JP2006226780A - Measuring device using total reflection attenuation, and its measuring method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To prevent occurrence of latency time by temperature regulation of a sensor unit in continuous measurement, in a measuring device using total reflection attenuation. <P>SOLUTION: A measuring machine 6 comprises a holder conveyance mechanism 71, a pickup mechanism 72, a measuring stage 73, and a stock section 74. Each of them is stored in case 75. A base plate 75a and side plate 75b of the case 75 have a jacket section 80 through which water circulates. By circulating water heated or cooled by a Peltier element 76 through each jacket section 80 with a circulation pump 77, the ambient temperature in the case 75 is adjusted to be constant. The stock section 74 is provided with three-stage shelf boards 92. A sensor unit 12 of a measurement waiting state after the completion of a fixing process, together with a holder 52, is mounted on each shelf board 92, thereby matching the temperature of each sensor unit 12 with the ambient temperature in the case 75. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a measuring apparatus using total reflection attenuation such as surface plasmon resonance and a measuring method thereof.

  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.

  In order to prevent denaturation and inactivation due to drying, 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. The SPR measurement device described in Patent Document 1 examines such interaction between biological samples, and a flow path for feeding a sample solution is provided on the sensor surface. The sensor surface is provided with a linker film for immobilizing the ligand sample. After injecting the ligand solution into the flow path to immobilize the ligand on the linker film (an immobilization process), the analyte solution is By injecting and contacting the ligand and the analyte (measurement process), the interaction is examined.

  In addition, the SPR measurement device includes a light source that emits light to the sensor unit and a detection unit that receives reflected light reflected by the sensor unit, and a measurement stage that performs SPR signal measurement is provided. The sensor unit is detachably set on the measurement stage.

In the SPR measurement apparatus described in Patent Document 2 below, a heater is provided on the measurement stage, and the temperature of the sample solution in the sensor unit is adjusted to a predetermined temperature by the heater to perform measurement. This is because the refractive index on the metal film that affects the resonance angle also changes depending on the temperature of the sample solution serving as a medium, so that the measurement signal changes under the influence of the temperature. For this reason, if the temperature at the time of each measurement is different among a plurality of sensor units that are set on the measurement stage in order and measured, it is impossible to accurately compare the acquired measurement data. Therefore, the temperature of the sample solution is adjusted by the heater.
Japanese Patent No. 3294605 Japanese Patent No. 3468091

  However, in the SPR measurement device described in Patent Document 2, since temperature adjustment by the heater is started after the sensor unit is set on the measurement stage, a waiting time is generated until the temperature adjustment is completed. Such a waiting time is not so much a problem if there are few sensor units that perform measurement, but if the number is large, it becomes a major factor that deteriorates the processing efficiency of the measurement work. Therefore, the temperature adjustment of the sensor unit in the measurement standby state is performed in advance by using a temperature adjustment device (for example, a thermostatic bath) separate from the SPR measurement device, and the temperature-adjusted sensor unit is removed from the temperature adjustment device. A method of sequentially taking out and setting on the measurement stage of the SPR measurement device is also conceivable. However, in this case, it is necessary to prepare a temperature control device in a separate housing from the SPR measurement device, which is not only disadvantageous in terms of cost and installation space, but also moves from the temperature control device to the SPR measurement device. There is concern that the temperature will change during the process. The temperature difference allowed between the plurality of sensor units is preferably within 0.1 ° C. However, in the method using the temperature control device of another casing, there is a problem that it is difficult to keep the temperature difference within the range. there were.

  The present invention has been made in view of the above problems, and prevents the occurrence of waiting time due to temperature adjustment and the temperature difference between the sensor units when measuring a plurality of sensor units continuously. An object of the present invention is to provide a measuring apparatus using the attenuated total reflection and a measuring method thereof.

  In order to achieve the above object, in the measuring apparatus using total reflection attenuation according to the present invention, one sensor unit for detecting a reaction state of a sample using the total reflection attenuation of light is set one by one. A light source that emits light so as to satisfy the total reflection condition, and a detection means that receives the reflected light from the sensor unit and photoelectrically converts it into an electrical signal, and measures the reaction status of the sample A measurement stage on which processing is executed, a stock unit for accumulating at least one sensor unit in a measurement standby state in which the measurement process is unprocessed, and at least one sensor unit taken out from the stock unit. A supply unit for supplying the sensor unit to the measurement stage, a housing for housing the measurement stage, the stock unit, and the supply unit, and an atmosphere in the housing. Characterized by comprising a temperature adjusting means for adjusting the air temperature.

  In addition, it is preferable that the said temperature control means consists of a circulation means to circulate a heat medium to the jacket part provided in the said housing | casing, and a heating or cooling means to heat or cool the said heat medium.

  Moreover, it is preferable that the said jacket part is provided in the inside of the base plate of the said housing | casing, and the inside of a side plate.

  The supply unit preferably includes a plate on which at least one sensor unit is placed, and a pickup mechanism that picks up the sensor unit from the plate and sends the sensor unit to the measurement stage.

  Furthermore, a liquid storage unit that stores a plurality of types of liquids including a sample solution in which the sample is dissolved in a solvent, a pipette that sucks liquid from the liquid storage unit and discharges the liquid to the sensor unit on the measurement stage, A pipette tip storage section for storing a plurality of pipette tips attached to the tip of the pipette in a replaceable manner, and the liquid storage section and the pipette tip storage section are arranged in the housing so that the liquid and the pipette tips It is preferable to adjust the temperature to the ambient temperature.

  In addition, it is preferable that a plurality of the sensor units are stored in the stock unit in a state of being accommodated in a holder, and are taken out from the stock unit in units of the holder and set in the supply unit.

  In addition, the stock unit is preferably formed of at least one shelf plate on which the sensor unit can be placed, and the shelf plate is formed of a metal material having high thermal conductivity and is attached to the inner side surface of the housing. It is preferred that

  Further, the sensor unit includes a long dielectric block formed with a thin film layer serving as a sensor surface on one surface, a sample solution in which the sample is dissolved in a solvent. It is preferable to comprise a flow path member in which a flow path for feeding liquid to the sensor surface is formed.

  In the measurement method using total reflection attenuation according to the present invention, the sensor unit for detecting the reaction state of the sample using the total reflection attenuation of light emits light from the light source so as to satisfy the total reflection condition. And at least one sensor unit in a measurement standby state in which the measurement process is unprocessed is stored. And a supply unit that holds at least one of the sensor units taken out from the stock unit and supplies the sensor unit to the measurement stage. The measurement process is performed on a plurality of the sensor units while adjusting the atmospheric temperature.

  According to the measurement apparatus using total reflection attenuation and the measurement method of the present invention, the light source and the detection means are arranged, the measurement stage for performing the measurement process for examining the reaction state of the sample, and at least the measurement standby state Since the stock unit for accumulating one sensor unit and the supply unit for supplying the sensor unit to the measurement stage are provided in a housing maintained at a constant atmospheric temperature by the temperature adjusting means, the temperature of each of these units is It can be adjusted to the ambient temperature in the body. Accordingly, by supplying the sensor unit accumulated in the stock unit and already temperature-adjusted to the measurement stage, a waiting time due to temperature adjustment does not occur even when continuous measurement is performed. Further, since the temperature adjustment is performed by the same temperature adjusting means, there is no temperature difference between the plurality of sensor units to be measured.

  FIG. 1 is a block diagram showing a schematic configuration of an SPR measurement device 2 as a measurement device using total reflection attenuation. The SPR measuring device 2 includes a fixing device 4 for fixing the ligand (fixing step), a measuring device 6 for adding an analyte to the immobilized ligand and measuring the reaction state of both (measurement step), and the measuring device 6 And a data analyzer 8 that performs analysis (data analysis process) of data obtained by the above. Further, the fixing step and the measuring step are performed on the sensor unit 12 which is a separate body, so that a plurality of samples can be measured smoothly.

  FIG. 2 is an exploded perspective view showing a schematic configuration of the sensor unit 12. The sensor unit 12 includes a prism (dielectric block) 14 having a metal film (thin film layer) 13 formed on the upper surface, a channel member 41 having a channel 16 for feeding a liquid to the metal film 13, and a prism. 14 and the bottom surface of the flow path member 41 are held in pressure contact with each other, and a lid member 43 is disposed above the holding member 42.

  The flow path member 41 is formed in a long shape, and three flow paths 16 are provided along the longitudinal direction thereof. The flow path 16 is a liquid feeding pipe bent into a substantially U-shape, and has an inlet 16a for injecting liquid and an outlet 16b for discharging it. The diameter of the channel 16 is about 1 mm, and the interval between the inlet 16a and the outlet 16b is about 10 mm. Further, the bottom of the flow path 16 is open, and this open part is covered and sealed with the metal film 13 when being pressed against the prism 14. Hereinafter, a portion of the metal film 13 surrounded by the open portion (see FIG. 3) is referred to as a sensor cell 17.

  The flow path member 41 is molded from, for example, an elastic material such as rubber or PDMS (polydimethylsiloxane) in order to improve the adhesion with the metal film 13. Thus, when the flow path member 41 is pressed against the upper surface of the prism 14, the flow path member 41 is elastically deformed to fill the gap between the contact surfaces with the metal film 13, and the open bottom portion of each flow path 16 is sealed together with the prism 14. Cover. In this example, an example in which the number of the flow paths 16 is three has been described. However, the number of the flow paths 16 is not limited to three, and may be one or two, or may be four or more. .

  On the upper surface of the prism 14, a strip-shaped metal film 13 facing each flow path 16 is formed by, for example, vapor deposition. For example, gold or silver is used for the metal film 13, and the film thickness is, for example, 50 nm. This film thickness is appropriately selected according to the material of the metal film 13, the wavelength of the irradiated light, and the like.

  A linker film 22 for fixing the ligand is formed at a position corresponding to each flow path 16 on the metal film 13 (in the sensor cell 17). Hereinafter, of the metal film 13, the surface on which the linker film 22 is formed is referred to as a sensor surface 13a, and the back surface (the surface in contact with the prism 14) is referred to as a light incident surface 13b. The prism 14 condenses incident light toward the light incident surface 13b, and the metal film 13 is formed on the sensor surface 13a when light is incident on the light incident surface 13b so as to satisfy the total reflection condition. Generate SPR.

  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 long side of the prism 14. 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. Further, protrusions 14 b are provided at both ends 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 engages with the holder 52 to position the sensor unit 12 at a predetermined storage position in the holder 52.

  The prism 14 may be made of, for example, optical glass represented by borosilicate crown (BK7) or barium crown (Bak4), polymethyl methacrylate (PMMA), polycarbonate (PC), amorphous polyolefin (APO), or the like. Representative optical plastics can be used.

  On the upper surface of the holding member 42, a receiving port 42b through which the tip of a dispensing means such as a pipette enters is formed at a position corresponding to the inlet 16a and the outlet 16b of each channel 16. These receiving ports 42b are formed in a funnel shape so that the liquid discharged from the pipette is guided to each inlet 16a. In addition, each receiving port 42 b comes into contact with each inlet 16 a and outlet 16 b and is connected to the channel 16 when the holding member 42 sandwiches the channel member 41 and engages with the prism 14.

  In addition, columnar bosses 42c are provided on both sides of each receiving port 42b. These bosses 42 c are fitted into 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 holes at positions corresponding to the receiving ports 42b and the bosses 42c.

  The lid member 43 covers the receiving port 42 b that communicates with the flow path 16, thereby preventing the liquid injected into the flow path 16 from evaporating. The lid member 43 is made 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. As a result, the lid member 43 elastically deforms the slit 43b to allow the pipette to be inserted, and when the pipette is removed, the lid member 43 maintains the initial state and closes the receiving port 42b.

  As shown in FIG. 3, the linker film 22 formed on the metal film 13 has a measurement region 22a (hereinafter referred to as an “act region”) in which a ligand is fixed and a reaction between the analyte and the ligand occurs, and a ligand. A reference region (hereinafter referred to as a “ref region”) 22b for obtaining a reference signal when measuring the signal in the act region 22a is provided without being fixed. The ref region 22b is formed when the linker film 22 is formed. As the formation method, for example, the linker film 22 is subjected to a surface treatment, and about a half of the linker film 22, the bonding group that binds to the ligand is deactivated, so that the half of the linker film 22 becomes the act region. 22a, and the other half becomes the ref region 22b.

  FIG. 4 is a perspective view schematically showing the configuration of the fixing machine 4. A mounting space 51 for mounting the plurality of sensor units 12 is secured on the housing base 50 that serves as a base of the fixed machine 4. The sensor unit 12 is subjected to a fixing process while being placed in the placement space 51.

  The sensor unit 12 is placed in the placement space 51 while being housed in the holder 52. The holder 52 can store a plurality of sensor units 12 (eight in this example). 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. In addition, an opening is formed in the bottom of the holder 52 except for support portions that support both ends of the sensor unit 12. As will be described later, when the sensor unit 12 is taken out from the holder 52 in the measurement process, a push-up member 85a (see FIG. 5) is inserted from the opening, and the sensor unit 12 is pushed up.

  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. Each pedestal 53 is formed with a positioning boss for positioning the holder 52.

  Further, the fixing machine 4 is provided with a pipette head 54 in which three pairs of pipettes 19 are connected. The pipette head 54 accesses the sensor units 12 arranged in the placement space 51 via the holder 52 to inject and discharge liquid. Each pair of pipettes 19 includes a pair of pipettes 19 a and 19 b (see FIG. 8), and each pipette 19 a and 19 b is inserted into each of the inlet 16 a and the outlet 16 b of each sensor unit 12. Each pipette 19a, 19b has a function of injecting liquid into the flow path 16 and sucking out from the flow path 16, and when one is performing an injection operation, the other performs a suction operation. So that they work together. Since three pipette pairs 19 are provided in the pipette head 54, liquid can be simultaneously injected into or discharged from the three flow paths 16 included in one sensor unit 12. Further, the fixing device 4 is provided with a controller (not shown). The suction and discharge operations of the pipette head 54 are controlled by this controller, and the suction amount, discharge amount, timing, etc. are determined for each pipette pair 19.

  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 motor, and the like. A Y-direction moving mechanism including a guide rail 58 to be held and an X-direction moving mechanism that holds the guide rail 58 at both ends and moves the pipette head 54 in the X direction together with the guide rail 58. The head moving mechanism 56 is controlled by a controller, and the controller drives the head moving mechanism 56 to control the front / rear / left / right / up / down positions of the pipette head 54.

  In addition to the mounting space 51, a plurality of liquid storage units 61 for storing various liquids to be injected into the flow path 16 and a pipette tip storage unit 63 for storing the pipette tips 62 are provided on the housing base 50. And a well plate 64 in which a plurality of well-shaped ridges are arranged in a matrix.

  The number of the liquid storage units 61 is determined according to the type of liquid to be used, such as a ligand solution, a cleaning liquid, a fixing buffer liquid, a drying prevention liquid, an activation liquid, and a blocking liquid. Each liquid storage unit 61 is provided with six insertion ports in a straight line. 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 the pipette head 54 injects the liquid into the flow path 16, the liquid storage unit 61 is accessed to suck in a desired liquid, and then moves to the placement space 51 to be injected into each flow path 16.

  The pipette tip 62 stored in the pipette tip storage unit 63 is detachably held by the pipette head 54. Since the pipette tip 62 is in direct contact with the liquid, it is exchanged for each liquid to be used so that different kinds of liquids are not mixed through the pipette tip 62. The pipette head 54 incorporates a mechanism for picking up and releasing the pipette tip 62 so that the pipette tip 62 is automatically replaced. When exchanging the pipette tip 62, the pipette head 54 first releases the used pipette tip 62 at a disposal unit (not shown), and then accesses the pipette tip storage unit 63 to access an unused pipette. The chip 62 is picked up.

  The well plate 64 is used for temporarily storing the liquid sucked up by the pipettes 19a and 19b, or for preparing a mixed liquid by mixing a plurality of types of liquids.

  Further, when the fixing device 4 performs the fixing process, the housing base 50 is covered with the cover, and the inside of the fixing device 4 including the placement space 51 is shielded. The sensor unit 12 is stored in the mounting space 51 for a predetermined time until the ligand immobilization is completed. At this time, the degree of progress of immobilization depends on the temperature. Therefore, the fixing machine 4 keeps the internal temperature at a predetermined temperature by a temperature controller (not shown) while fixing. The set temperature, standing time, and the like are appropriately determined according to the type of ligand to be immobilized.

  5 and 6 are perspective views schematically showing the configuration of the measuring instrument 6. FIG. The measuring machine 6 includes a holder transport mechanism 71, a pickup mechanism 72, a measurement stage 73, a stock unit 74, and the like. These units are accommodated in a housing 75. The base plate 75a and the side plate 75b of the housing 75 are formed in a hollow shape. In this internal space, a jacket portion 80 is formed that is partitioned by a plurality of partition plates 81 and allows water as a heat medium (hereinafter referred to as temperature-controlled water) to flow in a distorted manner along each partition plate 81. .

  The casing 75 is connected to a Peltier element (heating or cooling means) 76 that heats or cools the temperature-controlled water, and a circulation pump (circulation means) 77 that circulates the temperature-controlled water to each jacket portion 80. Yes. Thereby, the air in the housing 75 is warmed by heat radiation from the respective inner wall surfaces provided with the jacket portion 80, and the internal atmospheric temperature is adjusted to be constant (for example, 27.5 ° C.). Note that the amount of heat that the Peltier element 76 heats or cools may be determined according to the measurement result obtained by measuring the ambient temperature in the housing 75 or the temperature of the temperature-controlled water using a temperature sensor. Alternatively, it may be determined in advance by a calculation such as the above or a prior measurement.

  The structure of the jacket portion 80 is not limited to that of the partition plate 81 but may be a pipe or the like, or may be the internal space itself of the base plate 75a and the side plate 75b without being partitioned by the partition plate 81. . The base plate 75a and the side plate 75b are preferably formed of a metal material having a high thermal conductivity such as aluminum or copper in order to efficiently transmit the heat of the temperature-controlled water to the internal air. In addition, each side plate 75b may be provided with the jacket part 80 in all the four surfaces, and may be provided only in some of them. Further, the jacket portion 80 may be provided on the entire surface of the base plate 75a or the side plate 75b, or may be provided on only a part thereof.

  The holder conveyance mechanism 71 includes a conveyance belt 82, a carriage 83 attached to the conveyance belt 82, and a plate 84 attached to the carriage 83 for setting the holder 52. The holder conveyance mechanism 71 moves each sensor unit 12 accommodated in the holder 52 to a pickup position where the pickup mechanism 72 picks up by moving the plate 84 on which the holder 52 is placed in the X direction.

  The pickup mechanism 72 is a mechanism for picking up the sensor unit 12 from the holder 52, a push-up mechanism 85 that pushes up the sensor unit 12 accommodated in the holder 52 from below to above, and the push-up mechanism 85 above the holder 52. The handling head 86 is configured to sandwich and hold the pushed-up sensor unit 12 from both sides. As described above, an opening is provided in the bottom of the holder 52, and the plate 84 is formed in a frame shape so as to expose the opening. The push-up mechanism 85 enters the opening of the holder 52 through the plate 84, contacts the bottom surface of the sensor unit 12, pushes up the push-up member 85a, and push-up member that drives the push-up member 85a up and down. And a drive mechanism 85b.

  The handling head 86 is provided with a pair of claws that sandwich the sensor unit 12, and the sensor unit 12 is gripped by the claws. The handling head 86 has a head main body 86 a attached to a ball screw 88 via a nut 87, and is provided so as to be movable between the pickup position above the holder 52 and the measurement stage 73. The handling head 86 grips the sensor unit 12 at the pickup position, moves in the Y direction in this state, and conveys the sensor unit 12 to the measurement stage 73. Further, after the measurement is completed, the sensor unit 12 is released by returning to the pickup position, and the measured sensor unit 12 is returned to the holder 52. That is, the supply unit described in the claims includes a holder transport mechanism 71 and a pickup mechanism 72.

  The measurement stage 73 is provided with a measurement unit 31 including an illumination unit 32 and a detector (detection means) 33 below the position where the sensor unit 12 is disposed. The handling head 86 sequentially moves the sensor cells 17 to the measurement positions on the optical path of the illumination unit 32 by moving the sensor units 12 in the Y direction at the arrangement pitch of the sensor cells 17. As shown in FIGS. 5 and 3, the illumination unit 32 and the detector 33 are arranged such that the incident light beam irradiated on the sensor unit 12 and the reflected light beam reflected by the sensor surface 13 a and the direction in which the sensor cells 17 are arranged are arranged. That is, they are arranged so that the flow directions of the horizontal portions of the respective flow paths 16 are orthogonal to each other.

  The reaction state between the ligand and the analyte appears as a change in the resonance angle (the incident angle of light irradiated on the sensor surface 13a). The illumination unit 32 includes, for example, a light source 34 and an optical system 36 composed of a condenser lens, a diffuser plate, a polarizing plate, and the like (see FIG. 8), and the arrangement position and the installation angle are all incident angles of illumination light. It is adjusted to satisfy the reflection conditions.

  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. The light source 34 uses one such light emitting element, and irradiates light from the single light source toward each light incident surface 13b. 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 passes only p-polarized light (linearly polarized light having an oscillating electric field parallel to the incident surface) that causes SPR in the irradiated light. In addition, when using LD, when the direction of polarization of the irradiation light itself emitted from the light source 34 is uniform, the polarizing plate is unnecessary. Further, even when a light source with uniform polarization is used, if the direction of polarization becomes uneven due to passing through the diffusion plate, the direction of polarization is aligned using a polarizing plate. The light thus diffused and polarized is condensed by the condenser lens and applied to the prism 14. Thereby, the light which does not vary in light intensity and can have various incident angles can be incident on the light incident surface 13b.

  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 of various angles is incident on the light incident surface 13b, the light is reflected on the light incident surface 13b at a reflection angle corresponding to each incident angle. The detector 33 receives light of these various reflection angles. For example, a CCD area sensor or a photodiode array is used as the detector 33, and the reflected light reflected at various reflection angles on the light incident surface 13b is received and photoelectrically converted, and this is converted into an SPR signal as a data analyzer. 8 is output. In addition, in the measurement stage 73, the process until the reflected light from the sensor unit 12 of the light irradiated by the illumination unit 32 is received by the detector 33 and the photoelectrically converted SPR signal is output to the data analyzer 8 is claimed. This corresponds to the described measurement process.

  Further, as shown in FIG. 3, an act region 22 a and a ref region 22 b are formed on the linker film 22. 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 ref signal is used when the data analyzer 8 cancels noise caused by disturbance on the act signal.

  The illumination unit 32 and the detector 33 are respectively fixed to the metal block 90 so as to satisfy the above-described conditions. Each metal block 90 sandwiches a jacket plate 91 connected to the jacket portion 80 of the base plate 75a, and the temperature is strictly adjusted by conduction heat from the base plate 75a and the jacket plate 91. Thereby, expansion and contraction of the metal block 90 due to temperature change is suppressed, and the positional relationship of the measurement unit 31 is prevented from being shifted. The jacket plate 91 supports the sensor unit 12 carried by the handling head 86 to the measurement stage 73 from the bottom surface side, and strictly adjusts the temperature of the sensor unit 12 to prevent measurement errors due to temperature changes. . Note that, similarly to the base plate 75a and the side plates 75b, each metal block 90 is made of a metal material having a high thermal conductivity.

  The stock section 74 is provided with a three-stage shelf plate 92. A plurality of sensor units 12 in a measurement standby state in which the fixing process has been completed can be placed on each shelf 92 together with the holder 52. In addition, on the uppermost shelf 92, a plurality of liquid storage units 93 that store various buffer liquids, cleaning liquids, and the like are arranged in the same manner as the liquid storage units 61 of the fixing machine 4.

  Above the measurement stage 73, a shelf plate on which a well plate 94 for storing different types of analyte solutions in each well and a pipette tip storage unit 95 similar to the pipette tip storage unit 63 of the fixing machine 4 is placed. 96 is provided. The shelf plate 96 and each shelf plate 92 of the stock portion 74 are made of a metal material having high thermal conductivity and are formed integrally with the side plate 75b or fixed to the side plate 75b with bolts or the like. In this way, each sensor unit 12, each liquid storage unit 93, well plate 94, pipette chip storage unit 95, etc. that are placed inside the casing 75 also have conduction heat and radiation from each shelf plate 94, 96, In addition, the temperature is adjusted to a certain temperature according to the temperature of the temperature-controlled water circulating through each jacket portion 80 through the heat conduction from the air.

  As shown in FIG. 6, the measuring machine 6 is provided with a pipette pair 26 having the same function as the pipette pair 19 of the fixing machine 4. Openings 75d, 75e, and 75f are formed in the upper plate 75c of the housing 75 at positions corresponding to the sensor unit 12, each liquid storage unit 93, the well plate 94, and the pipette chip storage unit 95, respectively. . A head moving mechanism 78 configured almost in the same manner as the head moving mechanism 56 of the fixing machine 4 moves the pipette head 97 having the pipette pair 26 in three directions of X, Y, and Z, and opens the openings 75d, 75e, and 75f. The sensor unit 12, each liquid storage unit 93, the well plate 94, and the pipette chip storage unit 95 are accessed via the. The pipette head 97 accesses the flow path 16 of the sensor cell 17 to be measured, and injects and discharges various liquids. Since the pipette head 97 is for injecting and discharging liquid with respect to a specific sensor cell 17 to be measured, unlike the pipette head 54 of the fixing machine 4, only one pair of pipettes 26 is provided. ing.

  Shutters 98 and 99 are attached to the openings 75e and 75f, which are slidable between an open position for opening them and a closed position for closing them. By keeping the shutters 98 and 99 in the closed position and opening the pipette head 97 just before accessing the openings 75e and 75f, the inflow of outside air from the openings 75e and 75f is suppressed, and the heat retaining property is improved. Can be increased. The shutters 98 and 99 may be opened and closed manually or automatically using a motor or the like. Although not shown, it is assumed that a similar shutter is attached to the opening 75d.

  FIG. 7 is a block diagram schematically showing the configuration of the data analyzer 8. The data analyzer 8 includes, for example, an input device 100, a CPU (Central Processing Unit) 101, a ROM (Read Only Memory) 102, a RAM (Random Access Memory) 103, an HDD (Hard Disk Drive). 104, a monitor 105, a communication I / F 106, a signal processing unit 107, and the like. The input device 100 is, for example, a keyboard or a mouse, and inputs an instruction from the user to the data analyzer 8. The CPU 101 comprehensively controls each unit of the data analyzer 8. The ROM 102 stores a control program and various setting data. The RAM 103 is used as a working memory when the CPU 101 and the signal processing unit 107 perform calculations. The HDD 104 is a well-known data storage device, and various programs such as a control program and a data analysis program are stored in the HDD 104. In addition, various measurement data measured by the measuring device 6 are recorded in the HDD 104. The communication I / F 106 receives the SPR signal output from the detector 33. The signal processing unit 107 performs data analysis based on the acquired SPR signal. The monitor 105 displays the detected SPR signal and the analysis result based on the detected SPR signal.

  Next, the SPR measurement method of the SPR measurement device 2 having the above configuration will be described using the explanatory diagram shown in FIG.

  The fixing step of fixing the ligand to the linker film 22 is performed in a state where the sensor unit 12 is placed in the placement space 51 of the fixing machine 4 via the holder 52. The fixing device 4 drives the head moving mechanism 56 in response to a fixing start instruction from the user, and uses the pipette pair 19 to inject the ligand solution 21 in which the ligand 21a is dissolved in the solvent from the injection port 16a.

  As a pretreatment before performing the ligand immobilization process for injecting the ligand solution 21, the fixing machine 4 first feeds a fixing buffer solution to the linker film 22 to moisten the linker film 22, and then An activation process of the linker film 22 is performed to facilitate the binding of the ligand to the film 22. 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. The fixing device 4 cleans the flow path 16 with the fixing buffer solution after the activation process.

  Examples of the buffer solution for fixation and the solvent (diluent) of the ligand solution 21 include, for example, various buffer solutions (buffer solutions), physiological salt solutions represented by physiological saline, pure water, and the like. Is used. The type of each of these liquids, the pH value, the type of mixture, the concentration thereof, and the like are appropriately determined according to the type of ligand 21a. For example, when a biological sample is used as the ligand 21a, a physiological saline whose pH is adjusted to near neutral is often used. However, in the amine coupling method, since the linker film 22 is negatively (negatively) charged by carboxymethyldextran, the protein is positively (positively) charged so as to easily bind 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 performing the activation process and the washing, the fixing machine 4 injects the ligand solution 21 into the flow path 16 to perform the ligand immobilization process. When the ligand solution 21 is injected into the flow path 16, the ligand 21 a diffusing into the solution is precipitated and gradually deposited on the linker film 22. Thereby, the contacted ligand 21 a is bonded to the linker film 22, and the ligand 21 a is fixed on the linker film 22. The immobilization usually takes about one hour. During this time, the sensor unit 12 is stored in a state where environmental conditions such as temperature are set to predetermined conditions. Further, while the immobilization is proceeding, the ligand solution 21 in the flow channel 16 may be allowed to stand, but the ligand solution 21 in the flow channel 16 may be stirred and flowed. By doing so, the binding between the ligand 21a and the linker film 22 is promoted, and the amount of ligand 21a immobilized can be increased.

  When the fixation of the ligand 21a to the linker film 22 is completed, the fixing device 4 sucks out the ligand solution 21 by the pipette 19b and discharges it from the flow path 16, and then the cleaning liquid is injected into the flow path 16 to complete the fixation. The linker film 22 is washed. Moreover, the fixing machine 4 injects a blocking liquid as necessary, and performs a blocking process to deactivate the reactive group of the linker film 22 that has not been bonded to the ligand 21a. As the blocking liquid, for example, ethanolamine-hydrochloride is used. When this blocking process is performed, the channel 16 is washed again. After the final cleaning, the fixing device 4 injects a drying prevention liquid into the flow path 16. The sensor unit 12 is stored until measurement in a state where the linker film 22 is immersed in the anti-drying liquid.

  The measurement process starts from a state in which the holder 52 in which the stored sensor units 12 are fixed is placed on the plate 84 of the holder transport mechanism 71. The measuring device 6 moves the plate 84 to the pickup position in accordance with a measurement start instruction from the user and drives the pickup mechanism 72 to convey the predetermined sensor unit 12 to the measurement stage 73.

  The measuring machine 6 that has transported the sensor unit 12 to the measurement stage 73 drives the head moving mechanism 78 to first inject the measurement buffer solution 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 washed once before the measurement buffer solution is injected for the first time. Data reading by the detector 33 is started immediately after the measurement buffer solution is first injected in order to detect a reference signal level. After the analyte solution 27 is injected, the measurement buffer solution is injected again. Until it is done. Thereby, it is possible to measure the SPR signal until the detection of the reference level, the reaction state between the analyte and the ligand, and the desorption by the injection of the buffer solution for measurement of the bound analyte and the ligand.

  Examples of the buffer solution for measurement and the solvent (diluent) of the analyte solution 27 include various buffer solutions (buffer solutions), physiological salt solutions typified by physiological saline, and pure water. used. The type of each of these liquids, the pH value, the type of mixture and its concentration, etc. are appropriately determined according to the type of ligand or analyte. 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 detecting the reference level. Therefore, when the analyte solution 27 contains DMSO, the measurement buffer solution having a DMSO concentration comparable to the DMSO concentration should be used. Is preferred.

  The analyte solution 27 is often stored for a long time (for example, one year). In such a case, a difference in concentration occurs between the initial DMSO concentration and the DMSO concentration at the time of measurement due to a change with time. May end up. When it is necessary to perform strict measurement, it is preferable to estimate such a concentration difference from the level of the ref signal when the analyte solution 27 is injected, and to correct the measurement data (DMSO concentration correction).

  The correction data for correcting the DMSO concentration is obtained by injecting a plurality of types of measurement buffer solutions having different DMSO concentrations into the flow path 16 before injecting the analyte solution 27, and ref according to the DMSO concentration change at this time. It is obtained by examining the respective changes in the signal level and the act signal level.

  The reaction state between the ligand and the analyte appears as a transition of the attenuation position of the reflected light within the light receiving surface of the detector 33. 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. Then, when the analyte comes into contact with the ligand and starts a reaction, the resonance angle of the reflected light starts to change accordingly, and the attenuation position of the reflected light in the light receiving surface starts to move. The measuring device 6 outputs an SPR signal indicating the reaction state of the sample thus obtained to the data analyzer 8.

  In FIG. 8, for the sake of convenience, the direction of the incident light beam on the light incident surface 13 b and the reflected light beam reflected there are parallel to the direction of the liquid flowing in the flow channel 16 so that the configuration of the measuring instrument 6 is clear. The illumination unit 32 and the detector 33 are arranged so that, as shown in FIGS. 3 and 5, the directions of the incident light beam and the reflected light beam are actually orthogonal to the liquid flowing direction. The illumination part 32 and the detection part 33 are arrange | positioned so that it may irradiate in a direction (direction orthogonal to a paper surface). However, the measurement may be performed by arranging the measurement unit 31 as shown in FIG.

  In the data analysis step, the SPR signal obtained by the measuring device 6 is analyzed by the data analyzer 8 to quantitatively analyze the characteristics of the analyte. The data analyzer 8 receives the SPR signal from the detector 33 via the communication I / F 106 and sends this SPR signal to the signal processing unit 107.

  The signal processing unit 107 performs data analysis by obtaining the difference or ratio between the act signal and the ref signal obtained by the measuring device 6 based on the data analysis program stored in the ROM 102 or the HDD 104. For example, difference data between the act signal and the ref signal is obtained, and the difference data is used as measurement data, and analysis is performed based on the difference data. By doing so, it becomes possible to cancel noise caused by disturbances such as individual differences of the sensor unit 12 and the linker film 22 and mechanical fluctuations of the apparatus, and a highly accurate measurement with a good S / N ratio. It can be performed. The signal processing unit 107 displays the analysis result on the monitor 105 and records it in the HDD 104.

  Next, the operation of the SPR measurement device 2 of the present embodiment will be described with reference to the flowchart shown in FIG. As described above, the measurement process by the measuring machine 6 starts from a state where the holder 52 in which the stored sensor units 12 are fixed is placed on the plate 84 of the holder transport mechanism 71. At this time, apart from the holders 52 placed on the plates 84, the holders 52 in the measurement standby state after the fixing of the sensor units 12 are completed are placed on the shelf plates 92 of the stock unit 74.

  When the measurement of all the sensor units 12 stored in the holder 52 is completed, the holder 52 is removed from the plate 84, and any one of the holders 52 placed on each shelf plate 92 of the stock unit 74 It is placed on the plate 84. In the measurement process, the above procedure is repeated until the measurement of all the holders 52 placed on each shelf plate 92 is completed. The movement of the holder 52 between the plate 84 and each shelf plate 92 may be performed manually or automatically using a known transport mechanism or the like. In addition, when there is a holder 52 in which the sensor unit 12 in the measurement standby state is accommodated and cannot complete the shelf 92, the measurement for each holder 52 is finished and the holder 52 is moved from the shelf 92 to the plate 84. It is sufficient to replenish the vacant shelf 92 in order each time the is moved.

  The temperature of each sensor unit 12 placed on the stock unit 74 is measured by the shelf plate 92 or air-mediated conduction heat while measuring each sensor unit 12 placed on the plate 84. The ambient temperature in the body 75 is adjusted. By placing the holder 52 placed on the stock unit 74 on the plate 84, the sensor unit 12 that has already been temperature-adjusted can be supplied, so when measuring a plurality of sensor units 12 continuously. In addition, there is no waiting time due to temperature control. Further, since the temperature adjustment is based on the atmospheric temperature in the housing 75 that is kept constant by the temperature-controlled water circulating through each jacket portion 80, the holder 52 and each shelf plate 92 placed on the plate 84 are adjusted. There is no temperature difference between the plurality of sensor units 12 housed in each of the mounted holders 52.

  In the above embodiment, the temperature-controlled water is heated or cooled using the Peltier element 76, but is not limited to this, for example, another heat pump using a compressor or the like may be used. Only heating may be performed using a heating wire such as a nichrome wire or a heater made of an electric resistor. Further, the temperature is not limited to a method in which the temperature adjustment water is circulated through the jacket portion 80 to keep the ambient temperature in the casing 75 constant. For example, the ambient temperature is obtained by convection of the heated or cooled dry air in the casing 75. May be kept constant.

  Moreover, in the said embodiment, although the stock part 74 is comprised from the three-stage shelf board 92 and mounts the six holders 52 in total, the number of the stages of the shelf board 92 and the holder 52 is mounted. , And the structure thereof is not limited to this, and may be any structure as long as at least one sensor unit 12 to be measured next can be accumulated.

  Furthermore, in the above embodiment, each liquid storage unit 93, well plate 94, pipette chip storage unit 95, and the like are also adjusted to a constant temperature according to the temperature of the temperature-controlled water circulating through each jacket unit 80. Therefore, it is possible to prevent the temperature of the sensor unit 12 to be measured from being changed by the liquid injected by the pipette pair 26.

  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.

  Moreover, in the said embodiment, although the elongate sensor unit 12 in which each sensor cell 17 was arranged in a line is used, the shape of a sensor unit is not restricted to this, For example, as shown in FIG. The sensor unit 110 may be shaped like a ring. The sensor unit 110 includes a microtiter plate 114 in which a plurality of wells 112 containing sample solutions in which a sample is dissolved are arranged in a matrix, and convex portions 116 that fit into the wells 112 at positions corresponding to the respective wells 112. The sensor plate 118 is formed, the metal film 120 is formed on the surface of each projection 116, and the prism 122 is formed integrally with the sensor plate 118 on the opposite surface of each projection 116.

  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 a block diagram which shows schematic structure of a SPR measuring apparatus. It is a disassembled perspective view which shows schematic structure of a sensor unit. It is explanatory drawing which extracts and demonstrates one sensor cell. It is a perspective view which shows the structure of a fixing machine roughly. It is a perspective view which shows roughly the structure of the measuring machine which expanded a part of housing | casing. It is a perspective view which shows roughly the structure of the measuring machine covered with the housing | casing. It is a block diagram which shows the structure of a data analyzer roughly. It is explanatory drawing of a SPR measuring method. It is a flowchart which shows the procedure of a measurement process. It is explanatory drawing which shows the Example of a plate-shaped sensor unit.

Explanation of symbols

2 SPR measurement device (measurement device using total reflection attenuation)
4 Fixing machine 6 Measuring machine 8 Data analyzer 12 Sensor unit 13 Metal film (thin film layer)
14 Prism (dielectric block)
16 Flow path 32 Illumination part 33 Detector (detection means)
34 Light source 52 Holder 62 Pipette tip 71 Holder transport mechanism (supply unit)
72 Pickup mechanism (supply unit)
73 Measurement stage 74 Stock part 75 Case 75a Base plate 75b Side plate 76 Peltier element (heating or cooling means)
77 Circulation pump (circulation means)
78 Head moving mechanism 80 Jacket section 84 Plate 92 Shelf board 93 Liquid storage section 94 Well plate 95 Pipette tip storage section

Claims (12)

A sensor unit for detecting a reaction state of a sample using attenuation of total reflection of light is set one by one, and a light source for irradiating light so as to satisfy the total reflection condition for the sensor unit, and the sensor A detection stage that receives reflected light from the unit and photoelectrically converts the reflected light into an electrical signal, and a measurement stage that performs a measurement process for examining the reaction state of the sample;
A stock unit for accumulating at least one sensor unit in a measurement standby state in which the measurement process is not processed;
A supply unit for holding at least one sensor unit taken out from the stock unit and supplying the sensor unit to the measurement stage;
A housing for housing these measurement stage, stock section and supply section;
A measuring apparatus using total reflection attenuation, comprising temperature adjusting means for adjusting the ambient temperature in the casing to be constant.   The said temperature control means consists of a circulation means to circulate a heat medium to the jacket part provided in the said housing | casing, and a heating or cooling means to heat or cool the said heat medium. Measuring device using total reflection attenuation.   The measuring apparatus using total reflection attenuation according to claim 2, wherein the jacket portion is provided inside a base plate of the casing.   The measuring apparatus using total reflection attenuation according to claim 2, wherein the jacket portion is provided inside a side plate of the housing.   5. The supply unit according to claim 1, wherein the supply unit includes a plate on which at least one sensor unit is placed, and a pickup mechanism that picks up the sensor unit from the plate and sends the sensor unit to the measurement stage. A measuring apparatus using the total reflection attenuation described in any one of the above items.   A liquid storage unit that stores a plurality of types of liquids including a sample solution in which the sample is dissolved in a solvent, a pipette that draws liquid from the liquid storage unit and discharges the liquid to the sensor unit on the measurement stage; A pipette tip storage portion for storing a plurality of pipette tips attached to the tip in a replaceable manner, and the liquid storage portion and the pipette tip storage portion are arranged in the casing, and the temperature of the liquid and the pipette tip The measurement apparatus using total reflection attenuation according to any one of claims 1 to 5, wherein the temperature is adjusted to the ambient temperature.   2. The sensor unit according to claim 1, wherein a plurality of the sensor units are stored in the stock unit in a state of being housed in a holder, and are taken out from the stock unit in units of the holder and set in the supply unit. 7. A measuring apparatus using the total reflection attenuation described in any one of items 6 above.   The measurement apparatus using total reflection attenuation according to claim 1, wherein the stock unit includes at least one shelf plate on which the sensor unit can be placed.   The measuring apparatus using total reflection attenuation according to claim 8, wherein the shelf board is attached to an inner surface of the housing.   The measuring apparatus using total reflection attenuation according to claim 8 or 9, wherein the shelf board is made of a metal material having high thermal conductivity.   The sensor unit includes a long dielectric block having a thin film layer serving as a sensor surface on one surface, and a sensor solution in which a sample solution in which the sample is dissolved in a solvent is disposed facing the sensor surface. The measuring device using total reflection attenuation according to any one of claims 1 to 10, comprising a flow path member in which a flow path for feeding liquid is formed. By irradiating light from a light source so as to satisfy the total reflection condition for a sensor unit for detecting the reaction state of the sample using the total reflection attenuation of light, the reflected light is detected, In the measurement method using total reflection attenuation to examine the reaction status of the sample,
A measurement stage in which a measurement process for examining the reaction state is executed; a stock unit that accumulates at least one sensor unit in a measurement standby state in which the measurement process is unprocessed; and the sensor unit that is extracted from the stock unit A plurality of the sensors while holding at least one supply unit and supplying a supply unit for supplying the sensor unit to the measurement stage in the housing, and adjusting the ambient temperature in the housing to be constant by the temperature adjusting means. A measurement method using total reflection attenuation, wherein the measurement process is performed on a unit.

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