JP2008089389A - Measuring apparatus and measuring method - Google Patents

Abstract

[PROBLEMS] To suppress a decrease in measurement accuracy of a measuring apparatus.
The difference calculation unit 84 causes a corresponding reflection between the light intensity distribution indicated by the distribution information when the buffer solution is supplied and the light intensity distribution indicated by the distribution information when the sample is supplied. The area of the region surrounded by the curve representing the difference value between the angles and the straight line corresponding to zero of the difference value is calculated, and the change data deriving unit 88 stores the angle difference corresponding to the calculated area. It is derived by obtaining the angle difference of the reflection angle at which the dark line is generated from the relation information stored in the unit 86.
[Selection] Figure 15

Description

  The present invention relates to a measuring apparatus and a measuring method, and more particularly to a measuring apparatus that measures the characteristics of a sample using a surface plasmon resonance phenomenon.

  Conventionally, a surface plasmon sensor is known as one of measuring devices that measure the characteristics of a sample by using a surface plasmon resonance phenomenon (SPR). In general, a surface plasmon sensor includes a prism, a metallic thin film layer that is disposed on one surface of the prism and is in contact with a sample, a light source that generates a light beam, a light beam with respect to the prism, and a prism. An optical system that makes incident light at various angles so that a total reflection condition is obtained at the interface with the thin film layer, and a light detection means that detects a light intensity distribution for each reflection angle of the light beam totally reflected at the interface. A light beam is generated from a light source in a state where a reference sample to be measured and a physiologically active substance to be measured are individually in contact with a thin film layer, and the light beam is reflected at the interface by the light detection means. The light intensity distribution at each angle is detected separately for each of the reference sample and the physiologically active substance, and the total reflection attenuation due to the surface plasmon resonance phenomenon from the detected light intensity distribution of the reference sample and the physiologically active substance. Detecting reflected angle dark lines generated by the generator, by obtaining the angular difference between the reflected angle dark line occurs, and performs analysis of the characteristics of the bioactive substance.

By the way, as a technique for detecting the reflection angle at which the dark line is generated, for example, a technique for detecting the reflection angle having the smallest intensity in the detected light intensity distribution as the reflection angle at which the dark line is generated is known. Patent Document 1 discloses a technique for detecting a reflection angle at which a dark line is generated by obtaining a center of gravity of a recessed area of a dark line portion in a detected light intensity distribution.
JP 2006-98369 A

  By the way, in this type of measuring apparatus, it is detected by the light detection means due to a change over time in the light intensity distribution of the light beam emitted from the light source, dust adhering to the prism, scratches on the optical path of the optical system, and the like. Noise may occur at a specific reflection angle of the light intensity distribution.

  However, if the specific reflection angle at which this noise is generated is close to the reflection angle at which the dark line is generated, if the reflection angle having the smallest intensity is detected as a dark line, the noise may be erroneously detected as a dark line. In addition, the technique disclosed in Patent Document 1 also has a problem in that the measurement accuracy of the measuring device is lowered because the recessed area of the dark line portion changes and the position of the center of gravity changes. It was.

  The present invention has been made to solve the above-described problems, and an object thereof is to provide a measuring apparatus and a measuring method that can suppress a decrease in measurement accuracy.

  In order to achieve the above object, the invention according to claim 1 is transmissive to a P-polarized light beam, a thin film layer is formed in part, and a sample is contacted on the thin film layer. In such a manner that a reflective member, a reference sample serving as a measurement reference on the thin film layer, and a target sample to be measured are in contact with each other, a plurality of reflections are made at the interface between the light transmissive member and the thin film layer. An acquisition means for acquiring, for the reference sample and the target sample, distribution information indicating a light intensity distribution for each reflection angle of the P-polarized light beam that is incident on the light-transmissive member at the angle and is totally reflected at the interface; In a state where the target sample is in contact with the light intensity distribution indicated by the first distribution information which is the distribution information acquired by the acquisition means while the reference sample is in contact with the thin film layer. Acquired by the acquisition means Calculating means for calculating a difference amount from the light intensity distribution indicated by the second distribution information which is the distribution information, and the light intensity indicated by the first distribution information based on the difference amount calculated by the calculating means. Deriving means for deriving an angle difference between the reflection angle at which the total reflection attenuation occurs in the distribution and the reflection angle at which the total reflection attenuation occurs in the light intensity distribution indicated by the second distribution information.

  According to the first aspect of the present invention, the sample is brought into contact with the thin film layer of the light transmissive member which is transmissive to the light beam and partially formed with the thin film layer. With the means, the reference sample as a measurement reference and the target sample to be measured are brought into contact with each other on the thin film layer, and light is reflected at a plurality of angles so as to be totally reflected at the interface between the light transmissive member and the thin film layer. Distribution information indicating the light intensity distribution for each reflection angle of the P-polarized light beam incident on the transmissive member and totally reflected at the interface is acquired for the reference sample and the target sample.

  In the present invention, the light intensity distribution indicated by the first distribution information, which is the distribution information acquired by the acquisition means in a state where the reference sample is brought into contact with the thin film layer, and the target sample on the thin film layer are calculated by the calculation means. The amount of difference from the light intensity distribution indicated by the second distribution information, which is the distribution information acquired by the acquisition unit in a contacted state, is calculated, and the first unit is calculated by the derivation unit based on the amount of difference calculated by the calculation unit. An angle difference between the reflection angle at which the total reflection attenuation occurs in the light intensity distribution indicated by the distribution information and the reflection angle at which the total reflection attenuation occurs in the light intensity distribution indicated by the second distribution information is derived.

  As described above, according to the first aspect of the present invention, the light intensity distribution indicated by the first distribution information acquired in a state in which the reference sample is in contact with the thin film layer and the state in which the target sample is in contact with the thin film layer The amount of difference from the light intensity distribution indicated by the second distribution information acquired in step (b) is calculated. Based on the calculated amount of difference, the reflection angle at which total reflection attenuation occurs in the light intensity distribution indicated by the first distribution information and the first Since the angle difference from the reflection angle at which total reflection attenuation has occurred in the light intensity distribution indicated by the two distribution information is derived, it is possible to suppress a decrease in measurement accuracy.

  According to the first aspect of the present invention, as in the second aspect of the present invention, the calculation means uses the light intensity distribution indicated by the first distribution information and the light indicated by the second distribution information as the difference amount. An area of a region surrounded by a curve representing a difference value between corresponding reflection angles between the intensity distribution and a straight line corresponding to zero of the difference value is calculated, and a relationship between the area and the angle difference is calculated. Storage means for preliminarily storing the relationship information to be shown, and the derivation means obtains the angle difference from the relationship information stored in the storage means for an angle difference corresponding to the area calculated by the calculation means. It is good also as what derives by this.

  According to a first aspect of the present invention, as in the third aspect of the present invention, the calculating means uses the light intensity distribution indicated by the first distribution information and the light indicated by the second distribution information as the difference amount. Using one of the intensity distributions as a reference light intensity distribution, the ratio of the reflection angles corresponding to the other light intensity distribution with respect to the reference light intensity distribution is obtained, and a ratio distribution in which the ratio is indicated as a logarithmic value is obtained. A storage unit that calculates an area of a region surrounded by a distribution of the ratio and a straight line corresponding to a logarithmic value of the ratio of zero, and stores in advance relationship information indicating a relationship between the area and the angle difference; The derivation means may derive the angle difference by obtaining an angle difference corresponding to the area calculated by the calculation means from the relation information stored in the storage means.

  On the other hand, in order to achieve the above object, the measurement method according to claim 4 is transmissive to a P-polarized light beam, a thin film layer is formed in part, and a sample is brought into contact with the thin film layer. In a state where a reference sample as a measurement reference and a target sample to be measured are brought into contact with the thin film layer of the light transmissive member, total reflection is performed at the interface between the light transmissive member and the thin film layer. Distribution information indicating a light intensity distribution for each reflection angle of a P-polarized light beam incident on the light-transmissive member at a plurality of angles and totally reflected at the interface is acquired for the reference sample and the target sample, and the thin film A light intensity distribution indicated by the first distribution information, which is distribution information acquired in a state where the reference sample is in contact with the layer, and distribution information acquired in a state where the target sample is in contact with the thin film layer. Indicated by some second distribution information The amount of difference from the light intensity distribution is calculated, and based on the calculated amount of difference, the reflection angle at which total reflection attenuation occurs in the light intensity distribution indicated by the first distribution information and the light indicated by the second distribution information The angle difference from the reflection angle at which total reflection attenuation occurs in the intensity distribution is derived.

  Therefore, since the measuring method according to the fourth aspect operates in the same manner as the first aspect, it is possible to suppress a decrease in measurement accuracy.

  As described above, according to the present invention, the light intensity distribution indicated by the first distribution information acquired with the reference sample in contact with the thin film layer and the target sample on the thin film layer are acquired. The difference amount from the light intensity distribution indicated by the second distribution information is calculated, and based on the calculated difference amount, the reflection angle at which total reflection attenuation occurs in the light intensity distribution indicated by the first distribution information and the second distribution information Since the angle difference with respect to the reflection angle at which total reflection attenuation has occurred is derived in the light intensity distribution indicated by, the effect of suppressing a decrease in measurement accuracy can be obtained.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

[First Embodiment]
The biosensor 10 as a measuring apparatus according to the present embodiment is a so-called surface plasmon sensor that measures the interaction between the protein Ta and the sample A using the surface plasmon resonance phenomenon generated on the surface of the metal film. .

  As shown in FIGS. 1 to 4, the biosensor 10 includes a lower housing 11 and an upper housing 12. The upper housing 12 is made of a heat insulating member and covers the entire upper half of the biosensor 10. The interior of the upper housing 12 is insulated from the outside and the interior of the lower housing 11. The front side of the upper housing 12 can be opened upward, and a handle 13 is attached. A display 14 and an input unit 16 are installed outside the upper housing 12.

  2 is a view showing the inside of the biosensor 10 as seen from the back side of FIG. 1 with the upper case 12 removed, FIG. 3 is a view of the inside of the case from the top, and FIG. 4 is a front view of FIG. It is an internal side view seen from the side.

  Disposed inside the upper housing 12 are a dispensing head 20, a measurement unit 30, a sample stock unit 40, a pipette tip stock unit 42, a buffer stock unit 44, a cold insulation unit 46, a measurement tip stock unit 48, a radiator 60, and a radiator blower fan. 62, a horizontal blower fan 64 is provided.

  The sample stock unit 40 includes a sample stacking unit 40A and a sample setting unit 40B. In the sample stacking unit 40A, sample plates 40P for stocking analyte solutions as samples to be measured differently in individual cells are stacked and accommodated in the Z direction (vertical direction). One sample plate 40P is transported and set from the sample stacking section 40A by a transport mechanism (not shown) to the sample setting section 40B.

  The pipette tip stock portion 42 includes a pipette tip stacking portion 42A and a pipette tip setting portion 42B. In the pipette chip stacking part 42A, pipette chip stockers 42P for holding a plurality of pipette chips are stacked and accommodated in the Z direction. One pipette chip stocker 42P is transported and set from the pipette chip stacking section 42A by a transport mechanism (not shown) to the pipette chip setting section 42B.

  The buffer stock unit 44 includes a bottle storage unit 44A and a buffer supply unit 44B. A plurality of bottles 44C storing a buffer solution as a reference sample serving as a reference for measurement are stored in the bottle storage portion 44A. A buffer plate 44P is set in the buffer supply unit 44B. The buffer plate 44P is partitioned into a plurality of muscles, and buffer solutions having different concentrations are stored in each partition. Further, a hole H into which the pipette tip CP is inserted when the dispensing head 20 is accessed is formed in the upper part of the buffer plate 44P. The buffer liquid is supplied from the bottle 44C to the buffer plate 44P through the hose 44H.

  A correction plate 45 is disposed next to the buffer supply unit 44B, and a cold insulation unit 46 is disposed next to the correction plate 45. The correction plate 45 is a plate for adjusting the concentration of the buffer solution, and a plurality of cells are formed in a matrix. A sample that needs to be refrigerated is placed in the cold insulation unit 46. The cold insulation part is set to a low temperature, and the sample is kept at a low temperature.

  In the measurement chip stock portion 48, a measurement chip accommodation plate 48P is set. A plurality of measurement chips 50 are accommodated in the measurement chip accommodation plate 48P.

  A measurement chip transport mechanism 49 is provided between the measurement chip stock unit 48 and the measurement unit 30. The measuring chip transport mechanism 49 includes a holding arm 49A that sandwiches and holds the measuring chip 50 from both sides, a ball screw 49B that moves the holding arm 49A in the Y direction by rotation, and a transport rail on which the measuring chip 50 is placed. 49C. At the time of measurement, one measurement chip 50 is placed on the transport rail 49C from the measurement chip storage plate 48P by the measurement chip transport mechanism 49, and is moved and set to the measurement unit 30 while being held by the holding arm 49A. .

  As shown in FIGS. 5 and 6, the measurement chip 50 includes a dielectric block 52, a flow path member 54, and a holding member 56.

  The dielectric block 52 is made of a transparent resin or the like that is transparent to the light beam, and has a prism portion 52A having a trapezoidal cross section, and the prism portion 52A integrally with both ends of the prism portion 52A. A formed held portion 52B is provided. A metallic thin film 57 is formed on the upper surface on the wider side of the two parallel surfaces of the prism portion 52A. The dielectric block 52 functions as a so-called prism. When the measurement is performed by the biosensor 10, a light beam is incident from one of two opposing non-parallel sides of the prism portion 52A and the thin film 57 is incident from the other. A light beam totally reflected at the interface is emitted.

  On the surface of the thin film 57, a linker layer 57A for immobilizing the protein Ta on the thin film 57 is formed. Protein Ta is fixed on this linker layer 57A.

  Engaging convex portions 52C that are engaged with the holding member 56 are formed along the upper side edge on both side surfaces of the prism portion 52A. Further, a flange portion 52D that is engaged with the transport rail 49C is formed along the side end side below the prism portion 52A.

  As shown in FIG. 6, the flow path member 54 includes six base portions 54A, and four cylindrical members 54B are erected on each of the base portions 54A. In the base portion 54A, for each of the three base portions 54A, one upper portion of the standing cylindrical members 54B is connected by a connecting member 54D. The channel member 54 is made of a soft and elastically deformable material, for example, an amorphous polyolefin elastomer.

  As shown in FIG. 7, the base portion 54A has two substantially S-shaped channel grooves 54C formed on the bottom surface side. Each of the end portions of the flow channel 54C communicates with the hollow portion of one cylindrical member 54B. The bottom surface of the base portion 54A is in close contact with the upper surface of the dielectric block 52, and the liquid channel 55 is configured by the space formed between the channel groove 54C and the upper surface of the dielectric block 52 and the hollow portion. The Two liquid channels 55 are formed in one base portion 54A. In each liquid channel 55, an entrance / exit 53 of the liquid channel 55 is formed on the upper end surface of the cylindrical member 54B.

  Here, one of the two liquid channels 55 is used as the measurement channel 55A, and the other one is used as the reference channel 55R. The measurement is performed in a state where the protein Ta is fixed on the thin film 57 (on the linker layer 57A) of the measurement channel 55A and the protein Ta is not fixed on the thin film 57 (on the linker layer 57A) of the reference channel 55R. As shown in FIG. 7, light beams L1 and L2 are incident on the measurement channel 55A and the reference channel 55R, respectively. As shown in FIG. 8, the light beams L1 and L2 are applied to an S-shaped bent portion disposed on the center line M of the base portion 54A. Hereinafter, the irradiation region of the light beam L1 in the measurement channel 55A is referred to as a measurement region E1, and the irradiation region of the light beam L2 in the reference channel 55R is referred to as a reference region E2. The reference area E2 is an area for performing measurement for correcting data obtained from the measurement area E1 where the protein Ta is fixed.

  A holding member 56 (see FIG. 6) of the measurement chip 50 is long, and has a shape in which an upper surface member 56A and two side plates 56B are formed in a lid shape. The side plate 56B is formed with an engagement hole 56C to be engaged with the engagement protrusion 52C of the dielectric block 52, and a window 56D at a portion corresponding to the optical path of the light beams L1 and L2. The holding member 56 is attached to the dielectric block 52 with the engagement hole 56 </ b> C and the engagement protrusion 52 </ b> C engaged. The flow path member 54 is integrally formed with the holding member 56 and is disposed between the holding member 56 and the dielectric block 52.

  A receiving portion 59 is formed on the upper surface member 56A at a position corresponding to the cylindrical member 54B of the flow path member 54. The receiving part 59 is substantially cylindrical.

  On the other hand, as shown in FIG. 2, the dispensing head 20 is provided in the upper part of the upper housing 12 and can be moved in the arrow X direction by the horizontal drive mechanism 22. The horizontal drive mechanism 22 includes a ball screw 22A, a motor 22B, and a guide rail 22C. The ball screw 22A and the guide rail 22C are arranged in the X direction. Two guide rails 22C are arranged in parallel, and one of them is arranged below the ball screw 22A at a predetermined interval. The dispensing head 20 is moved in the X direction along the guide rail 22C by the rotation of the ball screw 22A.

  The dispensing head 20 is provided with a vertical drive mechanism 24 that moves the dispensing head 20 in the arrow Z direction. As shown in FIG. 9, the vertical drive mechanism 24 includes a motor 24A and a drive shaft 24B disposed in the Z direction, and moves the dispensing head 20 in the Z direction. As shown in FIG. 3, the cold storage unit 46, the correction plate 45, the buffer supply unit 44 </ b> B (buffer plate 44 </ b> P), and the measurement unit 30 (measurement chip 50) that are accessed by the dispensing head 20 to supply liquid. The sample setting unit 40B (sample plate 40P) and the pipette tip setting unit 42B (pipette tip stocker 42P) are arranged in this order in the X direction (movement direction of the dispensing head 20).

  As shown in FIG. 9, the dispensing head 20 includes 12 dispensing tubes 20A. Dispensing pipes 20A are arranged in a line along the arrow Y direction perpendicular to the X direction. Two adjacent pipes 20A are used as a pair, one for supplying liquid and the other for discharging liquid. A pipette tip CP is attached to the tip of the dispensing tube 20A. The pipette tip CP is stocked in the pipette tip stocker 42P and can be exchanged as necessary.

  At the time of measurement, a sample and a buffer solution are supplied to the measurement chip 50 through the dispensing tube 20A. These liquids are supplied by moving the dispensing head 20 onto the cold insulation unit 46, the sample setting unit 40A, and the buffer supply unit 44B, and using pipette tips CP attached to the six dispensing tubes 20A for supplying liquid. Aspirate sample and buffer solution. The suction amount at this time is an amount to be supplied to two channels. Then, the six pipette tips CP on the side of the pipe 20A that sucked the sample and the buffer solution are inserted into one of the inlet / outlet 53 (hereinafter referred to as “supply port 53A”) on the side of the measurement channel 55A of the measurement tip 50. The pipette tips CP attached to the six dispensing pipes 20A in the discharge row are inserted into the other inlet / outlet 53 (hereinafter referred to as “discharge outlet 53B”). Then, half of the liquid is discharged from the dispensing tube 20A on the supply port 53A side, and the liquid is sucked through the dispensing tube 20A on the discharge port 53B side. Subsequently, the remaining half amount of liquid of the pipette tip CP is also supplied to the reference channel 55R side in the same manner.

  As shown in FIG. 4, the measuring unit 30 includes an optical surface plate 32, a light emitting unit 34, and a light receiving unit 36. The optical surface plate 32 has an upper base 32A composed of a horizontal plane in the upper center as viewed from the side, an outgoing inclined portion 32B that decreases in a direction away from the upper base 32A, and an outgoing slope across the upper base 32A. A light receiving inclined portion 32C disposed on the side opposite to the portion 32B is formed. The measurement chip 50 is set on the upper base 32A along the Y direction. A light emitting portion 34 that emits the light beams L 1 and L 2 toward the measurement chip 50 is installed on the emission inclined portion 32 B of the optical surface plate 32. In addition, a light receiving portion 36 is installed in the light receiving inclined portion 32C. Next to the optical surface plate 32, a water cooling jacket 32J for cooling the optical surface plate 32 is provided.

  As shown in FIG. 10, the light emitting unit 34 includes a light source 34A and a lens unit 34B. Further, the light receiving unit 36 includes a lens unit 36A and a CCD 36B. The CCD 36B is connected to an image processing unit 38 to which a control unit 70 that controls the entire biosensor 10 is connected.

  A divergent light beam L is emitted from the light source 34A. The lens unit 34B has a built-in polarization beam splitter, which separates the P-polarized component and the S-polarized component of the light beam L incident from the light source 34A. The P-polarized component of the light beam L has a certain width with respect to the Z direction. Are divided into two relatively thick parallel light beams L1 and L2. The lens unit 34B then applies the two parallel light beams L1 and L2 at various incident angles that are greater than the total reflection angle with respect to the measurement region E1 and the reference region E2 at the interface between the thin film 57 and the dielectric block 52. Incident light is incident on the measurement region E1 and the reference region E2 so as to be in a convergent light state. Therefore, the light beams L1 and L2 incident on the measurement region E1 and the reference region E2 are totally reflected at various reflection angles at the interface between the dielectric block 52 and the thin film 57. The totally reflected light beams L1 and L2 are focused on the CCD 36B through the lens unit 36A. The CCD 36B is an area sensor having a light receiving surface having an area capable of receiving both of the totally reflected light beams L1 and L2, and generates and outputs image information indicating an image formed on the light receiving surface. . The output image information is input to the image processing unit 38. In the image processing unit 38, predetermined processing is performed based on the input image information, whereby refractive index change data in the measurement region E1 and the reference region E2 is obtained and output to the control unit 70.

The refractive index change data is obtained by individually supplying a sample and a buffer solution to the measurement chip 50 and emitting the light beam L from the light emitting unit 34 to irradiate the measurement region E1 and the reference region E2 with the light beams L1 and L2, respectively. When the reflection angles at which dark lines are generated in the light beams L1 and L2 totally reflected in the measurement region E1 and the reference region E2 are obtained, the reflection angles at which dark lines are generated in the measurement region E1 with the sample and the buffer solution are calculated. It is obtained based on the difference between the angle difference and the angle difference between the reflection angles at which dark lines are generated in the reference region E2. The light beams L1 and L2 incident on the interface between the thin film 57 and the protein Ta at a specific incident angle excite surface plasmons on the interface, and thereby the reflected light beams L1 and L2 incident on the specific incident angle are reflected. The intensity drops sharply and is observed as a dark line. The angle of incidence of the light beam L1, L2 as the dark lines are attenuated total reflection angle theta _SP, the refractive index change data is determined on the basis of the total reflection attenuation angle theta _SP change in according to the reaction between the protein Ta and the sample A It is done.

  The control unit 70 measures the reaction between the protein Ta and the sample A based on the refractive index change data, and displays the measurement result on the display 14.

  FIG. 11 is a functional block diagram showing a functional configuration of the image processing unit 38 according to the present embodiment.

  As shown in the figure, the image processing unit 38 performs a convolution operation on the two-dimensional image indicated by the image information acquired by the CCD 36B, and shows a one-dimensional light intensity distribution of the light beams L1 and L2. A convolution operation unit 80 for deriving information, a distribution information storage unit 82 for storing the distribution information of the derived light beams L1 and L2, and two distribution information of each distribution information stored in the distribution information storage unit 82 The difference amount calculation unit 84 that calculates the area of the difference region of the light intensity distributions indicated by the two distribution information as the difference amount indicating the degree of difference in the light intensity distribution indicated by, and the area of the difference region The relationship information storage unit 86 that stores relationship information indicating the relationship with the angle difference of the reflection angle at which the dark line is generated, and the angle difference corresponding to the area calculated by the difference amount calculation unit 84 are obtained from the relationship information. It includes a change data deriving unit 88 for deriving the refractive index change data, the Te.

  Note that the two-dimensional image indicated by the image information includes images of the two light beams L1 and L2 that are totally reflected at the interface. For this reason, the convolution operation unit 80 according to the present embodiment performs the convolution operation for each image region of the light beams L1 and L2 of the two-dimensional image indicated by the image information, and performs the one-dimensional operation of the light beams L1 and L2. The distribution information indicating the light intensity distribution is derived respectively.

  Next, the operation of the biosensor 10 according to the present embodiment when the refractive index change data is derived will be described.

  When the refractive index change data is derived, the biosensor 10 according to the present embodiment first supplies a buffer solution to the measurement chip 50 to be measured by the dispensing head 20 and transports the measurement chip 50 to the measurement chip 50. The measurement region E1 of the measurement flow channel 55A to be measured and conveyed to the upper base 32A by the mechanism 49 and the reference region E2 of the reference flow channel 55R are arranged at positions where the light beams L1 and L2 are incident, respectively. Then, the biosensor 10 emits a light beam from the light emitting unit 34 and irradiates the measurement region E1 and the reference region E2 with the light beams L1 and L2, respectively. These light beams L1 and L2 are totally reflected by the measurement region E1 and the reference region E2, and are emitted to the outside through the prism surface of the dielectric block 52 while diverging. The light beams L1 and L2 emitted to the outside are imaged on the light receiving surface of the CCD 36B through the lens unit 36A, and image information indicating the image formed on the light receiving surface is generated and output to the image processing unit 38.

  FIG. 12A shows an example of an image indicated by image information.

  As shown in the figure, the image indicated by the image information includes images of two light beams L1 and L2 totally reflected by the measurement region E1 and the reference region E2. In this image, the reflection angle direction is shown as the horizontal direction.

  The convolution operation unit 80 (see FIG. 11) of the image processing unit 38 includes an image of the area A including the image of the light beam L1 and an image of the light beam L2 of the image indicated by the input image information. Each of the images is subjected to a convolution operation to derive distribution information indicating a one-dimensional light intensity distribution of the light beams L1 and L2. FIG. 12B shows a one-dimensional light intensity distribution of the light beam L2 indicated by distribution information derived by performing a convolution operation on the image in the region B. In FIG. 12B, the horizontal axis indicates the reflection angle direction, and the position of the light beam imaged on the CCD 36B in the reflection angle direction corresponds to the reflection angle.

  The convolution operation unit 80 stores the derived distribution information of the light beams L1 and L2 in the distribution information storage unit 82.

  Next, the biosensor 10 supplies a sample to the measurement chip 50 to be measured by the dispensing head 20, and the measurement channel 55A to be measured by the measurement chip 50 is the same as in the case of the buffer solution described above. The measurement region E1 and the reference region E2 of the reference channel 55R are arranged at positions where the light beams L1 and L2 are incident, respectively. Then, the biosensor 10 emits a light beam from the light emitting unit 34 and irradiates the measurement region E1 and the reference region E2 with the light beams L1 and L2, respectively. These light beams L1 and L2 are totally reflected by the measurement region E1 and the reference region E2, are imaged on the light receiving surface of the CCD 36B through the lens unit 36A, and image information indicating the image formed on the light receiving surface is generated. The image is output to the image processing unit 38.

  As in the case of the buffer solution described above, the convolution operation unit 80 includes the image of the region A including the image of the light beam L1 and the image of the region B including the image of the light beam L2 indicated by the input image information. Are respectively convolved to derive distribution information indicating one-dimensional light intensity distributions of the light beams L1 and L2, and the distribution information storage unit 82 stores the derived distribution information of the light beams L1 and L2.

  As a result, the distribution information storage unit 82 supplies the sample and the buffer solution individually to the measurement chip 50 to be measured, and the distribution information of the light beams L1 and L2 totally reflected in the measurement region E1 and the reference region E2. Is memorized.

  The image processing unit 38 performs the following refractive index change data derivation process to derive refractive index change data.

  FIG. 13 is a flowchart showing the flow of refractive index change data derivation processing executed by the image processing unit 38 according to the first embodiment. Hereinafter, the refractive index change data deriving process will be described with reference to FIG.

  In step 102 in the figure, distribution information in the measurement region E1 and the reference region E2 when the sample and the buffer solution are respectively supplied from the distribution information storage unit 82 to the measurement chip 50 to be measured is read out. In this step 102, for each measurement region E1 and reference region E2, between the light intensity distribution indicated by the distribution information when the buffer solution is supplied and the light intensity distribution indicated by the distribution information when the sample is supplied. Curves representing the difference values between the corresponding reflection angles are obtained.

  For example, when the light intensity distribution indicated by the distribution information when the buffer solution is supplied in the measurement region E1 and the light intensity distribution indicated by the distribution information when the sample is supplied are shown in FIG. 14A, FIG. A curve representing a difference value between reflection angles as shown in B) is obtained.

  Next, in this step 102, for each measurement region E1 and reference region E2, a region surrounded by a curve representing a difference value between reflection angles and a straight line A corresponding to zero of the difference value (FIG. 14B). The area of the shaded area) is calculated.

  As a result, noise is generated at a specific reflection angle of the light beam emitted from the light emitting unit 34. For example, as shown in FIG. 15A, the distribution information when the buffer liquid is supplied is used. Even if noise occurs at a specific reflection angle of the light intensity distribution shown and the distribution information when the sample is supplied, the noise can be removed as shown in FIG.

That is, the light intensity distribution at the reflection angle θ when the buffer solution is supplied in a state where no noise is generated is P1 (θ), and the light intensity distribution at the reflection angle θ when the sample is supplied is P2 (θ). If a noise unevenness (θ) having a predetermined intensity occurs depending on the reflection angle θ, the light intensity distribution when the buffer liquid actually detected is supplied is expressed by P1 as shown in the following equation (1). '(Θ), and the light intensity distribution when a sample to be actually detected is supplied becomes P2' (θ) as shown in the following equation (2).
P1 (θ) ′ = P1 (θ) + unevenness (θ) (1)
P2 (θ) ′ = P2 (θ) + unevenness (θ) (2)
The difference between the corresponding reflection angles between the light intensity distribution P1 ′ (θ) when the buffer solution actually detected is supplied and the light intensity distribution P2 ′ (θ) when the sample is supplied is as follows ( It is required as in 3).
P1 (θ) ′ − P2 (θ) ′ = P1 (θ) −P2 (θ) (3)
Thereby, for example, the light intensity distribution of the light beams L1 and L2 is changed due to a change with time in the light intensity distribution of the light beam L emitted from the light source 34A, adhesion of dust to the lens unit 34B, scratch on the optical path, and the like. When noise unevenness (θ) occurs at a specific reflection angle, the influence of the noise unevenness (θ) can be removed.

  In step 102, area information indicating the areas calculated for each of the measurement area E1 and the reference area E2 of the measurement chip 50 to be measured is output to the change data deriving unit 88. Note that the processing of step 102 corresponds to the processing of the difference amount calculation unit 84.

  In addition, as shown in FIG. 14A, when the position of the dark line generated in the light intensity distribution indicated by the distribution information when the sample and the buffer solution are individually supplied is separated and the angle difference becomes large. The area of the hatched area shown in FIG. 14B also increases. That is, the angle difference of the reflection angle at which the dark line is generated can be obtained from the area of the shaded region.

  In the biosensor 10 according to the present embodiment, the angle difference of the dark line for each area of the hatched area is obtained and stored in advance in the relationship information storage unit 86 as an LUT (lookup table).

  In the next step 104, the relationship in which the angle difference corresponding to the area calculated for each of the measurement region E1 and the reference region E2 of the measurement chip 50 to be measured by the processing in step 102 is stored in the relationship information storage unit 86. The difference between the derived angle difference in the measurement region E1 and the derived angle difference in the reference region E2 is output to the control unit 70 as refractive index change data, and this refractive index change data derivation process is performed. finish. Note that the process of step 104 corresponds to the process of the change data deriving unit 88.

  The control unit 70 includes a storage unit (not shown) and stores refractive index change data in the storage unit. Further, the control unit 70 measures the reaction state between the protein Ta and the sample A based on the difference in angle difference between the dark line positions indicated by the refractive index change data stored in the storage unit, and displays the measurement result on the display 14. Let

  As described above, according to the present embodiment, the difference amount calculation unit 84 uses the light intensity distribution indicated by the distribution information when the buffer solution (reference sample) is supplied and the distribution information when the sample is supplied. The area of the region surrounded by the curve representing the difference value between the corresponding reflection angles between the light intensity distribution shown and the straight line corresponding to zero of the difference value is calculated, and the change data deriving unit 88 Since the angle difference corresponding to the calculated area is derived by obtaining the angle difference of the reflection angle at which the dark line is generated from the relation information stored in the relation information storage unit 86, the light intensity of the light beams L1 and L2 Even when noise occurs at a specific reflection angle of the distribution, it is possible to suppress a decrease in measurement accuracy of the measurement apparatus.

[Second Embodiment]
In the second embodiment, the intensity ratio between the light intensity distribution indicated by the distribution information when the buffer solution is supplied and the light intensity distribution indicated by the distribution information when the sample is supplied is obtained, and the intensity is obtained. An example in which the angle difference is derived based on the ratio will be described.

  Since the configuration of the biosensor 10 and the configuration of the image processing unit 38 according to the second embodiment are the same as those of the first embodiment (see FIGS. 1 to 11), description thereof is omitted here. To do.

  Note that the difference amount calculation unit 84 (see FIG. 11) of the image processing unit 38 according to the second embodiment supplies the light intensity distribution indicated by the distribution information and the sample supplied with the buffer solution supplied. The ratio of the reflection angles corresponding to the light intensity distributions indicated by the distribution information at is obtained, and the ratio distribution indicating the ratio as a logarithmic value is obtained, and the ratio distribution and the straight line corresponding to the logarithmic value of the ratio are The area of the region surrounded by is calculated.

  The relationship information storage unit 86 stores in advance an angle difference between dark lines generated in the light beams L1 and L2 for each area calculated by the difference amount calculation unit 84 as an LUT.

  FIG. 16 is a flowchart showing the flow of refractive index change data derivation processing executed by the image processing unit 38 according to the second embodiment. In the figure, the same processes as those in FIG. 13 are denoted by the same reference numerals as those in FIG.

  In step 103, the distribution information in the measurement region E1 and the reference region E2 when the sample and the buffer solution are respectively supplied from the distribution information storage unit 82 to the measurement chip 50 to be measured is read out. In step 103, for each of the measurement region E1 and the reference region E2, either the light intensity distribution indicated by the distribution information when the buffer solution is supplied or the light intensity distribution indicated by the distribution information when the sample is supplied. Using one as the reference light intensity distribution, the ratio of the reflection angles corresponding to the other light intensity distribution with respect to the reference light intensity distribution is obtained, and the distribution of the ratio indicated as the logarithmic value is obtained.

For example, when the light intensity distribution indicated by the distribution information when the buffer solution is supplied in the measurement region E1 and the light intensity distribution indicated by the distribution information when the sample is supplied are shown in FIG. As shown in FIG. 17B, the light intensity distribution at the time of supplying the liquid is set as the reference light intensity distribution, and the ratio between the reflection angles corresponding to the light intensity distribution at the time of supplying the sample with respect to the reference light intensity distribution is shown in FIG. Obtain the distribution of X. Then, assuming that the ratio X is, for example, Y = Log ₁₀ X, a distribution of ratios with the ratio as shown in FIG.

  Next, in this step 102, an area (shaded area in FIG. 17C) surrounded by the distribution of the ratio and the straight line A corresponding to the logarithmic value of the ratio as shown in FIG. Calculate the area. The relationship information storage unit 86 according to the present embodiment stores in advance an LUT obtained by determining the angle difference of the dark line for each calculated area.

That is, the light intensity distribution at the reflection angle θ when the buffer solution is supplied in a state where no noise is generated is P1 (θ), and the light intensity distribution at the reflection angle θ when the sample is supplied is P2 (θ). When the noise unevenness (θ) that depends on the reflection angle θ and is proportional to the intensity of the light beam occurs in the light beams L1 and L2, the light intensity distribution when the buffer solution to be detected is supplied is as follows ( P1 ′ (θ) as shown in the equation (5), and the light intensity distribution when the sample to be detected is supplied becomes P2 ′ (θ) as shown in the following equation (6).
P1 (θ) ′ = P1 (θ) × unevenness (θ) (4)
P2 (θ) ′ = P2 (θ) × unevenness (θ) (5)
The ratio between the corresponding reflection angles of the light intensity distribution P1 ′ (θ) when the buffer solution to be detected is supplied and the light intensity distribution P2 ′ (θ) when the sample is supplied is as shown in (6) below. Is required.
P1 (θ) ′ / P2 (θ) ′ = P1 (θ) / P2 (θ) (6)
Thereby, for example, the light intensity distribution of the light beams L1 and L2 is changed due to a change with time in the light intensity distribution of the light beam L emitted from the light source 34A, adhesion of dust to the lens unit 34B, scratch on the optical path, and the like. When noise unevenness (θ) in which the intensity of the light beam is proportional to a specific reflection angle occurs, the influence of the noise unevenness (θ) can be removed.

  In step 103, area information indicating the area calculated for each of the measurement region E 1 and the reference region E 2 of the measurement chip 50 to be measured is output to the change data deriving unit 88. Note that the processing of step 102 corresponds to the processing of the difference amount calculation unit 84.

  As a result, in the next step 104, the angle difference corresponding to the area calculated for each of the measurement region E1 and the reference region E2 of the measurement chip 50 to be measured by the processing of step 103 is stored in the relational information storage unit 86. It is derived by obtaining from the relationship information.

  As described above, according to the present embodiment, the difference amount calculation unit 84 calculates the light intensity distribution indicated by the distribution information when the buffer solution is supplied and the light intensity distribution indicated by the distribution information when the sample is supplied. Using either one as the reference light intensity distribution, the ratio of the reflection angles corresponding to the other light intensity distribution with respect to the reference light intensity distribution is obtained, and a ratio distribution in which the ratio is indicated as a logarithmic value is obtained. The area of the region surrounded by the distribution of the ratio and the straight line corresponding to the logarithmic value of the ratio is calculated, and the change data deriving unit 88 stores the angle difference corresponding to the calculated area in the relationship information storage unit 86. Since it is derived by obtaining the angle difference between the reflection angles at which dark lines are generated from the relationship information, the noise is proportional to the intensity of the light beam at a specific reflection angle of the light intensity distribution of the light beams L1 and L2. Even if that occurs, it is possible to suppress a decrease in measurement accuracy of the measuring device.

  In each of the above embodiments, the case where the relationship information storage unit 86 stores the relationship of the angle difference for each area as the LUT has been described. However, the present invention is not limited to this, and for example, the area is input. A conversion formula using the angle difference as an output value as a parameter may be stored, and the change data deriving unit 88 may derive the angle difference using the conversion formula.

  In each of the above embodiments, two parallel light beams L1 and L2 are simultaneously incident on the measurement area E1 and the reference area E2 by the lens unit 34B at various angles, so that the measurement area E1 and the reference area E2 are incident. Although the case where the light beams L1 and L2 totally reflected at various reflection angles at the interface are received by the CCD 36B at the same time has been described, the present invention is not limited to this. It is good also as what receives light.

  In each of the above embodiments, a case has been described in which distribution information indicating a one-dimensional light intensity distribution is acquired by performing a convolution operation on a two-dimensional image indicated by the image information generated by the CCD 36B. The present invention is not limited to this. For example, a relatively thin light beam is incident on the interface while changing the incident angle, and the light beam whose reflection angle changes according to the change in the incident angle of the incident light beam. May be detected by a small photodetector that moves in synchronization with the change in the reflection angle, and distribution information indicating the light intensity distribution of the light beam may be acquired.

  In addition, a leaky mode detector can be used as another biosensor using total reflection attenuation. The leakage mode sensor is composed of a dielectric, and a thin film composed of a clad layer and an optical waveguide layer that are sequentially layered thereon. One surface of the thin film serves as a sensor surface, and the other surface receives light. It becomes a surface. When light is incident on the light incident surface so as to satisfy the total reflection condition, a part of the light is transmitted through the cladding layer and taken into the optical waveguide layer. In this optical waveguide layer, when the waveguide mode is excited, the reflected light at the light incident surface is greatly attenuated. The incident angle at which the waveguide mode is excited varies according to the refractive index of the medium on the sensor surface, similarly to the surface plasmon resonance angle. By detecting the attenuation of the reflected light, the reaction on the sensor surface can be measured.

It is a perspective view of the whole biosensor which concerns on embodiment. It is a perspective view inside the biosensor which concerns on embodiment. It is a top view inside the biosensor which concerns on embodiment. It is an internal side view of the biosensor which concerns on embodiment. It is a perspective view of the measurement chip concerning an embodiment. It is a disassembled perspective view of the measuring chip concerning an embodiment. It is a figure which shows the state in which the light beam is injecting into the measurement area | region and reference area of the measurement chip which concerns on embodiment. It is the figure which looked at the channel member of the measuring chip concerning an embodiment from the lower side. It is a perspective view which shows the vertical drive mechanism of the dispensing head of the biosensor which concerns on embodiment. It is the schematic of the optical measurement part vicinity of the biosensor which concerns on embodiment. It is a block diagram which shows the function structure of the image process part which concerns on embodiment. (A) is a figure which shows an example of the image containing the image of the two light beams shown by image information, (B) is a graph which shows an example of light intensity distribution of a light beam. It is a flowchart which shows the flow of the refractive index change data derivation | leading-out process which concerns on 1st Embodiment. (A) is a graph which shows an example of light intensity distribution, (B) is a graph which shows an example of the curve showing the difference value between the corresponding reflection angles of light intensity distribution. (A) is a graph which shows an example of light intensity distribution containing noise, (B) is a graph which shows an example of the curve showing the difference value of reflection angles. It is a flowchart which shows the flow of the refractive index change data derivation | leading-out process which concerns on 2nd Embodiment. (A) is a graph which shows an example of light intensity distribution, (B) is a graph which shows an example of distribution of ratio of the reflection angle corresponding to light intensity distribution, (C) shows a ratio as a logarithmic value. It is a graph which shows an example of distribution of the ratio.

Explanation of symbols

10 Biosensor 52 Dielectric block (light transmissive member)
57 Thin film (thin film layer)
80 Convolution calculator (acquisition means)
84 Difference calculation unit (calculation means)
86 Relationship information storage unit (storage means)
88 Change data deriving section (derivation means)

Claims (4)

A light transmissive member having transparency to a P-polarized light beam, a thin film layer formed in part, and a sample contacting the thin film layer;
In a state in which a reference sample to be measured and a target sample to be measured are in contact with each other on the thin film layer, the light transmitting member and the thin film layer are totally reflected at the interface at a plurality of angles. An acquisition means for acquiring, for the reference sample and the target sample, distribution information indicating a light intensity distribution for each reflection angle of the P-polarized light beam incident on the light transmissive member and totally reflected at the interface;
In a state where the target sample is in contact with the light intensity distribution indicated by the first distribution information which is the distribution information acquired by the acquisition means while the reference sample is in contact with the thin film layer. Calculation means for calculating a difference amount from the light intensity distribution indicated by the second distribution information which is distribution information acquired by the acquisition means;
Based on the difference amount calculated by the calculation means, the reflection angle at which the total reflection attenuation occurs in the light intensity distribution indicated by the first distribution information and the total reflection attenuation in the light intensity distribution indicated by the second distribution information. Derivation means for deriving an angle difference from the generated reflection angle;
Measuring device. The calculation means, as the difference amount, a curve representing a difference value between corresponding reflection angles between the light intensity distribution indicated by the first distribution information and the light intensity distribution indicated by the second distribution information; Calculate the area of the area surrounded by the straight line corresponding to zero of the difference value,
Storage means for storing in advance relationship information indicating the relationship between the area and the angle difference;
The measuring apparatus according to claim 1, wherein the derivation unit derives the angle difference by obtaining an angle difference corresponding to the area calculated by the calculation unit from the relationship information stored in the storage unit. The calculation means uses the light intensity distribution indicated by the first distribution information and the light intensity distribution indicated by the second distribution information as a reference light intensity distribution as the difference amount, and the reference light intensity. The ratio of the corresponding reflection angles of the other light intensity distribution with respect to the distribution is obtained to obtain a ratio distribution indicating the ratio as a logarithmic value, and the ratio distribution and the logarithm value of the ratio are surrounded by a straight line corresponding to zero. Calculate the area of
Storage means for storing in advance relationship information indicating the relationship between the area and the angle difference;
The measuring apparatus according to claim 1, wherein the derivation unit derives the angle difference by obtaining an angle difference corresponding to the area calculated by the calculation unit from the relationship information stored in the storage unit. A reference sample serving as a reference for measurement on the thin film layer of a light transmissive member that is transparent to a P-polarized light beam, partially formed with a thin film layer, and in contact with the sample on the thin film layer; In a state in which the target sample to be measured is in contact, the light is transmitted to the light transmissive member at a plurality of angles so as to be totally reflected at the interface between the light transmissive member and the thin film layer, and is totally reflected at the interface. Distribution information indicating the light intensity distribution for each reflection angle of the P-polarized light beam is obtained for the reference sample and the target sample,
The light intensity distribution indicated by the first distribution information, which is the distribution information acquired when the reference sample is in contact with the thin film layer, and the distribution acquired when the target sample is in contact with the thin film layer Calculating the amount of difference from the light intensity distribution indicated by the second distribution information as information,
Based on the calculated difference amount, a reflection angle at which total reflection attenuation occurs in the light intensity distribution indicated by the first distribution information, and a reflection angle at which total reflection attenuation occurs in the light intensity distribution indicated by the second distribution information; Measuring method to derive the angular difference of.