JP2006292592A - Dispenser and measuring instrument utilizing total reflection attenuation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To prevent the falling-off of the pipette tip inserted in a nozzle. <P>SOLUTION: A nozzle 71 for sucking and discharging a liquid is provided on a head main body 70 and the pipette tip 62 is inserted in the nozzle 72. Two fixing plate 72 are pivotally provided on the head main body 70. Further, a solenoid 90 and a moving mechanism composed of a solenoid 90 and the connection member 92 connected to the movable iron core 91 of the solenoid 90 are provided on the head main body 70. The respective fixing plates are moved between a fixing position and a release position by the moving mechanism. The respective fixing plates present at the fixing position hold and fix the pipette tip 62 to prevent the falling-off of the pipette tip 62 from the nozzle 71. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a dispensing device that dispenses a liquid by inserting a pipette tip into a nozzle that sucks and discharges the liquid, and measurement using total reflection attenuation such as surface plasmon resonance equipped with the dispensing device. It relates to the device.

  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 dispenses a stored liquid such as a sample solution into 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 dispensed.

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 the liquid is fed into the flow path with a pipette, the tip of the pipette is inserted into the end of the exposed flow path, and the sucked liquid is discharged.
Japanese Patent No. 3294605

  The pipette tip is inserted into a nozzle formed in the head portion, and is held in the head portion by mechanical fitting with the nozzle. However, since the insertion direction of the pipette tip into the nozzle coincides with the insertion direction into the flow channel, when the pipette tip is pulled out from the flow channel, the pipette tip comes out of the nozzle and remains stuck in the flow channel. Sometimes it became. The pipette tip remaining in the flow path must be pulled out by the operator himself, which is time consuming, and if the process proceeds without noticing, not only normal measurement cannot be performed, but also the device and sensor. It may also cause damage to the unit.

  The present invention has been made in view of the above problems, and provides a dispensing device that prevents the pipette tip from coming out of the nozzle, and a measuring device that uses the total reflection attenuation provided with the dispensing device. For the purpose.

  In order to achieve the above object, the dispensing device of the present invention prevents a substantially cylindrical pipette tip inserted into a nozzle from a dispensing head provided with a nozzle for sucking and discharging liquid. It is provided with a retaining means.

  The retaining means includes a plurality of fixing plates arranged substantially orthogonal to the insertion direction of the pipette tip into the nozzle, and a fixing position for clamping and fixing the pipette tip inserted into the nozzle. It is preferable to include a moving mechanism that moves each of the fixing plates between a release position for releasing the clamping and fixing.

  Further, it is preferable that each of the fixing plates clamps and fixes the pipette tip when in the fixing position, and locks a part of the pipette tip with respect to a direction in which the pipette tip is removed.

  Furthermore, a notch that is an opening corresponding to the outer shape of the pipette tip when each of the fixing plates is in the fixing position is formed in a portion of the fixing plate that sandwiches and fixes the pipette tip. It is preferable.

  The retaining means is provided in the nozzle, a substantially cylindrical movable member movably accommodated in the nozzle, a moving mechanism for moving the movable member, and the pipette tip to the nozzle. An elastic member that is displaced between a retracted position that enables insertion and a protruding position that is pushed outward from the side surface of the nozzle by the movable member that has moved to the tip of the nozzle; The pipette tip may be prevented from coming off by pressing the inner surface of the pipette tip with the elastic member in position.

  Further, it is preferable that the elastic member at the projecting position presses the inner surface of the pipette tip and locks a part of the pipette tip with respect to the direction in which the pipette tip is removed.

  Furthermore, it is preferable that a plurality of the nozzles are formed in the dispensing head, and the retaining means prevent the plurality of pipette tips from being inserted into the plurality of nozzles.

  Further, the measuring device using total reflection attenuation according to the present invention includes a dielectric block having a thin film layer formed on one surface, and a flow path member having a flow path for supplying a sample solution to the thin film layer. A light source for irradiating the sensor unit with light so as to satisfy a total reflection condition; a detecting means for receiving reflected light from the sensor unit and photoelectrically converting it into an electrical signal; and suction and discharge of the sample solution A dispensing head for injecting the sample solution by inserting the tip of a substantially cylindrical pipette tip inserted into the nozzle into the inlet of the flow path, and the dispensing head. And a retaining means for preventing the pipette tip inserted into the nozzle from coming off.

  According to the dispensing device of the present invention and the measuring device using total reflection attenuation, since the dispensing head is provided with the retaining means for preventing the pipette tip inserted into the nozzle from coming off, for example, the pipette tip is allowed to flow. When the liquid is fed into the flow path by being inserted into the path, the pipette tip is prevented from fitting into the flow path and coming out of the nozzle.

  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 (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 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. 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 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 machine 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. . As a result, it is possible to measure the SPR signal until detection of the reference level (baseline), reaction state (binding state) of the analyte and ligand, and desorption of the combined analyte and ligand by injection of the buffer solution for measurement. it can.

  As the buffer solution for measurement and the solvent (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 (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 held in a state in which the flow path member 41 in which the flow path 16 is formed, the prism 14 with the metal film 13 formed on the upper surface, and the bottom surface of the flow path member 41 and the upper surface of the prism 14 are joined. And a lid member 43 disposed above the holding member 42.

  In the flow path member 41, for example, three flow paths 16 are formed. The flow path member 41 is formed in an elongated shape, and the three flow paths 16 are arranged 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. As a result, when the bottom surface of the flow path member 41 is pressed against the top surface of the prism 14, the flow path member 41 is elastically deformed to fill the gap between the joint surfaces with the metal film 13, and the open bottom of each flow path 16 is the prism. The upper surface of 14 is covered with water. In this example, the example in which the number of the flow paths 16 is three has been described. Of course, the number of the flow paths 16 is not limited to three, and may be one or two, or four or more. But you can.

  A metal film 13 is formed on the upper surface of the prism 14 by vapor deposition. The metal film 13 is formed in a strip shape so as to face the plurality of channels 16 formed in the channel member 41. Furthermore, a linker film 22 is formed on the upper surface (sensor surface 13 a) of the metal film 13 at a site corresponding to each flow path 16. 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, the 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 and the top surface of the prism 14 are in pressure contact with each other. Thus, the flow path member 41, the metal film 13, and the prism 14 are integrated.

  In addition, protrusions 14 b are provided at both ends in the short side direction of the prism 14. As will be described later, the sensor unit 12 is fixed in a state of being housed in a holder 52 (see FIG. 4). The protrusion 14 b is for positioning the sensor unit 12 at a predetermined storage position in the holder by fitting with the slit of the holder 52.

  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. The 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 the channel member 41 and engages with the prism 14, the lower surface of the receiving port 42 b is joined to the inlet 16 a and the outlet 16 b, and the receiving port 42 b and the channel 16 are connected.

  In addition, cylindrical bosses 42c are provided on both sides of each receiving port 42b. These bosses 42 c are for fitting the holes 43 a formed in the lid member 43 to position the lid member 43. The lid member 43 is affixed to the upper surface of the holding member 42 by a double-sided tape 44 having a hole in a position corresponding to the receiving port 42b and the boss 42c.

  The lid member 43 covers the receiving port 42 b communicating with the flow path 16, thereby preventing the liquid in the flow path 16 from evaporating. The lid member 43 is formed of, for example, an elastic material such as rubber or plastic, and a cross-shaped slit 43b is formed at a position corresponding to each receiving port 42b. Since the lid member 43 is for preventing evaporation of the liquid in the flow path 16, it is necessary to cover the receiving port 42b, but if it is completely covered, the pipette is inserted into the receiving port 42b. I can't. Therefore, by forming the slit 43b, the pipette can be inserted, and the receiving port 42b is closed when the pipette is not inserted. When the pipette is pushed into the slit 43b, the periphery of the slit 43b is elastically deformed (see FIG. 1), and the mouth of the slit 43b is wide open to receive the pipette. Then, when the pipette is pulled out, the slit 43b returns to the initial state by the elastic force and closes the receiving port 42b.

  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. .

  As shown in FIG. 4, in the fixing machine 10, a mounting space 51 for mounting a plurality of sensor units 12 is secured on a housing base 50 that is a base of the housing. The sensor unit 12 is subjected to all the fixing processes while being placed in the placement space 51. Accordingly, the mounting space 51 serves as a fixed stage that performs a fixing process on the sensor unit 12.

  The sensor unit 12 is set in the fixing machine 10 while being accommodated in the holder 52. The holder 52 can accommodate a plurality of (for example, eight) sensor units 12. The holder 52 is provided with a slit for positioning the sensor unit 12 by fitting with the protrusion 14 b of the sensor unit 12. Further, the bottom of the holder 52 is an opening except for support portions that support both ends of the sensor unit 12. In the measurement process, when the sensor unit 12 is taken out from the holder 52, a push-up member is inserted from the opening and the sensor unit 12 is pushed up from below.

  In the mounting space 51, for example, ten holders 52 can be arranged side by side, and pedestals 53 corresponding to the number of the holders 52 are provided. On each pedestal 53, a positioning boss for positioning the holder 52 is provided.

  The fixing machine 10 is provided with a pipette head (dispensing head) 54 in which three pipette pairs 19 are connected in series. The pipette head 54 accesses the sensor units 12 arranged in the placement space 51 to inject and discharge liquid. Since three pipette pairs 19 are provided in the pipette head 54, liquid can be injected (and discharged) simultaneously into the three sensor cells 17 included in one sensor unit 12. The fixed machine 10 is provided with a controller 60 for comprehensively controlling each part of the fixed machine 10, and the controller 60 controls the suction and discharge timing of each pipette pair 19, the suction amount and the discharge amount, and the like. Is done.

  The housing base 50 is provided with a head moving mechanism 56 that moves the pipette head 54 in three directions of X, Y, and Z. The head moving mechanism 56 is a known moving mechanism including, for example, a conveyance belt, a pulley, a carriage, and a motor. The head moving mechanism 56 moves up and down the pipette head 54, and moves the pipette head 54 in the Y direction together with the lifting mechanism. A Y-direction moving mechanism including a guide rail 58 that is freely held, and an X-direction moving mechanism that holds the guide rail 58 at both ends and moves the pipette head 54 together with the guide rail 58 in the X direction. The head moving mechanism 56 is controlled by a controller 60, and the controller 60 drives the head moving mechanism 56 to control the position of the pipette head 54 in the vertical and horizontal directions.

  A plurality of liquid storage units 61 for storing various liquids (ligand solution, cleaning liquid, fixing buffer liquid, anti-drying liquid, activation liquid, blocking liquid, etc.) injected into the flow path 16 on the housing base 50. Is provided. The number of the liquid storage units 61 is determined according to the type of liquid to be used. Each liquid storage unit 61 is provided with six insertion ports arranged side by side. The number of the insertion openings and the arrangement pitch are determined according to the number of pipettes of the pipette head 54 and the arrangement pitch. When injecting liquid into the sensor cell 17, the pipette head 54 accesses each liquid storage unit 61 to suck in a desired liquid, and then moves to the placement space 51 and injects it into the sensor unit 12.

  On the housing base 50, a pipette chip storage unit 63 for storing a plurality of pipette chips 62 and a well plate 64 in which a plurality of well-shaped ridges are arranged in a matrix are provided. The pipette tip 62 formed in a substantially conical cylinder shape is detachably attached to the pipette head 54. That is, each pipette 19a, 19b is configured by attaching the pipette tip 62 to the pipette head 54. Since the pipette tip 62 is in direct contact with the liquid, the pipette tip 62 is exchanged for each liquid to be used so that a mixture of different kinds of liquids does not occur through the pipette tip 62. The well plate 65 is used for temporarily storing the liquid sucked up by the pipettes 19a and 19b, or for adjusting a mixed liquid by mixing plural kinds of liquids.

  When fixing is started, the casing of the fixing machine 10 is covered with a cover (not shown), and the inside of the fixing machine 10 including the placement space 51 is shielded from the outside. The temperature in the fixing machine 10 can be adjusted by a temperature controller (not shown). After the ligand 21a is injected into the sensor cell 17, the sensor unit 12 is stored on the mounting space 51 for a predetermined time until the ligand 21a is fixed to the sensor surface 13a. During this storage, the ligand solution 21 in the flow path 16 is stirred as necessary. The degree of progress of immobilization during this period depends on the environmental conditions (temperature, etc.) of the sensor unit 12. Therefore, the internal temperature of the fixing machine 10 is kept at a predetermined temperature by the temperature controller. The set temperature, standing time, and the like are appropriately determined according to the type of the ligand 21a.

  When the fixing is completed, a buffer (cleaning liquid) is injected into the sensor cell 17. The buffer is injected into the sensor cell 17 by inserting the pipette 19 a sucking the buffer into the slit 43 b while the sensor cell 17 is filled with the ligand solution 21. When the buffer is discharged from the injection port 16a to the flow channel, the ligand solution 21 already injected into the flow channel 16 is pushed out toward the discharge port 16b and discharged. Then, the buffer to be discharged is sucked by the pipette 19b that performs the suction operation in conjunction with the injection operation of the pipette 19a. Thereby, replacement (replacement) of the liquid in the sensor cell 17 is performed.

  After the cleaning is completed, a drying prevention liquid for preventing the ligand 21a from drying may be injected into the sensor cell 17 by the same procedure as described above. This prevents the ligand from drying out until the measurement is started.

  FIG. 5 is an external perspective view showing the configuration of the pipette head 54. The pipette head 54 fixes a head main body 70 provided with a nozzle 71 for sucking and discharging liquid, two fixing plates 72 for holding and fixing a pipette tip 62 inserted into the nozzle 71, and the pipette tip 62. And a moving mechanism 73 for moving each fixing plate 72 between a fixing position for releasing and a releasing position for releasing the fixing and enabling the pipette tip 62 to be inserted and removed.

  The nozzle 71 is formed so as to protrude from the bottom surface of the head main body 70 in a substantially cylindrical shape, and its outer diameter and the inner diameter of the pipette tip 62 are substantially matched. When the pipette tip 62 is inserted into the nozzle 71, it is attached to the head main body 70 by mechanical fitting with the nozzle 71. Six nozzles 71 are formed in the X direction, and three pipette pairs 19 are configured by inserting pipette tips 62 into each of the nozzles 71. The nozzle 71 is connected to a pump (not shown) via the head body 70. The nozzle 71 transmits the pressure fluctuation from the pump to the pipette tip 62 and causes the pipette tip 62 to suck and discharge the liquid.

  Each fixed plate 72 bent in a substantially L shape has a first plate portion 75 that is parallel to the side surface of the head main body 70 when in a fixed position (the position shown in FIG. 5), and the first plate portion 75. A second plate portion 76 that is formed perpendicularly from one end and enters the bottom surface side of the head main body 70 so as to be substantially orthogonal to the insertion direction of the pipette tip 62 into the nozzle 71, and a predetermined amount on the other end of the first plate portion 75. The third plate portion 77 is formed to be inclined at an angle. The first plate portion 75 is provided with a hinge portion 80 in which a through hole is formed. The third plate portion 77 is provided with a plate-like link portion 81 in which a long hole 81a is formed so as to protrude vertically.

  At the four corners of the head main body 70, substantially long plate-like holding portions 82 protruding in the Y direction are formed. A round bar-shaped shaft 83 projects from the end of each holding portion 82. Each shaft 83 enters the through hole of each hinge portion 80. As a result, the head body 70 rotatably holds the fixed plates 72 via the holding portions 82, the shafts 83, and the hinge portions 80 of the fixed plates 72.

  The moving mechanism 73 provided on both side surfaces of the head main body 70 in the X direction includes a solenoid 90 attached to the head main body 70 and a long plate-like connecting member 92 attached to the movable iron core 91 of the solenoid 90. . For example, the solenoid 90 is electrically connected to the controller 60 of the stationary machine 10, and based on a drive signal from the controller 60, the storage position where the movable iron core 91 is stored in the main body and the protruding position where the movable core 91 protrudes from the main body are provided. Move between. In this example, a so-called push-type solenoid 90 that projects the movable iron core 91 while the drive signal is applied is used. Control of each position of the movable iron core 91 (ON / OFF of the drive signal) is performed so that the two solenoids 90 provided on both side surfaces are interlocked.

  At both ends of the connecting member 92, round bar-shaped shafts 93 project. Each shaft 93 enters the long hole 81 a of the link portion 81. The connecting member 92 moves in the Z direction according to the expansion and contraction of the movable iron core 91, and each shaft 93 is pivotally attached to the head main body 70 via each hinge portion 80 while sliding each shaft 93 in each elongated hole 81a. The fixed plate 72 is rotated. Thus, when the movable iron core 91 is in the retracted position, as shown in FIG. 6A, each fixed plate 72 is in the fixed position, and when the movable iron core 91 is in the protruding position, it is shown in FIG. As shown, each fixing plate 72 is in the release position.

  Each fixing plate 72 sandwiches the pipette tip 62 inserted into the nozzle 71 when in the fixing position. As shown in FIG. 5, a semicircular cutout 84 is formed at a portion of each fixing plate 72 that sandwiches each pipette tip 62. Each notch 84 is configured to substantially coincide with the outer diameter of the body portion 62a of the pipette tip 62 when the respective fixing plates 72 are in the fixed position and face each other. The pipette tip 62 is formed with a stepped portion 62b having a diameter larger than that of the body portion 62a. As shown in FIG. 6A, each fixing plate 72 in the fixing position sandwiches and fixes the body portion 62 a of the pipette tip 62 by the notch 84 and is larger than the circular opening formed by the notch 84. The step portion 62 b having a diameter is locked by the second plate portion 76. Thereby, when each fixing plate 72 is in the fixing position, the pipette tip 62 inserted into the nozzle 71 is reliably prevented from coming off.

  On the other hand, as shown in FIG. 6 (b), each fixing plate 72 in the release position rotates in accordance with the movement of the connecting member 92, and holds and fixes by the notch 84 and locking by the second plate portion 76. The pipette tip 62 can be attached and detached by releasing. The head body 70 is provided with a release mechanism (not shown). The release mechanism pushes down each pipette tip 62 inserted into each nozzle 71 to remove each pipette tip 62 from the head body 70.

  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 accommodated in the holder 52, and the holder 52 is set in the placement space 51. 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 60 applies a drive signal to each solenoid 90 in response to the fixation start instruction, moves the fixed plate 72 to the release position (see FIG. 6B) with the movable iron core 91 in the protruding position. The pipette head 54 in which each fixing plate 72 is set to the release position starts to move toward the pipette tip storage unit 63. The pipette head 54 moved to the pipette tip storage unit 63 inserts each nozzle 71 into the stored pipette tip 62 and attaches the pipette tip 62. The controller 60 having the pipette tip 62 attached to the pipette head 54 stops the application of the drive signal to each solenoid 90 and moves the movable iron core 91 to the storage position. When the movable iron core 91 moves to the storage position, each fixing plate 72 moves to the fixing position (see FIGS. 5 and 6A) via the connecting member 92 and the link portion 83. Each fixing plate 72 moved to the fixing position fixes each pipette tip 62 inserted into each nozzle 71.

  The pipette head 54 to which each pipette tip 62 is fixed moves to the liquid storage unit 61 that stores the activation liquid of the linker film 22, and causes each pipette 19a to suck the activation liquid. The pipette head 54 that has sucked the activation liquid moves toward the sensor unit 12 set in the placement space 51. The pipette head 54 that has moved to the sensor unit 12 inserts the tip of each pipette tip 62 into the inlet 16a and the outlet 16b of each channel 16, injects the activation liquid into each channel 16, The linker film 22 is activated. At this time, since each pipette tip 62 is fixed by each fixing plate 72, when the pipette tip 62 is pulled out from the flow path 16, the pipette tip 62 is restrained by each notch 84 and is engaged by each second plate portion 76. The pipette tip 62 is removed from the nozzle 71 and is prevented from being stuck in the flow path 16.

  The pipette head 54 into which the activation liquid has been injected moves to a discarding unit (not shown). The controller 60 that has moved the pipette head 54 to the discarding section puts each fixing plate 72 into the release position in the same procedure as described above, releases each pipette tip 62 from being fixed, and each pipette tip immersed in the activation liquid. 62 is released to the scrapping department. The pipette head 54 that has released each pipette tip 62 moves to the pipette tip storage unit 63 again, inserts the pipette tip 62 into the nozzle 71, and replaces the pipette tip 62 for the ligand solution 21 to be injected next. Thereafter, the fixing plates 72 are again set to the fixing positions by the same procedure as described above, and the pipette tips 62 are fixed. When each pipette tip 62 is released, each pipette tip 62 may be returned to the pipette tip storage unit 63 and a dedicated pipette tip 62 may be provided for each liquid to be injected.

  The pipette head 54 that has exchanged the pipette tip 62 moves to the liquid storage unit 61 that stores the ligand solution 21, and causes each pipette 19a to aspirate the ligand solution 21. The pipette head 54 that has aspirated the ligand solution 21 moves toward the sensor unit 12 set in the placement space 51. The pipette head 54 that has moved to the sensor unit 12 inserts the tip of each pipette tip 62 into the inlet 16a and outlet 16b of each channel 16, injects the ligand solution 21 into each channel 16, An immobilization process for immobilizing the ligand 21 a on the linker film 22 is performed.

  In the above-described embodiment, the moving mechanism 73 is configured by the solenoid 90 and the connecting member 92. However, the moving mechanism 73 is not limited thereto. For example, each fixing plate 72 may be directly rotated using a motor or the like. Then, each fixed plate 72 may be slid in the Y direction to switch between the fixed position and the release position. Further, the shape of each fixing plate 72 is not limited to the L shape, and for example, when the positions are switched by sliding movement, only the portion of the second plate portion 76 may be used.

  Moreover, in the said embodiment, although the level | step-difference part 62b is formed in the pipette tip 62 and it is made to latch on the 2nd board part 76 using the difference in diameter with the trunk | drum 62a, it does not restrict to this. For example, the second plate portion 76 may be locked by sandwiching the tapered portion of the pipette tip with the notch 84. Further, the frictional force generated when the body 62 a is clamped and fixed by the notch 84 is changed to the frictional force generated between the flow path 16 and the tip of the pipette tip 62 when the pipette tip 62 is pulled out from the flow path 16. The stepped portion 62b and the like need not be formed on the pipette tip 62 as long as they can sufficiently compete.

  In the above embodiment, an example in which the pipette tip 62 is sandwiched between the fixing plates 72 to prevent the removal is shown. However, as shown in FIG. 8, the pipette tip 62 is pressed from the inside to prevent the removal. Also good. As shown in FIGS. 8A and 8B, a ring member 100 is attached to the tip of the nozzle 71 via a connection member 101. The outer diameter and the wall thickness of the ring member 100 are matched with the nozzle 71. A rubber ring (elastic member) 102 is fitted in a gap between the nozzle 71 and the ring member 100 formed by the connection member 101. The inner diameter of the rubber ring 102 is slightly smaller than the nozzle 71 and the ring member 100. Further, the connection member 101 is formed with a notch that matches the gap. Thereby, the rubber ring 102 fitted in the gap enters the inner side of the inner surface of the nozzle 71 and the ring member 100 and contracts inward to press-contact with the connection member 101 (corresponding to the retracted position described in the claims). Held in.

  Inside the nozzle 71, a substantially cylindrical movable member 104 that moves up and down by the moving mechanism 103 is housed. The outer diameter of the movable member 104 is matched with the inner diameter of the nozzle 71. A slit 104 a for inserting the connection member 101 is formed on the side surface of the movable member 104. That is, the connection member 101 connects the nozzle 71 and the ring member 100 and also functions as a guide for the movable member 104. The moving mechanism 103 moves the movable member 104 in the nozzle 71 up and down a predetermined distance while guiding the connecting member 101. For the moving mechanism 103, for example, a known mechanism such as a solenoid or a rack and pinion may be used.

  As shown in FIG. 8C, the movable member 104 moved to the tip of the nozzle 71 by the moving mechanism 103 pushes the rubber ring 102 that has entered inside the inner surface of the nozzle 71. For this reason, the movable member 104 is formed with a taper whose diameter decreases toward the bottom surface so that the rubber ring 102 can be easily spread. The rubber ring 102 pushed and spread by the movable member 104 extends to a position (corresponding to a protruding position in claims) whose side surface protrudes outward from the side surfaces of the nozzle 71 and the ring member 100. The protruded side surface of the rubber ring 102 presses the inner surface of the pipette tip 105 inserted into the nozzle 71. As a result, the pipette tip 105 is pressed from the inside and is prevented from coming off from the nozzle 71.

  At this time, it is preferable that an overhanging portion 105 a projecting inward is formed in the vicinity of the insertion opening of the pipette tip 105 so as to be locked to the upper surface of the rubber ring 102 that has been spread out. In this example, the rubber ring 102 having a rectangular cross section is used as the elastic member. However, the present invention is not limited thereto, and for example, a rubber ring having a circular or elliptical cross section may be used. Furthermore, a plate-like member formed of a flexible metal or resin material may be used.

  Furthermore, in addition to the above two examples, as shown in FIG. 9, a nozzle 120 having a male screw 120a and a pipette tip 122 having a female screw 122a fitted to the male screw 120a are used. The pipette tip 122 may be fixed to the nozzle 120 by screwing to prevent the nozzle 120 from coming off.

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

  In the above-described embodiment, an example in which the pipette tips 62, 105, and 122 constituting each pipette pair 19 of the fixing device 10 are prevented from being detached is, of course, the pipette tip that constitutes the pipette pair 26 of the measuring device 11. You may make it prevent omission. Furthermore, the present invention may be applied to other dispensing devices that use pipette tips.

  Furthermore, in the above-described embodiment, an SPR measurement device is shown as an example of a measurement device using total reflection attenuation. However, for example, a leakage mode sensor is also known as a measurement device using total reflection attenuation. ing. The leakage mode sensor is composed of a dielectric, and a thin film composed of a clad layer and an optical waveguide layer that are sequentially layered thereon. One surface of the thin film serves as a sensor surface, and the other surface receives light. It becomes a surface. When light is incident on the light incident surface so as to satisfy the total reflection condition, a part of the light is transmitted through the cladding layer and taken into the optical waveguide layer. In this optical waveguide layer, when the waveguide mode is excited, the reflected light at the light incident surface is greatly attenuated. The incident angle at which the waveguide mode is excited changes according to the refractive index of the medium on the sensor surface, similar to the resonance angle of SPR. By detecting the attenuation of the reflection angle, the chemical reaction on the sensor surface is measured.

It is explanatory drawing 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 perspective view which shows the structure of a fixing machine roughly. It is an external appearance perspective view which shows the structure of a pipette head. It is explanatory drawing which shows the fixing position and cancellation | release position of a fixing plate. It is a flowchart which shows the procedure of a fixing process. It is explanatory drawing which shows the example which presses a pipette tip from an inner side. It is explanatory drawing which shows the example which fixes a pipette tip by screwing type.

Explanation of symbols

10 Fixing Machine 11 Measuring Machine 12 Sensor Unit 13 Metal Film (Thin Film Layer)
14 Prism (dielectric block)
32 Illumination part 33 Detector (detection means)
34 Light source 54 Pipette head (dispensing head)
62, 105, 122 Pipette tip 62b Stepped portion 71, 120 Nozzle 72 Fixed plate 73 Moving mechanism 84 Notch 102 Rubber ring (elastic member)
103 Moving mechanism 104 Movable member 105a Overhang portion

Claims (8)

In a dispensing apparatus equipped with a dispensing head provided with a nozzle for sucking and discharging liquid,
2. The dispensing apparatus according to claim 1, wherein the dispensing head includes a stopper means for preventing a substantially cylindrical pipette tip inserted into the nozzle from dropping out.   The retaining means includes a plurality of fixing plates arranged substantially perpendicular to the insertion direction of the pipette tip into the nozzle, a fixing position for holding and fixing the pipette tip inserted into the nozzle, and the clamping The dispensing apparatus according to claim 1, further comprising a moving mechanism that moves each of the fixing plates to and from a release position for releasing the fixation.   3. The fixing plate clamps and fixes the pipette tip when in the fixed position, and locks a part of the pipette tip with respect to a direction in which the pipette tip comes off. The dispensing device described.   A notch that is an opening corresponding to the outer shape of the pipette tip when each of the fixing plates is in the fixing position is formed in a portion of the fixing plate that sandwiches and fixes the pipette tip. The dispensing device according to claim 2 or 3, characterized in that   The retaining means is provided in a substantially cylindrical movable member movably accommodated in the nozzle, a moving mechanism for moving the movable member, and the nozzle, and the pipette tip is inserted into the nozzle. And an elastic member that is displaced between a retracted position that enables the nozzle and a protruding position that is spread outward from the side surface of the nozzle by the movable member that has moved to the tip of the nozzle, and is in the protruding position. The dispensing apparatus according to claim 1, wherein the elastic member presses an inner surface of the pipette tip to prevent the pipette tip from coming off.   6. The dispensing according to claim 5, wherein the elastic member at the projecting position presses the inner surface of the pipette tip and locks a part of the pipette tip with respect to a direction in which the pipette tip is removed. apparatus. A plurality of the nozzles are formed in the dispensing head,
The dispensing device according to any one of claims 1 to 6, wherein the retaining means prevents the plurality of pipette tips from being inserted into the plurality of nozzles. For a sensor unit comprising a dielectric block having a thin film layer formed on one surface and a flow path member having a flow path for supplying a sample solution to the thin film layer, light is transmitted so as to satisfy the total reflection condition. In a measuring device using total reflection attenuation, comprising: a light source that irradiates light; and a detecting means that receives reflected light from the sensor unit and photoelectrically converts the reflected light into an electrical signal.
A dispensing head which has a nozzle for sucking and discharging the sample solution, and injects the sample solution by inserting the tip of a substantially cylindrical pipette tip inserted into the nozzle into the inlet of the flow path; ,
A measuring device using total reflection attenuation, comprising: a stopper for preventing the pipette tip inserted into the nozzle from dropping out, provided in the dispensing head.

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