JP2007093395A - Sensor unit and measurement method using total reflection attenuation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To suppress the influence of non-specific adsorption without hindering high throughput of a sensor unit.
A sensor unit includes a flow path member having a plurality of flow paths and a prism having a metal film formed on an upper surface thereof. The main body 12a of the flow path member 12 is formed in a substantially rectangular parallelepiped shape. Each flow path 20 includes a substantially straight groove portion 20c formed on the bottom surface of the flow path member 12, and each feed 20a and 20b that penetrates the upper surface of the flow path member 12 from both ends of the groove portion 20c to form the respective entrances 20a and 20b. The drainage pipes 20d and 20e are formed in a substantially U shape. The channel 20 is formed so that the groove 20c is along the short direction of the main body 12a, and is arranged side by side at a predetermined interval in the longitudinal direction of the main body 12a.
[Selection] Figure 2

Description

  The present invention relates to a sensor unit comprising a dielectric block in which a thin film layer for detecting a reaction of a sample is formed, and a flow path member in which a flow path for feeding a sample solution in which the sample is dissolved in the thin film layer is formed. And a measuring method using total reflection attenuation for measuring the reaction of a sample using this sensor unit.

  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.

On the linker film, a measurement region having a binding group with a ligand and a reference region in which the binding group is deactivated 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 correcting the difference or ratio between the two signals obtained in this way, it is possible to obtain a highly accurate measurement result that cancels noise caused by disturbances such as individual differences in the sensor unit and changes in the temperature of the liquid. . In addition, in biological samples such as proteins and DNA, so-called non-specific adsorption, in which unintended substances are bound on the linker membrane, becomes a problem. Can also be suppressed.
Japanese Patent No. 3294605

  However, the measurement region having a binding group and the reference region in which the binding group is deactivated may have a difference in the amount of non-specific adsorption due to the difference in surface properties. As described above, the difference generated between the measurement region and the reference region cannot be removed even if the above-described correction is performed.

  In addition, when the measurement region and the reference region are formed separately and are sent through individual flow paths, the ligand solution can be sent only to the measurement region. As a result, it is possible to suppress the difference in the amount of non-specific adsorption by combining the surface properties of each region without deactivating the binding group in the reference region. Will lead to an increase in size. In an SPR measurement apparatus that handles biological samples, improvement of measurement accuracy and measurement of a large number of samples at one time, so-called high throughput is one of the major issues, and the increase in the size of the sensor unit hinders this high throughput. End up.

  The present invention has been made in view of the above problems, and an object thereof is to suppress the influence of non-specific adsorption without hindering high throughput.

  In order to achieve the above object, a sensor unit according to the present invention comprises a dielectric block having a thin film layer that attenuates the light intensity of reflected light when irradiated with light so as to satisfy the total reflection condition, A substantially rectangular parallelepiped main body disposed with the bottom surface in contact with the thin film layer, and a flow path member in which a plurality of flow paths for feeding a sample solution in contact with the thin film layer are provided. Each of the flow paths includes a substantially linear groove formed on the bottom surface along the short direction of the main body, and a pair of entrances connected to both ends of the groove from the upper surface of the main body. The main body is arranged side by side at a predetermined interval in the longitudinal direction of the main body.

  In addition, it is preferable that the center of each said groove part exists on the centerline of the longitudinal direction of the said main body.

  Moreover, it is preferable that a polymer film for fixing the sample is provided on the thin film layer facing each groove.

  Furthermore, the measurement method using total reflection attenuation according to the present invention includes a dielectric block formed with a thin film layer that attenuates the light intensity of the reflected light when light is irradiated so as to satisfy the total reflection condition; A substantially rectangular parallelepiped main body disposed with the bottom surface in contact with the thin film layer, and a flow path member formed with a plurality of flow paths for feeding a sample solution in which a sample is dissolved while contacting the thin film layer Each of the flow paths is configured by a substantially linear groove formed on the bottom surface along the short direction of the main body and a pair of entrances connected to both ends of the groove from the upper surface of the main body. In addition, a sensor unit that is arranged in the longitudinal direction of the main body at a predetermined interval is detachably set, and light that satisfies the total reflection condition is irradiated from the light source toward the thin film layer, and the reflected light is detected. Receive the light and convert it photoelectrically. When acquiring a measurement signal according to the reaction state of the sample on the thin film layer, the sample solution is sent to at least one of the flow paths to acquire the measurement signal, and the rest A reference signal for removing noise of the measurement signal is obtained using at least one of the flow paths.

  In addition, the sample includes a ligand that is preliminarily immobilized on the polymer film on the thin film layer, and an analyte that is brought into contact with the ligand to check a reaction, and the ligand is present in at least one of the channels. After injecting a dissolved ligand solution and fixing the ligand to the polymer membrane, the analyte solution in which the analyte is dissolved is injected to contact the ligand with the analyte, and the ligand and the analyte are contacted. A signal corresponding to the reaction state with the analyte is acquired as the measurement signal, the analyte solution is injected into at least one of the remaining channels, and only the analyte is brought into contact with the polymer film. It is preferable to acquire a signal at the time as the reference signal.

  The light source and the detector may be configured to perform irradiation of the light and reception of the reflected light on the two thin film layers facing at least two of the flow paths, and the measurement signal. And the reference signal are preferably acquired simultaneously.

  According to the present invention, the plurality of flow paths included in the flow path member are formed so that the substantially straight groove portions are along the short direction of the main body, and are arranged side by side with a predetermined interval in the longitudinal direction of the main body. Since it was made to do, it becomes possible to arrange | position each flow path in the main body of a flow path member at high density. As a result, even if the flow path is divided into the measurement area and the reference area, there is no fear of increasing the size of the sensor unit, so the influence of non-specific adsorption can be suppressed without hindering high throughput. Become.

  As shown in FIG. 1, the measurement method using SPR is roughly divided into three steps: a fixing step, a measurement step (data reading step), and a data analysis step. The measurement is performed using the sensor unit 10 that is an SPR sensor. The sensor unit 10 includes a metal film (thin film layer) 25 whose one surface becomes a sensor surface 25a on which SPR occurs, and a prism (dielectric block) 14 bonded to a light incident surface 25b on the back surface of the sensor surface 25a. The flow path member 12 is disposed opposite to the sensor surface 25a and has a flow path 20 through which a ligand and an analyte are fed.

  As the metal film 25, 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, the emission wavelength of the irradiated light, and the like. The prism 14 is a transparent dielectric having the metal film 25 formed on the upper surface thereof, and condenses the irradiated light on the light incident surface 25b so as to satisfy the total reflection condition. The flow path 20 is a liquid feeding pipe bent in a substantially U shape, and has inlets 20a and 20b through which liquid is injected and discharged. Further, the bottom of the flow path 20 is open, and this open portion is covered and sealed with the sensor surface 25a. As a result, the flow path 20 feeds the liquid injected from one of the inlets / outlets 20a, 20b while contacting the sensor surface 25a, and discharges it from the other of the inlets / outlets 20a, 20b. Hereinafter, a portion constituted by the flow path 20 and the sensor surface 25a is referred to as a sensor cell 18. As will be described later, the sensor unit 12 includes a plurality of such sensor cells 18 (see FIG. 2).

  The fixing step is a step of fixing the ligand to the sensor surface 25a. The SPR measuring device 30 is provided with a pipette pair 46 including a pair of pipettes 46a and 46b. The intervals between the pipettes 46a and 46b are adjusted so that the pipettes 46a and 46b are inserted into the entrances 20a and 20b, respectively. Each of the pipettes 46a and 46b has a function of injecting liquid into the flow channel 20 and sucking out from the flow channel 20, and when one of them performs the injection operation, the other performs the suction operation. So that they work together. By this pipette pair 46, a ligand solution LS in which a ligand is dissolved in a solvent is injected from one of the entrances 20a and 20b.

  A linker film (polymer film) 26 that binds to a ligand is formed in the approximate center of the sensor surface 25a corresponding to the sensor cell 18. The linker film 26 is formed in advance at the manufacturing stage of the sensor unit 10. Since the linker film 26 serves as a fixing group for fixing the ligand, it is appropriately selected according to the type of ligand to be fixed.

  Before performing the ligand immobilization process for injecting the ligand solution LS, first, the immobilization buffer solution is sent to the linker film 26, and the linker film 26 is moistened to facilitate the binding of the ligand film 26. Processing is performed. For example, in the amine coupling method, carboxymethyl dextran is used as the linker film 26, 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 this activation process, the flow path 20 is washed with the fixing buffer solution.

  As the buffer solution for fixation and the solvent (diluent) of the ligand solution LS, 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 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 amine coupling method, since the linker film 26 is negatively (negatively) charged by carboxymethyldextran, the protein is positively (positively) charged so as to be easily bonded to the linker film 26. In some cases, a phosphate buffer solution (PBS: phosphatic-buffered, saline) having a strong buffering action containing a high concentration of phosphate may be used.

  After such activation treatment and washing are performed, the ligand solution LS is injected into the flow path 20 to perform the ligand immobilization treatment. When the ligand solution LS is injected into the flow path 20, the ligand LG diffused in the solution gradually approaches the linker film 26 and binds thereto. In this way, the ligand LG is fixed to the sensor surface 25a. 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 LS in the flow path 20 may be allowed to stand while the immobilization proceeds, but it is preferable to stir and flow the ligand solution LS in the flow path 20. By doing so, the binding between the ligand LG and the linker film 26 is promoted, and the amount of ligand LG immobilized can be increased.

  When the immobilization of the ligand LG on the sensor surface 25a is completed, the ligand solution LS is discharged from the flow path 20. The ligand solution LS is sucked into and discharged from one of the pipettes 46a and 46b. When the immobilization is completed, the cleaning liquid is injected into the flow path 20 and the cleaning process of the sensor surface 25a is performed. After this washing, if necessary, a blocking solution is injected into the flow path 20 to perform a blocking process for inactivating the reactive group to which no ligand is bound in the linker film 26. As the blocking liquid, for example, ethanolamine-hydrochloride is used. After this blocking process, the flow path 20 is washed again. Thereafter, a drying preventing liquid is injected into the flow path 20. Thus, the sensor unit 10 is stored until measurement in a state where the sensor surface 25a is immersed in the anti-drying liquid.

  In the measurement step, first, a measurement buffer solution is injected into the flow path 20. Thereafter, an analyte solution AS 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 20 may be once cleaned before the measurement buffer solution is first injected. Data reading is started immediately after the measurement buffer is first injected to detect a reference signal level, and is performed after the injection of the analyte solution AS and until the measurement buffer is injected again. . 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 solution) of the analyte solution AS, for example, various buffer solutions (buffer solutions), physiological salt solutions represented by physiological saline, and pure water are used. Is done. 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 AS is often stored for a long period of time (for example, one year). In such a case, a concentration difference 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 AS is injected, and the measurement data is corrected (DMSO concentration correction). Although the details will be described later, the reference signal is an SPR signal corresponding to a reference region where the ligand is not fixed on the sensor surface 25a, and the measurement signal of the measurement region that causes a reaction with the analyte when the ligand is fixed; It is a signal to be compared and referenced. In the measurement, two signals of the measurement signal and the reference signal are detected.

  Correction data for correcting the DMSO concentration is obtained by injecting a plurality of types of buffer solutions for measurement having different DMSO concentrations into the flow path 20 before injecting the analyte solution AS, and according to the change in DMSO concentration at this time. It is obtained by examining the amount of change in each of the level of the reference signal and the level of the measurement signal.

  The measurement unit 31 includes an illumination 40 and a detector 42. Since the reaction state between the ligand and the analyte appears as a change in the resonance angle (the incident angle of the light applied to the light incident surface 25b), the illumination 40 is light having various incident angles that satisfy the total reflection condition. Is irradiated to the light incident surface 25b. The illumination 40 includes, for example, a light source 43 and an optical system 44 including a condensing lens (such as a cylindrical lens), a diffuser plate, and a polarizing plate. The arrangement position and the installation angle are such that the incident angle of illumination light is the total reflection described above. It is adjusted to satisfy the conditions.

  As the light source 43, 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 diffusion plate diffuses light from the light source 43 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. In addition, 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 irradiated onto the prism 14. As a result, light rays with no variation in light intensity and various incident angles are incident on the light incident surface 25b.

  The detector 42 receives the light reflected by the light incident surface 25b and outputs an electrical signal having a level corresponding to the light intensity. Since light rays are incident on the light incident surface 25b at various angles, the light rays are reflected on the light incident surface 25b at various reflection angles according to respective incident angles. The detector 42 receives light beams having these various reflection angles. When a change occurs in the medium on the sensor surface 25a, the refractive index changes, and the incident angle (resonance angle at which SPR occurs) of the light whose reflected light intensity attenuates also changes. When the analyte is fed onto the sensor surface 25a, the refractive index on the sensor surface 25a changes according to the reaction state between the analyte and the ligand, and the resonance angle also changes accordingly.

  The detector 42 uses, for example, a CCD area sensor or a photodiode array, receives reflected light reflected at various reflection angles on the light incident surface 25b, photoelectrically converts them, and outputs them as SPR signals. 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 contacts the ligand, the refractive index on the sensor surface 25a 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. As described above, in the measurement process, an SPR signal indicating a reaction state between the ligand and the analyte is acquired. In the data analysis process, the SPR signal obtained in the measurement process is analyzed to analyze the characteristics of the analyte.

  The detector 42 acquires a measurement signal corresponding to the measurement area on the sensor surface 25a and a reference signal corresponding to the reference area. In data analysis, the difference or ratio between the measurement signal thus obtained and the reference signal is obtained. In the data analysis step, for example, difference data between the measurement signal and the reference signal is obtained, and the difference data is used as measurement data, and analysis is performed based on the difference data. By doing so, it is possible to obtain a highly accurate measurement result in which noise caused by disturbances such as individual differences of the sensor unit 10, mechanical fluctuations of the apparatus, and temperature change of the liquid are canceled.

  FIG. 2 is an exploded perspective view schematically showing the configuration of the sensor unit 10. In the sensor unit 10, the flow path member 12 in which the six flow paths 20 are formed, the prism 14 in which the metal film 25 is formed on the upper surface, and the bottom surface of the flow path member 12 and the upper surface of the prism 14 are joined. It is comprised from the holding member 16 hold | maintained. In this sensor unit 10, the six sensor cells 18 are formed by the flow path member 12 and the prism 14, so that one SPR signal can be acquired by one sensor unit 10.

  The flow path member 12 has a main body 12a formed in a substantially rectangular parallelepiped shape. The flow paths 20 formed in the main body 12a are arranged side by side at a predetermined interval in the longitudinal direction of the main body 12a. Each channel 20 formed in a substantially U-shape has a substantially straight groove 20c formed on the bottom surface of the channel member 12, and an inlet / outlet that penetrates from one end of the groove 20c to the upper surface of the channel member 12. 20d is formed of a feeding / draining pipe 20d and a feeding / draining pipe 20e that penetrates from the other end of the groove 20c to the upper surface of the flow path member 12 to form an inlet / outlet 20b. To be along. Further, the respective channels 20 are arranged in a line on the main body 12a so that the center of the groove 20c is positioned on the center line CL in the longitudinal direction of the main body 12a. In addition, the pipe diameter of the flow path 20 is about 1 mm, for example, and the space | interval of each entrance / exit 20a, 20b is about 10 mm, for example.

  Each flow path 20 constitutes a sensor cell 18 (see FIG. 1) together with a metal film 25 bonded to the bottom surface thereof. Therefore, an elastic material such as rubber or PDMS (polydimethylsiloxane) is used for the flow path member 12 in order to improve the adhesion with the metal film 25. When the bottom surface of the flow path member 12 is brought into pressure contact with the upper surface of the prism 14, the flow path member 12 is elastically deformed to fill a gap in the joint surface with the metal film 25. As a result, the open bottom of each flow path 20 is covered with the upper surface of the prism 14 in a watertight manner.

  The metal film 25 formed on the upper surface of the prism 14 is formed into a strip shape by, for example, a vapor deposition method so as to face each flow path 20 formed in the flow path member 12. In addition, six linker films 26 corresponding to the respective flow paths 20 are provided on the upper surface of the metal film 25. In this example, the six linker films 26 corresponding to the respective flow paths 20 are formed. However, the present invention is not limited to this. For example, one linker film is formed on the entire surface of the metal film 25. You may make it do.

  Engaging claws 14 a that engage with the engaging portions 16 a of the holding member 16 are provided on both side surfaces of the prism 14 in the longitudinal direction. With these engagements, each flow path member 12 is sandwiched between the holding member 16 and the prism 14 and is held in a state where the bottom surface of the flow path member 12 and the top surface of the prism 14 are in pressure contact with each other. In this way, each part of each channel member 12, prism 14, and holding member 16 is integrated, and sensor unit 10 is constituted.

  The prism 14 is made of, for example, optical glass such as 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 16, receiving ports 16 b into which the tips of the pipettes 46 a and 46 b enter are formed at positions corresponding to the respective inlets 20 a and 20 b of the flow path 20. Each receiving port 16b has a funnel shape so as to guide the liquid discharged from each pipette 46a, 46b into each channel 20. When the holding member 16 sandwiches each flow path member 12 and engages with the prism 14, the lower surface of each receiving port 16 b and each of the entrances 20 a and 20 b are joined, and each receiving port 16 b and each flow path 20 are connected. The

  For example, an RFID (Radio Frequency IDentification) tag that is a non-contact type IC memory may be attached to the prism 14 or the holding member 16 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 simultaneously measuring a plurality of sensor units 10, 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. 3 is an explanatory diagram schematically showing the configuration of the SPR measurement device 30 as a measurement device using total reflection attenuation. The SPR measurement device 30 includes a measurement unit 31 that acquires an SPR signal corresponding to the reaction state of the sample on the metal film 25 of the sensor unit 10, and a pump that sends various liquids to each flow path 20 of the sensor unit 10. And a liquid head 32. Each of these units is comprehensively controlled by a controller (not shown).

  As described above, the measurement unit 31 includes the illumination 40 that irradiates the sensor unit 10 with light so as to satisfy the total reflection condition, and the detector that receives the light totally reflected by the sensor unit 10 and photoelectrically converts it into an electrical signal. 42. The illumination 40 and the detector 42 are fixed to a measurement stage (not shown). The measurement stage is, for example, a pedestal formed in a trapezoidal shape. The illumination stage 40 and the detector 42 are fixed at a predetermined angle that satisfies the total reflection condition, and the sensor unit 10 is detachably held to illuminate. The sensor unit 10 is positioned on 40 optical paths.

  The illumination 40 is a so-called line illumination that irradiates light having a width corresponding to the prism 14, and makes the illumination light enter the six sensor cells 18 included in the sensor unit 10 simultaneously. The detector 42 also has a width corresponding to the prism 14, and receives all the light emitted from the illumination 40 and reflected by the upper surface of the prism 14. For example, when a CCD area sensor is used as the detector 42, an SPR signal corresponding to each sensor cell 18 can be recognized by performing image processing on a signal obtained by receiving reflected light. Thus, the measurement unit 31 measures the SPR signal of each sensor cell 18 at the same time.

  In this example, each sensor cell 18 is irradiated with one illumination 40 that irradiates light according to the width of the prism 14. However, the present invention is not limited to this. A number of illuminations may be provided to irradiate each sensor cell 18, or light from a single light source may be dispersed to irradiate each sensor cell 18. In this example, the SPR signal of each sensor cell 18 is simultaneously measured by one detector 42. However, the number of detectors corresponding to each sensor cell 18 is provided, and the SPR signal of each sensor cell 18 is provided. May be measured simultaneously.

  The liquid feeding head 32 is provided with a pipette pair 46 including a pipette 46 a and a pipette 46 b that access the respective entrances 20 a and 20 b of the flow path 20. Although omitted in FIG. 3, the pipette pair 46 is provided corresponding to each sensor cell 18, and the liquid feeding head 32 simultaneously injects and discharges the liquid into the flow path 20 of each sensor cell 18. be able to. For example, a syringe pump is connected to each of the pipettes 46a and 46b, and suction and discharge of the liquid are controlled in accordance with driving of each syringe pump. A head moving mechanism 33 is connected to the liquid feeding head 32. The head moving mechanism 33 is a known moving mechanism including, for example, a conveyance belt, a pulley, a carriage, and a motor. The head moving mechanism 33 moves the liquid feeding head 32 in three directions, front, rear, left, right, up and down, under the control of a controller not shown. Move.

  The tip of each pipette 46a, 46b 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 46a and 46b. The SPR measurement device 30 is provided with a pipette tip storage unit (not shown) for storing a plurality of pipette tips. Replacement of the pipette tip is performed by driving the head moving mechanism 33 to allow the liquid feeding head 32 to access the pipette tip storage unit.

  In addition, the SPR measurement device 30 is provided with a well plate 34 for storing various liquids (ligand solution, analyte solution, washing solution, buffer solution, etc.) to be injected into each channel 20. The head moving mechanism 33 moves the liquid feeding head 32 to access the well plate 34, the sensor unit 10 set on the measurement stage, and the like.

  The liquid feeding head 32 incorporates a pipette projecting mechanism (not shown) that projects each of the pipettes 46a and 46b independently. When the liquid feeding head 32 accesses the well plate 34 and sucks the liquid in each well, as shown in FIG. 4A, only the pipette that sucks the liquid protrudes and is immersed in the liquid in the well. . This prevents, for example, the other pipette from entering the well and being immersed in a different liquid. In this example, different liquids are stored in each well of one well plate 34. However, an individual well plate may be provided for each stored liquid.

  On the other hand, when the liquid is fed to each flow path 20 by the liquid feed head 32, the pipettes 46a and 46b are inserted into the respective entrances 20a and 20b of the flow path 20, as shown in FIG. . In this state, the liquid feeding head 32 discharges the liquid sucked from one of the pipettes 46a and 46b, and sucks the fluid (air, liquid previously injected, etc.) in each channel 20 from the other, Liquid feeding is performed by replacing the fluid in each flow path 20.

  When performing the fixing process, the SPR measuring device 30 does not fix the ligand to at least one of the sensor cells 18. As a result, the linker film 26 of the sensor cell 18 to which the ligand is immobilized becomes the aforementioned measurement region, and the linker film 26 of the sensor cell 18 to which the ligand is not immobilized becomes the aforementioned reference region. Since the refractive index on the linker film 26 naturally changes depending on the feeding of the analyte solution, the number of required reference regions depends on whether the type of analyte is different.

  For example, when it is desired to see the reaction of one type of analyte to a plurality of types of ligands, such as drug screening, one reference region may be prepared. In other words, in this example, different types of ligands are fixed to the linker films 26 of the five sensor cells 18 so that each is used as a measurement region, and the linker film 26 of the remaining one sensor cell 18 is used as a reference region. In data analysis, one reference signal may be subtracted from the measurement signal in each measurement region.

  On the other hand, when it is desired to see the reactions of multiple types of analytes to one type of ligand, such as protein identification, a reference region is prepared for each analyte to be fed. In other words, in this example, the same ligand is fixed to the linker films 26 of the three sensor cells 18 to be used as measurement regions, and the linker films 26 of the remaining three sensor cells 18 are used as reference regions for each analyte. In the data analysis, the reference signal of the reference area where the same analyte solution is fed may be subtracted from the measurement signal. Note that the position and combination of the sensor cell 18 serving as the measurement region and the sensor cell 18 serving as the reference region may be arbitrary, and the measurement signal and the reference signal corresponding to the data analysis may be discriminated.

  Next, referring to the flowchart shown in FIG. 5, the operation of the SPR measurement device 30 configured as described above will be described by taking as an example the case of viewing the reactions of a plurality of types of analytes with respect to one type of ligand. When measuring the reaction state between the ligand and the analyte, first, the sensor unit 10 is set on the measurement stage and positioned so that each sensor cell 18 comes on the optical path of the illumination 40. As described above, the SPR measurement device 30 includes a fixing step of fixing a ligand to the sensor unit 10, a measuring 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 30 starts the fixing process in response to the input of the fixing start instruction from the operator. The controller of the SPR measurement device 30 drives the head moving mechanism 33 in response to the input of the fixing start instruction, and moves the liquid feeding head 32 to the well plate 34. The controller that has made the liquid feeding head 32 access the well plate 34 drives the pipette projecting mechanism to project the pipette 46a, and enters the pipette 46a into the well in which the ligand solution is stored. At this time, the controller causes only the three pipettes 46 a corresponding to the three sensor cells 18 to be set as the measurement area among the six pipettes 46 a included in the liquid feeding head 32 to protrude. Each pipette 46a that enters the well and is immersed in the ligand solution aspirates a predetermined amount of the ligand solution. The controller that has sucked the ligand solution into the pipette 46 a moves the liquid feeding head 32 to the sensor unit 10 and inserts the pipettes 46 a and 46 b into the inlets 20 a and 20 b of the channels 20.

  The controller into which each pipette 46a, 46b is inserted drives each pipette pair 46 that sucks and holds the ligand solution so that the pipette 46a discharges and the pipette 46b sucks, and the ligand solution is put into the corresponding flow path 20. inject. As a result, the ligand is fixed to the linker film 26 of the sensor unit 18 serving as the measurement region, and the fixing process is completed. In addition, before injecting the ligand solution into the flow path 20, as described above, the cleaning in each flow path 20 and the activation process of the linker film 26 are performed. The ligand solution in each channel 20 may be allowed to stand while the immobilization proceeds, but the suction and discharge of each pipette 46a, 46b are alternately driven to The ligand solution may be stirred and fluidized.

  The SPR measurement device 30 in which a ligand is fixed to the linker film 26 of each sensor cell 18 that becomes a measurement region holds the sensor unit 10 in a state where environmental conditions such as temperature are kept constant, and an instruction to start measurement is input from an operator. In response to this, the measurement process is started. In response to the input of the measurement start instruction, the controller starts data reading by the illumination 40 and the detector 42. At the same time, the head moving mechanism 33 is driven to move the liquid feeding head 32 to the well plate 34. The controller that has moved the liquid feeding head 32 to the well plate 34 drives the pipette projecting mechanism to project each pipette 46a, and causes each pipette 46a to suck a predetermined amount of the analyte solution. At this time, the controller uses the pipette 46a corresponding to the measurement region and the pipette 46b corresponding to the reference region as one set, and sucks three different types of analyte solutions for each set of pipettes 46a.

  The controller that sucks each analyte solution into each pipette 46 a moves the liquid feeding head 32 to the sensor unit 10 and inserts each pipette 46 a, 46 b into the inlet / outlet 20 a, 20 b of each flow path 20. The controller into which the pipettes 46 a and 46 b are inserted drives each pipette pair 46 so that the pipette 46 a discharges and the pipette 46 b performs suction, and injects the analyte solution into each flow path 20.

  On the linker film 26 for the measurement region on which the ligand is fixed, the ligand and the analyte start to react in response to the injection of the analyte solution. Thereby, an SPR signal corresponding to the reflected light from each linker film 26 for the measurement region is acquired as a measurement signal. On the other hand, the reaction of the analyte naturally does not occur on the linker film 26 for the reference region where the ligand is not fixed. As a result, an SPR signal corresponding to the reflected light from each linker film 26 for the reference region is acquired as a reference signal corresponding to a change in the refractive index on the linker film 26 accompanying the feeding of the analyte solution.

  The SPR measurement device 30 that has acquired each signal stops data reading by the illumination 40 and the detector 42 and ends the measurement process. Although omitted in the present example, in the measurement step, after starting the data reading, the measurement buffer solution is sent to each channel 20 before and after the analyte solution is sent. Thereby, the detector 42 acquires each SPR signal 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 30 that has completed the measurement process calculates measurement data by subtracting each corresponding reference signal from each measurement signal, and analyzes the reaction state between the ligand and the analyte based on this measurement data. At this time, in this example, since the measurement signal and the reference signal are obtained from the linker film 26 of each individual sensor cell 18, each signal is obtained from the linker film 26 having the same surface property, and between each signal. It is possible to suppress the occurrence of non-specific adsorption differences. Thereby, by calculating the measurement data, the influence of non-specific adsorption is surely prevented.

  In addition, each channel 20 is formed so that the groove 20c is along the short direction of the main body 12a, and is arranged side by side with a predetermined interval in the longitudinal direction of the main body 12a. It can be arranged in the flow path member 12 with high density. As a result, even when the flow path 20 is divided and the measurement signal and the reference signal are acquired, the sensor unit 10 is prevented from being enlarged, so that high throughput is not hindered.

  Further, since each channel 20 is formed so that the center of the groove 20c is positioned on the longitudinal center line CL of the main body 12a, the force when inserting each pipette pair 46 into each channel 20 is equal to the left and right. It is possible to add a good balance and prevent tilting with the longitudinal direction of the sensor unit 10 as an axis, thereby suppressing variations in each SPR signal due to tilting.

  In addition, although the sensor unit 10 of the said embodiment has the six sensor cells 18, the sensor cell 18 should just have at least 2 for measurement area | regions and for reference areas. In the above embodiment, the SPR signals of the six sensor cells 18 are simultaneously acquired by the illumination 40 and the detector 42. However, the present invention is not limited to this, and the illumination 40 and the detector 42 are respectively measured signals. A plurality of sensor cells 18 included in the sensor unit 10 may be measured while moving the sensor unit 10 or the measurement unit 31 by providing only two channels for the reference signal and the reference signal.

  In the above embodiment, each step is performed by one SPR measuring device 30, 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.

  In the above-described embodiment, the prism 14 is shown as the dielectric block. However, the dielectric block may be a plate made of optical glass, optical plastic, or the like, or those plate-like ones. What integrated the prism with the optical surface smoothing agent (for example, optical matching oil) shall be included.

  Furthermore, in the above-described embodiment, 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 also known as a measurement device using total reflection attenuation. ing. 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 explanatory drawing which shows roughly the measuring method using SPR. 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 explaining operation | movement of a liquid feeding head. It is a flowchart which shows a measurement procedure roughly.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Sensor unit 12 Flow path member 12a Main body 14 Prism (dielectric block)
18 sensor cell 20 flow path 20a entrance / exit 20b entrance / exit 20c groove part 25 metal film (thin film layer)
26 Linker membrane (polymer membrane)
30 SPR measuring device 40 Illumination 42 Detector 43 Light source

Claims (6)

When light is irradiated so as to satisfy the total reflection condition, a dielectric block having a thin film layer formed to attenuate the light intensity of the reflected light, and a substantially rectangular parallelepiped arranged with the bottom surface in contact with the thin film layer In a sensor unit comprising a channel member in which a plurality of channels for feeding a sample solution in which a sample is dissolved in contact with the thin film layer is formed in a shaped body,
Each of the flow paths includes a substantially linear groove formed on the bottom surface along the short direction of the main body, and a pair of entrances connected to both ends of the groove from the upper surface of the main body. A sensor unit, which is arranged side by side at a predetermined interval in the longitudinal direction of the main body.   The sensor unit according to claim 1, wherein the center of each of the grooves is on a longitudinal center line of the main body.   The sensor unit according to claim 1, wherein a polymer film for fixing the sample is provided on the thin film layer facing each groove. When light is irradiated so as to satisfy the total reflection condition, a dielectric block having a thin film layer formed to attenuate the light intensity of the reflected light, and a substantially rectangular parallelepiped arranged with the bottom surface in contact with the thin film layer And a flow path member in which a plurality of flow paths for feeding a sample solution in which a sample is dissolved is brought into contact with the thin film layer, and the flow paths are arranged along the short direction of the main body. And a pair of doorways connected to both ends of the groove portion from the top surface of the main body, and arranged at a predetermined interval in the longitudinal direction of the main body. Set the placed sensor unit to be detachable
Irradiating light from the light source toward the thin film layer satisfying the total reflection condition,
In the measurement method using total reflection attenuation that receives the reflected light with a detector and photoelectrically converts it to obtain a measurement signal according to the reaction state of the sample on the thin film layer,
Reference for removing the noise of the measurement signal using at least one of the remaining flow paths while obtaining the measurement signal by sending the sample solution to at least one of the flow paths A measurement method using total reflection attenuation, characterized by acquiring a signal. The sample includes a ligand that is preliminarily immobilized on the polymer film on the thin film layer, and an analyte that is contacted with the ligand to examine a reaction.
After injecting a ligand solution in which the ligand is dissolved into at least one of the channels and fixing the ligand to the polymer membrane, injecting an analyte solution in which the analyte is dissolved Contacting the analyte, obtaining a signal corresponding to a reaction state of the ligand and the analyte as the measurement signal;
The analyte solution is injected into at least one of the remaining channels, and a signal when only the analyte is brought into contact with the polymer film is obtained as the reference signal. 4. A measuring method using total reflection attenuation described in item 4. The light source and the detector are configured to perform irradiation of the light and reception of the reflected light to the two thin film layers facing at least two of the flow paths, and the measurement signal 6. The measuring method using total reflection attenuation according to claim 4, wherein the reference signal is acquired simultaneously.

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