JP2006322896A - Liquid feeder, liquid feed method, and measuring instrument using total reflection attenuation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To prevent contamination caused by defective liquid feeding in a pipette. <P>SOLUTION: A liquid level sensor 60 comprising an LD 61 and a PD 62 is provided in a fixing machine 10. The LD 61 emits detection light DB toward a face of a liquid injected into a flow passage 16. The PD 62 is arranged to receive the detection light DB reflected on the liquid face, when the liquid face of the liquid injected into the flow passage 16 is located in a proper position. The liquid sensor 60 detects the propriety as to the proper position of a height of the liquid face, by changing a photoreception situation of the detection light DB, in response to the height of the liquid face. The occurrence of the defective liquid feeding is recognized by an operator, by detecting the height of the liquid face by this manner, since the height of the liquid face is proportional to the liquid injected into the flow passage 16. The contamination is thereby prevented from resulting from the defective liquid feeding in the pipette. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a liquid feeding apparatus for feeding a sample solution into a flow path using a pipette, a liquid feeding method thereof, and a measuring apparatus using total reflection attenuation such as surface plasmon resonance provided with the liquid feeding apparatus. Is.

  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 flow path and the prism are arranged on a measurement stage provided in the apparatus main body, and the above-described measurement can be performed by mounting a chip-type sensor unit having a metal film formed on a glass substrate on the measurement stage. Done.

  In Patent Document 1, the sample solution is directly fed into the flow path from a container for storing the sample solution via a pipe (tube) connected to a pump, a valve, or the like. There is a problem that so-called contamination is likely to occur, in which the sample 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. 2004-287615). 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. The flow path is a liquid feed pipe formed by penetrating the flow path member in a substantially U shape, and both ends thereof are exposed on the upper surface of the flow path member. When liquid is fed into the flow path using a pipette, the tip of the pipette is inserted into or pressed against each of both ends of the flow path. After connecting the two pipettes in this way, while sucking with one pipette to discharge the liquid (or air) in the flow path, the liquid is discharged with the other pipette and the fluid in the flow path is replaced. Is done.
Japanese Patent No. 3294605

  However, in the method using a pipette, more liquid than the predetermined amount has flowed into the flow path due to poor connection of the pipette such as insertion and over-pressing, and liquid leakage from the tip. Sometimes it was injected. Since the amount of liquid injected into and discharged from the flow path is controlled to be essentially constant, the amount that has been inadvertently injected cannot be completely discharged when the next liquid is fed, It will remain. For this reason, different types of liquids fed before and after are mixed in the flow path, which is a cause of so-called contamination.

  The present invention has been made in view of the above problems, and provides a liquid feeding device that prevents contamination caused by poor liquid feeding of a pipette, a liquid feeding method thereof, and a measuring device that uses total reflection attenuation. The purpose is to do.

  In order to achieve the above object, in the liquid delivery device of the present invention, a sensor unit in which a flow path having a plurality of inlets and outlets and a sensor surface for detecting a reaction of a sample are opposed to each other is detachably set. A pipette that is inserted into at least one of the inlets and outlets, injects a sample solution in which the sample is dissolved into the flow path, and sends the sample to the thin film layer; and a liquid containing the sample solution into the flow path Liquid level detecting means for detecting whether or not the height of the liquid level seen from the inlet / outlet into which the pipette is inserted is an appropriate position when injected is provided.

  In addition, it is preferable that the said liquid level detection means consists of the light emission part which irradiates a detection light toward the said liquid level, and the light-receiving part which light-receives the said detection light reflected on the said liquid level. Moreover, it is preferable that the light emitting unit has directivity.

  Further, when the liquid level detection means detects that the liquid level is not at an appropriate position, an abnormality control means for performing an abnormality detection process different from a normal process may be provided. At this time, it is preferable that the abnormality control means performs any one of abnormality notification, operation stop, and injection process re-execution as the abnormality detection process.

  In the liquid feeding method of the present invention, at least one of the respective inlets and outlets is provided for a sensor unit provided with a flow path having a plurality of inlets and outlets and a sensor surface for detecting the reaction of the sample. After inserting the pipette into the pipette and injecting the sample solution in which the sample is dissolved into the flow path, it is detected whether the liquid level seen from the inlet / outlet into which the pipette is inserted is at an appropriate position. Then, only when it is detected that the liquid is in an appropriate position, the next liquid is fed into the flow path.

  Further, when it is detected that the liquid level of the liquid is not an appropriate position, an abnormality detection process including at least one of an abnormality notification, an operation stop, and a reinjection process is performed. Is preferred.

  Furthermore, the measuring apparatus using total reflection attenuation according to the present invention is provided with a flow path having a plurality of entrances and a dielectric block on which a thin film layer for detecting a reaction of a sample is formed on one surface. A light source for irradiating the sensor unit with light so as to satisfy the total reflection condition; a light detecting means for receiving the reflected light from the sensor unit and photoelectrically converting it into an electrical signal; and at least one of the respective entrances / exits A pipette that is inserted into the flow path and injects the sample solution in which the sample is dissolved into the flow path and feeds the sample into the thin film layer; and when the liquid containing the sample solution is injected into the flow path, the pipette The liquid level detection means for detecting whether or not the height of the liquid level seen from the inlet / outlet inserted is an appropriate position is provided.

  In the present invention, when the liquid containing the sample solution is injected into the flow path, the liquid level detecting means detects whether or not the height of the liquid level seen from the inlet / outlet into which the pipette is inserted is an appropriate position. . Since the height of the liquid level is proportional to the amount of liquid injected into the flow path, if the liquid level is not at the proper position, liquid feeding failure occurs when the liquid is injected into the flow path with a pipette. Can be determined to have occurred. This makes it possible for the operator to recognize the occurrence of a liquid feeding failure, so that it is possible to prevent contamination caused by a pipetting liquid feeding failure by stopping the operation or restarting the liquid feeding. it can.

  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 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 (thin film layer) 13 whose one surface becomes a sensor surface 13a on which SPR occurs, and a prism (dielectric block) 14 bonded to the light incident surface 13b on the back surface of the sensor surface 13a. And a flow path member 41 which is disposed to face the sensor surface 13a and in which a flow path 16 through which a ligand or an analyte is fed is formed.

  As the metal film 13, 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 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 flow path 16 is a liquid feeding pipe bent in a substantially U shape, and has an inlet (inlet / outlet) 16a for injecting liquid and an outlet (inlet / outlet) 16b for discharging the liquid. 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. As will be described later, the sensor unit 12 includes a plurality of such sensor cells 17 (see FIG. 3).

  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 process for injecting the ligand solution 21, first, the immobilization buffer solution is sent to the linker film 22, and the linker film 22 is moistened to facilitate the binding of the ligand film 22. Processing is performed. 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 with the fixing buffer solution.

  As the buffer solution for fixing 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 The type of each of these liquids, the pH value, the type of mixture and its concentration, etc. are appropriately determined according to the type of ligand. For example, when a biological substance is used as a ligand, physiological saline whose pH is adjusted to near neutral is often used. However, in the above-described amine coupling method, the 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. Immobilization usually takes about one hour, and during this time, the sensor unit 12 is stored in a state where environmental conditions including 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, the drying preventing liquid is injected into the flow path 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 instrument 11. The measuring machine 11 is also provided with a pipette pair 26 similar to the pipette pair 19 of the fixing machine 10. By the pipette pair 26, various liquids are injected into the flow path 16 from the injection port 16a. In the measurement process, first, a measurement buffer solution 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 solution is injected again. Note that the flow path 16 may be once cleaned before the measurement buffer solution is first injected. 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. . Thus, it is possible to measure the SPR signal until detection of the reference level (baseline), the reaction state (binding state) of the analyte and the ligand, and the desorption of the bound analyte and the ligand by injection of the measurement buffer solution. it can.

  As the buffer solution for measurement and the solvent (diluted solution) of the analyte solution 27, 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 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 level of the reference signal (ref signal) when the analyte solution 27 is injected, and correction (DMSO concentration correction) is performed on the measurement data. Is called.

  Here, as will be described later, the reference signal (ref signal) is an SPR signal corresponding to a reference region provided on the sensor surface where the ligand is not fixed, and a measurement region where the ligand is fixed and a reaction with the analyte occurs. It is a signal that is compared and referenced with the measurement signal (act signal). At the time of measurement, two signals of the measurement signal and the reference signal are detected, and at the time of data analysis, for example, a difference between the two SPR signals is taken and analyzed as measurement data. By doing so, for example, noise caused by disturbances such as individual differences between a plurality of sensor cells and liquid temperature changes can be canceled, and a signal with a good S / N ratio can be obtained. .

  The correction data for correcting the DMSO concentration is obtained by injecting a plurality of types of measurement buffer solutions having different DMSO concentrations into the sensor cell 17 before injecting the analyte solution 27, and changing the ref according to the change in DMSO concentration at this time. It is obtained by examining the amount of change in each of the signal level and the act signal level.

  The measurement unit 31 includes an illumination unit 32 and a detector (light detection means) 33. As described above, since the reaction state between the ligand and the analyte appears as a change in the resonance angle (incident angle of the light irradiated to the light incident surface), 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 the single light source toward one sensor cell 17. When measuring a plurality of sensor cells 17 at the same time, the light from a single light source may be dispersed and irradiated to the plurality of sensor cells 17, or one light emitting element is provided for each sensor cell 17. A plurality of light emitting elements may be used side by side so as to be allocated. 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 on the sensor surface 13a, the refractive index changes, and the incident angle (resonance angle at which SPR occurs) of the light whose reflected light intensity is attenuated also changes. When the analyte is fed onto the sensor surface 13a, the refractive index on the sensor surface 13a changes according to the reaction state between the analyte and the ligand, and the resonance angle also changes accordingly.

  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 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 13a 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. 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 characteristics of 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, the directions of the incident light beam and the reflected light beam are actually orthogonal to the flow direction. The illumination unit 32 and the detector 33 are arranged so as to be irradiated. 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. 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, as described above, 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 can be canceled. Is possible.

  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 illuminating 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 shifted by 180 degrees on the optical path is arranged, and this disk is rotated to rotate the incident timing of each channel. 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 exploded perspective view of the sensor unit 12. The sensor unit 12 is a state in which the flow path member 41 in which the flow path 16 is formed, the prism 14 in which the metal film 13 is formed on the upper surface by vapor deposition, and the bottom surface of the flow path member 41 and the upper surface of the prism 14 are joined. And a holding member 42 to be held. A plurality of (six in this example) linker films 22 for immobilizing ligands are provided on the surface of the metal film 13. Each linker film 22 is provided at a predetermined interval along the longitudinal direction of the long prism 14 and the metal film 13. A flow path member 41 is prepared for each linker film 22 and arranged side by side on the metal film 13 so that each flow path 16 and each linker film 22 face each other. In this example, the number of linker films 22 and flow path members 41 is six. However, the number is not limited to six, and may be five or less, or may be seven or more. In this example, six flow path members 41 each having one flow path 16 are used. However, a long flow path member in which six flow paths 16 are formed side by side is used. May be.

  The channel member 41 is formed in a substantially rectangular parallelepiped shape, and the channel 16 is formed in a substantially U shape along the longitudinal direction thereof. This flow path 16 constitutes a sensor cell 17 (see FIG. 1) together with the metal film 13 bonded to the bottom surface thereof. Therefore, the flow path member 41 is formed of an elastic material such as rubber or PDMS (polydimethylsiloxane) in order to improve the adhesion with the metal film 13. When the bottom surface of the flow path member 41 is brought into pressure contact with the top surface of the prism 14, the flow path member 41 is elastically deformed to fill a gap in the joint surface with the metal film 13. As a result, the open bottom of the flow path 16 is covered with the upper surface of the prism 14 in a watertight manner.

  Engaging claws 14 a that engage with the engaging portions 42 a of the holding member 42 are provided on both side surfaces of the prism 14 in the longitudinal direction. By these engagements, each flow path member 41 is sandwiched between the holding member 42 and the prism 14, and is held in a state where the bottom surface thereof and the upper surface of the prism 14 are pressed against each other. In addition, protrusions 14 b are provided at both ends in the short side direction of the prism 14. The sensor unit 12 is set in the fixing device 10 or the measuring device 11 in a state of being housed in a holder (not shown). The protrusion 14b engages with the slit of the holder to position the sensor unit 12 at a predetermined storage position in the holder.

  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 42, a receiving port 42b into which the tip of the pipette (19a, 19b, 26a, 26b) enters is formed at a position corresponding to the inlet 16a and the outlet 16b of each channel 16. Each receiving port 42b has a funnel shape so that the liquid discharged from the pipette is guided to each inlet 16a. When the holding member 42 sandwiches each flow path member 41 and engages with the prism 14, the lower surface of each reception port 42 b is joined to the injection port 16 a and the discharge port 16 b, and each reception port 42 b is connected to the flow channel 16. Is done.

  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 42 of the sensor unit 12. For example, the sensor unit 12 can be identified by writing a unique ID number for each sensor unit 12 in a read-only RFID tag and reading this ID number before performing each process. Thereby, even when fixing and measuring the plurality of sensor units 12 at the same time, it is possible to prevent the occurrence of problems such as erroneous injection of 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. 4 is a configuration diagram schematically showing the configuration of the fixing device 10. The fixing machine 10 is configured to detachably hold a pipette tip 50 formed in a substantially conical cylindrical shape, and to suck or discharge liquid into the pipette tip 50 by pressurizing or depressurizing the inside of the pipette tip 50. A pump 52, a head moving mechanism 53 that moves the pipette head 51 in three directions, front, rear, left, right, and up, and a controller 54 that controls each part of the fixing machine 10 in an integrated manner.

  The pipette head 51 is formed with two nozzles 55 protruding in a substantially cylindrical shape. The outer diameter of each nozzle 55 substantially matches the inner diameter of the pipette tip 50. When the pipette tip 50 is inserted into each nozzle 55, the pipette tip 50 is held by the pipette head 51 by mechanical fitting with the nozzle 55. That is, each pipette 19a, 19b is configured by inserting the pipette tip 50 into each nozzle 55. Further, the pipette head 51 is provided with a release mechanism (not shown), and the pipette tip 50 inserted into each nozzle 55 is pushed down to remove the pipette tip 50 from the pipette head 51. Since the pipette tip 50 is in direct contact with the liquid to be fed, the pipette tip 50 is exchanged every time the liquid is fed so as not to cause a mixed liquid of different kinds of liquids through the pipette tip 50.

  As the two pumps 52 provided for each pipette 19a, 19b, for example, a so-called syringe pump composed of a cylinder and a piston can be used. Each pump 52 is connected to each nozzle 55 via a pipe 56 and a pipette head 51. Each pump 52 causes the pipettes 19a and 19b to suck the liquid by depressurizing the inside of the pipe path leading to each nozzle 55, and discharges the liquid sucked to each pipette 19a and 19b by pressurizing the inside of the pipe path. Let Each pump 52 is electrically connected to, for example, the controller 54. The controller 54 transmits a drive signal to each pump 52 via a pump driver (not shown), and controls the suction and discharge timing, the suction amount and the discharge amount, and the like.

  The head moving mechanism 53 is a known moving mechanism including, for example, a conveyance belt, a pulley, a carriage, and a motor. The head moving mechanism 53 moves the pipette head 51 in three directions, front, rear, left, right, up and down, under the control of the controller 54. The fixing device 10 includes a plurality of liquid storage units for storing various liquids (ligand solution, washing solution, fixing buffer solution, anti-drying solution, activation solution, blocking solution, etc.) to be injected into the flow path 16. A pipette tip storage unit for storing the pipette tips 50 (not shown) is installed. The head moving mechanism 53 causes the pipette head 51 to access these parts and the sensor unit 12 set in the fixing machine 10.

  Further, the fixing machine 10 includes a laser diode (corresponding to a light emitting unit described in claims, hereinafter referred to as “LD”) 61 and a photodiode (corresponding to a light receiving unit described in claims, hereinafter referred to as “PD”) 62. A liquid level sensor (liquid level detection means) 60 is provided. The liquid level sensor 60 is provided corresponding to each of the inlet 16a and the outlet 16b, and when the liquid is injected into the flow path 16, the liquid level that can be seen from the inlet 16a and the outlet 16b. Detects whether the height is an appropriate position.

  Each LD 61 irradiates the detection light DB toward the liquid level LS of the liquid injected into the flow path 16 through the receiving port 42b of the holding member 42, the injection port 16a, and the discharge port 16b. On the other hand, as shown in FIG. 5A, each PD 62 receives the detection light DB reflected by the liquid surface LS when the liquid surface LS of the liquid injected into the flow channel 16 is at an appropriate position. Is arranged. As shown in FIG. 5B, when the height of the liquid level LS is higher than the appropriate position, the detection light DB reflected by the liquid level LS deviates from the light receiving surface of the PD 62. Further, as shown in FIG. 5C, when the height of the liquid level LS is lower than an appropriate position, the detection light DB is blocked by the holding member 42 and the like. The liquid level sensor 60 detects whether or not the height of the liquid level LS is an appropriate position by changing the light receiving state of the detection light DB in accordance with the height of the liquid level LS.

  Each LD 61 and each PD 62 are electrically connected to the controller 54, for example. The controller 54 transmits a drive signal to each LD 61 to cause each LD 61 to emit light and monitor the output voltage of each PD 62. Since the PD 62 outputs a higher voltage as the amount of incident light increases, the PD 62 outputs a lower voltage while detecting that the liquid level LS is not in an appropriate position, receives the detection light DB, and the liquid level LS is appropriate. A high voltage is output in response to the detection of being in the correct position. The controller 54 monitors the output voltage. For example, when the output voltage of the PD 62 exceeds a predetermined threshold value, the liquid level sensor 60 detects that the liquid level LS is in an appropriate position. to decide. Such a comparison operation circuit (comparator or the like) may be provided in the liquid level sensor 60 in advance, and digitally output whether or not the position is appropriate.

  Next, the operation of the fixing machine 10 having the above configuration will be described with reference to the flowchart shown in FIG. When performing the fixing process on the sensor unit 12, first, the sensor unit 12 is stored in a holder (not shown), and the holder is set in a predetermined placement space of the fixing machine 10. After the sensor unit 12 is set, when a fixing start instruction is input from the operator, the fixing machine 10 starts the fixing process.

  The controller 54 drives the head moving mechanism 53 in response to the input of the fixation start instruction, and moves the pipette head 51 to the liquid storage unit that stores the activation liquid of the linker film 22. The controller 54 that has moved the pipette head 51 to the liquid storage unit drives the pump 52 of the pipette 19a via the pump driver, and causes the pipette 19a to suck a predetermined amount of the activation liquid. The controller 54 that has caused the pipette 19a to suck the activation liquid moves the pipette head 51 to the sensor unit 12, and inserts each of the pipettes 19a and 19b into the inlet 16a and the outlet 16b of the flow path 16, respectively.

  The controller 54 having the pipettes 19a and 19b inserted into the flow path 16 drives the pumps 52 so that the pipette 19a performs discharge and the pipette 19b performs suction. The pipette 19a discharges the activation liquid held in the pipette tip 50 and injects it into the flow path 16, and the pipette 19b sucks the air in the flow path 16 or the previously injected cleaning liquid, etc. 16 is discharged. As a result, the fluid in the flow path 16 is replaced with the activation liquid from the air or the cleaning liquid, and the linker film 22 is activated.

  The controller 54 that has injected the activation liquid into the flow path 16 drives the head moving mechanism 53 to raise the pipette head 51, and pulls out the pipettes 19 a and 19 b from the flow path 16. The controller 54 that has pulled out each pipette 19a, 19b from the flow path 16 transmits a drive signal to each LD 61 of the liquid level sensor 60 to cause each LD 61 to emit light, and also monitors the output voltage of each PD 62, thereby It is detected whether or not the height of the liquid level LS of the activation liquid injected into the liquid is at an appropriate position. Since the height of the liquid level LS is proportional to the amount of the activation liquid injected into the flow path 16, when the height of the liquid level LS is not at an appropriate position, the activation liquid is supplied by the pipettes 19 a and 19 b. When injecting into the flow path 16, it can be determined that a liquid feeding failure has occurred.

  When each liquid level sensor 60 detects that the height of the liquid level LS is at an appropriate position, the controller 54 moves the pipette head 51 to the pipette tip storage unit. The pipette head 51 moved to the pipette tip storage unit releases each pipette tip 50 immersed in the activation liquid, and inserts an unused pipette tip 50 into each nozzle 55. In this way, by exchanging the pipette tip 50, contamination of the ligand solution 21 to be fed next and the activation liquid through the pipette tip 50 is prevented. When the liquid discharged from the flow path 16 is held in the pipette 19b, the pipette head 51 is moved to a waste liquid tank (not shown) before replacing the pipette tip 50, and the pipette 19b. The liquid inside is discharged into the waste liquid tank.

  The controller 54 that has exchanged the pipette tip 50 moves to a liquid storage unit for storing the ligand solution 21 and causes the pipette 19a to aspirate the ligand solution 21. The controller 54 that has aspirated the ligand solution 21 moves the pipette head 51 to the sensor unit 12, inserts it into the flow channel 16 of each pipette 19 a, 19 b in the same procedure as the activation liquid, injects the ligand solution 21, In addition, the pipettes 19 a and 19 b are pulled out from the flow path 16, and an immobilization process for fixing the ligand 21 a to the linker film 22 is performed. In addition, before pulling out each pipette 19a, 19b, it is also possible to stir the ligand solution 21 injected into the flow path 16 by alternately repeating suction and discharge to each pipette 19a, 19b. 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.

  On the other hand, if any one of the liquid level sensors 60 detects that the height of the liquid level LS is not an appropriate position, the controller 54 notifies the operator of the abnormality by, for example, a warning sound or a warning light. An abnormality detection process different from a normal process, such as stopping the operation or discharging the activation liquid in the flow path 16 once and reinjecting it, is performed. In addition to these, the fact that an abnormality has been detected is recorded in, for example, the RFID tag (not shown) of the controller 54 or the sensor unit 12, and the presence or absence of the abnormality can be determined later. The same processing as when appropriate may be continued.

  By performing the abnormality detection process in this manner, when each pipette 19a, 19b injects the activation liquid into the flow path 16, it is possible to make the operator recognize that a liquid feeding failure has occurred. Contamination between the activation liquid and the ligand solution 21 due to the poor liquid feeding of 19a and 19b is prevented. Of course, after injecting the ligand solution 21, it may be detected whether the height of the liquid level LS of the ligand solution 21 is an appropriate position.

  In the above embodiment, the liquid level sensor 60 is provided corresponding to each of the inlet 16a and the outlet 16b. However, the liquid level sensor 60 is not limited to this, and each liquid level sensor 60 is moved to You may make it detect whether the height of the liquid level LS seen from the injection port 16a and the discharge port 16b is an appropriate position. Moreover, in the said embodiment, each pipette 19a, 19b is inserted in each of the injection port 16a and the discharge port 16b, and discharge and suction | inhalation of each pipette 19a, 19b are mutually interlocked, A liquid is supplied to the flow path 16. Although the fixing device 10 for injection is shown, the present invention is not limited to this, and the present invention may be applied to, for example, a fixing device 80 in which only the injection side is pipetted as shown in FIG. Components that are the same in function and configuration as those in the above embodiment are given the same reference numerals, and detailed description thereof is omitted.

  The pipette head 81 of the fixing machine 80 includes a pipette 82 provided corresponding to the injection port 16a and a suction pipe 83 provided corresponding to the discharge port 16b. The pipette 82 includes a nozzle 84 that protrudes from the pipette head 81 in a substantially cylindrical shape, and a pipette tip 85 that is replaceably fitted into the nozzle 84, similarly to the pipettes 19 a and 19 b of the above embodiment. The pipette 82 performs suction and discharge of the liquid according to the drive of the connected pump 52, and injects the sucked liquid into the flow path 16 when inserted into the injection port 16a.

  The suction tube 83 is a substantially cylindrical tube that protrudes from the pipette head 81, and is formed so that the length thereof is substantially the same as the pipette 82. When the suction pipe 83 is inserted into the discharge port 16 b, the liquid injected by the pipette 82 according to the drive of the connected pump 52 is sucked out from the flow path 16, and the waste liquid tank is connected via the pump 52 and the pipe 86. To 87. The pump 52 connected to the suction pipe 83 moves the fluid in each pipe in one direction. For example, a plunger pump or a centrifugal pump can be used.

  The pipette head 81 inserts the pipette 82 and the suction tube 83 into the injection port 16a and the discharge port 16b, respectively, injects with the pipette 82, and discharges with the suction tube 83, thereby sending the liquid to the flow path 16. . At this time, after injecting the liquid with the pipette 82, the liquid level sensor 60 detects whether the level of the liquid level seen from the injection port 16a is an appropriate position, thereby obtaining the same effect as the above embodiment. Can do.

  In the above two embodiments, the LD 61 is used as the light emitting part of the liquid level detecting means, but an LED or the like can be used as the light emitting part. Furthermore, although a light bulb, a fluorescent lamp, or the like may be used, it is preferable to use a light source having high directivity, such as an LD or an LED, in order to suppress the influence of scattered light.

  In the above-described two embodiments, the PD 62 is used as the light receiving unit of the liquid level detection unit. However, the present invention is not limited to this, and an image sensor such as a CCD line sensor may be used. In the PD 62, the detection light DB is received only when the liquid level LS is at an appropriate position to detect whether or not the height of the liquid level LS is at an appropriate position. However, when a CCD line sensor or the like is used. In this case, the light receiving position of the detection light DB is changed according to the height of the liquid level LS, and it is possible to detect whether or not the height of the liquid level LS is an appropriate position based on the light receiving position.

  Further, as the liquid level detection means, for example, a liquid level gauge can be used in addition to the liquid level sensor 60 composed of the LD 61 and the PD 62. The liquid level gauge instructs to raise or lower the liquid level by floating the float on the liquid level. It may be possible to detect whether or not the liquid level is at an appropriate position by inserting the liquid level gauge through the inlet 16a and the outlet 16b and measuring the height of the liquid level.

  In the two embodiments described above, the liquid is injected into the flow path 16 by inserting the tips of the pipettes 19a, 19b, and 82 into the inlet 16a and the outlet 16b. However, the present invention is not limited to this. The pipette 19a, 19b, 82 may be injected into the flow channel 16 with the tip of the pipette 19a pressed against the inlet 16a, the outlet 16b.

  In the above-described two embodiments, the prism 14 is shown as the dielectric block. However, in addition to the dielectric block, optical glass, optical plastic, or the like, or those plate-like shapes are used. It is intended to include those obtained by integrating an object and a prism with an optical surface smoothing agent (for example, optical matching oil).

  Furthermore, in the above two embodiments, an SPR measurement device is shown as an example of a measurement device using total reflection attenuation. However, as a measurement device using total reflection attenuation, for example, a leakage mode sensor is used. Are known. 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 of a SPR measuring method. It is explanatory drawing which extracts and demonstrates one sensor cell. It is a disassembled perspective view which shows schematic structure of a sensor unit. It is a block diagram which shows the structure of a fixing machine roughly. It is explanatory drawing which shows the change of the light reception condition of PD according to the height of a liquid level. It is a flowchart which shows the procedure of a fixing process. It is a block diagram which shows other embodiment of a fixing machine.

Explanation of symbols

10, 80 Fixing machine 11 Measuring machine 12 Sensor unit 13 Metal film (thin film layer)
14 Prism (dielectric block)
16 Channel 16a Inlet (entrance / exit)
16b Outlet (entrance / exit)
19 Pipette pair 19a Pipette 19b Pipette 32 Illumination part 33 Detector (light detection means)
34 Light source 54 Controller (Abnormal control means)
60 Liquid level sensor (Liquid level detection means)
61 Laser diode (light emitting part)
62 Photodiode (light receiving part)
82 Pipette 83 Suction tube

Claims (8)

A sensor unit provided with a flow path having a plurality of inlets and outlets and a sensor surface for detecting the reaction of the sample facing each other is detachably set, and by a pipette inserted into at least one of the inlets and outlets, In a liquid feeding device for feeding the sample to the sensor surface by injecting the sample solution in which the sample is dissolved into the flow path,
When a liquid containing the sample solution is injected into the flow path, liquid level detection means is provided for detecting whether or not the height of the liquid level seen from the inlet / outlet into which the pipette is inserted is an appropriate position. A liquid feeding device characterized by.   2. The transmission according to claim 1, wherein the liquid level detection unit includes a light emitting unit that emits detection light toward the liquid level and a light receiving unit that receives the detection light reflected by the liquid level. Liquid device.   The liquid delivery device according to claim 2, wherein the light emitting unit has directivity.   The abnormality control means for performing an abnormality detection process different from a normal process when the liquid level detection means detects that the height of the liquid level is not an appropriate position. 4. The liquid feeding device according to any one of 1 to 3.   5. The liquid feeding device according to claim 4, wherein the abnormality control means performs any of abnormality notification, operation stop, and injection process re-execution as the abnormality detection process. A pipette is inserted into at least one of the inlets and outlets with respect to a sensor unit provided with a flow path having a plurality of inlets and outlets and a sensor surface for detecting the reaction of the sample. In the liquid feeding method of feeding the sample to the sensor surface by injecting the sample solution in which the sample is dissolved into the flow path,
After injecting the liquid containing the sample solution into the flow path, detect whether the height of the liquid level seen from the inlet / outlet into which the pipette is inserted is an appropriate position,
Only when it is detected that the liquid is in an appropriate position, the next liquid is fed into the flow path.   When it is detected that the liquid level of the liquid is not an appropriate position, an abnormality detection process including at least one of an abnormality notification, an operation stop, and a reinjection process is performed. The liquid feeding method according to claim 6. Light is applied so that the total reflection condition is satisfied with respect to a sensor unit in which a flow path having a plurality of entrances and exits and a dielectric block having a thin film layer for detecting a reaction of a sample formed on one surface are opposed to each other. A light source for irradiating; a light detecting means for receiving reflected light from the sensor unit and photoelectrically converting it into an electrical signal; and a sample solution in which the sample is dissolved and inserted into at least one of the inlets and outlets. In a measuring device using total reflection attenuation, comprising a pipette that is injected into the pipette and feeds the sample into the thin film layer,
When a liquid containing the sample solution is injected into the flow path, liquid level detection means is provided for detecting whether or not the height of the liquid level seen from the inlet / outlet into which the pipette is inserted is an appropriate position. Measuring device using total reflection attenuation.

Images