JP2008241475A - Measuring instrument - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a measuring instrument capable of accurately correcting a drift phenomenon by which the index of refraction changes with time. <P>SOLUTION: A plurality of kinds of analyte solution used for measuring the property of protein Ta on a thin film 57 and buffer liquid of predetermined concentration as measurement reference are alternately supplied to the adhesion region of the protein Ta, and the index of refraction of the protein Ta adhered on the thin film 57 is measure a plurality of times. Based on each index of refraction measured with supplying the buffer liquid, when the index of refraction is increased by more than a predetermined value ΔS than the index measured in the previous measurement, predetermined times of the measurement results of the index of refraction after the measurement of the index increased by more than the predetermined value ΔS are excluded, the change information indicating a change with time of the index of refraction is derived, and the measurement result is corrected based on the derived change information. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a measuring apparatus, and more particularly, to a measuring apparatus that measures changes in the refractive index of an analyte substance by individually supplying various solutions to the analyte substance to be measured.

  Conventionally, a liquid flow path is formed so that liquid can flow, and various solutions are respectively applied to the liquid flow path of the measurement chip in which the analyte substance to be measured is attached to the wall surface in the liquid flow path. There is known a measuring apparatus that measures the characteristics of a specimen material by separately supplying and measuring the change in the refractive index of the specimen material.

  For example, in Patent Document 1, various solutions are individually supplied to a flow path, and a light beam is incident at various angles on an interface of an attachment portion to which an analyte is attached, and is totally reflected at the interface. By detecting the light intensity distribution at each reflection angle, and detecting the reflection angle at which the dark line is generated by the occurrence of total reflection attenuation due to the surface plasmon resonance phenomenon (SPR) from the detected light intensity distribution. A measuring apparatus for measuring a change in refractive index to measure a characteristic of an analyte is disclosed.

  By the way, in this type of measuring apparatus, the attachment state of the specimen material changes due to the solution flowing through the liquid flow path, etc., the thickness of the specimen substance decreases with time, and the refractive index of the specimen substance changes with time. There is a case where a phenomenon called drift that changes with time is generated.

  For example, a dark line is generated when a measurement device supplies a reference solution serving as a measurement reference and various types of solutions alternately and individually to a liquid flow path, and supplies the solution after the reference solution. When the change in the refractive index of the specimen substance is measured by determining the difference in the generated reflection angles and the characteristics of the specimen substance are measured, as shown in FIG. 23, the refractive index of the specimen substance changes with time. As a result, a drift phenomenon occurs in which the reflection angle detected when the reference solution and each solution are supplied decreases with time, and the change in the refractive index of the sample substance measured from the angle difference between the reflection angles occurs. As shown in FIG. 24, the measurement result is shifted to the minus side from the estimated measurement result. Note that the vertical axis in FIG. 23 indicates the reflection angle (1RU = 1/10000 °) where the dark line is generated when the reference solution and the solution are individually supplied after the reference solution, and the horizontal axis indicates time. Yes.

As a technique for correcting this drift, Patent Document 1 calculates the rate of change of refractive index with respect to time in a reference period in which the refractive index of a specimen substance after supplying a solution is estimated to show a constant value, A technology is disclosed in which a component whose refractive index changes at a rate of change calculated over the entire measurement period is used as a drift component, and the measurement result is corrected so as to remove this drift component, based on the moment when the current is supplied. ing.
JP 2006-105914 A

  Therefore, by applying the technique of Patent Document 1, for example, as shown in FIG. 25, change information indicating a change in refractive index with time based on each reflection angle when each reference solution is supplied (in FIG. 25, change information). FIG. 26 shows that the refractive index change due to the drift phenomenon can be corrected when the reflection angle when the reference solution and each solution are supplied is corrected based on the derived change information. As a result, the measurement accuracy of the angle difference of the reflection angle is improved.

  However, when the solution and the sample substance react when the solution is supplied to the liquid flow path, the ratio of the sample substance changes, and the sample substance reacts with the solution in the measurement result when the reference solution is supplied next. As a result, the influence of the change in the refractive index remains, and the derived change information includes the influence of the change in the refractive index. Therefore, there is a problem in that the drift phenomenon may not be accurately corrected.

  The present invention has been made to solve the above problems, and an object of the present invention is to provide a measuring apparatus capable of accurately correcting a drift phenomenon in which the refractive index changes with time.

  In order to achieve the above object, the invention described in claim 1 has a light transmission property, and a light transmission in which a thin film layer is partially formed and a sample substance to be measured is attached on the thin film layer. And a supply means for supplying a plurality of types of solutions used for measuring the characteristics of the analyte substance and a predetermined reference solution serving as a measurement reference to the adhesion area of the analyte substance on the thin film layer, Each of the plurality of types of solutions is individually supplied by the supply means, and the reference solution is supplied every time the solution is supplied a predetermined number of times, and the refractive index information of the analyte substance adhered on the thin film layer is supplied a plurality of times. Based on the refractive index information measured by the measuring means for measuring and the refractive index information measured by the measuring means in a state where the reference solution is supplied by the supplying means, the refractive index information is more predetermined than the refractive index information measured once before. Has increased more than the value In this case, the derivation means for deriving change information indicating temporal change of the refractive index information by excluding the measurement result of the refractive index information for a predetermined number of times after the measurement of the refractive index information increased by the predetermined value or more. And correction means for correcting the measurement result by the measurement means based on the change information derived by the derivation means.

  According to the first aspect of the present invention, a thin film layer is formed on a part of a light transmissive member having light transmissivity, and an analyte substance to be measured is attached on the thin film layer. A plurality of types of solutions used for measuring the characteristics of the sample substance and a predetermined reference solution serving as a measurement reference are respectively supplied to the adhesion area of the sample substance on the layer. Each is supplied individually, and the reference solution is supplied every time the solution is supplied a predetermined number of times, and the refractive index information of the analyte substance adhered on the thin film layer is measured a plurality of times.

  And this invention is based on each refractive index information measured by the measurement means in the state by which the reference | standard solution was supplied by the supply means by the derivation | leading-out means, and refractive index information is more than the refractive index information measured 1 time ago. In the case where the refractive index information is increased by a predetermined value or more, the measurement result of the refractive index information for a predetermined number of times after the measurement of the refractive index information increased by the predetermined value or more is excluded, and the change in refractive index information with time is shown The change information is derived, and the correction result is corrected by the correction means based on the change information derived by the derivation means.

  Thus, according to the first aspect of the present invention, the plurality of types of solutions used for measuring the characteristics of the specimen substance are individually supplied to the adhesion area of the specimen substance on the thin film layer, and the solution is supplied a predetermined number of times. Refractive index information of the sample substance adhering to the thin film layer is measured multiple times by supplying a predetermined reference solution as a measurement reference each time, and each refractive index measured with the reference solution supplied Based on the information, when the refractive index information is increased by a predetermined value or more than the refractive index information measured one time before, the refraction for a predetermined number of times after the time when the refractive index information increased by the predetermined value or more is measured. Since the change information indicating the change in refractive index information over time is derived by excluding the measurement result of the refractive index information, and the measurement result is corrected based on the derived change information, the refractive index information changes over time. It is possible to correct the drift phenomenon That.

  The measuring means of the present invention is incident on 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. The refractive index information of the specimen substance may be measured by detecting a reflection angle at which a dark line is generated due to total reflection attenuation from the light intensity distribution of the light beam totally reflected at the interface.

  Further, in the invention of claim 2, as in the invention of claim 3, the thin film layer is provided with an attachment region to which the sample substance is attached and an unattached region to which the sample substance is not attached. The measuring means measures a reflection angle at which a dark line is generated from a light intensity distribution of a light beam totally reflected at each of the attached region and the non-attached region at the interface between the light transmissive member and the thin film layer; The refractive index information of the specimen substance may be measured by calibrating the reflection angle at which the dark line is generated in the attached region with the reflection angle at which the dark line is generated in the non-attached region.

  Moreover, the measuring means of this invention may supply the said reference | standard solution, whenever the kind of the said solution switches by the said supply means, when measuring refractive index information like invention of Claim 4.

  As described above, according to the present invention, each of a plurality of types of solutions used for measuring the characteristics of the specimen material is individually supplied to the adhesion area of the specimen material on the thin film layer, and the measurement is performed each time the solution is supplied a predetermined number of times. Measure the refractive index information of the sample substance adhering to the thin film layer multiple times by supplying a predetermined reference solution that is the reference for the reference, and based on each refractive index information measured with the reference solution supplied When the refractive index information is increased by a predetermined value or more than the refractive index information measured one time before, the refractive index information for a predetermined number of times after the measurement of the refractive index information increased by the predetermined value or more is performed. Since the measurement information is excluded, change information indicating the change in refractive index information over time is derived, and the measurement result is corrected based on the derived change information, so the drift phenomenon in which the refractive index information changes over time Can be corrected accurately. Effect can be obtained.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. In the following, the case where the present invention is applied to a biosensor that measures the characteristics of a specimen substance using a surface plasmon resonance (SPR) will be described.

  As shown in FIGS. 1 to 4, the biosensor 10 according to the present embodiment 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 section 40A, sample plates 40P for stocking different analyte solutions as samples used for measuring the characteristics of the specimen substance are stacked in the Z direction (vertical direction) and stored in individual cells. 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, there is formed a linker layer 57A for attaching protein Ta on the thin film 57 as a sample substance to be measured. Protein Ta is attached 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. In this manner, by configuring the flow path member 54 with a material that can be elastically deformed, the adhesion with the dielectric block 52 is enhanced, and the sealing performance of the liquid flow path 55 configured between the dielectric block 52 is improved. Secured.

  The holding member 56 is long and has a shape in which the upper surface member 56A and the 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 beam. 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.

  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 The capacity of the liquid channel 55 according to the present embodiment is 7 μl.

  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 attached on the thin film 57 (on the linker layer 57A) of the measurement channel 55A and the protein Ta is not attached 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 to which the protein Ta is attached.

  FIG. 9 shows a detailed configuration of the dispensing head 20.

  The dispensing head 20 includes 12 dispensing tubes 20A. Each dispensing tube 20A is held by a holding member 20B so as to be arranged in a line along an arrow Y direction orthogonal 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.

  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 when the ball screw 22A is rotated by the rotational drive of the motor 22B. By this movement in the X direction, the dispensing head 20 is cooled, the correction plate 45, the buffer supply unit 44B (buffer plate 44P), the measurement unit 30 (measurement chip 50), the sample setting unit 40B (sample plate 40P), The pipette tip set unit 42B (pipette tip stocker 42P) can be moved to a position facing it.

  As shown in FIG. 9, the dispensing head 20 is provided with a vertical drive mechanism 24 that moves the dispensing head 20 in the arrow Z direction. The vertical drive mechanism 24 includes a motor 24A and a drive shaft 24B disposed in the Z direction, and the drive shaft 24B is rotated by the rotational drive of the motor 24A, thereby moving the dispensing head 20 in the Z direction. By this movement in the Z direction, the dispensing head 20 has a pipette tip stocker 42P set in the pipette tip setting portion 42B, a sample plate 40P set in the sample setting portion 40B, a buffer plate 44P set in the buffer supply portion 44B, The correction plate 45, the plate set in the cold insulation unit 46, the measurement chip 50 set in the measurement unit 30, and the like can be accessed.

  As shown in FIG. 10, a suction / discharge drive unit 26 is connected to the dispensing head 20. The intake / exhaust drive unit 26 includes a first pump 27 and a second pump 28. The first pump 27 and the second pump 28 are provided corresponding to the pair of dispensing pipes 20A described above. The first pump 27 includes a syringe pump, and includes a first cylinder 27A, a first piston 27B, and a first motor 27C that drives the first piston 27B. The first cylinder 27A is connected to the dispensing head 20 via a pipe 27H. The second pump 28 is also a syringe pump, and includes a second cylinder 28A, a second piston 28B, and a second motor 28C that drives the second piston 28B. The second cylinder 28A is connected to the dispensing head 20 via a pipe 28H.

  The dispensing head 20 controls the rotational drive of the first motor 27C and the second motor 28C, and controls the drive of the first piston 27B and the second piston 28B. In addition, the speed of the solution when sucking and discharging is adjustable.

  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 40B, and the buffer supply unit 44B, and using pipette tips CP attached to the six dispensing tubes 20A for liquid supply. 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.

  On the other hand, 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. 11, 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.

  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. .

  FIG. 12 shows the configuration of the electrical system of biosensor 10 according to the present embodiment.

  As shown in the figure, the biosensor 10 includes a CPU 70A, a ROM 70B, a RAM 70C, an HDD 70D, and the like. The biosensor 10 controls the operation of the entire apparatus, the rotational drive of the motor 22B and the motor 24A, and the first By controlling the rotational drive of the first motor 27C and the second motor 28C, the dispensing head 20 moves in the X direction and the Z direction, and the pipette tip CP attached to each dispensing pipe 20A of the dispensing head 20 A dispensing chip drive control unit 72 that controls the suction and discharge of the sample and the buffer solution, and the rotation of a motor (not shown) that rotates the holding arm 49A and rotates the ball screw 49B, thereby controlling the measurement chip transport mechanism. A measurement chip conveyance control unit 74 that controls the operation of 49, a CCD control unit 76 that controls the imaging operation of the CCD 36B, By controlling the power supply to the source 34A, and includes a lighting control unit 78 for controlling the lighting of the light source 34A, a.

  A dispensing head drive control unit 72, a measurement chip transport control unit 74, a CCD control unit 76, a lighting control unit 78, a display 14, and an input unit 16 are connected to the control unit 70.

  Therefore, the control unit 70 controls the movement of the dispensing head 20 via the dispensing head drive control unit 72 and the suction and discharge of the sample and buffer liquid to the pipette tip CP, and the measurement chip conveyance control unit 74. Control of conveyance of the measurement chip 50, control of the imaging operation of the CCD 36B via the CCD controller 76, control of lighting of the light source 34A via the lighting controller 78, operation screens for the display 14, various messages, etc. Control of display of various types of information can be performed. Further, the control unit 70 can grasp the operation content for the input unit 16.

  The control unit 70 performs predetermined processing based on the image information input via the CCD control unit 76, and derives refractive index change data in the measurement region E1 and the reference region E2.

The refractive index change data is obtained by individually supplying a sample and a buffer solution to the measurement chip 50, emitting the light beam L from the light emitting unit 34, and irradiating the measurement region E1 and the reference region E2 with the light beams L1 and L2. This is obtained based on the angle difference between the reflection angle at which the dark line of the light beam L1 totally reflected in the measurement region E1 is generated and the reflection angle at which the dark line of the light beam L2 totally reflected in the reference region E2 is generated. The light beams L1 and L2 incident on the interface between the thin film 57 and the dielectric block 52 at a specific incident angle excite surface plasmons on the interface, thereby reflecting the light beams L1 and L2 incident on the specific incident angle. The intensity of light sharply decreases and is observed as a dark line. The incident angles of the light beams L1 and L2, which are dark lines, are the total reflection attenuation angle θ _SP , and the total reflection attenuation angle θ _SP detected in the measurement region E1 and the reference region E2 when the buffer liquid is supplied to the measurement chip 50. And the difference between the total reflection attenuation angle θ _SP detected in the measurement region E1 and the reference region E2 when the sample is supplied to the measurement chip 50 is the refractive index change data.

  Next, the operation of the biosensor 10 according to the present embodiment will be described.

  When measuring the characteristics of the protein Ta, the control unit 70 controls the dispensing head 20 via the dispensing head drive control unit 72 and applies a predetermined concentration from the dispensing head 20 to the measurement chip 50 to be measured. The buffer solution and various types of analyte solutions are alternately supplied separately.

  Further, the control unit 70 controls the measurement chip transport mechanism 49 via the measurement chip transport control unit 74 so that the measurement chip 50 supplied with the buffer solution or the analyte solution is supplied to the upper base 32A at every predetermined measurement cycle. The measurement region E1 of the measurement channel 55A to be measured and the reference region E2 of the reference channel 55R are respectively arranged at positions where the light beams L1 and L2 are incident. Then, the control unit 70 controls the lighting control unit 78 to turn on the light source 34A, emits a light beam from the light emitting unit 34, and applies the light beams L1 and L2 to the measurement region E1 and the reference region E2, respectively. Irradiate. 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 focused on the light receiving surface of the CCD 36B through the lens unit 36A.

  The control unit 70 controls the imaging operation of the CCD 36B via the CCD control unit 76, causes the image formed on the light receiving surface of the CCD 36B to be captured, and outputs image information indicating the image from the CCD 36B. The output image information is input to the control unit 70 via the CCD control unit 76.

  FIG. 13A 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.

  When the image information is input, the control unit 70 performs the following dark line position derivation process.

  FIG. 14 is a flowchart showing the flow of dark line position derivation processing executed by the control unit 70. Hereinafter, the dark line position deriving process will be described with reference to FIG.

  In step 100 in the figure, 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 (see also FIG. 13A) are displayed. The respective convolution operations are performed on each of them to derive distribution information indicating the one-dimensional light intensity distributions of the light beams L1 and L2. FIG. 13B 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 of the region B. In FIG. 13B, the horizontal axis indicates the reflection angle direction, and each position on the horizontal axis corresponds to the reflection angle.

  In the next step 102, dark line positions are detected from the light intensity distributions indicated by the derived distribution information readings, so that dark lines are derived from the light intensity distributions of the light beams totally reflected in the measurement area E1 and the reference area E2. Each of the reflection angles at which is generated is detected.

  In step 102, the reflection angle at which the dark line is generated is detected by performing the processing shown in FIGS. 15A to 15E on each distribution information reading.

  That is, first, a smoothing process is performed on the light intensity distribution (FIG. 15A) indicated by the distribution information to obtain a smoothed light intensity distribution (FIG. 15B). Next, a difference intensity distribution is derived by subtracting the smoothed light intensity distribution from the light intensity distribution indicated by the distribution information (FIG. 15C). Then, a predetermined threshold value is added to each intensity indicated by the derived difference intensity distribution (FIG. 15D). Finally, a region where the intensity is zero or less in the difference intensity distribution to which the threshold is added is identified (FIG. 15F), and a dark line is generated by obtaining the barycentric position of the area of the region where the intensity is zero or less. Detect the reflection angle.

  In the next step 104, the reflection angle at which the dark line is generated in the reference area E2 is subtracted from the reflection angle at which the dark line is generated in the measurement area E1, and angle difference information indicating the angle difference of the reflection angle after the subtraction is stored in the HDD 70D. Then, the dark line position derivation process ends. As described above, by subtracting the reflection angle at which the dark line is generated at the reference region E2 from the reflection angle at which the dark line is generated at the measurement region E1, the change in the reflection angle at which the dark line is generated due to the influence of the supplied solution can be calibrated. it can.

  As a result, the HDD 70D is supplied with a predetermined concentration of buffer solution and various types of analyte solutions alternately from the dispensing head 20 to the measurement chip 50 to be measured, and is obtained every predetermined measurement cycle. The angle difference information is stored.

  When the user performs a predetermined operation for instructing the input unit 16 to derive the refractive index change data, the control unit 70 performs the following refractive index change data derivation process to derive the refractive index change data.

  FIG. 16 shows a flowchart showing the flow of refractive index change data derivation processing executed by the control unit 70. Hereinafter, the refractive index change data deriving process will be described with reference to FIG.

  In step 150 of the figure, each angle difference information of the measurement chip 50 to be measured is read from the HDD 70D, and the angle differences of the reflection angles indicated by the angle difference information are arranged in the order in which each angle difference information was obtained.

  FIG. 17 shows the time difference of the reflection angle obtained by alternately supplying a buffer solution of a predetermined concentration and various types of analyte solutions from the dispensing head 20 to the measuring chip 50 to be measured. An example of a typical change is shown. In FIG. 17, since the solution in the liquid channel 55 becomes unstable at the timing when the supply of the buffer solution and the analyte solution is switched, noise is generated.

  In the next step 152, an average angle difference obtained by averaging the angle difference of the reflection angles is obtained for each period when the buffer solution of FIG. 17 is supplied and for each period when each analyte solution is supplied.

  In FIG. 18, each period in which the buffer solution and the analyte solution in FIG. 17 are supplied is sent to the liquid channel 55 once, and the buffer is sequentially added to the liquid channel 55 of the measurement chip 50 from the start of measurement. A graph is shown in which the average angle difference is plotted for each number of times the liquid or the analyte solution has been fed.

  Here, as shown in FIG. 18, in the graph of the average angle difference, the refractive index of the protein Ta changes over time due to the change in the attachment state of the protein Ta and the decrease in the film thickness. There is a drift phenomenon that decreases with time. Further, when a specific analyte solution (see arrow t in FIG. 18) is supplied, the average angle difference increases due to the increase in the refractive index due to the analyte contained in the analyte solution adhering to the protein Ta. Since the analyte adhering to the protein Ta is peeled off little by little by the subsequent supply of the buffer solution or the analyte solution, the drift component when the buffer solution is supplied remains affected.

  Therefore, in the next step 154, each average angle difference is compared in time order, and when the average angle difference is increased by a predetermined value ΔS or more than the average angle difference measured one time before, the predetermined value ΔS or more. The measurement results of the refractive index for a predetermined number of times after the time when the increased average angle difference is measured (in this embodiment, 6 times) are excluded (see FIG. 19). It should be noted that the predetermined value ΔS is determined that the refractive index has increased due to an analyte adhering to the protein Ta after taking into account various errors such as errors in manufacturing the apparatus and errors due to changes in measurement conditions. Set a value that can be used. In addition, the predetermined number of times may be set to the number of times of liquid feeding that is separated to the extent that the analyte attached to the protein Ta satisfies the required measurement accuracy.

  Then, in the next step 156, as shown in FIG. 20, among the average angle differences remaining without being excluded, the average angle difference obtained in the period during which the buffer solution is supplied is specified, and the buffer solution is supplied. Based on the average angle difference of the determined period, change information indicating a change in refractive index with time is derived.

  In step 156, as the change information, as shown in FIG. 21, for example, an approximate straight line connecting the reflection angles is derived by the least square method.

  In the next step 158, the measurement result is corrected based on the derived approximate straight line as shown in FIG. 22, and in the next step 160, the buffer solution and the analyte solution after the buffer solution are corrected in the corrected measurement result. The change (difference) in the average angle difference at the time of supply is measured, the difference is stored in the HDD 70D as refractive index change data, and the refractive index change data deriving process is terminated.

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

  As described above, according to the present embodiment, a plurality of types of analyte solutions used for measuring the characteristics of protein Ta and a buffer solution having a predetermined concentration serving as a reference for measurement are alternately applied to the protein Ta adhesion region on the thin film 57. The refractive index of the protein Ta adhering to the thin film 57 is measured several times, and the refractive index is measured once before based on each refractive index measured with the buffer solution supplied. When the refractive index is increased by a predetermined value ΔS or more than the measured refractive index, the refractive index measurement result for a predetermined number of times after the measurement of the refractive index increased by the predetermined value ΔS is excluded. Since the change information indicating the change is derived and the measurement result is corrected based on the derived change information, the drift phenomenon in which the refractive index changes with time can be accurately corrected.

  In addition, according to the present embodiment, the analyte solution and the buffer solution are alternately supplied to the adhesion region of the protein Ta on the thin film 57, so even if the protein Ta and the analyte react, Therefore, the reaction state when each analyte solution is supplied can be accurately detected.

  In the present embodiment, the case where the analyte solution and the buffer solution are alternately supplied to the protein Ta adhesion region on the thin film 57 has been described. However, the present invention is not limited to this, for example, The buffer solution may be supplied every time the analyte solution is supplied a predetermined number of times (for example, three times).

  In the present embodiment, the case where an approximate line is derived as change information has been described. However, the present invention is not limited to this, and approximation may be performed using a specific function.

  In the present embodiment, 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 a light beam whose reflection angle changes according to a change in the incident angle of the incident light beam. Alternatively, distribution information indicating the light intensity distribution of the light beam may be acquired by detection with a small photodetector that moves in synchronization with the change in the reflection angle.

  In the present embodiment, the case where the dark line position is detected by subtracting the smoothed light intensity distribution from the light intensity distribution indicated by the distribution information has been described, but the present invention is not limited to this. For example, reference data indicating the light intensity distribution of a light beam in a state where total reflection attenuation has not occurred is stored in the dark line position detection unit 84 in advance, and the dark line position detection unit 84 stores the light intensity distribution indicated by the distribution information. The difference intensity distribution may be derived by obtaining a difference or division from the light intensity distribution indicated by the stored reference data.

  In the present embodiment, the case where the dark line position is detected by subtracting the smoothed light intensity distribution from the light intensity distribution indicated by the distribution information has been described, but the present invention is not limited to this. For example, when the range where the dark line is generated in the light intensity distribution can be specified, the dark line position may be detected from the area of the area within the range where the intensity of the light intensity distribution indicated by the distribution information is equal to or less than the threshold and the dark line is generated. Good.

  Furthermore, a leaky mode detector can be given as another measuring apparatus using the total reflection attenuation of the present embodiment. 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.

  In addition, the configuration of the measurement apparatus described in this embodiment (see FIGS. 1 to 12) is merely an example, and it is needless to say that the measurement apparatus can be appropriately changed without departing from the gist of the present invention.

  Further, the flow of the dark line position deriving process (see FIG. 14) and the refractive index change data deriving process (see FIG. 16) described in the present embodiment is also an example, and within the scope not departing from the gist of the present invention. Needless to say, it can be changed as appropriate.

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 a schematic block diagram of the liquid suction / discharge part of the biosensor according to the 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 structure of the electric system of the biosensor 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 dark-line position derivation | leading-out process which concerns on embodiment. (A)-(E) are figures which show the flow at the time of detecting a dark line. It is a flowchart which shows the flow of the refractive index data derivation | leading-out process which concerns on embodiment. It is a graph which shows an example of a time-dependent change of the angle difference of the reflection angle calculated | required by supplying a buffer solution and various kinds of analyte solutions separately separately. It is the graph which plotted the average angle difference for every period when the buffer solution and the analyte solution were supplied. It is a graph which shows an example of the result which excluded the average angle difference of exclusion from FIG. It is a graph which shows the average angle difference calculated | required in the period when the buffer solution was supplied among each average angle difference shown in FIG. It is a graph which shows the derivation | leading-out result of the approximate expression which connects each reflection angle. It is a graph which shows the result of having corrected each average angle difference shown in FIG. It is a graph which shows an example in the state where the drift phenomenon in which the refractive index of the conventional specimen substance decreased with time occurred. It is a figure which shows an example of the measurement result estimated with the measurement result at the time of measuring the change of the refractive index of the conventional specimen substance. It is a graph which shows an example of the state which correct | amended the conventional drift phenomenon. It is a graph which shows an example of the measurement result which correct | amended the conventional drift phenomenon and measured the refractive index of the specimen substance.

Explanation of symbols

10 Biosensor (measuring device)
20 dispensing head (supply means)
52 Dielectric block (light transmissive member)
70 Control unit (measuring means, deriving means, correcting means)

Claims (4)

A light-transmitting member having a light-transmitting property, in which a thin-film layer is formed in part and a specimen substance to be measured is attached on the thin-film layer;
Supply means for supplying each of a plurality of types of solutions used for measuring the characteristics of the specimen substance and a predetermined reference solution serving as a measurement reference to the specimen substance adhesion region on the thin film layer;
A plurality of types of solutions are individually supplied by the supply means, and the reference solution is supplied each time the solution is supplied a predetermined number of times, so that a plurality of pieces of refractive index information of the analyte substance adhering to the thin film layer are supplied. Measuring means for measuring times,
Based on each refractive index information measured by the measuring means in a state where the reference solution is supplied by the supplying means, the refractive index information is increased by a predetermined value or more than the refractive index information measured once before. In this case, a derivation means for deriving change information indicating a change in refractive index information over time by excluding a predetermined number of refractive index information measurement results after the measurement of the refractive index information increased by a predetermined value or more. When,
Correction means for correcting the measurement result by the measurement means based on the change information derived by the derivation means;
Measuring device. The measuring means is a light intensity distribution of a light beam incident on the light transmissive member at a plurality of angles and totally reflected at the interface so as to be totally reflected at the interface between the light transmissive member and the thin film layer. The measurement apparatus according to claim 1, wherein the refractive index information of the specimen substance is measured by detecting a reflection angle at which a dark line is generated due to total reflection attenuation. The thin film layer is provided with an attachment region to which the specimen material is attached and an unattached region to which the specimen material is not attached,
The measuring means measures a reflection angle at which a dark line is generated from a light intensity distribution of a light beam totally reflected in each of the adhesion region and the non-attachment region at the interface between the light transmissive member and the thin film layer, The measuring apparatus according to claim 2, wherein the refractive index information of the specimen substance is measured by calibrating the reflection angle at which the dark line is generated in the adhesion region with the reflection angle at which the dark line is generated in the non-attached region. The measurement apparatus according to claim 1, wherein the measurement unit supplies the reference solution every time the type of the solution is switched by the supply unit when measuring refractive index information.