JP2007010538A - Liquid sending system, liquid sending method, flow channel unit and measuring method using total reflection return loss - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To perform measurement of high precision by preventing the non-specific bonding of a sample. <P>SOLUTION: A flow channel 30 is formed into an almost chevron shape by the groove part 30d formed to the base of a flow channel member 20, and three liquid sending and draining pipes 30e, 30f and 30g which pierce the upper surface of the flow channel member 20 from the groove part 30d to form entrance and exits. A liquid sending head 43 is provided with three pipettes 50a, 50b and 50c fitted in the respective entrance and exits. Pumps allowing the respective pipettes 50a, 50b and 50c to suck or discharge a liquid under pressure or reduced pressure are connected to the respective pipettes 50a, 50b and 50c. The individual sending of the liquid and the simultaneous sending of the liquid are selectively changed over with respect to the respective linker films 32 and 33 provided on the metal film 31 by controlling the pressurization, depressurization and stopping of the respective pumps. The liquid is individually sent to the respective linker films 32 and 33 to prevent the non-specific bonding of the sample. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a liquid-feeding device for injecting a sample solution into a flow path in which a plurality of inlets / outlets are formed and feeding the sample solution to a sensor surface for detecting a reaction of the sample, and a liquid-feeding method thereof, and these The present invention relates to a measurement method that uses total reflection attenuation using the.

  2. Description of the Related Art There are known measuring apparatuses that measure the reaction of a sample by using total reflection attenuation when measuring an interaction between biochemical substances such as proteins and DNA, screening drugs, and the like.

  One of the measuring devices that use such total reflection attenuation is a measuring device that uses a surface plasmon resonance phenomenon (hereinafter referred to as an SPR measuring device). The surface plasmon is generated by the collective vibration of free electrons in the metal, and is a density wave of free electrons traveling along the surface of the metal.

  For example, in an SPR measurement apparatus that employs a Kretschmann arrangement known from Patent Document 1 or the like, a surface of a metal film formed on a transparent dielectric (hereinafter referred to as a prism) is used as a sensor surface, and a sample is formed on the sensor surface. After reacting, the metal film is irradiated from the back side of the sensor surface through the prism so as to satisfy the total reflection condition, and the reflected light is measured.

  Of the light irradiated to the metal film so as to satisfy the total reflection condition, a small amount of light called an evanescent wave passes through the metal film without being reflected and oozes out to the sensor surface side. At this time, if the frequency of the evanescent wave coincides with the frequency of the surface plasmon, SPR is generated and the intensity of the reflected light is greatly attenuated. In addition, the incident angle (resonance angle) of light at which this attenuation occurs varies depending on the refractive index on the metal film. That is, the SPR measurement device measures the reaction state of the sample on the sensor surface by capturing the reflected light from the metal film and detecting the resonance angle.

  By the way, biological samples such as proteins and DNA are often handled as sample solutions dissolved in a solvent such as physiological saline, pure water, or various buffer solutions in order to prevent denaturation and inactivation due to drying. The SPR measurement device described in Patent Document 1 is for examining such interaction between biological samples, and a flow path for feeding a sample solution is provided on the sensor surface. The sensor surface is provided with a linker film for immobilizing the ligand sample. After injecting the ligand solution into the flow path to immobilize the ligand on the linker film (an immobilization process), the analyte solution is By injecting and contacting the ligand and the analyte (measurement step), the interaction is measured.

  The flow path and the prism are arranged on a measurement stage provided in the apparatus main body. The above-described measurement is performed by setting a chip-type sensor unit in which a metal film is formed on a glass substrate on a measurement stage. A pump is connected to the flow path via pipes (including rubber tubes) and valves, and the sample solution stored in the container is sent into the flow path by this pump. However, there has been a problem that so-called contamination easily occurs, in which a sample adhering to the pipe is mixed into a sample solution to be injected later.

  In order to solve this problem, the present applicant uses a pipette consisting of a substantially conical cylindrical pipette tip with a small hole formed at the tip and a head portion that detachably holds the pipette tip, and attaches it to the container. An SPR measurement device that sends a liquid such as a stored sample solution to a flow path has been proposed (see, for example, Japanese Patent Application No. 2004-288534). In this SPR measurement device, contamination occurring when liquid is fed into the flow path can be prevented by exchanging the pipette tip for each liquid to be fed.

  Further, in this SPR measurement device, the flow path member in which the flow path is formed, the prism having the metal film formed on the upper surface, and the bottom surface of the flow path member and the upper surface of the prism are joined (the flow path and the metal A sensor unit including a holding member that holds the film in a state of facing the film is used. A linker film similar to that described above is also provided on the metal film of the sensor unit, and measurement is performed by feeding a sample solution such as a ligand solution or an analyte solution into the flow path with a pipette.

In the linker film, a measurement region that binds to the ligand and a reference region that does not bind are formed. The above-described SPR measurement device irradiates light from a light source to a measurement region and a reference region, and photoelectrically converts reflected light from each region with a detector to obtain a measurement signal and a reference signal. By obtaining and analyzing the difference or ratio between the two signals thus obtained, it is possible to obtain a highly accurate measurement result in which noise caused by disturbances such as individual differences of the sensor units and liquid temperature changes is canceled.
Japanese Patent No. 3294605

  The reference region is formed by forming a film in the same manner as the measurement region and then deactivates the fixing group with the ligand, and the ligand should not be bound by nature. However, it is difficult to completely suppress the binding of the ligand to the reference region simply by deactivation, and there is a problem that a part of the ligand binds to the reference region. In addition, when the analyte solution is fed and the analyte is brought into contact with the linker film, the analyte may be bound to the reference region. Such non-specific binding of the sample causes a measurement error, and therefore a countermeasure for preventing this is desired.

  The present invention has been made in view of the above problems, and an object thereof is to prevent non-specific binding of a sample and perform highly accurate measurement.

  In order to achieve the above object, the liquid feeding device of the present invention has a flow path for feeding the sample solution injected through the three inlets and outlets through which the sample solution containing the sample is injected and discharged, between the inlets and outlets. A stage on which a flow path unit is formed, the flow path unit for contacting the sample solution flowing in the flow path with the sensor surface by bringing the bottom surface into contact with the sensor surface for detecting the reaction of the sample, A liquid feeding head provided with three pipettes to be fitted in each of the respective inlets and outlets, provided corresponding to each of the pipettes, and pressurizing or depressurizing the inside of each pipette to each sample pipe Three pumps for feeding the sample solution into the flow path by sucking or discharging the flow path, and the flow path from the groove portion formed on the bottom surface and from both ends of the groove portion, respectively. A first feeding / draining pipe and a second feeding / draining pipe that penetrate the upper surface of the path unit to form the inlet / outlet; and a third feeding / drainage that penetrates the upper surface from the approximate center of the groove to form the inlet / outlet. A first path from the first feed / drain pipe to the third feed / drain pipe via the groove by controlling the pressurization, decompression, and stop of each pump, A second path from the second feed / drain pipe through the groove to the third feed / drain pipe; and from the first feed / drain pipe through the groove to the second feed / drain pipe. Drive control means is provided for selectively switching the flow path of the flow path with the third path to reach.

  The drive control means is configured such that the pump corresponding to the pipette inserted into the first feed / drain pipe is pressurized, the pump corresponding to the pipette fitted into the second feed / drain pipe is stopped, The pump corresponding to the pipette inserted into the third feed / drain pipe is driven to select the first path so that the pump is depressurized, and the pipette fitted into the first feed / drain pipe is The corresponding pump is stopped, the pump corresponding to the pipette fitted in the second feed / drain pipe is depressurized, and the pump corresponding to the pipette fitted in the third feed / drain pipe is pressurized. The pump is driven to select the second path, the pump corresponding to the pipette fitted into the first feed / drain pipe is pressurized, and the pipette fitted into the second feed / drain pipe is The corresponding pump is depressurized, the 3 the pump corresponding to the pipette fitted in the liquid supply and discharge tube stop, it is preferable that the selecting the third path to drive each pump to.

  Further, it is preferable that each pump shuts off the flow of fluid from the inlet / outlet into which the pipette is inserted when stopped.

  Furthermore, on the sensor surface corresponding to the first path, there is provided a measurement polymer film for fixing the sample and acquiring a measurement signal representing a reaction state of the sample, and the second path It is preferable that a reference polymer film for obtaining a reference signal used for removing noise of the measurement signal is provided on the sensor surface corresponding to the above.

  The liquid feeding device of the present invention is formed with a flow path for feeding the sample solution injected through a plurality of inlets and outlets where a sample solution containing a sample is injected and discharged, between the inlets and outlets. A stage on which a flow channel unit is detachably set to bring the sample solution flowing in the flow channel into contact with the sensor surface by bringing the bottom surface into contact with a sensor surface for detecting the reaction of A liquid-feeding head provided with a plurality of pipettes to be fitted in each of the pipettes, and a pipette corresponding to each of the pipettes. The sample solution is sucked into each pipette by pressurizing or depressurizing the inside of each pipette. Or a plurality of pumps for feeding the sample solution into the flow path by discharging, the groove formed in the bottom surface, and the groove portion penetrating from the groove portion to the top surface of the flow path unit. each A plurality of liquid supply passages formed by each of the liquid discharge / drain pipes and the groove portion by controlling the pressurization, pressure reduction, and stop of the pumps. A drive control means for selectively switching between them may be provided.

  In the liquid feeding method of the present invention, a flow path for feeding the sample solution injected through the three inlets and outlets where the sample solution containing the sample is injected and discharged is formed between the inlets and outlets. A flow path unit is set on the stage by bringing the bottom surface into contact with the sensor surface for detecting the reaction of the sample and contacting the sample solution flowing in the flow path with the sensor surface, and is provided with three pipettes. The head is accessed to the flow path unit, the pipettes are inserted into the inlets and outlets, and the inside of each pipette is pressurized or depressurized by three pumps provided corresponding to the pipettes. When the sample solution is fed into the flow path by sucking or discharging the sample solution to the pipettes, the flow path is formed from a groove formed on the bottom surface and both ends of the groove. A first feed / drain pipe for penetrating the upper surface of the knit to form the inlet / outlet; and a third feed / drain pipe for penetrating the upper face from substantially the center of the groove portion to form the inlet / outlet. The third liquid supply / discharge path from the first liquid supply / discharge pipe through the groove is driven by a drive control means for controlling pressurization, pressure reduction, and stop of each pump. A first path to the liquid pipe, a second path from the second feed / drain pipe through the groove to the third feed / drain pipe, and a first path from the first feed / drain pipe through the groove. And selectively switching to the third path leading to the second feed / drain pipe.

  Furthermore, the measurement method using total reflection attenuation according to the present invention includes a dielectric block having a thin film layer formed on one surface, and the sample injected through three inlets and outlets in which a sample solution containing the sample is injected and discharged. A sensor unit comprising: a channel member for feeding a solution to the thin film layer; and a channel member for contacting a bottom surface of the thin film layer and contacting the sample solution flowing in the channel with the thin film layer. And a stage provided with a light source for irradiating the thin film layer with light so as to satisfy the total reflection condition and a detecting means for receiving the reflected light from the thin film layer and photoelectrically converting it into an electrical signal. The liquid feeding head provided with two pipettes is accessed to the sensor unit, the pipettes are inserted into the inlets and outlets, and the three pumps provided corresponding to the pipettes are connected to the pipettes. When measuring the reaction state of the sample on the thin film layer by feeding the sample solution into the flow path by pressurizing or depressurizing the inside of the pipe and sucking or discharging the sample solution to each pipette , A groove portion formed on the bottom surface, a first feed / drain liquid pipe that forms the inlet / outlet through the top surface of the flow path member from each of both ends of the groove portion, and the groove portion Drive control means for controlling the pressurization, depressurization, and stop of each pump in the liquid supply path of the flow path constituted by the third liquid supply / drainage pipe penetrating from the approximate center to the upper surface to form the inlet / outlet A first path from the first feed / drain pipe through the groove to the third feed / drain pipe, and a third path from the second feed / drain pipe through the groove to the third feed / drain pipe. A second path leading to the second and the second delivery / discharge from the first delivery / drain pipe through the groove. Selectively switching the third path to the tube, characterized in that for feeding the sample solution.

  On the thin film layer corresponding to the first path, a measurement polymer film for fixing the sample and acquiring a measurement signal indicating the reaction state of the sample corresponds to the second path. On the thin film layer, a reference polymer film for obtaining a reference signal used for removing noise of the measurement signal is provided, and when the sample solution is fed to the channel, It is preferable that the first path is selected by the drive control means, the sample solution is fed to the first path, and the sample solution is brought into contact only with the measurement polymer membrane.

  In addition, after the sample is fixed to the measurement polymer membrane, the third path is selected by the drive control means, and a test liquid containing a sample to be reacted with the sample is sent to the third path. The measurement signal representing the reaction state between the sample and the analyte, and the measurement signal corresponding to the measurement polymer membrane and the reference polymer membrane. The reference signal is preferably measured by the light source and the detection means.

  Furthermore, before the test solution is brought into contact with the reference polymer film, the second path is selected by the drive control means to prevent the analyte from binding to the reference polymer film. It is preferable that a blocking liquid containing a blocking agent is sent to the second path, the blocking liquid is brought into contact with the reference polymer film, and the blocking agent is bonded to the reference polymer film.

  According to the present invention, the flow path includes a groove portion formed on the bottom surface of the flow path unit, and a first feed / drain pipe that forms an inlet / outlet through each of the both ends of the groove portion to the upper surface of the flow path unit, 2 pumping and draining pipes and a third feeding and draining pipe that penetrates from the approximate center of the groove to the upper surface to form an inlet / outlet, and pressurizes and depressurizes each pump that sucks or discharges the sample solution to each pipette. By controlling the stop, the first path from the first feed / drain pipe through the groove to the third feed / drain pipe, and the second feed / drain pipe through the groove to the third feed / drain pipe Drive control means is provided for selectively switching the flow path of the flow path between the second path leading to the second path and the third path from the first feed / drain pipe through the groove to the second feed / drain pipe. Therefore, when the sample solution is fed by switching to the first path or the second path, the sensor surface that faces the groove portion is set to each path. The sample solution only a portion of response can be brought into contact with. On the other hand, when the sample solution is sent by switching to the third path, the sample solution can be brought into contact with the entire sensor surface facing the groove. Thus, for example, by forming a measurement region on the sensor surface corresponding to the first path and forming a reference region on the sensor surface corresponding to the second path, the sample solution is applied only to the region where the sample is to be combined. The liquid can be fed, and non-specific binding of the sample to each region can be prevented, so that highly accurate measurement can be performed.

  FIG. 1 is an exploded perspective view of a sensor unit (channel unit) 10 used for measurement using SPR. The sensor unit 10 includes a flow path member 20 in which a flow path 30 is formed, a prism (dielectric block) 21 in which a metal film (sensor surface, thin film layer) 31 is formed on an upper surface, a bottom surface of the flow path member 20, It comprises a holding member 22 that holds the prism 21 in a state where it is bonded to the upper surface thereof.

  For example, two flow paths 30 are formed in the flow path member 20. The flow path member 20 is formed in a long shape, and the two flow paths 30 are arranged along the longitudinal direction thereof. Each flow channel 30 has three ports, a first port 30a, a second port 30b, and a third port 30c, on the upper surface of the flow channel member 20, and a groove 30d formed on the bottom surface of the flow channel member 20. The first feeding / drainage pipe 30e, the second feeding / draining pipe 30f that penetrate the upper surface of the flow path member 20 from both ends of the groove 30d to form the first inlet / outlet 30a, the second inlet / outlet 30b, and the groove 30d It is formed in a substantially chevron shape by a third feeding / draining pipe 30g that penetrates from the approximate center to the upper surface of the flow path member 20 and forms a third inlet / outlet 30c. In addition, the pipe diameter of the flow path 30 is about 1 mm, for example, and each space | interval of each entrance / exit 30a, 30b, 30c is about 10 mm, for example.

  The bottom of the flow path 30 is open, and this open part is covered with a metal film 31 and sealed. For this reason, for the flow path member 20, for example, an elastic material such as rubber or PDMS (polydimethylsiloxane) is used so as to improve the adhesion with the metal film 31. As a result, when the bottom surface of the flow path member 20 is pressed against the top surface of the prism 21, the flow path member 20 is elastically deformed to fill the gap between the joint surfaces with the metal film 31, and the open bottom of each flow path 30 is the prism. The upper surface of 21 is covered watertight. Hereinafter, a portion surrounded by the flow path 30 and the upper surface of the prism 21 is referred to as a sensor cell 23 (see FIG. 2). In this example, the number of flow paths 30 (the number of sensor cells 23) has been described as two examples. However, the number is not limited to two, and may be one or three or more. Good.

  The prism 21 is a transparent dielectric having a metal film 31 formed on the upper surface thereof, and condenses the light irradiated from the bottom surface side so as to satisfy the total reflection condition on the upper surface (metal film 31). The metal film 31 is formed into a strip shape by, for example, a vapor deposition method so as to face the two flow paths 30 formed in the flow path member 20. As the metal film 31, for example, gold or silver is used, and the film thickness is, for example, 50 nm. This film thickness is appropriately selected according to the material of the metal film 31, the wavelength of light applied to the prism 21, and the like.

  In addition, on the upper surface of the metal film 31, a measurement linker film (measurement polymer film) 32 and a reference linker film (reference polymer film) 33 are provided corresponding to each flow path 30. ing. The measurement linker film 32 is formed at a position facing the groove 30d between the first entrance 30a and the third entrance 30c. On the other hand, the reference linker film 33 is formed at a position facing the groove 30d between the second entrance 30b and the third entrance 30c. The measurement linker film 32 is a fixed group to which a ligand (sample) is fixed, and serves as a region for measuring a reaction between the fixed ligand and an analyte (analyte). The reference linker film 33 is formed by forming the film in the same manner as the measurement linker film 32 and then inactivating the fixing group. As a result, the ligand is not fixed to the reference linker film 33.

  As will be described in detail later, an SPR signal generated corresponding to the measurement linker film 32 is measured as a measurement signal, and an SPR signal generated corresponding to the reference linker film 33 is measured as a reference signal. The difference of the SPR signal is taken. By doing so, it becomes possible to cancel noise caused by disturbances such as individual differences of the sensor units 10 and temperature changes of the liquid, and a signal having a good S / N ratio can be obtained. The linker films 32 and 33 are formed in advance at the manufacturing stage of the sensor unit 10. As each linker film 32 and 33, carboxymethyl dextran etc. are used, for example. These types are appropriately selected according to the type of ligand immobilized on the measurement linker film 32.

  Engaging claws 21 a that engage with the engaging portions 22 a of the holding member 22 are provided on both side surfaces of the prism 21 in the longitudinal direction. By these engagements, the flow path member 20 is sandwiched between the holding member 22 and the prism 21 and is held in a state where the bottom surface thereof and the top surface of the prism 21 are in pressure contact with each other. Thus, the flow path member 20, the prism 21, and the holding member 22 are integrated to form the sensor unit 10.

  The prism 21 may be made of, for example, optical glass represented by borosilicate crown (BK7) or barium crown (Bak4), polymethyl methacrylate (PMMA), polycarbonate (PC), amorphous polyolefin (APO), or the like. Representative optical plastics can be used.

  On the upper surface of the holding member 22, a receiving port 22b into which the tip of the pipette (see FIG. 2) enters is formed at a position corresponding to each of the entrances 30a, 30b, and 30c of the flow path 30. Each receiving port 22 b has a funnel shape so as to guide the liquid discharged from the pipette into the flow path 30. When the holding member 22 sandwiches the flow path member 20 and engages with the prism 21, the lower surface of each receiving port 22b and the respective entrances 30a, 30b, and 30c are joined, and the receiving port 22b and the flow path 30 are connected. .

  For example, an RFID (Radio Frequency IDentification) tag that is a non-contact type IC memory may be attached to the prism 21 or the holding member 22 of the sensor unit 10. For example, the sensor unit 10 can be identified by writing a unique ID number for each sensor unit 10 in a read-only RFID tag and reading this ID number before performing each process. Thereby, even when fixing or measuring to a plurality of sensor units 10 at the same time, it is possible to prevent the occurrence of problems such as injection of wrong analytes and mistaken measurement results. Furthermore, using a readable / writable RFID tag, for example, the type of immobilized ligand, the date and time when the ligand was immobilized, and the type of analyte reacted may be written for each step. .

  FIG. 2 is an explanatory diagram schematically illustrating the configuration of the SPR measurement device 12 as a measurement device using total reflection attenuation. The SPR measurement device 12 includes a measuring unit 14 that acquires an SPR signal corresponding to a reaction state of a sample on the metal film 31 of the sensor unit 10, and a liquid supply that supplies various liquids to the flow path 30 of the sensor unit 10. Part (liquid feeding device) 16. Each of these units is comprehensively controlled by a controller (not shown).

  The measurement unit 14 includes an illuminator (light source) 40 that irradiates the sensor unit 10 with light so as to satisfy the total reflection condition, and a detector that receives the light totally reflected by the sensor unit 10 and photoelectrically converts it into an electrical signal ( Detection means) 41 and a measurement stage 42 for fixing the illuminator 40 and the detector 41. The measurement stage 42 is, for example, a pedestal formed in a trapezoidal shape. The measurement stage 42 fixes the illuminator 40 and the detector 41 at a predetermined angle that satisfies the total reflection condition, and also includes a sensor unit on the optical path of the illuminator 40. 10 is positioned. The illuminator 40 irradiates the prism 21 with light having various incident angles that satisfy the total reflection condition. The illuminator 40 includes, for example, an optical system including a condensing lens, a diffusion plate, a polarizing plate, and the like, and a light source, and the arrangement position and the installation angle satisfy the total reflection condition with respect to the incident angle of the irradiated light. To be adjusted.

  As the light source of the illuminator 40, for example, a light emitting element such as an LED (Light Emitting Diode), an LD (Laser Diode), or an SLD (Super Luminescent Diode) is used. The illuminator 40 uses one such light emitting element, and irradiates light from the single light source toward one sensor cell 23. In the case where a plurality of sensor cells 23 (two in this example) provided in the sensor unit 10 are measured at the same time, the light from a single light source may be dispersed and irradiated to each of the plurality of sensor cells 23. Alternatively, a plurality of light emitting elements may be used side by side so that one light emitting element is assigned to each sensor cell 23. The diffusion plate diffuses light from the light source and suppresses unevenness in the amount of light in the light emitting surface. The polarizing plate allows only p-polarized light that causes SPR to pass through. In addition, when using LD, when the direction of polarization of the light itself emitted from the light source is uniform, the polarizing plate is unnecessary. Further, even when a light source with uniform polarization is used, if the direction of polarization becomes uneven by passing through the diffusion plate, the direction of polarization is aligned using a polarizing plate. The light thus diffused and polarized is condensed by the condenser lens and applied to the prism 21. As a result, light having various incident angles without variation in light intensity can be incident on the sensor cell 23.

  For example, a CCD area sensor or a photodiode array is used for the detector 41. Light incident from one side of the long side of the prism 21 passes through the prism 21 and is condensed from the inside onto the upper surface of the prism 21 (the back surface of the metal film 31), totally reflected on the upper surface, and reflected on the other side surface. Exit. Since light of various angles is incident on the prism 21, the incident light is reflected on the upper surface of the prism 21 at a reflection angle corresponding to each incident angle. The detector 41 receives the reflected light of these various angles, photoelectrically converts them, and outputs them as an SPR signal having a level corresponding to the light intensity.

  The detector 41 outputs an SPR signal corresponding to the measurement linker film 32 as a measurement signal, and outputs an SPR signal corresponding to the reference linker film 33 as a reference signal. The illuminator 40 and the detector 41 are configured to perform two-channel measurement of these measurement signals and reference signals. For example, when the illuminator 40 is composed of a single light emitting element, irradiation light is applied to a plurality of light beams incident on the measurement linker film 32 and the reference linker film 33 using a reflection mirror or the like. Spectroscopy. Each light beam is received by a detector 41 configured by a plurality of photodiode arrays for each channel.

  On the other hand, when a CCD area sensor is used as the detector 41, the measurement signal and the reference signal can be recognized by performing image processing on the signal obtained by simultaneously receiving the reflected light of each channel. it can. However, when such a method using image processing is difficult, the timing at which light is incident on the measurement linker film 32 and the reference linker film 33 is shifted by a minute time to receive the signal of each channel. Good. As a method for shifting the incident timing, for example, a disk in which two holes are formed at a position where the arrangement angle is shifted by 180 degrees is arranged on the optical path of the illuminator 40, and this disk is rotated. Each hole is arranged at a position where the distance from the center differs by the distance between the linker films 32 and 33. When one hole enters the optical path, the light is incident on the measurement linker film 32 and the other When the hole enters the optical path, a light beam is incident on the reference linker film 33. Thereby, the incident timing to each channel is shifted.

  The liquid feeding unit 16 includes a liquid feeding head 43 provided with three pipettes of a first pipette 50a, a second pipette 50b, and a third pipette 50c that access the respective entrances 30a, 30b, and 30c, and the liquid feeding head. And a head moving mechanism 44 for moving 43. The head moving mechanism 44 is a known moving mechanism including, for example, a conveyance belt, a pulley, a carriage, and a motor. The head moving mechanism 44 moves the liquid feeding head 43 in three directions, front, rear, left, right, up and down, under the control of a controller (not shown). Move. The SPR measurement device 12 is provided with a plurality of liquid storage units (not shown) that store various liquids (ligand solution, analyte solution, cleaning liquid, buffer liquid, etc.) injected into the flow path 30. The head moving mechanism 44 causes the liquid feeding head 43 to access these liquid storage units, the sensor unit 10 set on the measurement stage 42, and the like.

  Each pipette 50a, 50b, 50c of the liquid feeding head 43 has a substantially conical cylindrical shape with a small hole formed at the tip, and the interval between the pipettes 50a, 50b, 50c corresponds to the interval between the entrances 30a, 30b, 30c. ing. The tip of each pipette 50a, 50b, 50c has a replaceable tip shape (hereinafter referred to as “pipette tip”). Since the pipette tip is in direct contact with the liquid to be fed, the pipette tip is exchanged every time the liquid is fed so as not to cause a mixed liquid of different kinds of liquids through the pipettes 50a, 50b, 50c. The SPR measurement device 12 is provided with a pipette tip storage unit (not shown) that stores a plurality of pipette tips. Replacement of the pipette tip is performed by driving the head moving mechanism 44 to allow the liquid feeding head 43 to access the pipette tip storage unit.

  A first pump 45a, a second pump 45b, and a third pump 45c are connected to each of the pipettes 50a, 50b, and 50c via a liquid feeding head 43 and a pipe that is not shown. Each of the pumps 45a, 45b, and 45c reduces the pressure in the pipe path leading to the liquid feeding head 43, thereby causing the corresponding pipettes 50a, 50b, and 50c to suck the liquid and pressurizing the pipe path to increase the pressure in the pipe path. The sucked liquid is discharged into the pipettes 50a, 50b and 50c. Each pump 45 a, 45 b, 45 c is connected to a pump driver (drive control means) 46. The pump driver 46 is electrically connected to, for example, a controller of the SPR measurement device 12, and independently drives each pump 45a, 45b, 45c based on a control signal from this controller, and each pipette 50a, 50b. , 50c suction and discharge timing, suction amount and discharge amount, and the like are controlled. In addition, as each pump 45a, 45b, 45c, what is called a syringe pump etc. which consist of a cylinder and a piston can be used, for example. Further, when a syringe pump is used for each of the pumps 45a, 45b, 45c, a known slide moving mechanism that reciprocates the piston in the cylinder may be used as the pump driver 46.

  The liquid feeding head 43 inserts the tips of the pipettes 50 a, 50 b, 50 c into the inlets 30 a, 30 b, 30 c, and injects and discharges various liquids into the flow path 30. At this time, if the liquid is not supplied so that the pressurization of the flow path 30 by the liquid injection and the pressure reduction of the flow path 30 by the suction of the liquid are balanced, the liquid to be discharged may remain in the flow path 30. This may cause liquid feeding problems such as liquid overflowing from the flow path 30. For this reason, the liquid feed head 43 is fitted with the tips of the pipettes 50a, 50b, 50c so that no gaps are formed between the inlets 30a, 30b, 30c (see FIGS. 3 and 4), and the pumps 45a, The pressure fluctuation due to 45b and 45c is accurately transmitted to the flow path 30.

  After fitting the pipettes 50a, 50b, 50c into the respective entrances 30a, 30b, 30c, the pump driver 46 pressurizes the first pump 45a, stops the second pump 45b, and depressurizes the third pump 45c. , The liquid is injected with the first pipette 50a, and the liquid is discharged with the third pipette 50c. At this time, if the second pump 45b is stopped and the flow of fluid from the second inlet / outlet 30b is blocked, the groove 30d between the second feed / drain pipe 30f and the third feed / drain pipe 30g. Since there is no escape space for the fluid inside and the fluid inside the second feeding / draining pipe 30f, the liquid does not flow into the reference linker film 33, as shown in FIG. The liquid can be fed (corresponding to the first path described in the claims).

  On the other hand, the first pump 45a is stopped by the pump driver 46, the second pump 45b is depressurized, and the third pump 45c is pressurized so that liquid is injected by the third pipette 50c and discharged by the second pipette 50b. If it does, as shown in FIG.3 (b), it will become possible to send a liquid only to the reference linker film | membrane 33, without flowing a liquid into the measurement linker film | membrane 32 (2nd path | route of Claim) Equivalent). In addition, the pump driver 46 drives the first pump 45a to pressurize, the second pump 45b to depressurize, and the third pump 45c to stop, and the first pipette 50a injects liquid and the second pipette 50b discharges. As shown in FIG. 4, it is possible to send a liquid to both the linker films 32 and 33 (corresponding to the third path described in the claims). As described above, the liquid feeding unit 16 controls the driving of the pumps 45a, 45b, and 45c, so that the path for individually feeding the linker films 32 and 33 and the path for simultaneously feeding the liquid are provided. The 30 liquid supply paths can be selectively switched. In the three liquid feeding paths described above, the relationship between the pressurization and the pressure reduction (direction of liquid feeding) may be reversed.

  The measurement using SPR is roughly divided into three steps: a fixing step, a measurement step (data reading step), and a data analysis step. The fixing step is a step of injecting a ligand solution (sample solution) in which the ligand is dissolved into the flow channel 30 to fix the ligand to the measurement linker film 32. Before performing the ligand immobilization process for injecting the ligand solution, first, the immobilization buffer solution is sent to the measurement linker film 32, and the measurement linker film 32 is moistened to facilitate the binding of the ligand. An activation process of the film 32 is performed. For example, in the amine coupling method, carboxymethyl dextran is used as the linker films 32 and 33, and the amino group in the ligand is directly covalently bonded to the dextran. As the activation liquid in this case, a mixed liquid of N ′-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) is used. After the activation process, the flow path 30 is washed with the fixing buffer solution.

  As the buffer solution for fixing and the solvent (diluent) of the ligand solution, for example, various buffer solutions (buffer solutions), physiological salt solutions represented by physiological saline, and pure water are used. . The type of each of these liquids, the pH value, the type of mixture and its concentration, etc. are appropriately determined according to the type of ligand. For example, when a biological substance is used as a ligand, physiological saline whose pH is adjusted to near neutral is often used. However, in the above-described amine coupling method, the measurement linker film 32 is negatively (negatively) charged by carboxymethyldextran, so that the protein is positively (positively) charged so as to be easily bonded to the measurement linker film 32. Therefore, a phosphate buffer solution (PBS: phosphatic-buffered, saline) having a strong buffering action containing a high concentration phosphate which is not physiological may be used.

  After such activation processing and washing, the ligand solution is injected into the flow path 30 to perform the ligand immobilization processing. When the ligand solution is injected into the flow path 30, the ligand diffused in the solution gradually approaches the measurement linker film 32 and binds thereto. In this way, the ligand is fixed to the measurement linker film 32. Immobilization usually takes about one hour, and during this time, the sensor unit 10 is stored in a state where environmental conditions including temperature are set to predetermined conditions. The ligand solution in the flow path 30 may be allowed to stand while the immobilization proceeds, but it is preferable that the ligand solution in the flow path 30 is stirred and flowed. By doing so, the binding between the ligand and the measurement linker film 32 is promoted, and the amount of ligand immobilized can be increased.

  When the immobilization of the ligand to the measurement linker film 32 is completed, the ligand solution is discharged from the flow path 30. The ligand solution is sucked and discharged by each pipette 50a, 50b, 50c. When the ligand solution is sucked out, a cleaning liquid is injected into the flow path 30 and a cleaning process is performed. Thereafter, the drying preventing liquid is injected into the flow path 30. Thus, the sensor unit 10 is stored until measurement in a state where the linker films 32 and 33 are immersed in the anti-drying liquid.

  In the measurement process, first, a measurement buffer solution is injected into the flow path 30. Thereafter, an analyte solution (test solution) in which the analyte is dissolved in a solvent is injected, and then the measurement buffer solution is injected again. Note that the flow path 30 may be once cleaned before the measurement buffer solution is first injected. Data reading by the illuminator 40 and the detector 41 is started immediately after the measurement buffer is first injected in order to detect a reference signal level. After the analyte solution is injected, the measurement buffer is read again. This is done until it is injected. As a result, it is possible to measure the SPR signal until detection of the reference level (baseline), reaction state (binding state) of the analyte and ligand, and desorption of the combined analyte and ligand by injection of the buffer solution for measurement. it can.

  As the buffer solution for measurement and the solvent (diluent) of the analyte solution, for example, various buffer solutions (buffer solutions), physiological salt solutions represented by physiological saline, and pure water are used. The The type of each of these liquids, the pH value, the type of mixture, its concentration, etc. are appropriately determined according to the type of ligand. For example, DMSO (dimethyl-sulfo-oxide) may be included in physiological saline in order to facilitate the dissolution of the analyte. This DMSO greatly affects the signal level. As described above, the measurement buffer solution is used for detection of the reference level. Therefore, when DMSO is contained in the solvent of the analyte, the measurement buffer solution having a DMSO concentration similar to the DMSO concentration should be used. Is preferred.

  The analyte solution is often stored for a long period (for example, one year). In such a case, a difference in concentration occurs between the initial DMSO concentration and the DMSO concentration at the time of measurement due to a change over time. May end up. When strict measurement is required, such a concentration difference is estimated from the level of the reference signal when the analyte solution is injected, and the measurement data is corrected (DMSO concentration correction).

  The correction data for correcting the DMSO concentration is obtained by injecting a plurality of types of measurement buffer solutions having different DMSO concentrations into the flow path 30 before injecting the analyte solution, and measuring according to the change in DMSO concentration at this time. It is obtained by examining the amount of change in each of the signal level and the reference signal level.

  When a change occurs in the medium on the measurement linker film 32, the refractive index changes, and the incident angle of light at which the light intensity of the reflected light attenuates (resonance angle at which SPR occurs) also changes. When the analyte is fed onto the measurement linker film 32, the refractive index on the measurement linker film 32 changes according to the reaction state between the analyte and the ligand, and the resonance angle also changes accordingly. The reaction state of the ligand and the analyte appears as a transition of the attenuation position of the reflected light in the light receiving surface. For example, before and after the analyte comes into contact with the ligand, the refractive index on the measurement linker film 32 is different, and the resonance angle at which SPR occurs is different. When the analyte comes into contact with the ligand and starts the reaction, the resonance angle starts to change accordingly and the attenuation position of the reflected light in the light receiving surface starts to move.

  In the data analysis process, the SPR signal representing the reaction state thus obtained is analyzed to analyze the characteristics of the analyte. As described above, the detector 41 outputs the SPR signal corresponding to the measurement linker film 32 as a measurement signal, and outputs the SPR signal corresponding to the reference linker film 33 as a reference signal. These measurement signal and reference signal are measured almost simultaneously from the detection of the standard level to the desorption through the binding reaction. Data analysis is performed by obtaining a difference or ratio between the measurement signal and the reference signal. For example, difference data between the measurement signal and the reference signal is obtained, the difference data is used as measurement data, and analysis is performed based on the difference data. By doing so, noise due to disturbances such as individual differences of the sensor unit 10 and each sensor cell 23, mechanical fluctuations of the apparatus, and temperature change of the liquid are canceled, and highly accurate measurement is possible.

  Next, the operation of the SPR measurement device 12 configured as described above will be described with reference to the flowcharts shown in FIGS. When measuring the reaction state between the ligand and the analyte, first, the sensor unit 10 is set on the measurement stage 42, and one sensor cell 23 is aligned with the measurement position located on the optical path of the illuminator 40. As described above, the SPR measurement device 12 includes a fixing step of fixing a ligand to the sensor unit 10, a measurement step of bringing an analyte into contact with the fixed ligand, and acquiring an SPR signal at that time, and the acquired SPR signal. And a data analysis step for analyzing the reaction state, and the reaction state between the ligand and the analyte is measured.

  After the sensor unit 10 is set, the SPR measurement device 12 starts the fixing process in response to the input of the fixing start instruction from the operator. The controller of the SPR measurement device 12 drives the head moving mechanism 44 in response to the input of the fixation start instruction, and moves the liquid feeding head 43 to the liquid storage unit that stores the activation liquid of the measurement linker film 32. Let The controller that has made the liquid feeding head 43 access the liquid storage unit drives the first pump 45a via the pump driver 46, and causes the first pipette 50a to suck a predetermined amount of the activation liquid. The controller that has caused the first pipette 50a to suck the activation liquid moves the liquid feeding head 43 to the sensor unit 10 and inserts the pipettes 50a, 50b, and 50c into the inlets 30a, 30b, and 30c to be measured. The flow path 30 of the sensor cell 23 and the pipettes 50a, 50b, 50c are connected. At this time, the head moving mechanism 44 adjusts the height of the liquid feeding head 43 so that no gap is generated between each pipette 50a, 50b, 50c and each of the inlets 30a, 30b, 30c as described above.

  The controller in which each pipette 50a, 50b, 50c is connected to the flow path 30 transmits a control signal to the pump driver 46, the first pump 45a is pressurized, the second pump 45b is stopped, and the third pump 45c is decompressed. So that each is driven. The first pipette 50a discharges the activation liquid held inside and injects it into the flow path 30, and the third pipette 50c sucks the air in the flow path 30 or the cleaning liquid previously injected. The fluid is discharged from the channel 30.

  At this time, since the second pump 45b is stopped and the flow of the fluid from the second inlet / outlet 30b is blocked, the groove 30d between the second feed / drain pipe 30f and the third feed / drain pipe 30g. There is no escape space for the fluid inside and the fluid inside the second feed / drain pipe 30f, and as shown in FIG. 3A, the activation liquid is fed only to the measurement linker film 32. As a result, the fluid in the flow passage 30 corresponding to the measurement linker film 32 is replaced with the activation liquid from air or a cleaning liquid, and the measurement linker film 32 is activated.

  When the activation of the measurement linker film 32 is completed, the controller drives the third pump 45 c so that the third pipette 50 c performs suction, and discharges the activation liquid from the flow path 30. The controller that has discharged the activation liquid moves the liquid feeding head 43 to a waste liquid tank (not shown), discards the activation liquid held in the third pipette 50c, and then has each immersed in the activation liquid. Replace pipette tips of pipettes 50a, 50b, 50c. By exchanging the pipette tip in this way, the mixed solution of the ligand solution and the activation solution to be sent next through the pipettes 50a, 50b, and 50c is prevented.

  The controller that has exchanged the pipette tip moves the liquid feeding head 43 to the liquid storage unit that stores the ligand solution, and causes the first pipette 50a to suck the ligand solution. The controller that sucks the ligand solution moves the liquid feeding head 43 to the sensor unit 10, sends the ligand solution only to the measurement linker film 32 in the same procedure as the activation liquid, and then sends the ligand solution to the measurement linker film 32. A fixing process is performed to fix the. Note that the ligand solution injected into the flow path 30 may be agitated by alternately aspirating and discharging the first pipette 50a and the third pipette 50c. By doing so, the binding between the ligand and the measurement linker film 32 is promoted, and the amount of ligand immobilized can be increased.

  The controller in which the ligand is fixed to the measurement linker film 32 performs a block process for preventing the analyte from adsorbing to the reference linker film 33. The controller that has discharged the ligand solution from the flow path 30 and replaced the pipette tip moves the liquid feeding head 43 to a liquid storage unit that stores the block liquid. The blocking agent corresponding to the reference linker film 33 is dissolved in the blocking liquid. As this blocking agent, for example, serum albumin represented by HSA (Human Serum albumine) and BSA (Bovine Serum albumine), and casein can be used. The blocking agent is appropriately selected according to the type of analyte, the film forming material of each linker film 32, 33, and the like.

  The controller that has moved the liquid feeding head 43 to the liquid storage unit drives the third pump 45c via the pump driver 46, and causes the third pipette 50c to suck a predetermined amount of block liquid. The controller that causes the third pipette 50 c to suck the block liquid moves the liquid feeding head 43 to the sensor unit 10 and connects each pipette 50 a, 50 b, 50 c to the flow path 30.

  The controller in which each pipette 50a, 50b, 50c is connected to the flow path 30 transmits a control signal to the pump driver 46 so that the first pump 45a is stopped, the second pump 45b is depressurized, and the third pump 45c is pressurized. Then, as shown in FIG. 3B, the block solution is fed only to the reference linker film 33. The blocking agent dissolved in the blocking solution contacts and binds to the reference linker film 33. In this way, by binding the blocking agent to the reference linker film 33 in advance, nonspecific binding of the analyte fed during measurement to the reference linker film 33 is prevented.

  As described above, the fixing step is completed by binding the ligand to the measurement linker film 32 and the blocking agent to the reference linker film 33. In this example, the drive of each pump 45a, 45b, 45c is controlled so that the respective solutions are sent only to one of the linker films 32, 33, so that the ligand adheres to the reference linker film 33, Nonspecific binding such as attachment of a blocking agent to the measurement linker film 32 can be reliably prevented. In this example, the block process is performed after the ligand immobilization process. However, the block process may be performed first. Furthermore, when an analyte having a low possibility of binding to the reference linker film 33 is sent, the blocking process may not be performed.

  The SPR measurement device 12 that has completed the fixing process holds the sensor unit 10 in a state in which environmental conditions such as temperature are kept constant, and starts the measurement process in response to an input of a measurement start instruction from the operator. The controller starts reading data by the illuminator 40 and the detector 41 in response to the input of the measurement start instruction. At the same time, the head moving mechanism 44 is driven to move the liquid feeding head 43 to the liquid storage unit for storing the measurement buffer liquid. The controller that has moved the liquid feeding head 43 to the liquid storage unit drives the first pump 45a via the pump driver 46, and causes the first pipette 50a to suck a predetermined amount of the buffer solution for measurement.

  The controller having sucked the buffer solution for measurement into the first pipette 50a moves the liquid feeding head 43 to the sensor unit 10, and connects each pipette 50a, 50b, 50c to the flow path 30 of the sensor cell 23 to be measured. . The controller which connected each pipette 50a, 50b, 50c to the flow path 30 transmits a control signal to the pump driver 46 so that the first pump 45a is pressurized, the second pump 45b is depressurized, and the third pump 45c is stopped. As shown in FIG. 4, the buffer solution for measurement is fed to both of the linker films 32 and 33 at once.

  The controller injecting the measurement buffer liquid into the flow path 30 moves the liquid feeding head 43 to the liquid storage section storing the analyte solution after exchanging the pipette tip, and so on. And a measurement buffer solution for desorption reaction are sequentially sent to the flow path 30. At this time, since the blocking process is performed on the reference linker film 33, the binding of the analyte to the reference linker film 33 is prevented. The controller that has injected the measurement buffer solution for desorption reaction into the flow path 30 stops data reading by the illuminator 40 and the detector 41. Thereby, the detector 41 acquires SPR signals from detection of the reference level (baseline), reaction state of the analyte and ligand (binding state), and desorption of the bound analyte and ligand.

  The SPR measurement device 12 that has acquired the SPR signal calculates the measurement data by subtracting the reference signal corresponding to the reference linker film 33 from the measurement signal corresponding to the measurement linker film 32, and based on this measurement data, the ligand and Analyze the reaction with the analyte. Thus, the measurement for one sensor cell 23 is completed. When a plurality of sensor cells 23 are included in one sensor unit 10 as in this example, each sensor cell 23 is measured in the same procedure as described above.

  In the above embodiment, the sensor unit 10 including the metal film 31 is the flow path unit described in the claims. However, for example, only the flow path member 20 in which the flow path 30 is formed may be the flow path unit. Further, when only the flow path member 20 is used as the flow path unit, the flow path member 20 and the prism 21 may be individually set in the SPR measurement device 12.

  In the above embodiment, each step is performed by one SPR measuring device 12, but the device may be divided for each step. By doing so, it becomes possible to process a plurality of sensor units 10 at the same time, and the processing efficiency can be improved.

  Furthermore, in the above-described embodiment, since the liquid supply path overlaps at the portion of the third liquid supply / drain pipe 30g, the measurement linker film 32 and the reference linker film 33 are separately formed. The linker film 32 and the reference linker film 33 may be formed continuously.

  In the above-described embodiment, the substantially mountain-shaped flow path 30 including the groove portion 30d and the three supply / discharge liquid pipes 30e, 30f, and 30g is shown, but the number of the supply / discharge liquid pipes is limited to three. Without limitation, a larger number of liquid supply / drainage pipes may be provided. The flow path member 101 of the sensor unit 100 shown in FIG. 7 is formed with a flow path 102 including a groove portion 102a and five supply / discharge liquid tubes 102b formed at equal intervals. On the metal film 104 formed on the upper surface of the prism 103, four linker films 105 are provided so as to be positioned between the respective liquid supply / drain pipes 102b.

  The liquid feeding head 110 is provided with pipettes 111 corresponding to the respective liquid feeding / draining pipes 102b. Each pipette 111 is connected to a corresponding pump 112 as in the above embodiment. Each pump 112 is driven by a pump driver 113 and pressurizes or depressurizes the inside of a pipe path leading to the pipette 111 to cause each pipette 111 to suck or discharge a liquid.

  In the sensor unit 100 and the liquid feeding head 110 configured in this way, each pump 112 is driven so that any one of the pumps 112 is pressurized, any one is depressurized, and all the rest are stopped, and the flow path 102 is reached. The liquid is injected and discharged by one pipette 111 each. Thereby, for example, if the liquid is injected and discharged by the two pipettes 111 located at both ends, and the flow of the fluid from the three feeding / draining pipes 102b located between them is blocked, each linker film 105 can be fed simultaneously. In addition, if the two adjacent pipettes 111 inject and discharge liquid and block the flow of fluid from the other three liquid discharge pipes 102b, one pipet 111 is positioned between the two pipettes 111. Liquid can be fed only to the linker film 105. In this way, by controlling the driving of each pump 112, liquid is fed to all the linker films 105 simultaneously, individually fed to each linker film 105, or two or three adjacent ones Highly versatile liquid feeding can be performed by selectively switching the liquid feeding path of the flow path 102, such as feeding to the linker film 105.

  Each linker film 105 may be alternately arranged for measurement and reference, or may form a measurement region and a reference region in each linker film 105. Furthermore, if it is not necessary to remove noise from the measurement signal, such as when high accuracy is not required, only the measurement linker film may be arranged.

  In each of the above embodiments, the prisms 21 and 103 are shown as the dielectric block. However, in addition to the dielectric block, optical glass, optical plastic, or the like, or these plate shapes are used. And a prism integrated with an optical surface smoothing agent (for example, optical matching oil).

  Further, in each of the above embodiments, an SPR measurement device is shown as an example of a measurement device using total reflection attenuation. However, for example, a leakage mode sensor is known as a measurement device using total reflection attenuation. It has been. 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 changes according to the refractive index of the medium on the sensor surface, similar to the resonance angle of SPR. By detecting the attenuation of the reflection angle, the chemical reaction on the sensor surface is measured.

It is a disassembled perspective view which shows schematic structure of a sensor unit. It is explanatory drawing which illustrates the structure of a SPR measuring apparatus roughly. It is explanatory drawing which shows the example which liquid-feeds to each linker film | membrane separately. It is explanatory drawing which shows the example which liquid-feeds simultaneously to each linker film | membrane. It is a flowchart (1/2) which shows the procedure of a measurement by a SPR measuring device. It is a flowchart (2/2) which shows the procedure of a measurement by a SPR measuring device. It is explanatory drawing which shows the example which increased the number of sending / draining pipes.

Explanation of symbols

10, 100 Sensor unit (flow path unit)
12 SPR measurement device (measurement device using total reflection attenuation)
14 Measuring unit 16 Liquid feeding unit (Liquid feeding device)
20, 101 Channel member 21, 103 Prism (dielectric block)
30, 102 Channel 30a First inlet / outlet 30b Second inlet / outlet 30c Third inlet / outlet 30d Groove 30e First feeding / draining pipe 30f Second feeding / draining pipe 30g Third feeding / draining pipe 30h Thin wall part 31, 104 Metal film (sensor Surface, thin film layer)
32 Linker membrane for measurement (polymer membrane for measurement)
33 Linker membrane for reference (polymer membrane for reference)
40 Illuminator (light source)
41 Detector (detection means)
42 Measurement stage
43, 110 Liquid feed head 45a First pump 45b Second pump 45c Third pump 46 Pump driver (drive control means)
50a First pipette 50b Second pipette 50c Third pipette 111 Pipette 102a Groove 102b Feed / drain pipe 112 Pump

Claims (10)

A flow path for feeding the sample solution injected through the three inlets and outlets where the sample solution containing the sample is injected and discharged is formed between the inlets and outlets, and a sensor surface for detecting the reaction of the sample is formed. A stage on which a flow path unit for bringing the sample solution flowing in the flow path into contact with the sensor surface is detachably set by contacting a bottom surface;
A liquid-feeding head provided with three pipettes to be fitted into the respective entrances and exits;
Three pumps provided corresponding to each of the pipettes, for feeding the sample solution into the flow path by pressurizing or depressurizing the inside of each pipette to cause the pipette to suck or discharge the sample solution In a liquid feeding device provided with
The flow path includes a groove formed on the bottom surface, a first feed / drain pipe that forms the inlet / outlet through the top surface of the flow path unit from both ends of the groove, and a second feed / drain pipe, And a third feed / drain pipe that penetrates the upper surface from the approximate center of the groove and forms the inlet / outlet,
By controlling the pressurization, decompression, and stop of each pump, a first path from the first feed / drain pipe to the third feed / drain pipe through the groove, and the second feed / drain liquid A second path from a pipe to the third feed / drain pipe via the groove and a third path from the first feed / drain pipe to the second feed / drain pipe via the groove A liquid delivery apparatus comprising drive control means for selectively switching the liquid delivery path of the flow path. The drive control means is configured to pressurize the pump corresponding to the pipette inserted into the first feeding / draining pipe, stop the pump corresponding to the pipette fitted into the second feeding / draining pipe, Selecting the first path by driving the pumps so that the pumps corresponding to the pipettes fitted in the liquid discharge pipes are depressurized;
The pump corresponding to the pipette inserted into the first feed / drain pipe is stopped, the pump corresponding to the pipette fitted into the second feed / drain pipe is decompressed, and is inserted into the third feed / drain pipe. Drive each pump so that the pump corresponding to the pipette pressurizes, selects the second path,
The pump corresponding to the pipette fitted in the first feeding / draining pipe is pressurized, the pump corresponding to the pipette fitted in the second feeding / draining pipe is depressurized, and fitted into the third feeding / draining pipe The liquid feeding device according to claim 1, wherein the third path is selected by driving each pump so that the pump corresponding to the pipette is stopped.   3. The liquid feeding device according to claim 1, wherein each pump shuts off a fluid flow from the inlet / outlet into which the pipette is inserted when the pump is stopped. On the sensor surface corresponding to the first path, there is provided a measurement polymer film for fixing the sample and obtaining a measurement signal representing a reaction state of the sample,
The reference polymer film for acquiring a reference signal used when removing noise of the measurement signal is provided on the sensor surface corresponding to the second path. 4. The liquid feeding device according to any one of items 1 to 3. A flow path for feeding the sample solution injected through a plurality of inlets and outlets through which sample solution containing a sample is injected and discharged is formed between the inlets and outlets, and a sensor surface for detecting the reaction of the sample is formed. A stage on which a flow path unit for bringing the sample solution flowing in the flow path into contact with the sensor surface is detachably set by contacting a bottom surface;
A liquid-feeding head provided with a plurality of pipettes to be fitted into the respective entrances and exits;
A plurality of pumps provided corresponding to each of the pipettes, and sending the sample solution into the flow path by pressurizing or depressurizing the inside of each pipette to cause the pipette to suck or discharge the sample solution. In a liquid delivery device comprising:
The flow path is composed of a groove portion formed on the bottom surface, and a plurality of liquid discharge / drain pipes that penetrate the upper surface of the flow path unit from the groove portion to form the respective outlets.
Drive control means for selectively switching a plurality of liquid supply paths formed by the respective liquid supply / drain pipes and the grooves by controlling pressurization, pressure reduction and stop of each pump is provided. Liquid feeding device. A flow path for feeding the sample solution injected through the three inlets and outlets where the sample solution containing the sample is injected and discharged is formed between the inlets and outlets, and a sensor surface for detecting the reaction of the sample is formed. Set the flow path unit on the stage to bring the sample solution flowing in the flow path into contact with the sensor surface by contacting the bottom surface,
A liquid feeding head provided with three pipettes is allowed to access the flow path unit, and the pipettes are inserted into the respective inlets and outlets.
The three sample pumps provided corresponding to each of the pipettes pressurize or depressurize the inside of each pipette, and the sample solution is sucked or discharged into each pipette, thereby allowing the sample solution to flow into the channel. In the liquid feeding method for feeding
An abbreviation of the groove portion formed on the bottom surface, a first feeding / draining pipe, a second feeding / draining pipe that penetrates the upper surface of the flow path unit from both ends of the groove portion to form the inlet / outlet, and the groove portion Drive control means for controlling the pressurization, decompression, and stop of the respective pumps, the liquid supply path of the flow path constituted by the third supply / drainage pipe penetrating from the center to the upper surface to form the inlet / outlet A first path from the first feed / drain pipe through the groove to the third feed / drain pipe, and a second path from the second feed / drain pipe to the third feed / drain pipe through the groove. The sample solution is fed selectively by switching to the second path leading to the second path and the third path leading from the first feeding / draining pipe through the groove to the second feeding / draining pipe. Liquid feeding method. A dielectric block having a thin film layer formed on one surface, and a flow path for feeding the sample solution injected through the three inlets and outlets for injection and discharge of the sample solution including the sample to the thin film layer are formed, A sensor unit comprising a flow path member for bringing the sample solution flowing in the flow path into contact with the thin film layer by bringing a bottom surface into contact with the thin film layer is configured to emit light so as to satisfy the total reflection condition on the thin film layer. Set on a stage provided with a light source to irradiate and detection means for receiving reflected light from the thin film layer and photoelectrically converting it into an electrical signal,
A liquid feeding head provided with three pipettes is made to access the sensor unit, and the pipettes are inserted into the inlets and outlets,
The three sample pumps provided corresponding to each of the pipettes pressurize or depressurize the inside of each pipette, and the sample solution is sucked or discharged into each pipette, thereby allowing the sample solution to flow into the channel. In the measurement method using total reflection attenuation to measure the reaction state of the sample on the thin film layer by sending
An abbreviation of the groove portion formed on the bottom surface, a first feeding / draining pipe, a second feeding / draining pipe that penetrates the upper surface of the flow path member from both ends of the groove portion to form the inlet / outlet, and the groove portion Drive control means for controlling the pressurization, decompression, and stop of the respective pumps, the liquid supply path of the flow path constituted by the third supply / drainage pipe penetrating from the center to the upper surface to form the inlet / outlet A first path from the first feed / drain pipe through the groove to the third feed / drain pipe, and a second path from the second feed / drain pipe to the third feed / drain pipe through the groove. The sample solution is fed selectively by switching to the second path leading to the second path and the third path leading from the first feeding / draining pipe through the groove to the second feeding / draining pipe. Measuring method using total reflection attenuation. On the thin film layer corresponding to the first path, a measurement polymer film for fixing the sample and obtaining a measurement signal indicating a reaction state of the sample is the thin film corresponding to the second path. On each layer, a reference polymer film for obtaining a reference signal used for removing noise of the measurement signal is provided, respectively.
When sending the sample solution to the flow path, the drive control means selects the first path,
The measurement method using total reflection attenuation according to claim 7, wherein the sample solution is sent to the first path, and the sample solution is brought into contact only with the measurement polymer film. After fixing the sample to the measurement polymer membrane, the drive control means selects the third path,
Sending a test solution containing a sample to be reacted with the sample to the third path, bringing the test solution into contact with the measurement polymer membrane and the reference polymer membrane;
9. The whole measurement signal according to claim 8, wherein the measurement signal representing a reaction state between the sample and the analyte and the reference signal corresponding to the measurement signal are measured by the light source and the detection means. Measurement method using return loss. Before bringing the test liquid into contact with the reference polymer membrane, the drive control means selects the second path,
Sending a block solution containing a blocking agent for preventing the analyte from binding to the reference polymer membrane to the second path, bringing the block solution into contact with the reference polymer membrane;
The measurement method using total reflection attenuation according to claim 9, wherein the blocking agent is bonded to the reference polymer film.

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