustrates the relationship between the light intensity for each incident angle θ of the light beam 13 totally reflected at the interface 50f (or 50g) and the outputs of the differential amplifiers 18a, 18b, 18c. Here, it is assumed that the relationship between the incident angle θ of the light beam 13 to the interface 50f (or 50g) and the light intensity I is as shown in the graph of FIG.

Light incident at a specific incident angle θ _SP at the interface 50f (or 50g) excites surface plasmons at the interface between the metal film 55 and the buffer 72, and the reflected light intensity I sharply decreases for this light. That theta _SP is attenuated total reflection angle, the reflected light intensity I in the angle theta _SP takes a minimum value. This decrease in the reflected light intensity I is observed as a dark line in the reflected light, as indicated by D in FIG.

  (2) in FIG. 10 shows the direction in which the photodiodes 17a, 17b, 17c... Are arranged, and as described above, the positions in the direction in which these photodiodes 17a, 17b, 17c. It uniquely corresponds to the incident angle θ.

  The relationship between the positions of the photodiodes 17a, 17b, 17c..., That is, the incident angle θ, and the output I ′ (differential value of the reflected light intensity I) of the differential amplifiers 18a, 18b, 18c. As shown in FIG.

Based on the value of the differential value I ′ input from the A / D converter 24, the signal processing unit 20 selects the differential corresponding to the total reflection attenuation angle θ _SP from among the differential amplifiers 18a, 18b, 18c. After selecting the output that is closest to the value I ′ = 0 (in the example of FIG. 10, the differential amplifier 18d), the differential value I ′ that it outputs is subjected to a predetermined correction process, The value is displayed on the display unit 21. In some cases, there may be a differential amplifier that outputs a differential value I ′ = 0. In this case, the differential amplifier is naturally selected.

Thereafter, the differential value I ′ output from one of the differential amplifiers selected as described above every time a predetermined time elapses is displayed on the display unit 21 after receiving a predetermined correction process. The differential value I'has a dielectric constant clogging material in contact with the metal film 55 of the measuring chip refractive index changes, the attenuated total reflection angle theta _SP is changed, the left and right curve shown in FIG. 10 (1) If it changes in a moving direction, it will move up and down accordingly. Therefore, by continuously measuring this differential value I ′ with the passage of time, the refractive index change of the buffer 72 (or the ligand 73) in contact with the metal film 55 can be examined.

Incidentally, the total reflection eliminates angle theta _SP angular change measured in the detection region of the device is strictly accurately reflect changes in the refractive index caused in response to changes in binding state of the ligand 73 and the analyte Instead, as described above, an error due to individual differences in the measurement sensitivity of the apparatus is included.

  Therefore, in the measurement apparatus of the present invention, preliminary measurement is performed on four or more buffers having known refractive indexes in the reference region, and based on the result, the refractive index of the buffer and the dark line as shown by the solid line in FIG. A tertiary calibration curve representing the relationship with the position is calculated by the calculation means, this calibration curve is stored in the storage means, and the calibration is performed based on the calibration curve stored in the storage means when the actual measurement is performed. The result of the actual measurement is calibrated by means, and it is possible to calibrate measurement errors caused by individual differences in measurement sensitivity of the measuring device.

  In this embodiment, there are two regions, a detection region and a reference region, for performing the reference method, but the result of the main measurement in any region should be calibrated from the result of the preliminary measurement in the reference region. Is possible.

  Of course, preliminary measurement may be performed in the detection region, and the result of the main measurement in each region may be calibrated based on the result of the preliminary measurement in the detection region, or preliminary measurement may be performed in each of the detection region and the reference region. The result of the main measurement may be calibrated based on the result of the preliminary measurement in each region.

  The calibration curve may be a higher-order calibration curve of the fourth order or higher, but the higher the order, the more sensitive the measurement error during the preliminary measurement, and the more complicated the shape of the calibration curve deviates from the original curve. Since the calculation amount for calculating the calibration curve increases, it is preferable to use a tertiary calibration curve.

  In this embodiment, in order to perform the reference method, there are two areas, a detection area and a reference area, and since these two areas are measured simultaneously, the difference between the measured values in the two areas is obtained. Thus, a measurement result can be obtained in which the measurement error caused by the influence of the temperature change of the buffer 72 is offset.

  Further, nothing is fixed to the reference region on the metal film 55, but it is preferable that the reference region has a function of not binding to the analyte in the buffer 72. In order to achieve such an embodiment, for example, alkylthiol, amino alcohol, amino ether or the like may be fixed on the metal film 55. Alternatively, an organic film having no ligand fixing function or a protein known not to react with an analyte used for measurement may be used as a reference surface.

  In addition, the measuring device is not limited to a mode in which a plurality of surface plasmon measurement systems simultaneously measure all the channels provided in the measurement unit, and includes only one surface plasmon measurement system, It is good also as an aspect which measures the several flow path provided in the measurement unit sequentially by moving the position of a measurement unit relatively with respect to a measurement system.

  Next, a second embodiment of the present invention will be described with reference to FIG. In FIG. 11, the same elements as those in FIG. 3 are denoted by the same reference numerals, and description thereof will be omitted unless particularly required. The measurement unit of the second embodiment corresponds to a leaky mode sensor, and the measurement system has the same configuration as the surface plasmon sensor of the first embodiment.

  On one surface (upper surface in the figure) of the dielectric block 50 of the measurement unit 10 ′, a clad layer 56 and an optical waveguide layer 57 as a thin film layer are sequentially laminated. The dielectric block 50 is formed using, for example, synthetic resin or optical glass such as BK7. On the other hand, the cladding layer 56 is formed in a thin film shape using a dielectric having a lower refractive index than that of the dielectric block 50 or a metal such as gold. The optical waveguide layer 57 is also formed into a thin film using a dielectric having a higher refractive index than the cladding layer 56, such as PMMA. The thickness of the cladding layer 56 is, for example, 36.5 nm when formed from a gold thin film, and the thickness of the optical waveguide layer 57 is, for example, about 700 nm when formed from PMMA.

  In the leakage mode sensor configured as described above, when the light beam 13 emitted from the laser light source 14 is incident on the cladding layer 56 through the dielectric block 50 at an incident angle equal to or greater than the total reflection angle, the light beam 13 and the dielectric block 50 Although the light is totally reflected at the interfaces 50f and 50g with the clad layer 56, light of a specific wave number that is transmitted through the clad layer 56 and incident on the optical waveguide layer 57 at a specific incident angle propagates through the optical waveguide layer 57 in a waveguide mode. Will come to do. When the waveguide mode is excited in this way, most of the incident light is taken into the optical waveguide layer 57, and total reflection attenuation occurs in which the intensity of light totally reflected at the interfaces 50f and 50g sharply decreases.

  Since the wave number of guided light in the optical waveguide layer 57 depends on the refractive index of the buffer 72 or the ligand 73 on the optical waveguide layer 57, knowing the specific incident angle at which total reflection attenuation occurs, the buffer 72 or the ligand The refractive index of 73 can be known. Further, it is possible to examine the change in the binding state between the ligand 73 and the analyte in the buffer 72 based on the differential value I ′ output from each differential amplifier of the differential amplifier array 18.

  Also in the second embodiment, the same effect as in the first embodiment can be obtained.

The top view which shows schematic structure of the surface plasmon sensor by the 1st Embodiment of this invention Plan view of the measurement system of the above surface plasmon sensor Side view of the measurement system of the above surface plasmon sensor Perspective view of the measurement unit of the surface plasmon sensor Exploded perspective view of the measurement unit Top view of the above measurement unit VII-VII line sectional view in FIG. VIII-VIII sectional view in FIG. Block diagram showing the electrical configuration of the measurement system of the surface plasmon sensor Schematic showing the relationship between the light beam incident angle and the detected light intensity and the relationship between the light beam incident angle and the differential value of the light intensity detection signal in the measurement system of the surface plasmon sensor. Side view of measurement system of leaky mode sensor according to second embodiment of the present invention Graph showing the relationship between the refractive index of the buffer and the position of the dark line

Explanation of symbols

10 Measuring unit
13 Light beam
14 Laser light source
15 Optical system
16 Collimator lens
17 Photodiode array
17a, 17b, 17c …… Photodiode
18 Differential amplifier array
18a, 18b, 18c ... Differential amplifier
19 Drivers
20 Signal processor
21 Display
22a, 22b, 22c ... Sample hold circuit
23 Multiplexer
24 A / D converter
25 Drive circuit
26 Controller
50 dielectric block
51 Channel member
52 Holding member
53 Double-sided tape
54 Evaporation prevention member
55 Metal film
56 Clad layer
57 Lightwave layer
60 channels
61 entrance
62 Supply channel
63 Measurement unit
64 Discharge channel
65 Exit
70, 71 pipettes
72 Liquid sample
73 Ligand

Claims (4)

The light beam is incident on the dielectric block on which the thin film layer is formed at various angles so that the total reflection condition is obtained at the interface between the dielectric block and the thin film layer,
Detecting the position of the dark line in the light beam totally reflected at various reflection angles at the interface;
In a measurement method for measuring characteristics depending on the refractive index of a substance in contact with the thin film layer based on the detection result,
Perform preliminary measurements on at least four buffers of known refractive index,
Based on the result of the preliminary measurement, a high-order calibration curve representing the relationship between the refractive index of the buffer and the position of the dark line is calculated,
A measurement method characterized by calibrating the result of the main measurement based on the calibration curve.   The measurement method according to claim 1, wherein the higher-order calibration curve is a third-order calibration curve. A dielectric block in which a thin film layer is formed, and a measurement unit including a sample holding mechanism for holding a sample on the thin film layer;
A light source that generates a light beam;
An incident optical system that causes the light beam to enter the dielectric block at various angles so that a total reflection condition is obtained at an interface between the dielectric block and the thin film layer;
Dark line position detection means for detecting the position of the dark line in the light beam totally reflected at the interface;
In a measuring apparatus comprising measuring means for measuring characteristics depending on the refractive index of the substance in contact with the thin film layer based on the detection result by the dark line position detecting means,
A calculation means for calculating a high-order calibration curve representing the relationship between the refractive index of the buffer and the position of the dark line based on the result of the preliminary measurement for at least four buffers having a known refractive index;
Storage means for storing the calibration curve calculated by the calculation means;
A measuring apparatus comprising calibration means for calibrating the result of the main measurement based on the calibration curve.   The measuring apparatus according to claim 3, wherein the higher-order calibration curve is a third-order calibration curve.

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