JP2007033289A - Measuring apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To enable the selection of a method for acquiring a proper position of a dark line in accordance with a measurement mode, in a measuring apparatus which generates an evanescent wave at an interface between a dielectric block and a thin-film layer contacting with an object to be measured such as a sample by using a total reflection of a light beam and analyzes the sample by measuring a change in intensity of the light beam after the total reflection. <P>SOLUTION: In the measuring apparatus, a signal processing section 18 simultaneously executes computations by using two dark line position acquiring means which are a wide range-use dark line position acquiring means (center of gravity method) and a narrow range-use dark line position acquiring means (difference method), based on signal values obtained by applying an A/D conversion to signals output from photodiodes 17a, 17b and 17c, .... In a selection means, if a precise computation executed by the narrow range-use dark line position acquiring means (difference method) requires time, a result with a normal precision obtained by the wide range-use dark line position acquiring means (center of gravity method) is output at a high rate, and then a highly precise result is output when the computation executed by the narrow range-use dark line position acquiring means (difference method) is completed. <P>COPYRIGHT: (C)2007,JPO&INPIT

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. Thus, the present invention relates to 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. 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 can be obtained. That is, when the surface plasmon wave number is K _SP , the surface plasmon angular frequency is ω, c is the speed of light in vacuum, εm and εs are metal, and the dielectric constant of the sample is as follows.

Knowing the dielectric constant εs of the sample, the refractive index or the like of the sample is found based on a predetermined calibration curve or the like, after all, by knowing the total reflection attenuation angle theta _SP, dielectric constant, that of the sample related to the refractive index Characteristics 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, in a measuring device such as a surface plasmon sensor as described above, a relatively thick light beam is focused on the interface between the dielectric block and the thin film layer so as to include components incident on the light beam at various angles. Alternatively, it is detected by an area sensor that is incident in a divergent light state and extends in a direction in which all light beams reflected at various reflection angles can be received, and the total reflection attenuation angle θ _SP , that is, the interface is based on the detection result by the area sensor. In the case of calculating the position of the dark line where the reflected light intensity at the local minimum is generally low, it generally takes a long time to calculate the position of the dark line with high accuracy, and when trying to calculate the position of the dark line at high speed There is a problem that accuracy decreases. It is also conceivable that the optimum method for calculating the position of the dark line differs depending on the measurement object.

  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 To provide a measuring apparatus capable of selecting an appropriate method for obtaining a dark line position according to a measurement mode in a measuring apparatus for analyzing a sample by measuring a change in the intensity of a totally reflected light beam. Objective.

  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. On the other hand, the incident optical system is incident at various angles so that the total reflection condition is obtained at the interface between the dielectric block and the thin film layer, and the intensity of the light beam totally reflected at various reflection angles at the interface is detected. In the measurement apparatus comprising: a light detection means that performs the calculation, and a calculation means that obtains the position of the dark line where the reflected light intensity at the interface is a minimum value based on a detection result by the light detection means, the calculation means are different from each other. A plurality of dark line position acquisition means for acquiring a dark line position by an acquisition method; and a selection means for selecting one dark line position acquisition means from the plurality of dark line position acquisition means. It is an butterfly.

  In the measurement apparatus according to the present invention, the calculation means includes a wide-range dark line position acquisition means for acquiring a dark line position based on a detection result of the wide detection area of the light detection means, and detection of a narrow detection area of the light detection means. It is good also as what was provided with the dark line position acquisition means for narrow areas which acquires the position of a dark line based on a result.

  In this case, it is preferable that the wide-range dark line position acquisition unit acquires the position of the dark line by the center of gravity method, and the narrow range dark line position acquisition unit acquires the position of the dark line by the difference method. preferable.

  Here, the center-of-gravity method, for example, as described in Meas. Sci. Technol. 11 (2000) p1632, acquires the position of the dark line by acquiring the position of the center of gravity based on a predetermined expression from the detection result. Means the method.

  The difference method means a method of acquiring the position of the dark line based on the difference value between the outputs of two adjacent sensors, as described in, for example, Japanese Patent Application Laid-Open No. 09-292334.

  In addition, the measuring apparatus of the present invention can be configured as a so-called surface plasmon sensor in which the thin film layer is made of a metal film and performs measurement using the effect of the surface plasmon resonance described above. 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.

  In addition, the above apparatus detects the reflected light at the interface of the light beam incident at various incident angles with respect to the interface between the dielectric block and the thin film layer, and calculates the total reflection attenuation angle or its angle change. It may be one that measures the refractive index or refractive index change by detection, and DVNoort, K. johansen, C.-F. Mandenius, Porous Gold in Surface Plasmon Resonance Measurement, EUROSENSORS XIII, 1999, As described in pp.585-588, a light beam having a plurality of wavelengths is incident on the interface at an incident angle at which a total reflection condition is obtained, and the intensity of the light beam totally reflected at the interface is determined for each wavelength. The refractive index or refractive index change may be measured by measuring and detecting the degree of total reflection attenuation for each wavelength.

  According to the apparatus of the present invention, a dielectric block in which a thin film layer is formed, 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, and the light beam as a dielectric The incident optical system is incident at various angles so that the total reflection condition is obtained at the interface between the dielectric block and the thin film layer, and the intensity of the light beam totally reflected at the various reflection angles at the interface. In a measuring apparatus comprising: a light detecting means for detecting; and a calculating means for acquiring a position of a dark line at which the reflected light intensity at the interface is a minimum value based on a detection result by the light detecting means. Since a plurality of dark line position acquisition means for acquiring the position of the dark line by different acquisition methods and a selection means for selecting one dark line position acquisition means from the plurality of dark line position acquisition means are provided. It is possible to select the method for acquiring the position of the appropriate dark line in accordance with the embodiment.

  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 signal processing unit 18 connected to the two photodiode arrays 17, and a display unit 19 connected to the signal processing unit 18. The signal processing unit 18 also functions as a calculation unit that acquires the position of the dark line. 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 portion 51a is formed surrounding the outlet of the supply passage 62 and the inlet of the discharge passage 64, and the inside of the seal portion 51a becomes the measurement portion 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 the configuration of the signal processing unit 18 of the surface plasmon sensor. As shown in the figure, the signal processing unit 18 includes a multiplexer 18a to which outputs of the photodiodes 17a, 17b, 17c,... Are input, an A / D conversion unit 18b for A / D converting the output of the multiplexer 18a, The calculation unit 18c is configured to process the signal output from the D conversion unit 18b.

  The calculation unit 18c includes a wide-range dark line position acquisition unit that acquires the position of the dark line by the barycentric method based on the wide-range detection result of the photodiode array 17, and a differential method based on the narrow-range detection result of the photodiode array 17. It has a function as a narrow range dark line position acquisition unit for acquiring a dark line position and a selection unit for selecting one dark line position acquisition unit from these two dark line position acquisition units.

  Here, a dark line calculation method by the wide range dark line position acquisition unit (center of gravity method) and a dark line calculation method by the narrow range dark line position acquisition unit (difference method) will be described.

  FIG. 10A is a graph showing the light intensity at each incident angle θ of the light beam 13 totally reflected at the interface 50f (or 50g). The vertical axis indicates the light intensity I, and the horizontal axis indicates the incident angle θ. FIG. 10 (2) 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.

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.

When calculating the position of the dark line by the dark line position acquisition means for wide area (centroid method), the signal value after A / D conversion of the signal output from each photodiode is Y _i , and the array direction of each photodiode is X _i The position of the dark line can be calculated by calculating the center of gravity position C (X) by the equation (2).

Incidentally, .alpha.i is an element of the weighting coefficients such .alpha.i = 1 For example, if simple, if complex, for example .alpha.i uses functions such as X _i Y _i.

  Next, a case where the dark line position is calculated by the narrow range dark line position acquisition means (difference method) will be described. FIG. 10 (3) shows a difference value when a difference is obtained between signal values of adjacent photodiodes in the signal values after A / D conversion of the signals output from the photodiodes 17a, 17b, 17c. It is a graph which shows I '. The vertical axis indicates the difference value I ′, and the horizontal axis indicates the incident angle θ.

When the dark line position is calculated by the narrow range dark line position acquisition means (difference method), the difference value corresponding to the total reflection attenuation angle θ _SP among the plurality of difference values I ′ based on the value of the difference value I ′. The output that is closest to I ′ = 0 is obtained (in the example of FIG. 10, the difference value I ′ between the photodiodes 17d and 17e is selected), and a predetermined correction process is performed on the difference value I ′. After that, the value is displayed on the display unit 19.

  When the dark line position is calculated by the wide-area dark line position acquisition means (centroid method), the dark line position accuracy depends on the arrangement pitch of the photodiodes, but the dark line position can be calculated at high speed. Conversely, when dark lines are calculated by the narrow range dark line position acquisition means (difference method), the dark line position can be calculated with higher accuracy than the arrangement pitch of the photodiodes. When the detected photodiode is changed, it takes time to calculate the dark line as compared with the wide-area dark line position acquisition means (centroid method).

  In the calculation unit 18c, based on the signal value after A / D conversion of the signals output from the photodiodes 17a, 17b, 17c..., The wide-range dark line position acquisition means (centroid method) and the narrow-range dark line position acquisition Calculation is performed simultaneously by the two dark line position acquisition means of the means (difference method). In the selection means, when the calculation of the dark line position by the narrow-line dark line position acquisition means (difference method) is finished when the calculation of the dark line position by the wide-area dark line position acquisition means (center of gravity method) is finished, the narrow range The result calculated by the dark line position acquisition means (difference method) is output. If the calculation of the dark line position by the narrow range dark line position acquisition means (difference method) is not completed when the calculation of the dark line position by the wide area dark line position acquisition means (center of gravity method) is completed, first, the wide area dark line is calculated. The result calculated by the position acquisition unit (center of gravity method) is output, and this result is output when the calculation of the dark line position by the narrow range dark line position acquisition unit (difference method) is completed.

  By adopting the above-described aspect, even if it takes time to calculate with high accuracy by the dark line position acquisition unit for narrow range (difference method), the result of normal accuracy by the dark line position acquisition unit for wide area (center of gravity method) can be obtained at high speed. In addition, it is possible to output a highly accurate result at the time when the calculation by the narrow-line dark line position acquisition means (difference method) is completed, so that the convenience of measurement can be improved.

  When accuracy is not required, only data obtained by the dark line position acquisition means for wide area (centroid method) is used, and when accuracy is required, it is obtained by the dark line position acquisition means for narrow range (difference method). It is also possible to use the data separately.

  In this embodiment, there are two regions, a detection region and a reference region, for performing the reference method, and these two regions are measured at the same time. Since the error due to disturbance can be calibrated, the measurement accuracy of the apparatus can be improved.

  A linker layer (not shown) is formed on the surface of the metal film 55. The linker layer is a layer for immobilizing the ligand on the metal film 55. As a method for forming the reference region, for example, a surface treatment is applied to half of the linker layer to deactivate the binding group that binds to the ligand. Thereby, the area | region which deactivated the bonding group of the half of the linker layer becomes a reference area, and the other half becomes a detection area. In this way, ethanolamine-hydrochloride may be used to deactivate the bonding group. As another constitution method of the reference region, if an alkylthiol is arranged in the reference region, an alkyl group can be arranged on the surface, and this alkyl group cannot be ligand-bonded by the amino coupling method. Can be used as a reference area. 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 the reference region.

  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.

  In addition, when the dark line position is acquired by the dark line position acquisition unit for the wide range (center of gravity method) and the dark line position acquisition unit for the narrow range (difference method), the A / D conversion of the signal output from each photodiode as described above is performed. It is not limited to a mode in which the dark line position is calculated by software based on the later signal value, but in the case of a wide range dark line position acquisition means (center of gravity method), for example, a digital circuit or the like In this case, the dark line position may be acquired using hardware such as a difference amplifier.

  Further, the selection method of the dark line position acquisition unit by the selection unit is not limited to the above, and the selection may be performed based on the following determination criteria. First, the measurement of the dark line position is started by the dark line position acquisition means for wide area (centroid method), and when the amount of change in the dark line position per second becomes less than 10 RU, the dark line position acquisition means for the narrow area (difference method) is switched. At the time of measurement by the narrow line dark line position acquisition means (difference method), when the amount of change in dark line position per second becomes 10 RU or more, switching to the wide area dark line position acquisition means (center of gravity method) is performed. Thereafter, the dark line position acquisition means is switched every time the above condition is satisfied. Even in such an aspect, it is possible to improve the convenience of measurement while achieving both accuracy and calculation time.

  Furthermore, the light detection means is not limited to a photodiode array, and a CCD or the like may be used.

  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. To come. 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. The block diagram which shows the structure of the signal processing part of the said 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

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 Signal processor
19 Display
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)

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;
Light detecting means for detecting the intensity of the light beam totally reflected at various reflection angles at the interface;
In a measuring apparatus comprising: an arithmetic unit that acquires a position of a dark line at which the reflected light intensity at the interface becomes a minimum value based on a detection result by the light detection unit;
The calculation means includes a plurality of dark line position acquisition means for acquiring the position of the dark line by different acquisition methods, and a selection means for selecting one dark line position acquisition means from the plurality of dark line position acquisition means. A measuring device characterized by being a thing.   The calculation means is based on the detection result of the dark line position for the wide area based on the detection result of the wide detection area of the light detection means, and the detection result of the narrow detection area of the light detection means. The measuring apparatus according to claim 1, further comprising: a narrow range dark line position acquisition unit configured to acquire the position of the dark line.   The measuring apparatus according to claim 1 or 2, wherein the wide-area dark line position acquisition means acquires the position of the dark line by a center of gravity method.   The measuring apparatus according to claim 1, wherein the narrow range dark line position acquisition unit acquires the position of the dark line by a difference method.

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