JP2006090985A - Measuring apparatus utilizing total reflection attenuation and method therefor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a measuring apparatus utilizing total reflection attenuation, which can carry out a highly precise measurement with a small amount of sample. <P>SOLUTION: Air and an analyte solution are alternately sucked by a pipette 26, and then they are injected into a fluid channel in order of the air 51b, the analyte solution 27b, the air 51a and the analyte solution 27a. A measurement buffer 52 which is previously injected into the fluid channel 16a, is pushed out by the headmost air 52b and discharged therefrom. The pipette 26 continues its discharging operation until a portion of the analyte solution 27b is discharged, and then it carries out a sucking operation, thereby causing the analyte solution 27 and the air 51 to flow backward. Therefore, the analyte solution 27 and the air 51 are moved back and forth on a sensor surface 13a, and liquid displacements of the analyte solution 27 and the measurement buffer 52 are carried out on the sensor surface 13a without unevenly distributed between upstream and downstream sides. As a result, respective signals corresponding to an active region and a reference region on the sensor surface 13a can be compared, whereby measurement accuracy is improved. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a measuring apparatus and method using total reflection attenuation, and more particularly to an apparatus and method for performing measurement by sending an analyte solution to a sensor surface on which a ligand is fixed.

  A chemical reaction of the sample is generated on the sensor surface, which is one surface of the thin film formed on the transparent dielectric, and light is incident so as to satisfy the total reflection condition on the light incident surface on the back surface of the sensor surface, A measuring device using total reflection attenuation for measuring the chemical reaction based on the attenuation state of the reflected light is known, and one of them is a measuring device using a surface plasmon resonance phenomenon.

  In a metal, free electrons collectively vibrate to generate a dense wave called a plasma wave. Plasmon is an expression obtained by quantizing this dense wave, and among these, the dense wave generated on the surface of the metal is called surface plasmon. This surface plasmon travels along the surface of the metal. A measuring device using the surface plasmon resonance phenomenon (hereinafter referred to as SPR measuring device) uses a metal film as a thin film formed on a transparent dielectric, and one surface of the metal film is a sensor. As a surface, this is a device that generates surface plasmon resonance (hereinafter referred to as SPR) on the sensor surface and measures the reaction state of a substance generated by detecting the surface plasmon resonance phenomenon.

  When light is irradiated from the back side of the sensor surface of the metal film so as to satisfy the total reflection condition (at an incident angle greater than the critical angle), total reflection occurs at the light incident surface, but only a small amount of incident light is It passes through the metal film without reflection and oozes out to the sensor surface. This light wave that oozes out is called an evanescent wave. When the frequencies of the evanescent wave and the surface plasmon coincide and resonate (when SPR occurs), the intensity of the reflected light is greatly attenuated. The SPR measurement device detects the SPR generated on the sensor surface on the back side by capturing the attenuation of the reflected light reflected by the light incident surface.

  The incident angle (resonance angle) of light for generating SPR depends on the refractive index of the medium through which the evanescent wave and the surface plasmon propagate. In other words, if the refractive index of the medium changes, the resonance angle that generates SPR changes. The substance in contact with the sensor surface becomes a medium for propagating evanescent waves and surface plasmons. For example, when a chemical reaction such as bonding or dissociation between two types of molecules occurs on the sensor surface, it is determined by the refractive index of the medium. It appears as a change, and the resonance angle changes. The SPR measurement device measures the interaction between molecules by capturing the change in the resonance angle.

  This SPR measurement device is used for various researches represented in the biochemical field, such as examining the interaction of biochemical substances such as proteins and DNA, and screening drugs. In biochemical research, proteins, DNA, drugs, etc. are used as ligands and analytes. For example, when screening for drugs, biological substances such as proteins are used as ligands, and a plurality of types of drugs serving as analytes are brought into contact with the sensor surface to examine their interaction.

  The SPR measurement device described in Patent Document 1 below employs a Kretschmann arrangement as an optical system for making light incident on a metal film. In the Kretschmann arrangement, for example, a sensor in which a metal film is formed on a glass substrate that is a transparent dielectric is used, and the glass substrate and the prism are bonded to face the light incident surface of the metal film. The prism condenses the light irradiated toward the light incident surface so as to satisfy the total reflection condition. A ligand is fixed on the sensor surface, and a flow path for flowing an analyte solution obtained by dissolving an analyte in a solvent is disposed at a position facing the sensor surface. An analyte solution is fed into this flow path, the analyte and the ligand are brought into contact with each other, and the interaction between them is measured by detecting the attenuation of the reflected light at that time.

  Before the analyte solution is injected into the channel, a buffer is injected in advance, and this buffer is pushed out by injecting the analyte solution and discharged from the channel. Thereby, liquid replacement in the flow path is performed. When the reaction between the analyte and the ligand is stopped, the buffer is again injected into the flow path and the analyte solution is discharged. The signal measurement starts before the analyte solution is injected, and is performed until a predetermined time elapses after the buffer is injected again. By doing so, it is possible to measure the signal baseline from the reaction between the reaction of the analyte and the ligand to the desorption while measuring the signal baseline at the time of buffer injection.

The feeding of the analyte solution to the sensor surface is performed, for example, by continuously flowing the analyte solution through the flow path (continuous feeding) during the measurement. However, when such continuous liquid feeding is performed, a large amount of analyte solution is required. Since the analyte is generally expensive, it is preferable that the amount used is as small as possible in consideration of the experimental cost. Therefore, a so-called flow-and-stop method is started in which the feeding of the analyte solution is started and the feeding is stopped when a predetermined amount of feeding is completed.
JP-A-6-167443

  However, as shown in FIG. 6, the analyte solution 82 is injected into the flow path 81 by, for example, a pipette 85, and the liquid replacement with the already injected buffer 84 is performed. Since the injected analyte solution 82 has a velocity gradient from the central portion in the flow path 16 toward the tube wall, the buffer 84 and the analyte are more downstream in the flow direction in the sensor surface 83 than in the upstream. The liquid replacement with the light solution 82 is delayed. For this reason, the binding reaction between the analyte and the ligand could not be accurately measured.

  This is because the sensor surface 83 is provided with a measurement region (act region) 86 where a ligand is fixed and a reaction with an analyte occurs, and a reference region (ref region) 87 where a ligand is not fixed. The regions 86 and 87 are arranged side by side along the flow direction of the flow path 16. Signal detection is performed simultaneously on each of the regions 86 and 87, and is acquired as an act signal and a ref signal, respectively. Then, for example, the difference between the two channel signals thus detected is obtained, and the difference data is used as measurement data. By doing so, various factors that cause errors such as individual differences between different sensors and the concentration of the solution can be canceled, so that highly accurate measurement can be performed.

  However, since the signal detection of the two channels is performed at the same time, if the progress of the liquid replacement in each of the regions 86 and 87 is not at the same level at the time of detection, it is meaningless to compare the two channels. The expected effect cannot be obtained by signal detection. In the flow-and-stop method, the liquid measurement is stopped after the injection and the analyte solution is retained in the flow path. Compared with the continuous liquid flow with high fluidity of the analyte solution in the flow path. Then, the progress of the liquid replacement is poor, and the difference in the progress of the liquid replacement between the upstream portion and the downstream portion that occurs immediately after the injection is difficult to be solved. For this reason, it has been difficult to obtain highly accurate measurement data even if signal detection of two channels is performed.

  An object of the present invention is to provide a measuring apparatus using total reflection attenuation capable of performing highly accurate measurement with a small number of samples.

  The measuring apparatus using total reflection attenuation according to the present invention uses a sensor including a thin film formed on a transparent dielectric, one surface serving as a sensor surface to which a ligand is fixed, and the other surface serving as a light incident surface. Then, an analyte solution in which an analyte is dissolved in a solvent is injected into a flow path arranged opposite to the sensor surface, and the solution is fed onto the sensor surface, so that the light incident surface satisfies the total reflection condition. In the measuring apparatus using total reflection attenuation for measuring the interaction between the ligand on the sensor surface and the analyte by detecting the attenuation of the reflected light, the flow path of the analyte solution is measured. A first feeding / discharging means capable of discharging to and sucking from the first solution, and using the first feeding / discharging means, the analyte solution is once fed to the sensor surface by discharging, and then the analyte solution is discharged. Suck By flow, said on the sensor surface is reciprocated at least once the analyte solution, thereafter, characterized by re-feeding the analyte solution to the sensor surface.

  For example, another liquid is injected into the channel before the analyte solution is injected, and the other liquid is pushed out of the channel and discharged by the injection of the analyte solution. Thus, liquid replacement in the flow path is performed.

  The first feeding / discharging means alternately discharges the analyte solution and air to the flow path, passes the sensor solution on the sensor surface, and then sucks back the analyte solution and the air. It is preferable.

  Of the plurality of portions of the analyte solution divided by the air, the portion previously injected into the flow path is preferably discharged out of the flow path after reciprocating on the sensor surface.

  As the first feeding / discharging means, for example, a pipette is used. Further, a liquid reservoir that retains the liquid overflowing from the discharge port may be provided outside the discharge port of the flow path. In addition, a second feeding / discharging is performed at the discharge port of the flow path by performing a suction operation in conjunction with the discharging operation of the first feeding / discharging means and performing a discharging operation in conjunction with the suction operation of the first feeding / discharging means. Means may be arranged.

  The measurement method of the present invention uses a sensor provided with a thin film formed on a transparent dielectric material, one surface serving as a sensor surface to which a ligand is fixed, and the other surface serving as a light incident surface. Then, an analyte solution in which an analyte is dissolved in a solvent is injected into the flow path arranged and sent onto the sensor surface, and light is incident on the light incident surface so as to satisfy the total reflection condition. In the measurement method using the total reflection attenuation for measuring the interaction between the ligand on the sensor surface and the analyte by detecting the attenuation of the analyte, the analyte solution is discharged once into the flow path. The analyte solution is reciprocated at least once on the sensor surface by performing at least one suction after the liquid is fed to the sensor surface and causing the analyte solution to flow backward, and then the analyte solution is Characterized by feeding into the liquid again the sensor surface.

  It is preferable that another liquid is injected into the flow path before the analyte solution is injected, and the other liquid is pushed out of the flow path and discharged by the injection of the analyte solution. .

  Preferably, the analyte solution and air are alternately discharged into the flow path and passed over the sensor surface, and then suction is performed to reversely flow the analyte solution and the air.

  Of the plurality of portions of the analyte solution divided by the air, the portion previously injected into the flow path is preferably discharged out of the flow path after reciprocating on the sensor surface. .

  In the measurement using the total reflection attenuation, the present invention is configured to discharge the analyte solution into the flow path and once send the solution to the sensor surface, and then perform at least one suction to make the analyte solution flow backward. Since the light solution is reciprocated at least once on the sensor surface, and then the analyte solution is again sent to the sensor surface, even when the solution is sent by the flow and stop method, the sensor Liquid replacement can be performed without any deviation in the plane. For this reason, it is possible to perform highly accurate measurement with a small number of samples.

  As shown in FIG. 1, the measurement method using SPR is roughly divided into three steps: a fixing step, a measurement processing step (data reading step), and a data analysis step. The SPR measuring device includes a fixing machine 10 that performs a fixing process, a measuring machine 11 that performs a measuring process, and a data analyzer that analyzes data obtained by the measuring machine 11.

  The measurement is performed using the sensor unit 12 that is an SPR sensor. The sensor unit 12 has a metal film 13 having one surface serving as a sensor surface 13a on which SPR occurs, a prism 14 bonded to a light incident surface 13b on the back surface of the sensor surface 13a, and the sensor surface 13a. And a flow path member 41 having a flow path 16 through which a ligand and an analyte are fed.

  As the metal film 13, for example, gold is used, and the film thickness is, for example, 500 angstroms. 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 13 formed on the upper surface thereof, and condenses the light irradiated so as to satisfy the total reflection condition toward the light incident surface 13b. The channel 16 is a liquid feeding pipe bent in a substantially U shape, and has an inlet 16a for injecting liquid and an outlet 16b for discharging it. The tube diameter of the channel 16 is, for example, about 1 mm, and the interval between the inlet 16a and the outlet 16b is, for example, about 10 mm.

  The bottom of the channel 16 is open, and this open part is covered and sealed with the sensor surface 13a. A sensor cell 17 is constituted by the flow path 16 and the sensor surface 13a.

  The fixing step is a step of fixing the ligand to the sensor surface 13a. The fixing process is performed by setting the sensor unit 12 to the fixing machine 10. The fixing machine 10 is provided with a pipette pair 19 including a pair of pipettes 19a and 19b. In the pipette pair 19, the pipettes 19a and 19b are inserted into the inlet 16a and the outlet 16b, respectively. Each of the pipettes 19a and 19b has a function of injecting liquid into the flow channel 16 and sucking out from the flow channel 16, and when one of them performs the injection operation, the other performs the suction operation. So that they work together. Using this pipette pair 19, a ligand solution 21 in which a ligand is dissolved in a solvent is injected from the injection port 16a.

  A linker film 22 that binds to the ligand is formed at substantially the center of the sensor surface 13a. The linker film 22 is formed in advance at the manufacturing stage of the sensor unit 12. Since the linker film 22 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 treatment for injecting the ligand solution 21, as a pretreatment, first, a buffer solution for fixing is sent to the linker film 22 to wet the linker film 22, and then the linker film 22 is moved to the linker film 22. In order to facilitate the binding of the ligand, the linker film 22 is activated. For example, in the amine coupling method, carboxymethyl dextran is used as the linker film 22, 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 16 is washed by the fixing buffer.

  As the buffer for fixation and the solvent (diluent) of the ligand solution 21, 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, the concentration thereof, and the like are appropriately determined according to the type of ligand. For example, when a biological substance is used as a ligand, a physiological saline in which ph is adjusted to near neutral is often used. However, in the amine coupling method, the linker film 22 is negatively (negatively) charged by carboxymethyl dextran. Therefore, the protein is positively (positively) charged so as to be easily bonded to the linker film 22. 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 processing and cleaning, the ligand solution 21 is injected into the sensor cell 17 and the ligand immobilization processing is performed. When the ligand solution 21 is injected into the flow path 16, the ligand 21 a diffusing in the solution gradually approaches the linker film 22 and binds. In this way, the ligand 21a is fixed to the sensor surface 13a. The immobilization usually takes about one hour, and during this time, the sensor unit 12 is stored in a state where the environmental conditions including the temperature are set to predetermined conditions. The ligand solution 21 in the channel 16 may be allowed to stand while the immobilization proceeds, but the ligand solution 21 in the channel 16 is preferably stirred and flowed. By doing so, the binding between the ligand and the linker film 22 is promoted, and the amount of the ligand immobilized can be increased.

  When the immobilization of the ligand 21a on the sensor surface 13a is completed, the ligand solution 21 is discharged from the flow path 16. The ligand solution 21 is sucked and discharged by the pipette 19b. After the immobilization, the sensor surface 13a is subjected to a cleaning process by injecting a cleaning liquid into the channel 16. After this washing, if necessary, a blocking liquid is injected into the flow path 16 to perform a blocking process for inactivating the reactive group to which no ligand is bound in the linker film 22. As the blocking liquid, for example, ethanolamine-hydrochloride is used. After this blocking process, the channel 16 is washed again. Thereafter, as will be described later, an anti-drying liquid is injected into the channel 16. Thus, the sensor unit 12 is stored until measurement in a state in which the sensor surface 13a is immersed in the anti-drying liquid.

  The measurement process is performed with the sensor unit 12 set on the measuring machine 11. The measuring device 11 is provided with a pipette 26, and various liquids are injected from the inlet 16 a into the flow path 16 using the pipette 26. The pipette 26 can be adjusted in suction amount and discharge amount, and is driven by the drive unit 25. The diameter of the tip of the pipette 26 is substantially the same as the inner diameter of the injection port 16a. When the tip of the pipette 26 is arranged at the injection port 16a, the injection port 16a is closed. This prevents the entry of air from the inlet 16a at the time of injection, so that the analyte solution 27 is smoothly injected from the pipette 26 into the flow path 16.

  In the measurement (data reading) step, first, a measurement buffer is injected into the flow path 16. Thereafter, an analyte solution 27 in which the analyte is dissolved in a solvent is injected, and then the measurement buffer is injected again. Note that the flow path 16 may be once cleaned before the measurement buffer is first injected. A predetermined amount of the analyte solution 27 is injected into the channel 16. Then, the binding reaction with the ligand is examined while the analyte solution 27 is retained in the flow path 16 for a predetermined time.

  Data reading is started immediately after the measurement buffer is first injected in order to detect a reference signal level, and is performed after the analyte solution 27 is injected and until the measurement buffer is injected again. . As a result, it is possible to measure the SPR signal from detection of the reference level (baseline), reaction state of the analyte and ligand (binding state), and desorption of the combined analyte and ligand by injection of the measurement buffer.

  As the buffer for measurement and the solvent (diluent) of the analyte solution 27, 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, the concentration thereof, and the like 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 is used for detection of the reference level. Therefore, when DMSO is contained in the analyte solvent, it is preferable to use a measurement buffer having a DMSO concentration comparable to that DMSO concentration. .

  The analyte solution 27 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 ref signal level when the analyte solution 27 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 buffers having different DMSO concentrations into the sensor cell 17 before injecting the analyte solution 27, which will be described later according to the change in DMSO concentration at this time. It is obtained by examining the amount of change in each of the ref signal level and the act signal level.

  The measurement unit 31 includes an illumination unit 32 and a detector 33. As described above, 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), so that the illumination unit 32 can perform various incidents that satisfy the total reflection condition. The light of the corner is irradiated to the light incident surface 13b. The illumination unit 32 includes, for example, a light source 34 and an optical system 36 including a condensing lens, a diffuser plate, and a polarizing plate. The arrangement position and the installation angle satisfy the total reflection condition in terms of the incident angle of illumination light. To be adjusted.

  As the light source 34, 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. One such light emitting element is used, and light is emitted from this single light source toward one sensor cell. When measuring a plurality of sensor cells at the same time, the light from a single light source may be dispersed and irradiated to the plurality of sensor cells, or one light emitting element may be assigned to each sensor cell. A plurality of light emitting elements may be used side by side. The diffusion plate diffuses light from the light source 34 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, it is possible to cause the light incident surface 13b to enter the light rays having various incident angles without variation in light intensity.

  The detector 33 receives the light reflected by the light incident surface 13b and outputs an electrical signal having a level corresponding to the light intensity. Since light rays are incident on the light incident surface 13b at various angles, the light rays are reflected on the light incident surface 13b at various reflection angles according to respective incident angles. The detector 33 receives light beams having these various reflection angles. When a change occurs in the medium (surface plasmon) on the sensor surface 13a, the refractive index changes, and the reflection angle at which the light intensity attenuates (the resonance angle at which SPR occurs) also changes. When the analyte is fed onto the sensor surface 13a, the resonance angle changes according to the reaction state between the analyte and the ligand, so the reflection angle at which the light intensity attenuates also changes.

  The detector 33 uses, for example, a CCD area sensor or a photodiode array, receives reflected light reflected at various reflection angles on the light incident surface 13b, photoelectrically converts them, and outputs them as SPR signals. The reaction state between 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 sensor surface 13a is different, and the resonance angle (attenuation position of reflected light) at which SPR occurs is different. When the analyte comes into contact with the ligand and starts to react, the resonance angle of the reflected light starts to change accordingly, and the attenuation position of the reflected light in the light receiving surface starts to move. An SPR signal representing the reaction situation thus obtained is output to the data analyzer. In the data analysis step, the SPR signal obtained by the measuring instrument 11 is analyzed to analyze the interaction between the ligand and the analyte.

  For the sake of convenience, in FIG. 1, the direction of the incident light beam on the light incident surface 13 b and the reflected light beam reflected there is the flow direction of the liquid in the flow channel 16 so that the configuration of the measurement unit 31 becomes clear. Although the illumination unit 32 and the detector 33 are arranged so as to be parallel to each other, as shown in FIG. 2, in this embodiment, the directions of the incident light beam and the reflected light beam are orthogonal to the flow direction. The illumination part 32 and the detector 33 are arrange | positioned so that it may irradiate to a direction. Of course, the measurement unit 31 may be arranged and measured as shown in FIG.

  As shown in FIG. 2, on the linker film 22, a measurement region (act region) 22 a where a ligand is immobilized and a reaction between the analyte and the ligand occurs, and the ligand is not immobilized. A reference region (ref region) 22b for obtaining a reference signal is formed. These regions 22a and 22b are arranged side by side along the flow direction so that one is on the upstream side in the flow direction of the flow path 16 and the other is on the downstream side. In this example, the act region 22a is arranged on the upstream side, and the ref region 22b is arranged on the downstream side.

  The ref region 22b is formed when the linker film 22 described above is formed. As a formation method, for example, the linker film 22 is subjected to a surface treatment, and a binding group that binds to a ligand is deactivated in a region about half of the linker film 22. Thereby, half of the linker film 22 becomes the act region 22a, and the other half becomes the ref region 22b.

  The detector 33 outputs an SPR signal corresponding to the act region 22a as an act signal, and outputs an SPR signal corresponding to the ref region 22b as a ref signal. The act signal and the ref signal are measured almost simultaneously from the detection of the reference level to the desorption through the binding reaction. Data analysis is performed by obtaining the difference or ratio between the act signal and the ref signal thus obtained. For example, the data analyzer obtains difference data between the act signal and the ref signal, uses the difference data as measurement data, and performs analysis based on the difference data. By doing this, it becomes possible to cancel noise caused by disturbances such as individual differences between sensor units and sensor cells, mechanical fluctuations of the device, and temperature changes of the liquid, and a signal with a good S / N ratio. Is obtained.

  The illumination unit 32 and the detector 33 are configured so as to be able to measure two channels of these act signals and ref signals. For example, the illumination unit 32 splits one light emitting element into a plurality of light beams incident on the act region 22a and the ref region 22b using a reflection mirror or the like. Each light beam is received by a detector 33 constituted by a plurality of photodiode arrays for each channel.

  When a CCD area sensor is used as the detector 33, the reflected light of each channel simultaneously received can be recognized as an act signal and a ref signal by image processing. However, when such a method using image processing is difficult, the timings of incidence on the act region 22a and the ref region 22b may be shifted by a minute time to receive the signal of each channel. As a method of shifting the incident timing, for example, a disk with two holes formed at a position where the arrangement angle is shifted by 180 ° C. is arranged on the optical path, and the incident timing of each channel is rotated by rotating the disk. Is shifted. Each hole is arranged at a position where the distance from the center is different by the distance between the regions 22a and 22b, so that when one hole enters the optical path, a light beam enters the act region 22a and the other When the hole enters the optical path, the light beam enters the ref region 22b.

  FIG. 3 is an explanatory diagram showing a procedure for injecting the analyte solution 27 into the flow channel 16 during measurement. As shown in FIG. 3 (A), the pipette 26 first sucks the analyte solution 27 from the well plate arranged in the measuring instrument 11, and then the tip thereof is arranged in the injection port 16a. Since the diameter of the tip of the pipette 26 and the inner diameter of the inlet 16 a are almost the same, the inlet 16 a is closed by the pipette 26.

  In the suction of the analyte solution 27, the analyte solution 27 and the air 51 are alternately sucked. For example, first, the amount of the analyte solution 27a necessary to fill the flow path 16 (for example, 20 μl) is sucked, then 2 μl of the air 51a is sucked, and then about half of the first sucked amount. Aspirate (10 μl) of the analyte solution 27b. Finally, 2 μl of air 51b is sucked. After the preparation for injection is thus completed, signal measurement is started before the analyte solution 27 is injected.

  Then, after a predetermined time has elapsed after the start of measurement, as shown in FIG. 3B, the analyte solution 27 sucked by the pipette 26 and the air 51 are discharged, and injection into the flow channel 16 is started. . Air 51b, analyte solution 27b, air 51a, and analyte solution 27a are injected into the channel 16 in this order. Since the injection port 16a is blocked by the pipette 26, the air 51 in the pipette 26 does not leak outside. As a result, the leading air 51b passes through the sensor surface 13a and pushes the measurement buffer 52 to the discharge port 16b. Further, by injecting the air 51a prior to the analyte solution 27, the mixed solution of the analyte solution 27 and the measurement buffer 52 is suppressed.

  And as shown in FIG.3 (C), the pipette 26 continues discharge, and discharges the head air 51b from the discharge port 16b. This discharge operation continues until the air 51a injected later passes through the sensor surface 13a and a part of the analyte solution 27b is discharged from the discharge port 16b.

    As a result, the measurement buffer 52 and the analyte solution 27 are replaced in the flow channel 16. However, in this state, a part of the measurement buffer 52 remains on the sensor surface 13a, and the residual amount is larger in the downstream portion than in the upstream portion due to the velocity gradient of the fluid.

  Therefore, as shown in FIG. 3D, the pipette 26 starts the suction operation to cause the analyte solution 27 and the air 51 to flow backward. This suction operation is continued until the analyte solution 27b passes through the sensor surface 13a following the analyte solution 27a and air 51a, as shown in FIG. Thus, the analyte solution 27 and the air 51 are reciprocated on the sensor surface 13a. Then, as shown in FIG. 3 (F), the pipette 26 performs the discharge operation again to inject the analyte solution 27 and the air 51 into the flow path 16. This discharging operation continues until the analyte solution 27b and the air 51a pass through the sensor surface 13a and are discharged from the discharge port 16b, and the flow path 16 is filled with the analyte solution 27a. In this way, after the channel solution 16 is filled with the analyte solution 27a, the analyte solution 27a is retained in the channel 16 for a predetermined time, and measurement is performed. After a predetermined time has elapsed, the measurement buffer 52 is injected again, and the measurement is completed.

  Thus, since the analyte solution 27 and the air 51 are allowed to pass through the sensor surface 13a a plurality of times, the measurement buffer 52 remaining on the sensor surface 13a can be discharged. Moreover, since the reciprocating flow causes a flow in the forward direction and in the reverse direction, it is possible to eliminate the uneven amount of the residual amount generated on the sensor surface 13a due to the velocity gradient. As a result, the progress of the liquid replacement of the act region 22a and the ref region 22b can be made uniform, so that the effect expected by the two-channel measurement of the act signal and the ref signal can be obtained, and as a result, the measurement accuracy can be improved. Can do. In addition, due to the reciprocation, the fluidity of the analyte solution 27 is increased, and the contact amount between the analyte and the ligand is improved.

  Further, by injecting the analyte solution 27 and the air 51b, a higher replacement efficiency can be obtained as compared with the case of injecting only the analyte solution 27. In addition, the analyte solution 27 is divided into a plurality of portions by the air 51a, and a portion (analyte solution 27b) that is first injected into the flow path 16 reciprocates on the sensor surface 13a, and then the outside of the flow path 16. To be discharged. As a result, the channel 16 is washed with the analyte solution 27b, and the analyte solution 27a, which is less mixed than the previously injected analyte solution 27b, can be retained in the channel 16. Highly accurate signals can be detected.

  The amount of the analyte solution and air injected in the above embodiment is an example, and is appropriately determined according to the volume of the flow path and the like. Further, air and the analyte solution are alternately injected twice, but this number may be two times or more.

  In the above embodiment, air is injected into the flow path together with the analyte. However, air is not always necessary, and only the analyte solution may be injected and reciprocated on the sensor surface. When only an analyte solution is injected without using air at all, various effects (such as an effect of preventing liquid mixture and further improvement of liquid replacement efficiency) cannot be expected. However, even if air is not used, the effect of eliminating the unevenness of the residual amount of the measurement buffer on the sensor surface can be obtained by generating both the forward flow and the reverse flow of the analyte solution.

  In addition, air is injected in two portions, such as injecting air once before injecting the analyte solution, and then injecting air again after injecting the analyte solution. The air may be used only to divide the analyte solution into a plurality of portions without using the second air. If the first air is not used, the washing analyte solution for co-washing and the measurement buffer are in direct contact. For this reason, naturally, the amount of the measurement buffer mixed in the analyte solution for cleaning increases, leading to a reduction in the cleaning effect. However, since the cleaning analyte solution is discharged, the amount of decrease in the cleaning effect that is manifested as signal noise is not so large, and the influence on the measurement is considered to be small. The presence / absence of such air and the degree of use may be appropriately selected depending on the required measurement accuracy.

  In the above embodiment, the liquid once discharged from the discharge port of the flow path is discarded as it is and does not enter the flow path again. However, as shown in FIG. 4, a liquid reservoir 56 for retaining the discharged liquid is provided outside the discharge port 16 b of the flow path 16, and once the discharged analyte solution 27 flows back into the flow path 16. Good. In this way, a larger amount of the analyte solution 27 is flowed back, so that the liquid replacement efficiency is improved.

  Further, as shown in FIG. 5, the analyte solution 27 may be injected by arranging a pipette 58 that is interlocked with the operation of the pipette 26 at the discharge port 16 b. The pipette 58 performs a discharge operation in conjunction with the suction operation of the pipette 26 and performs a suction operation in conjunction with the discharge operation of the pipette 26. By disposing such a pipette 58, the flow rate of the analyte solution 27 in the flow channel 16 can be increased, so that the liquid replacement efficiency is improved.

  In addition, a pipette is used as a delivery / discharge unit that discharges and injects the liquid into the flow path and sucks and discharges the injected liquid. However, the pipette may not be a pipette. You may comprise from the other pump. Moreover, these may be used together, such as using a pipette on the injection side and using a pump on the discharge side.

  In this embodiment, the SPR sensor that generates SPR on the sensor surface and detects the attenuation of the reflected light at that time has been described as an example. However, the present invention is not limited to the SPR sensor, and the measurement is performed using total reflection attenuation. The present invention can also be applied when measuring the fixed amount of other sensors used. As a sensor using total reflection attenuation, for example, a leakage mode sensor is known in addition to the SPR sensor. 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 passes through the cladding layer and is taken into the optical waveguide layer. When a surface acoustic wave (SAW) is generated in this optical waveguide layer, the reflected light at the light incident surface is greatly attenuated. The incident angle at which the surface acoustic wave is generated varies 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 reflected light, the chemical reaction on the sensor surface is measured.

It is explanatory drawing of a SPR measuring method. It is explanatory drawing of the act area | region and ref area | region on a linker film | membrane. It is explanatory drawing which shows the injection | pouring procedure of the analyte solution in the case of a measurement. It is explanatory drawing of the example which provided the liquid storage part in the discharge port. It is explanatory drawing of the example which has arrange | positioned the pipette in the discharge port. It is explanatory drawing of the replacement method of an analyte solution and a buffer.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Fixing machine 11 Measuring machine 12 Sensor unit 13 Metal film 13a Sensor surface 16 Flow path 17 Sensor cell 22 Linker film 22a Act area 22b ref area 27 Analyte solution 31 Measuring part 32 Illumination part 33 Detector 51 Air 52 Measurement buffer

Claims (11)

A flow path that is formed on a transparent dielectric and uses a sensor with a thin film with one surface serving as a sensor surface to which a ligand is fixed and the other surface serving as a light incident surface. By injecting an analyte solution in which the analyte is dissolved in a solvent and feeding the solution onto the sensor surface, the light is incident on the light incident surface so as to satisfy the total reflection condition, and the attenuation of the reflected light is detected. In a measuring device using total reflection attenuation for measuring the interaction between the ligand on the sensor surface and the analyte,
First delivery / discharge means capable of discharging the analyte solution into the flow path and suctioning from the flow path is provided, and the analyte solution is once discharged onto the sensor surface by discharge using the first delivery / discharge means. After the liquid has been fed, the analyte solution is aspirated and back-flowed so that the analyte solution is reciprocated at least once on the sensor surface, and then the analyte solution is again fed to the sensor surface. Measuring device using total reflection attenuation.   Before the analyte solution is injected into the flow path, another liquid is injected, and when this other liquid is pushed out of the flow path by the injection of the analyte solution and discharged, 2. The measuring apparatus using attenuated total reflection according to claim 1, wherein liquid replacement in the flow path is performed.   The first feeding / discharging means alternately discharges the analyte solution and air to the flow path, passes the sensor solution on the sensor surface, and then sucks back the analyte solution and the air. The measuring apparatus using total reflection attenuation according to claim 1 or 2.   Of the plurality of portions of the analyte solution divided by the air, a portion previously injected into the flow path is discharged out of the flow path after reciprocating on the sensor surface. The measuring apparatus using the total reflection attenuation according to claim 3.   The measuring apparatus using total reflection attenuation according to any one of claims 1 to 4, wherein a pipette is used as the first feeding / discharging means.   The measuring apparatus using total reflection attenuation according to any one of claims 1 to 5, wherein a liquid reservoir for retaining the liquid overflowing from the discharge port is provided outside the discharge port of the flow path. .   A second feeding / discharging unit that performs a suction operation in conjunction with the discharging operation of the first feeding / discharging unit and a discharging operation in conjunction with the suction operation of the first feeding / discharging unit is provided at the discharge port of the flow path. The measuring apparatus using total reflection attenuation according to claim 1, wherein the measuring apparatus is disposed. A flow path that is formed on a transparent dielectric and has a thin film with one surface serving as a sensor surface to which a ligand is fixed and the other surface serving as a light incident surface, and is disposed opposite to the sensor surface. By injecting an analyte solution in which the analyte is dissolved in a solvent and feeding the solution onto the sensor surface, the light is incident on the light incident surface so as to satisfy the total reflection condition, and the attenuation of the reflected light is detected. In the measurement method using total reflection attenuation for measuring the interaction between the ligand on the sensor surface and the analyte,
The analyte solution is discharged on the sensor surface at least once on the sensor surface by discharging the analyte solution into the flow path and once feeding the sensor solution to the sensor surface and performing at least one suction to reverse the analyte solution. A measurement method using total reflection attenuation, wherein the analyte solution is reciprocated once and then the analyte solution is again fed to the sensor surface.   Another liquid is injected into the flow path before the analyte solution is injected, and the other liquid is pushed out of the flow path and discharged by the injection of the analyte solution. A measuring method using total reflection attenuation according to claim 8.   9. The analyte solution and the air are alternately discharged into the flow path and passed over the sensor surface, and then suction is performed to reversely flow the analyte solution and the air. Alternatively, a measurement method using the total reflection attenuation described in 9. Of the plurality of portions of the analyte solution divided by the air, the portion previously injected into the flow path is reciprocated on the sensor surface and then discharged out of the flow path. The measurement method using the total reflection attenuation according to claim 10.

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