JP2006266850A - Measuring method and biosensor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To measure the interaction of the interaction state with another substance in a reaction liquid in a state that a molecule to be analyzed and a certain substance are allowed to interact with each other. <P>SOLUTION: In a step S30, the reaction liquid A is supplied to a liquid flow channel 45 during measurement by a head 78B and, in a step S32, the reaction liquid A is set to a standby state until a predetermined reaction time T1 is elapsed. Measurement processing is carried out even during the reaction time T1. After the elapse of the reaction time T1, in a step S36, the supply indicating signal of a reaction liquid B is outputted to the head 78B and the reaction liquid B is supplied to the liquid flow channel 45 by the head 78B during the measurement due to the supply of the reaction liquid A. By the supply of the reaction liquid B, a state that the molecule M to be analyzed and the substance A are allowed to interact with each other and the interaction with a substance B can be observed. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a biosensor that detects and measures a substance that interacts with an analyte molecule fixed on a thin film layer, and a measurement method using this biosensor.

  Conventionally, a surface plasmon sensor is known as one of measuring devices using an evanescent wave (see Patent Document 1). In general, a surface plasmon sensor includes a prism, a metal film disposed on one surface of the prism, to which a molecule to be analyzed is fixed, a light source that generates a light beam, and the light beam to the prism. Based on the detection result of the light detection means, and an optical system that makes the light incident at various angles so that a total reflection condition is obtained at the interface with the light detection means, and a light detection means that detects the intensity of the light beam totally reflected at the interface. Thus, the analysis of the molecule to be analyzed is performed.

  In the measurement using the surface plasmon sensor, a reaction solution is supplied to the molecule to be analyzed immobilized on the metal film, and a light beam is applied to the opposite side of the metal film to which the reaction solution is supplied. The interaction between the molecule to be analyzed and the substance in the reaction solution is measured based on the refractive index information obtained from the incident and reflected light.

  In the technique described in Patent Document 1, a flow path member for feeding a reaction liquid is attached to the apparatus, and a measurement cell in which a metal film is formed separately from the flow path member is attached to the measurement apparatus. Yes. The metal film is disposed so as to be exposed to the liquid flow path formed in the flow path member, and the reaction liquid is always sent on the molecule to be analyzed during the measurement. By the way, in the apparatus described in Patent Document 1, when switching a reaction solution to be sent from a certain reaction solution H1 to a reaction solution H2 different from this reaction solution, a buffer solution is sent during the switching. It is controlled. That is, after the completion of the feeding of a certain reaction solution H1, the buffer solution is always sent before the next reaction solution H2 is sent.

However, if a buffer solution is sent after sending a certain reaction solution H1, the interaction between the substance J in the reaction solution H1 and the molecule to be analyzed may be canceled by the buffer solution. The reaction state cannot be measured while supplying the next reaction solution H2 while maintaining the complete interaction state with the substance J in H1.
Japanese Patent No. 3294605

  The present invention has been made in consideration of the above-mentioned facts. In a state where an analyte molecule and a substance can interact with each other, the analyte molecule under the interaction state and the substance and the other substance in another reaction solution. It is an object of the present invention to provide a biosensor capable of measuring an interaction with a substance and a measurement method using the biosensor.

  The measurement method according to claim 1 supplies a reaction liquid for inspecting the interaction with the molecule to be analyzed to a liquid channel that can be fed onto the thin film layer on which the molecule to be analyzed is fixed, The light beam is incident on the surface of the thin film layer opposite to the liquid flow path, and the interaction between the molecule to be analyzed and the substance in the reaction solution based on the information on the refractive index change obtained from the reflected light. A biosensor for measuring a first sensor, wherein a first reaction solution is supplied to the liquid channel, and a first measurement method measures the interaction between the molecule to be analyzed and a substance in the first reaction solution. A measurement step, supplying a second reaction liquid different from the first reaction liquid to the liquid flow channel to which the first reaction liquid is supplied, replacing the first reaction liquid with the second reaction liquid, A second unit for measuring an interaction between the molecule to be analyzed and a substance in the second reaction solution; It includes a measuring step.

  In addition, the biosensor according to claim 6 supplies a reaction solution for inspecting an interaction with the molecule to be analyzed to a liquid channel that can be fed onto the thin film layer on which the molecule to be analyzed is fixed. In addition, a light beam is incident on the surface of the thin film layer opposite to the liquid channel, and based on the information on the refractive index change obtained from the reflected light, the molecule to be analyzed and the substance in the reaction solution A biosensor for measuring an interaction, comprising: a first reaction liquid supply unit that supplies a first reaction liquid to the liquid flow path; and the molecule to be analyzed and the first reaction liquid in a state where the first reaction liquid is supplied. A first measuring means for measuring an interaction with a substance in one reaction liquid; and a second reaction liquid different from the first reaction liquid is supplied to the liquid channel to which the first reaction liquid is supplied A second reaction liquid supply means for replacing the first reaction liquid with the second reaction liquid; 2 the reaction liquid is configured to include a, a second measuring means for measuring the interaction between a material of the second reaction solution with the analyte molecules solution in a state of being supplied.

  In the measurement method of claim 1 and the biosensor of claim 6, first, the first reaction solution is supplied to the liquid flow path, and the interaction between the analyte molecule and the substance in the first reaction solution is measured. . Then, the second reaction solution different from the first reaction solution is supplied to the liquid flow path to which the first reaction solution is supplied without interposing the supply of the buffer solution, and the first reaction solution is replaced with the second reaction solution. Is done. If the substance in the first reaction solution interacts with the molecule to be analyzed, the second reaction solution is supplied while the interaction state is maintained. Therefore, the interaction between the interacted state and the substance in the second reaction solution can be measured.

  Moreover, in the measuring method of Claim 1 and the biosensor of Claim 6, since it can measure, supplying a reaction liquid, the interaction from the initial stage of reaction can be measured correctly.

  The measurement method according to claim 2 is the measurement method according to claim 1, wherein the measurement of at least one of the first measurement step and the second measurement step stops the flow of the supplied liquid. The present invention is characterized in that the present invention is also carried out in a state in which it is allowed to occur.

  The biosensor according to claim 7 is the biosensor according to claim 6, wherein the measurement of at least one of the first measurement unit and the second measurement unit stops the flow of the supplied liquid. The present invention is characterized in that the present invention is also carried out in a state in which it is allowed to occur.

  According to the measurement method of claim 2 and the biosensor of claim 7, since the measurement can be performed even when the flow of the supplied reaction solution is stopped, it is used in comparison with the case where the reaction solution is kept flowing. The amount of reaction solution to be reduced can be reduced. In addition, noise due to the flow of the reaction liquid can be reduced.

  The measurement method according to claim 3 is the measurement method according to claim 1 or 2, wherein the thin film layer is formed on a flat surface of a transparent body capable of transmitting a light beam, and the liquid channel. Is configured between the channel member attached to the transparent body and the thin film layer, and the thin film layer, the transparent body, and the channel member configure a measurement cell separate from the biosensor. It is characterized by that.

  The biosensor according to claim 8 is the biosensor according to claim 6 or 7, wherein the thin film layer is formed on a flat surface of a transparent body that can transmit a light beam. The liquid channel is configured between a channel member attached to the transparent body and the thin film layer, and the thin film layer, the transparent body, and the channel member are separate from the biosensor. It constitutes a body measuring cell.

  In the measurement method according to claim 3 and the biosensor according to claim 8, the liquid flow path is configured in a measurement cell separate from the biosensor by the transparent body on which the thin film layer is formed and the flow path member. Therefore, it is not necessary to provide a flow path for supplying a liquid to the biosensor side, and the biosensor can have a simple configuration.

  The measurement method according to claim 4 and the transparent body of the biosensor according to claim 9 are configured by a prism having two surfaces that are not parallel to each other in addition to the flat surface on which the thin film layer is formed. It can also be characterized.

  Thus, by forming a thin film layer on the prism itself, no other member is interposed between the prism and the thin film layer, which is optically advantageous.

  A measurement method according to a fifth aspect is the measurement method according to the third or fourth aspect, wherein a supply port capable of supplying a liquid and a supplied liquid are provided to the flow path member constituting the liquid flow path. A discharge port capable of being discharged is formed, and the supply of the first reaction liquid to the liquid flow path is performed from the supply port using a pipette filled with the first reaction liquid, Supply to the liquid flow path is performed using a pipette filled with the second reaction liquid from the supply port.

  The biosensor according to claim 10 is the biosensor according to claim 8 or 9, further comprising a reservoir capable of storing the first reaction liquid and the second reaction liquid, respectively, and the liquid The flow path member constituting the flow path is formed with a supply port capable of supplying a liquid and a discharge port capable of discharging the supplied liquid, and the first reaction liquid supply means and the second reaction liquid supply means include In addition, the storage unit sucks the first reaction liquid or the second reaction liquid, and includes a supply head that supplies the sucked first reaction liquid or the second reaction liquid to the liquid flow path. It is characterized by this.

  In the measurement method of claim 5 and the biosensor of claim 10, since the liquid is supplied from the supply port of the flow path member by the supply head, a long flow path from the place where the reaction liquid is stored to the measurement position is provided. It becomes unnecessary, and the biosensor can have a simple configuration.

  Since the present invention has the above-described configuration, the molecule to be analyzed can interact with a certain substance, and the molecule to be analyzed and the interaction between the certain substance and the substance in another reaction solution under this interaction state. Can be measured.

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

  The biosensor 70 of the present embodiment is a so-called surface plasmon sensor that measures the characteristics of the molecule M to be analyzed using surface plasmons generated on the surface of a metal film. In the biosensor 70, various reaction liquids are supplied to the molecule M to be analyzed, and the presence / absence of bonding between the molecule M to be analyzed and the substance in the reaction liquid, the bonding speed, the bonding amount, and the like are measured. As the analyzed molecule M, physiologically active substances such as proteins, nucleic acids, and fatty acids can be analyzed.

  As shown in FIGS. 1 and 2, the biosensor 70 includes a tray holding unit 72, a transport unit 74, a reaction liquid plate holding unit 76, a liquid supply unit 78, an optical measurement unit 80, and a control unit 90. .

  The tray holding unit 72 includes a mounting table 72A and a belt 72B. The mounting table 72A is attached to a belt 73 spanned in the direction of arrow I, and is movable in the direction of arrow I by the rotation of the belt 72B. Two trays 30 are positioned and placed on the mounting table 72A. Eight sensor sticks 40 are stored in the tray 30. The sensor stick 40 is a chip on which the analyte molecule M to be measured is fixed, and details will be described later. Under the mounting table 72A, a push-up mechanism 72D that pushes up the sensor stick 40 to a position of a stick holding member 74C described later is disposed.

  The sensor stick 40 is to which the molecule M to be analyzed is fixed. As shown in FIGS. 3 and 4, the sensor stick 40 includes a dielectric block 42, a flow path member 44, a holding member 46, an adhesive member 48, and an evaporation preventing member 49.

  The dielectric block 42 is made of transparent resin or the like that is transparent to the light beam, and has a prism portion 42A having a trapezoidal cross section, and the prism portion 42A integrally with both ends of the prism portion 42A. The formed held portion 42B is provided. A metal film 50 is formed on the upper surface on the wide side of the two parallel surfaces of the prism portion 42A as shown in FIG. Analytical molecules to be analyzed by the biosensor 70 are fixed on the metal film 50. The dielectric block 42 also functions as a so-called prism, and in the measurement by the biosensor 70, the light beam is incident from one of the two opposite side surfaces of the prism portion 42A that are not parallel to each other, and the other is a metal from the other side. A light beam totally reflected at the interface with the film 50 is emitted.

  As shown in FIG. 6, as shown in FIG. 6, for fixing the molecule M to be analyzed, a part of the surface of the metal film 50, which is located on the downstream side of the part exposed to the liquid channel 45 described later. An immobilization carrier 50A is arranged. The molecule to be analyzed M is immobilized on this portion by covalent bonding with the reactive group of the immobilization carrier 50A. As the immobilization carrier 50A, dextran, carboxymethyl group, NTA or the like can be used, and is selected according to the molecule M to be analyzed.

  A region located on the upstream side of the portion exposed to the liquid channel 45 on the metal film 50 is a reference region E2. The reference region E2 is a region provided for correcting the refractive index data of light obtained from the measurement region E1 where the analyte molecule M is fixed, and the light beam is also incident on the reference region E2. The reference region E2 is preferably treated with, for example, alkyl thiol, amino alcohol, amino ether or the like so as to prevent binding with various analytes.

  On both side surfaces of the prism portion 42A, engaging convex portions 42C that are engaged with the holding member 52 along the upper edge are virtual surfaces perpendicular to the upper surface of the prism portion 42A along the lower edge. Vertical protrusions 42D configured on the extension are formed at seven locations, respectively. Further, an engagement groove 42E is formed in the center portion along the longitudinal direction of the lower surface of the dielectric block 42.

  The flow path member 44 has a rod shape with substantially the same length as the prism portion 42A of the dielectric block 42, and forms a liquid flow path 45 between the metal film 50 of the dielectric block 42 as shown in FIG. To do. The liquid channel 45 is configured by being surrounded by the channel wall 44 </ b> A of the channel member 44 and the metal film 50, and the supplied liquid can be stored on the metal film 50. The flow path member 44 is formed with a supply port 45 </ b> A and a discharge port 45 </ b> B that communicate with the liquid flow path 45. Six liquid channels 45 are arranged in the longitudinal direction of the channel member 44. Accordingly, six independent liquid channels 45 are formed in one sensor stick 40. On the side wall of the flow path member 44, a convex portion 44 </ b> B is formed that is press-fitted into a concave portion (not shown) inside the holding member 46 to ensure adhesion with the holding member 46.

  In addition, since it is assumed that the liquid containing the protein is supplied to the liquid channel 45, the material of the channel member 44 is a non-protein as a material for preventing the protein from adhering to the channel wall 44A. It is preferable not to have specific adsorptivity. The flow path member 44 preferably has elasticity in order to ensure adhesion with the metal film 50 and prevent liquid leakage. Specifically, silicon, polypropylene, or the like can be used.

  The holding member 46 is long and includes an upper plate 46A and two side plates 46B. The side plate 46B is formed with an engagement hole 46C that is engaged with the engagement convex portion 42C of the dielectric block 42. The holding member 46 is attached to the dielectric block 42 with the engagement hole 46 </ b> C and the engagement protrusion 42 </ b> C engaged with the flow path member 44 interposed therebetween. Thereby, the flow path member 44 is attached to the dielectric block 42. In the upper surface plate 46A, a tapered pipette insertion hole 46D that narrows toward the flow path member 44 is formed at a position facing the supply port 45A and the discharge port 45B of the flow path member 44. A positioning boss 46E is formed between the adjacent pipette insertion hole 46D and the pipette insertion hole 46D.

  An evaporation preventing member 49 is bonded to the upper surface of the holding member 46 via an adhesive member 48. A pipette insertion hole 48D is formed at a position facing the pipette insertion hole 46D of the adhesive member 48, and a positioning hole 48E is formed at a position facing the boss 46E. Further, a slit 49D, which is a cross-shaped cut, is formed at a position facing the pipette insertion hole 46D of the evaporation preventing member 49, and a positioning hole 49E is formed at a position facing the boss 46E. By inserting the boss 46E into the holes 48E and 49E and bonding the evaporation preventing member 49 to the upper surface of the holding member 52, the slit 49D of the evaporation preventing member 49 and the supply port 45A and the discharge port 45B of the flow path member 44 are formed. Configured to face each other. When the pipette tip CP is not inserted, the slit 49D covers the supply port 45B, and the liquid supplied to the liquid channel 45 is prevented from evaporating.

  The evaporation preventing member 54 is also preferably made of an elastic material so that the pipette tip CP can be inserted from the slit 49D, and specifically silicon or polypropylene can be used.

As shown in FIGS. 1 and 2, the transport unit 74 of the biosensor 70 includes an upper guide rail 74A, a lower guide rail 74B, and a stick holding member 74C. The upper guide rail 74 </ b> A and the lower guide rail 74 </ b> B are horizontally disposed in the arrow J direction orthogonal to the arrow I direction, above the tray holding unit 72 and the optical measurement unit 80. A stick holding member 74C is attached to the upper guide rail 74A. The stick holding member 74C can hold the held portions 42B at both ends of the sensor stick 40, and can move along the upper guide rail 74A. When the engagement groove 42E of the sensor stick 40 held by the stick holding member 74C and the lower guide rail 74B are engaged and the stick holding member 74C moves in the direction of arrow J, as shown in FIG. 40 is conveyed to the measuring unit 82 on the optical measuring unit 80. The measurement unit 82 measures the analyte molecule M fixed to the sensor stick 40. In the measurement unit 82, the lower guide rail 74B is partially separated for measurement. The reaction solution plate holding unit 76 includes a mounting table 76A and a guide rail 76B. The guide rail 76B is arranged along the arrow J direction. The mounting table 76A is attached to the guide rail 76B, and is capable of mounting a reaction solution plate 77 on which a reaction solution is set. The mounting table 76A is movable along the guide rail 76B.

  The liquid supply unit 78 is configured to include a cross rail 78A and a head 78B that are bridged in the direction of arrow I above the upper guide rail 74A and the guide rail 76B. The head 78B is attached to the cross rail 78A, and is movable between the measurement unit 82 and the reaction solution plate 77 as shown in FIG. Further, the head 78B can be moved in the vertical direction (arrow Z direction) by a drive mechanism (not shown). The head 78B has a replaceable pipette tip CP attached to the tip, and has a function as a so-called pipette.

  As shown in FIG. 7, the optical measurement unit 80 includes a light source 80A, a first optical system 80B, a second optical system 80C, a light receiving unit 80D, and a signal processing unit 80E. A light beam L is emitted from the light source 80A. The light beam L becomes two light beams L1 and L2 via the first optical system 80B, and is incident on the measurement region E1 and the reference region E2 of the dielectric block 42 disposed in the measurement unit 82. In the measurement region E1 and the reference region E2, the light beams L1 and L2 are incident on the interface between the metal film 50 and the dielectric block 42 with various incident angle components and at an angle equal to or greater than the total reflection angle. . The light beams L 1 and L 2 are totally reflected at the interface between the dielectric block 42 and the metal film 50. The totally reflected light beams L1 and L2 are also reflected with various reflection angle components. The totally reflected light beams L1 and L2 are received by the light receiving unit 80D through the second optical system 80C, are photoelectrically converted, and a light detection signal is output to the signal processing unit 80E. In the signal processing unit 80E, predetermined processing is performed based on the input light detection signal, and the total reflection attenuation angles of the measurement region E1 and the reference region E2 are obtained as refractive index data. The refractive index data is output to the control unit 90.

  The control unit 90 has a function of controlling the entire biosensor 70, and is connected to a light source 80A, a signal processing unit 80E, and a drive system (not shown) of the biosensor 70 as shown in FIG. As shown in FIG. 8, the control unit 90 includes a CPU 90A, a ROM 90B, a RAM 90C, a memory 90D, and an interface I / F 90E, which are connected to each other via a bus B, and display various types of information. 92, is connected to an input unit 94 for inputting various instructions and information.

  Various programs for controlling the biosensor 70 and various data are recorded in the memory 90D.

  Next, a procedure for measuring the molecule M to be analyzed by the biosensor 70 will be described. Here, first, the interaction between the analyte molecule M and the substance A is measured, and then the state in which the analyte molecule M and the substance A interact with each other is determined as to Measure whether the effect is shown.

  First, the tray 30 containing the sensor stick 40 with the analyte molecule M immobilized thereon is set on the mounting table 72A of the biosensor 70. Further, the reaction solution plate 77 containing the reaction solution is set on the mounting table 76A. The reaction solution plate 77 stores a reaction solution A containing the substance A and a reaction solution B containing the substances A and B.

  At the time of measurement, one sensor stick 40 set on the plate 30 is pushed up to the position of the stick holding member 74C by the push-up mechanism 72D and held by the stick holding member 74C. The stick holding member 74 </ b> C moves along the lower guide rail 74 </ b> B while holding the sensor stick 40, and conveys the sensor stick 40 to the measurement unit 82.

  In the measurement unit 82, the measurement of the sensor stick 40 is sequentially performed for each liquid channel 45. When the liquid channel 45 to be measured is arranged at the measurement position, the control unit 90 executes the measurement process shown in FIG.

  First, in step S12, an output instruction signal for the light beam L is output to the light source 80A. Thereby, the light beam L is emitted from the light source 80A. The emitted light beam L becomes two light beams L1 and L2 by the first optical system 80B, and is incident on the measurement region E1 and the reference region E2 of the liquid channel 45, respectively. In step S14, an operation instruction signal is output to the light receiving unit 80D and the signal processing unit 80E. Thus, the light beams L1 and L2 that are totally reflected by the measurement region E1 and the reference region E2 and pass through the second optical system 80C are received by the light receiving unit 80D, and the received light is received for each of the measurement region E1 and the reference region E2. The photoelectric conversion is performed and a light detection signal is output to the signal processing unit 80E. In the signal processing unit 80E, predetermined processing is applied to the light detection signal, refractive index data is generated, and the refractive index data is continuously output to the control unit 90.

  In step S16, the controller 90 determines whether or not a predetermined time has elapsed. After the predetermined time has elapsed, the controller 90 stores the input refractive index data in the memory 90D in step S18. In step S20, the refractive index data obtained from the photodetection signal from the measurement region E1 is corrected with the refractive index data obtained from the photodetection signal from the reference region E2, etc. The binding state data indicating the binding state with the substance in the inside is generated. In step S22, the combined state data is output to the display unit 92. As a result, the coupling state data for each predetermined time is stored in the memory 90D and displayed on the display unit 92. As shown in FIG. 10, the combined state data for each time is graphed and output to the display unit 92. This measurement process is continued until a measurement process end signal is received.

  On the other hand, the reaction solution is supplied to the liquid channel 45 during the above measurement process. The control unit 90 executes the supply process shown in FIG. 11 at a predetermined timing during the measurement process.

  First, at step S30, a supply instruction signal for the reaction liquid A is output to the head 78B. Thereby, the reaction liquid A is supplied to the liquid channel 45 under measurement by the head 78B. Specifically, the reaction solution A is supplied as follows. First, the head 78B moves to the upper part of the reaction solution plate 77 in which the reaction solution A is set and descends, and the tip of the pipette chip CP attached to the head 78B is inserted into the cell in which the reaction solution A is stored. Aspirate the reaction solution A into the pipette tip CP. Next, the head 78 </ b> B is lifted and moved to the upper portion of the measuring unit 82 and lowered to insert the tip of the pipette tip CP into the supply port 45 </ b> A of the liquid channel 45. Then, the sucked reaction liquid A is injected into the liquid channel 45 (see FIG. 12A). Thereby, the substance A is supplied to the molecule M to be analyzed, and the interaction can be observed. When the molecule to be analyzed M and the substance A are bonded, the bonding proceeds with the passage of time as shown in FIG.

  When the injection of the reaction solution A is completed, a replacement instruction signal for the pipette tip CP is output to the head 78B in step S32. As a result, the head 78B moves to the upper portion of the pipette tip stocker (not shown) to remove the attached pipette tip CP and attach a new pipette tip CP.

  Thereafter, in step S34, the process waits until a predetermined reaction time T1 elapses. This reaction time T1 is a time sufficient for the interaction between the substance A contained in the reaction solution A and the molecule M to be analyzed to be saturated, and may be recorded in the memory 90D in advance. Whether the interaction is saturated may be determined based on the binding state data. The measurement process is also executed during the reaction time T1. At this time, in the liquid flow path 45, the measurement process is performed in a state where the flow of the supplied reaction solution A is stopped (a state in which no reaction solution A is fed).

  After the elapse of the reaction time T1, in step S36, a supply instruction signal for the reaction liquid B is output to the head 78B. As a result, the reaction liquid B is supplied to the liquid channel 45 under measurement to which the reaction liquid A is supplied by the head 78B. The supply of the reaction solution B is performed in the same manner as the reaction solution A. By supplying the reaction solution B, it is possible to observe the state in which the analyte molecule M and the substance A are interacting with each other and the interaction with the substance B. For example, when the substance B in the reaction solution B binds to the substance A in a state of being bound to the molecule M to be analyzed, the binding proceeds with the passage of time after the injection of the reaction solution B as shown in FIG.

  Here, when the reaction liquid B is injected into the liquid channel 45 from the supply port 45A, the reaction solution A supplied to the liquid channel 45 is discharged from the discharge port 45B as shown in FIG. Is done. That is, the liquid in the liquid channel 45 is replaced with the reaction liquid B from the reaction liquid A. The amount of the reaction liquid B to be injected is set to be sufficiently larger than the volume of the liquid flow path 45 so that the replacement with the reaction liquid A is sufficiently performed. The discharged reaction solution A overflows on the upper surface of the sensor stick 40.

  Next, in step S38, a recovery instruction signal for the discharged reaction solution A is output to the head 78B. As a result, the pipette tip CP of the head 78B sucks the discharged liquid containing the reaction liquid A overflowing the upper surface of the sensor stick 40 (see FIG. 12C), and the sucked discharged liquid is discharged to a waste liquid cell (not shown). Discarded.

  Thereafter, in step S40, the process waits until a predetermined reaction time T2 elapses. This reaction time T2 is a time sufficient to measure what kind of interaction the substance B contained in the reaction solution B exhibits with respect to the interaction state between the molecule M to be analyzed and the substance A. It may be recorded in the memory 90D, or it may be determined whether the interaction has reached saturation based on the binding state data. The measurement process is also executed during the reaction time T2. At this time, in the liquid flow path 45, the measurement process is performed in a state where the flow of the supplied reaction solution B is stopped (a state in which no reaction solution B is fed).

  After the elapse of the reaction time T2, in step S42, a measurement end instruction signal is output, and the supply process is ended.

  On the other hand, the measurement process is also terminated in response to the measurement end instruction signal.

  According to this embodiment, the reaction liquid A is supplied to the liquid channel 45 to cause the substance A and the molecule to be analyzed M to interact with each other, and then the buffer solution is not supplied to the liquid channel 45. B is supplied. Accordingly, by supplying the reaction liquid B while the interaction state between the analyte molecule M and the substance A is maintained, how the reaction liquid B corresponds to the interaction state between the substance A and the analyte molecule M. Can be measured.

  Moreover, in this embodiment, since the reaction liquid can be supplied while performing the measurement process, the interaction from the initial stage of the reaction can be accurately measured.

  Further, in this embodiment, after supplying the reaction solution, the measurement can be performed even when the flow of the supplied reaction solution is stopped. Can be reduced. In addition, measurement noise due to the flow of the reaction liquid can be reduced.

  In this embodiment, since the liquid flow path 45 is configured in the sensor stick 40 that is separate from the biosensor 70, there is no need to provide a flow path for supplying liquid to the biosensor 70 side. The sensor 70 can have a simple configuration.

  In this embodiment, since the reaction solution is supplied using the replaceable pipette tip CP, the reaction solution can be washed as compared with the case where the reaction solution is supplied using one needle. Troubles caused by insufficientness can be avoided.

  In the present embodiment, the liquid discharged from the liquid channel 45 is recovered by moving the pipette tip CP supplied with the liquid to the discharge port 45B and sucking it. It can also be done by the method. For example, as shown in FIG. 13, one pipette tip CP is inserted from the supply port 45A to supply the liquid, and another pipette tip CP is inserted to the discharge port 45B to suck the liquid. Liquid replacement in the passage 45 can be performed.

  In the present embodiment, the surface plasmon sensor is described as an example of the biosensor, but the biosensor is not limited to the surface plasmon sensor. Others, for example, quartz crystal microbalance (QCM) measurement technology, optical measurement technology using functionalized surface from colloidal gold particles to ultrafine particles, supply of reaction solution for measurement with any biosensor and It can be applied to measurement procedures.

It is a whole perspective view of the biosensor of this embodiment. In the whole perspective view of the biosensor of this embodiment, it is a figure showing the state where the head is located near the measurement part. It is a perspective view of the sensor stick of this embodiment. It is a disassembled perspective view of the sensor stick of this embodiment. It is sectional drawing of the 1 liquid flow path part of the sensor stick of this embodiment. It is a figure which shows the state in which the light beam is injecting into the measurement area | region and reference area | region of the sensor stick of this embodiment. It is the schematic of the optical measurement part vicinity of the biosensor of this embodiment. It is a schematic block diagram of the control part of this embodiment, and its periphery. It is a flowchart of the measurement process of this embodiment. It is a graph which shows an example of the measurement result in this embodiment. It is a flowchart of the supply process of this embodiment. It is a figure explaining the reaction liquid supply procedure of the liquid channel in this embodiment. It is explanatory drawing which shows the other method of liquid replacement.

Explanation of symbols

40 sensor stick 42 dielectric block 44 flow path member 45 liquid flow path 70 biosensor 77 reaction liquid plate 78 liquid supply section 78B head 80 optical measurement section 80A light source 80D light receiving section 80E signal processing section 82 measurement section 90D memory 90 control section M Analyte molecule

Claims (10)

The liquid flow path of the thin film layer is supplied to a liquid flow path which can be fed onto the thin film layer on which the molecule to be analyzed is fixed, and a reaction liquid for inspecting the interaction with the molecule to be analyzed is supplied. And a biosensor that measures the interaction between the molecule to be analyzed and the substance in the reaction solution based on the information on the refractive index change obtained from the reflected light. A measuring method,
A first measurement step of supplying a first reaction liquid to the liquid channel and measuring an interaction between the analyte molecule and a substance in the first reaction liquid;
Supplying a second reaction liquid different from the first reaction liquid to the liquid flow channel to which the first reaction liquid is supplied, replacing the first reaction liquid with the second reaction liquid, and the molecule to be analyzed A second measuring step for measuring an interaction between the first reaction liquid and a substance in the second reaction solution;
Measuring method.   The measurement according to claim 1, wherein the measurement in at least one of the first measurement step and the second measurement step is performed even in a state where the flow of the supplied liquid is stopped. Method. The thin film layer is formed on a flat surface of a transparent body capable of transmitting a light beam,
The liquid channel is configured between a channel member attached to the transparent body and the thin film layer,
The measurement method according to claim 1, wherein the thin film layer, the transparent body, and the flow path member constitute a measurement cell separate from the biosensor.   The measurement method according to claim 3, wherein the transparent body includes a prism having two surfaces that are not parallel to each other in addition to the flat surface on which the thin film layer is formed. In the flow path member constituting the liquid flow path, a supply port capable of supplying a liquid and a discharge port capable of discharging the supplied liquid are formed,
The supply of the first reaction liquid to the liquid flow path is performed using a pipette filled with the first reaction liquid from the supply port, and the supply of the second reaction liquid to the liquid flow path is The measurement method according to claim 3, wherein the measurement is performed using a pipette filled with the second reaction liquid from the supply port. A reaction solution for inspecting the interaction with the molecule to be analyzed is supplied to a liquid channel that can be fed onto the thin film layer on which the molecule to be analyzed is fixed, and is opposite to the liquid channel of the thin film layer. A biosensor that measures the interaction between the molecule to be analyzed and a substance in the reaction solution based on information on a change in refractive index obtained from the reflected light by making a light beam incident on the side surface,
First reaction liquid supply means for supplying a first reaction liquid to the liquid flow path;
First measuring means for measuring an interaction between the analyte molecule and a substance in the first reaction liquid in a state where the first reaction liquid is supplied;
Second reaction liquid supply means for supplying a second reaction liquid different from the first reaction liquid to the liquid flow channel to which the first reaction liquid is supplied and replacing the first reaction liquid with the second reaction liquid. When,
Second measuring means for measuring an interaction between the molecular liquid to be analyzed and a substance in the second reaction liquid in a state where the second reaction liquid is supplied;
Biosensor equipped with.   The biosensor according to claim 6, wherein the measurement of at least one of the first measurement unit and the second measurement unit is performed even when the flow of the supplied liquid is stopped. The thin film layer is formed on a flat surface of a transparent body capable of transmitting a light beam,
The liquid channel is configured between a channel member attached to the transparent body and the thin film layer,
The biosensor according to claim 6 or 7, wherein the thin film layer, the transparent body, and the flow path member constitute a measurement cell separate from the biosensor.   The biosensor according to claim 8, wherein the transparent body includes a prism having two surfaces that are not parallel to each other in addition to the flat surface on which the thin film layer is formed. A storage section capable of storing each of the first reaction liquid and the second reaction liquid;
In the flow path member constituting the liquid flow path, a supply port capable of supplying a liquid and a discharge port capable of discharging the supplied liquid are formed,
The first reaction liquid supply means and the second reaction liquid supply means suck the first reaction liquid or the second reaction liquid in the reservoir, and suck the first reaction liquid or the second reaction liquid. The biosensor according to claim 8, wherein the biosensor is configured to include a supply head for supplying the liquid to the liquid flow path.

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