JP2006133220A - Sample supply method and sample supply device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To enhance maintainability during measurements, regarding a sample supply method and a sample supply device for supplying a sample to a sample supply flow passage that a measuring unit of a measuring apparatus such as a surface plasmon sensor has. <P>SOLUTION: The sample supply device is used comprising a body formed with an inlet pipette chip mounting part and an outlet pipette chip mounting part; an inlet piston in communication with the inlet pipette tip mounting part for sucking and discharging air from the inlet pipette tip mounting part; an operating part for operating the inlet piston; an inlet pipette chip 90 for mounting on the inlet pipette chip mounting part; and an outlet pipette chip 91 for mounting on the outlet pipette chip mounting part. The inlet pipette chip 90 and the outlet pipette chip 91 are inserted respectively into the inlet 61 and the outlet 65 of the flow passage 60 of the measuring unit and the sample is fed from the inlet pipette chip 90 toward the outlet pipette chip 91. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a sample supply method and a sample supply apparatus for supplying a sample to a flow path for a sample provided in a measurement unit of a measurement apparatus such as a surface plasmon sensor.

  Conventionally, a surface plasmon sensor is known as one of measuring devices using an evanescent wave. In the metal, free electrons collectively vibrate to generate a dense wave called a plasma wave. A quantized version of this dense wave generated on the metal surface is called surface plasmon. The surface plasmon sensor analyzes the characteristics of a sample using a phenomenon in which the surface plasmon is excited by a light wave, and various types of sensors have been proposed. Among them, one that uses a system called Kretschmann configuration is well known (for example, see Patent Document 1).

  A surface plasmon sensor using the above system basically includes, for example, a dielectric block formed in a prism shape, a metal film formed on one surface of the dielectric block and brought into contact with a sample, and a light source that generates a light beam. An optical system that causes the light beam to be incident on the dielectric block at various angles so that a total reflection condition is obtained at the interface between the dielectric block and the metal film, and a light beam that is totally reflected at the interface A light detecting means for detecting the intensity of the light and a measuring means for measuring the surface plasmon resonance state based on the detection result of the light detecting means.

  In order to obtain various incident angles as described above, a relatively thin light beam may be incident on the interface by changing the incident angle, or a component incident on the light beam at various angles is included. As described above, a relatively thick light beam may be incident on the interface in a convergent light state or a divergent light state. In the former case, a light beam whose reflection angle changes according to the change in the incident angle of the incident light beam is detected by a small photodetector that moves in synchronization with the change in the reflection angle, or the direction in which the reflection angle changes Can be detected by an area sensor extending along the line. On the other hand, in the latter case, it can be detected by an area sensor extending in a direction in which all light beams reflected at various reflection angles can be received.

In the surface plasmon sensor having the above configuration, when a light beam is incident on the metal film at a specific incident angle θ _SP that is equal to or greater than the total reflection angle, an evanescent wave having an electric field distribution is generated in the sample in contact with the metal film, This evanescent wave excites surface plasmons at the interface between the metal film and the sample. When the wave number vector of the evanescent light is equal to the wave number of the surface plasmon and the wave number matching is established, both are in a resonance state and the energy of the light is transferred to the surface plasmon, so that the entire energy is transferred to the interface between the dielectric block and the metal film. The intensity of the reflected light decreases sharply. This decrease in light intensity is generally detected as a dark line by the light detection means.

  The resonance described above occurs only when the incident beam is p-polarized light. Therefore, it is necessary to set in advance so that the light beam is incident as p-polarized light.

When the wave number of the surface plasmon is found from a specific incident angle θ _SP (hereinafter referred to as total reflection attenuation angle θ _SP ) that is greater than or equal to the total reflection angle at which the light intensity is reduced, the dielectric constant of the sample is obtained. That is, when the surface plasmon wave number is K _SP , the surface plasmon angular frequency is ω, c is the speed of light in vacuum, εm and εs are metal, and the dielectric constant of the sample is as follows.

Knowing the dielectric constant εs of the sample, the refractive index or the like of the sample is found based on a predetermined calibration curve or the like, after all, by knowing the total reflection attenuation angle theta _SP, dielectric constant, that of the sample related to the refractive index Characteristics can be obtained.

  Further, a leak mode sensor is also known as a similar sensor using an evanescent wave (see, for example, Non-Patent Document 1). This leakage mode sensor is basically a dielectric block formed in a prism shape, for example, a clad layer formed on one surface of the dielectric block, and formed on the clad layer to be brought into contact with a sample. An optical waveguide layer, a light source that generates a light beam, and the light beam are incident on the dielectric block at various angles so that a total reflection condition is obtained at the interface between the dielectric block and the cladding layer. An optical system, a light detecting means for measuring the intensity of the light beam totally reflected at the interface, and a measuring means for measuring the excited state of the waveguide mode based on the detection result of the light detecting means. is there.

In the leaky mode sensor having the above configuration, when a light beam is incident on the cladding layer through the dielectric block at an incident angle greater than the total reflection angle, the light waveguide layer transmits a specific wave number after passing through the cladding layer. Only light having a specific incident angle is propagated in the guided mode. When the waveguide mode is excited in this way, most of the incident light is taken into the optical waveguide layer, resulting in total reflection attenuation in which the intensity of light totally reflected at the interface is sharply reduced. Since the wave number of the waveguide light depends on the refractive index of the sample on the optical waveguide layer, by knowing the total reflection attenuation angle theta _SP, it can be analyzed refractive index of the sample and the properties of the sample related thereto .

In addition, the surface plasmon sensor and leakage mode sensor described above may be used for random screening to find an analyte that binds to a desired ligand in the field of drug discovery research. In this case, the thin film layer (surface plasmon sensor) is used. In the case of a sensor, it is a metal film. In the case of a leak mode sensor, a ligand is fixed on a clad layer and an optical waveguide layer), and buffers (liquid samples) containing various analytes are supplied onto the ligand. every time elapses measures the ATR angle theta _SP described above. If the analyte in the buffer is one that binds to the ligand, the binding causes the refractive index of the ligand to change over time. Therefore, the attenuated total reflection angle theta _SP was measured every predetermined time, by a change in the attenuated total reflection angle theta _SP to measure whether occurring, whether binding between the analyte and ligand have been made That is, it can be determined whether or not the analyte is a specific substance that binds to the ligand. Examples of such a combination of an analyte and a ligand include an antigen and an antibody or an antibody and an antibody. As a specific measurement related to such an analyte, for example, a ligand is a rabbit anti-human IgG antibody, Examples include detection of the presence or absence of binding to a human IgG antibody, which is a light, and quantitative analysis thereof.

In order to measure a binding state between the analyte and ligand in buffer, it is not always necessary to detect the angle itself of an attenuated total reflection angle theta _SP. For example after first measuring the baseline as a reference by using the buffer without analyte was measured angle variation of the attenuated total reflection angle theta _SP at the time of supplying the buffer containing the analyte on the ligand Thus, the coupling state can be measured based on the magnitude of the angle change amount.
JP-A-6-167443 “Spectroscopy” Vol. 47, No. 1 (1998)

  By the way, in the measuring apparatus such as the surface plasmon sensor as described above, there is known one that performs measurement by continuously supplying a buffer using a flow path mechanism on a thin film layer on which a ligand is fixed. With this type of sensor, when measuring the binding state between the ligand and the analyte, a new buffer is always supplied onto the thin film layer, so the concentration of the analyte in the buffer does not change, and the binding state Measurement can be performed with high accuracy. In addition, after the binding state of the ligand and the analyte is measured, when the binding is performed, by flowing a buffer not containing the analyte on the thin film layer on which the conjugate is fixed, The dissociation state between the ligand and the analyte can be measured. Furthermore, for example, when a gas is used as a sample or when a buffer in which a gas is dissolved is used, the sample can be easily supplied onto the thin film layer using the flow path mechanism.

  As a method for supplying a sample to such a flow path mechanism, a pipe for waste liquid treatment is inserted on the outlet side of the flow path mechanism, and a pipette or a sample supply pipe is inserted on the inlet side of the flow path mechanism. However, in any case, it was necessary to use piping. The flow path mechanism used in a measuring device such as a surface plasmon sensor has a very narrow internal diameter of the flow path of several millimeters or less, and it is necessary to use a pipe with a narrow internal diameter. When piping is used to supply a sample to the path mechanism, problems such as clogging in the piping may occur, so the maintainability during measurement is not very good.

  The present invention has been made in view of such a problem. In the sample supply method and the sample supply apparatus for supplying a sample to a sample supply channel provided in a measurement unit of a measurement device such as a surface plasmon sensor, An object of the present invention is to provide a sample supply method and a sample supply apparatus that eliminates the above-mentioned problem.

  The sample supply method of the present invention comprises a dielectric block having a thin film layer formed on a smooth surface and a flow path member in close contact with the thin film layer of the dielectric block. The flow path member faces the thin film layer. And a discharge path from the other end of the measurement unit to the outlet on the flow channel member, from the inlet to the outlet through the measurement unit A sample supply method for supplying a sample to a measurement unit having a flow path through which a sample can flow, wherein an inlet pipette tip with a sample sucked is attached to an inlet, an outlet pipette tip is attached to an outlet, and the inlet In this method, the sample sucked into the pipette tip is sent out from the inlet, and the fluid pushed out from the flow path to the outlet by the sent sample is collected by the outlet pipette tip.

  Here, mounting the pipette tip may be a mode in which only the sample in the pipette tip is injected into the flow channel by bringing the tip of the pipette tip into close contact with the peripheral edge of the inlet or outlet of the flow channel member. A mode in which the tip portion is inserted into the inlet or the outlet of the flow channel member and the sample in the pipette tip is injected into the flow channel may be employed. Note that the tip of the pipette tip is closely attached to the inlet periphery or the outlet periphery of the flow path member is not limited to the case where the pipette tip and the flow path member are directly contacted, but another member between the pipette tip and the flow path member. This includes the case where these pipette tips, other members, and flow path members are all brought into close contact with each other.

  In addition, “collecting the fluid pushed out from the flow path into the outlet by the sample with the pipette tip for the exit” means that there is some fluid such as a liquid sample or air in the flow path. When a sample is sent out from the inlet side, it is pushed out to the outlet side by this sample, which means that such fluid is collected by the pipette tip for outlet. Of course, if a sample with an amount larger than the volume of the flow path is sent from the inlet, the fluid in the flow path is completely discharged and the sent sample is further discharged. The sample delivered from the inlet together with the fluid may also be collected.

  Furthermore, the sample collected by the outlet pipette tip is sent out from the outlet, and the fluid pushed out from the flow path to the inlet by the sent sample is collected by the inlet pipette tip, that is, from the outlet toward the inlet. The sample may be made to flow backward.

  The sample supply device of the present invention comprises a dielectric block having a thin film layer formed on a smooth surface and a flow path member in close contact with the thin film layer of the dielectric block. A measuring section facing the inlet, a supply path from the inlet on the flow path member to one end of the measuring section, and a discharge path from the other end of the measuring section to the outlet on the flow path member. A sample supply device for supplying a sample to a measurement unit having a flow path through which a sample can flow to an outlet, an inlet pipette tip for mounting on the inlet, an outlet pipette tip for mounting on the outlet, The inlet pipette tip mounting portion to which the inlet pipette tip can be attached and detached, the outlet pipette tip mounting portion to which the outlet pipette tip can be attached and detached, and the inlet pipette tip mounting portion are communicated with each other. It is to characterized in that it comprises an inlet for a piston for pumping the chromatography.

  In the sample supply device according to the present invention, it is preferable that the outlet pipette tip mounting portion includes a check valve for preventing the backflow of the sample entering from the outlet pipette tip side.

  In addition, it is further provided with an outlet piston that communicates with the outlet pipette tip mounting portion and sucks and discharges air from the outlet pipette tip mounting portion, so that the inlet piston and the outlet piston can be controlled independently. It is preferable to do.

  Furthermore, a plurality of sets of inlet pipette tips, outlet pipette tips, inlet pipette tips mounting portions, and outlet pipette tips mounting portions may be provided. In this case, an inlet piston may be provided for each inlet pipette tip mounting portion, or a plurality of inlet pipette tip mounting portions may be communicated with one inlet piston.

  The measurement unit in the sample supply method and the sample supply apparatus of the present invention is used for a so-called surface plasmon sensor in which a thin film layer is made of a metal film and measurement is performed using the effect of the surface plasmon resonance described above. It may be configured as a measurement unit. In addition, the thin film layer is composed of a clad layer formed on the one surface of the dielectric block and an optical waveguide layer formed on the clad layer, and measurement is performed by using the effect of waveguide mode excitation in the optical waveguide layer. It can be configured as a measurement unit used for a so-called leakage mode sensor.

  According to the sample supply method of the present invention, a dielectric block having a thin film layer formed on a smooth surface and a flow path member in close contact with the thin film layer of the dielectric block. A measuring section facing the inlet, a supply path from the inlet on the flow path member to one end of the measuring section, and a discharge path from the other end of the measuring section to the outlet on the flow path member. For a measurement unit equipped with a flow channel that allows the sample to flow to the outlet, the inlet pipette tip with the sample sucked is attached to the inlet, the outlet pipette tip is attached to the outlet, and the inlet pipette tip is sucked into the inlet pipette tip. Since the sample pushed out from the inlet and the fluid pushed out from the flow path to the outlet by the delivered sample is collected by the pipette tip for the outlet, both the inlet side and the outlet side are piped when the sample is supplied. The Without necessity, also because it is possible that none pipette tip for inlet and for the outlet to use a disposable type, it is possible to improve the maintainability of making the measurement.

  In addition, according to the sample supply device of the present invention, it is composed of a dielectric block having a thin film layer formed on a smooth surface, and a flow path member in close contact with the thin film layer of the dielectric block. It consists of a measurement section facing the thin film layer, a supply path from the inlet on the flow path member to one end of the measurement section, and a discharge path from the other end of the measurement section to the outlet on the flow path member. A sample supply device for supplying a sample to a measurement unit having a flow path through which a sample can flow to an outlet, an inlet pipette tip for mounting at the inlet, and an outlet pipette tip for mounting at the outlet; An inlet pipette tip mounting portion to which an inlet pipette tip can be attached and detached, an outlet pipette tip mounting portion to which an outlet pipette tip can be attached and detached, and an inlet pipette tip mounting portion that communicate with the inlet pipette tip mounting portion In addition, the inlet and outlet pipes are not required for supplying the sample, and both the inlet and outlet pipette tips are disposable. Since a thing can be used, the maintainability at the time of measuring can be improved.

  Also, the outlet pipette tip mounting part is equipped with a check valve that prevents the backflow of the sample that has entered from the outlet pipette tip side, so that the outlet pipette tip can be removed when the outlet pipette tip is removed from the flow path. The sample can be prevented from flowing back into the flow path from the pipette tip.

  In addition, it is further provided with an outlet piston that communicates with the outlet pipette tip mounting portion and sucks and discharges air from the outlet pipette tip mounting portion, so that the inlet piston and the outlet piston can be controlled independently. Thus, when supplying the sample into the flow path, the sample can be smoothly supplied by discharging air from the inlet piston and sucking air from the outlet piston. Further, when the sample is stored in the outlet pipette tip, the sample can be reversely fed by discharging air from the outlet piston and sucking air from the inlet piston. In addition, since the outlet pipette tip can be made negative pressure by providing such an outlet piston, the sample stored in the outlet pipette tip when the outlet pipette tip is removed from the flow path. Can be prevented from flowing back into the flow path.

  In addition, by providing multiple sets of inlet pipette tips, outlet pipette tips, inlet pipette tips mounting sections, and outlet pipette tips mounting sections, samples can be supplied to multiple channels simultaneously in parallel. It becomes possible to do.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. The sample supply apparatus according to the first embodiment of the present invention supplies a sample to the measurement unit of the measurement apparatus described below. The measurement apparatus will be described first. This measuring apparatus is a surface plasmon sensor capable of simultaneously analyzing a plurality of samples by making light beams incident on a plurality of measuring sections of a measuring unit in parallel. FIG. 1 shows a sample according to the present embodiment. FIG. 2 is a plan view of the measurement system of the surface plasmon sensor, FIG. 3 is a side view of the measurement system of the surface plasmon sensor, and FIG. 9 is a side view of FIG. It is the IX-IX sectional view taken on the line.

  As shown in FIG. 1, the surface plasmon sensor 1 has a surface that can simultaneously analyze a plurality of samples by allowing a light beam to enter in parallel for each of a plurality of measurement units provided in the measurement unit 10. A plasmon sensor, which is composed of a plurality of surface plasmon measurement systems 1A, 1B,. The configuration of each measurement system will be described by omitting the symbols A, B,.

  As shown in FIGS. 2, 3, and 9, each measurement system measures a light source 14 (hereinafter referred to as a laser light source 14) composed of a semiconductor laser or the like that generates one light beam 13 and the light beam 13. Through the unit 10, the light is incident in parallel on the two interfaces 50d and 50e between the dielectric block 50 and the metal film 55 below the flow channel 60 (measurement unit) so that various incident angles can be obtained. An optical system 15, two collimator lenses 16 for collimating the light beams 13 totally reflected by the interfaces 50d and 50e, and two photodiode arrays 17 for detecting the collimated light beams 13, respectively. A differential amplifier array 18 connected to the two photodiode arrays 17, a driver 19, a signal processing unit 20 including a computer system, and a display unit 21 connected to the signal processing unit 20. Yes. The signal processing unit 20 includes a storage unit (not shown) that stores data of a preliminary measurement result used in a correction operation described later, and also functions as a correction unit.

  First, the measurement unit 10 will be described. 4 is a perspective view of the measurement unit 10, FIG. 5 is an exploded perspective view of the measurement unit, FIG. 6 is a top view of the measurement unit, and FIG. 7 is a sectional view taken along the line VII-VII in FIG.

  The measurement unit 10 is transparent to the light beam and is in close contact with the dielectric block 50 in which a metal film 55 as a thin film layer is formed on a smooth upper surface 50a, and the metal film 55 of the dielectric block 50. The flow path member 51 and the holding member 52 that engages with the dielectric block 50 and holds the flow path member 51 on the upper surface 50a of the dielectric block 50 are configured.

  The dielectric block 50 is made of, for example, a transparent resin, and has a trapezoidal body whose section perpendicular to the longitudinal direction is shorter at the lower base than at the upper base, and at both ends in the longitudinal direction of the main body. A holding portion 50b having a width smaller than that of the main body when viewed from the upper surface (or lower surface) direction is formed, and a light beam emitted from a light source of a measuring apparatus described later is applied to the dielectric block 50 and the metal film. A prism portion is formed integrally so as to be incident on the interface with 55 and to emit the light beam totally reflected on the interface toward the light detection means of the measuring apparatus. On both side surfaces in the longitudinal direction of the main body are engaging convex portions 50c for engaging with engaging holes 52c formed in a holding member 52, which will be described later, and vertical convex portions 50d whose side surfaces are formed vertically. Sliding grooves 50e extending in parallel with the longitudinal direction are formed on the bottom surface.

  The flow path member 51 includes a plurality of flow paths 60 including a supply path 62 extending from the inlet 61 to the measurement section 63 and a discharge path 64 extending from the measurement section 63 to the outlet 65 in the longitudinal direction of the flow path member 51. The plurality of flow paths 60 are arranged in a straight line.

  As shown in FIG. 7, an inlet 61 and an outlet 65 are opened on the upper surface of the flow path member 51, an outlet of the supply path 62 and an inlet of the discharge path 64 are opened at the lower portion of the flow path member 51, and A seal portion 51a surrounding the outlet of the supply passage 62 and the inlet of the discharge passage 64 is formed in a region in contact with the surface of the metal film 55 located on the lower surface of the flow path member 51, and the inside of the seal portion 51a is measured. It becomes part 63. For this reason, when the flow path member 51 is brought into intimate contact with the metal film 55 of the dielectric block 50, the measurement section 63 in the seal section 51a functions as a flow path. Through the outlet 65, the buffer can flow in a substantially square shape. The seal portion 51a may be integrally formed with the upper portion of the flow path member 51, or may be formed of a material different from the upper portion and attached later, for example, O A ring or the like attached to the lower portion of the flow path member 51 may be used.

  In a measuring device such as a surface plasmon sensor using the measuring unit of the present invention, it is assumed that a liquid sample containing a protein is used. However, if the protein in the liquid sample is fixed in the flow channel 60, the measurement is performed. Therefore, it is preferable that the material of the flow path member 51 does not have non-specific adsorptivity to proteins, and specifically, silicon, polypropylene, or the like may be used. In addition, since the flow path member 51 is made of such an elastic material, the flow path member 51 can be reliably brought into close contact with the metal film 55, thereby preventing liquid leakage of the liquid sample from the contact surface. can do. Furthermore, a hydrophilization treatment may be performed in the flow path 60 in order to prevent adsorption of proteins and the like.

  The holding member 52 is made of an elastic material such as polypropylene, and the cross section in the direction orthogonal to the longitudinal direction has a substantially square shape, and the inlet 61 of the flow path member 51 of the upper plate (holding plate portion) of the holding member 52. Further, a tapered pipette insertion hole 52a that narrows toward the flow path member 51 is formed at a position facing the outlet 65, and pipette insertion at the middle of each pipette insertion hole 52a on the upper surface of the holding member 52 and at both ends A positioning boss 52b is formed on the outer side of the hole 52a.

  Further, an evaporation preventing member 54 is attached to the upper surface of the holding member 52 with a double-sided tape (adhesive member) 53. As shown in FIG. 5, a pipette insertion hole 53a is formed at a position facing the pipette insertion hole 52a of the double-sided tape 53, and a positioning hole 53b is formed at a position facing the boss 52b. Similarly, a slit 54a is formed at a position facing the pipette insertion hole 52a of the evaporation preventing member 54, and a positioning hole 54b is formed at a position facing the boss 52b. In a state where the hole 53b and the hole 54b of the evaporation preventing member 54 are inserted, the evaporation preventing member 54 is attached to the upper surface of the holding member 52, whereby the slit 54a of the evaporation preventing member 54, the inlet 61 and the outlet 65 of the flow path member 51 are obtained. Are configured to face each other. The evaporation preventing member 54 needs to be made of a material having elasticity so that a pipette can be inserted from the slit 54a, and specifically silicon or polypropylene may be used. The holding member 52 and the evaporation preventing member 54 may be integrally formed, and in addition to this, the flow path member 51 may be integrally formed.

  Furthermore, an engagement hole 52c for engaging with an engagement protrusion 50c formed in the dielectric block 50 is formed in the longitudinal side plate of the holding member 52, and the engagement hole 52c is engaged with the engagement protrusion 52c. In a state where the holding member 52 and the dielectric block 50 are engaged with each other by the portion 50c, the flow path member 51 is sandwiched between the holding member 52 and the dielectric block 50, and the flow path member 51 is the dielectric block. 50 is configured to be held on the upper surface 50a.

  As shown in FIG. 7, in a state where the flow path member 51 is sandwiched between the holding member 52 and the dielectric block 50, the inlet 61 and the outlet 65 of the flow path member 51 are separated from the outside air by the slit 54a of the evaporation preventing member 54. The liquid sample that is blocked and injected into the flow path 60 is configured to prevent evaporation.

  The incident optical system 15 includes a collimator lens 15a that collimates the light beam 13 emitted from the laser light source 14 in a divergent light state, a half mirror 15c that separates the collimated light beam 13, and a half mirror 15c. The light beam 13 reflected by the mirror 15d is reflected in the direction of the measurement unit 10, the light beam 13 transmitted through the half mirror 15c, and the light beam 13 reflected by the mirror 15d are converged on the interfaces 50d and 50e, respectively. It is composed of two condenser lenses 15b.

  Since the light beam 13 is condensed as described above, the light beam 13 includes components incident on the interfaces 50d and 50e at various incident angles θ. In addition, this incident angle (theta) shall be an angle more than a total reflection angle. Therefore, the light beam 13 is totally reflected at the interfaces 50d and 50e, and the reflected light beam 13 includes components reflected at various reflection angles. The optical system 15 may be configured to allow the light beam 13 to enter the interfaces 50d and 50e in a defocused state. By doing so, errors in surface plasmon resonance state detection are averaged, and measurement accuracy is improved.

  The light beam 13 is incident on the interfaces 50d and 50e as p-polarized light. In order to do so, the laser light source 14 may be disposed in advance so that the polarization direction thereof is a predetermined direction. In addition, the direction of polarization of the light beam 13 may be controlled with a wave plate.

As shown in FIG. 9, in the present embodiment, the measurement unit 63 of each flow path 60 of the measurement unit 10 is used.
The light beam 13 is incident on the two interfaces 50d and 50e in parallel, but the metal film 55 on one of the interfaces 50d is used as a reference region where nothing is fixed, and the other interface is used. The metal film 55 on 50e is used as a detection region in which the ligand 73 is fixed so that the measurement result can be calibrated by the reference method described later.

  The sample supply apparatus according to the present embodiment supplies a sample to each channel 60 of the measurement unit 10 of the surface plasmon sensor 1 configured as described above, and the sample supply apparatus will be described with reference to the drawings. FIG. 8 is an external view of this sample supply apparatus.

  The sample supply device 80 includes a main body 81 in which an inlet pipette tip mounting portion 83 and an outlet pipette tip mounting portion 84 are formed, and is built in the main body 81 and communicates with the inlet pipette tip mounting portion 83, and is used for the inlet. An inlet piston (not shown) that sucks and discharges air from the pipette tip mounting portion 83 and a built-in body 81, communicates with the outlet pipette tip mounting portion 84, and stores the sample that has entered from the outlet pipette tip mounting portion 84. A storage portion (not shown), an operation portion 82 for operating the inlet piston, an inlet pipette tip 90 attached to the inlet pipette tip attachment portion 83, and an outlet pipette tip attachment portion 84 are attached. And an outlet pipette tip 91. In addition, the outlet pipette tip mounting portion 84 includes a check valve (not shown) that prevents the backflow of the sample that has entered from the outlet pipette tip 91 side.

  The distance between the center of the inlet pipette tip mounting portion 83 and the center of the outlet pipette tip mounting portion 84 is configured to be substantially equal to the distance between the center of the inlet 61 and the center of the outlet 65 of the flow path 60. With the inlet pipette tip 90 and the outlet pipette tip 91 attached to the inlet pipette tip attachment portion 83 and the outlet pipette tip attachment portion 84, the tips of both pipette tips are connected to the inlet 61 and outlet 65 of the flow channel 60. By inserting, the sample can be moved from the inlet pipette tip 90 to the outlet pipette tip 91.

  The inlet pipette tip 90 and the outlet pipette tip 91 are disposable pipette tips that are detachable from the inlet pipette tip mounting portion 83 and the outlet pipette tip mounting portion 84, respectively.

  Further, the outer diameters of the tip portions of the inlet pipette tip 90 and the outlet pipette tip 91 are substantially equal to the inner diameters of the inlet 61 and outlet 65 of the flow path 60 (or the inner diameter of the pipette insertion hole 52a of the holding member 52). By doing so, when the sample supply device 80 is inserted into the measurement unit 10, it is possible to prevent unexpected bubble mixing and sample evaporation.

  Hereinafter, sample analysis by the surface plasmon sensor 1 having the above-described configuration will be described. Prior to the measurement, the measurement unit 10 is moved from the temperature-controlled room 2 toward the measurement position on the chip holder 11. The chip holding portion 11 is formed with a rail 11a that engages with a sliding groove 50e formed in the dielectric block 50, so that high positional accuracy can be ensured when the measuring unit 10 is moved. ing. Further, after the measurement unit 10 is placed on the chip holding part 11, the vertical convex part 50d formed on the dielectric block 50 is clamped by a fixing mechanism (not shown) and fixed at the measurement position on the chip holding part 11. Is done. Thereafter, as shown in FIG. 9, after inserting the inlet pipette tip 90 and the outlet pipette tip 91 into which the buffer 72 containing the analyte as a liquid sample of the flow path member 51 is inserted into the inlet 61 and the outlet 65, respectively, By operating the part 82, the buffer 72 is sent from the inlet pipette tip 90, and the buffer 72 is supplied to the measuring part 63 of the flow path 60. At this time, since the protective solution 75 (or the buffer for the previous measurement, etc.) for preventing the ligand 73 from drying is injected into the flow path 60 before the injection of the buffer 72, this protective solution 75 In order to prevent mixing of the buffer 72 used for the new measurement (or the buffer for the previous measurement) with the bubble 72 in the tip of the inlet pipette tip 90, the buffer 72 is allowed to flow. If bubbles 74 enter the channel 60 first when injected into the channel 60, the bubbles 72 separate the buffer 72 and the protective liquid 75 (or the buffer for the previous measurement, etc.). The mixture of the two can be prevented, and the protective liquid 75 (or the buffer for the previous measurement or the like) is pushed out by the bubbles 74 and all discharged into the outlet pipette tip 91. In addition, although the case of exchanging the buffer 72 and the protective liquid 75 was described here, the exchange from the reference buffer to the buffer containing the analyte, or the exchange from the buffer containing the analyte to the reference buffer, etc. When the liquid such as the sample in the flow channel 60 is exchanged, the liquid can be prevented from being mixed before and after the exchange by the above method. Of course, a liquid such as a sample may be directly injected without using the above method.

  After supplying the buffer 72 to the measurement unit 63 of the flow channel 60 as described above, the measurement is started. Note that the sample supply device 80 is removed from the measurement unit 10 during measurement in order to prevent the surface plasmon sensor 1 from being adversely affected by unnecessary vibrations and the like.

  As shown in FIG. 3, the light beam 13 emitted from the laser light source 14 in a divergent light state is caused on the interfaces 50d and 50e between the dielectric block 50 and the metal film 55 under the measuring unit 63 by the action of the optical system 15. Converge. At this time, the light beam 13 includes components incident on the interfaces 50d and 50e at various incident angles θ. In addition, this incident angle (theta) shall be an angle more than a total reflection angle. Therefore, the light beam 13 is totally reflected at the interfaces 50d and 50e, and the reflected light beam 13 includes components reflected at various reflection angles.

  After total reflection at the interfaces 50d and 50e, the two light beams 13 respectively collimated by the two collimator lenses 16 are detected by the two photodiode arrays 17, respectively. In the photodiode array 17 in this example, a plurality of photodiodes 17a, 17b, 17c... Are arranged in a line, and in the traveling direction of the collimated light beam 13 in the plane shown in FIG. On the other hand, the photodiodes are arranged in a direction in which the parallel arrangement direction of the photodiodes is substantially a right angle. Therefore, different photodiodes 17a, 17b, 17c,... Receive each component of the light beam 13 totally reflected at various reflection angles at the interfaces 50d and 50e.

  FIG. 10 is a block diagram showing an electrical configuration of the surface plasmon sensor. As shown in the figure, the driver 19 samples and holds the outputs of the differential amplifiers 18a, 18b, 18c,... Of the differential amplifier array 18, and these sample-hold circuits 22a, 22b. , 22c... Are input to the multiplexer 23, the A / D converter 24 which digitizes the output of the multiplexer 23 and inputs it to the signal processing unit 20, the multiplexer 23 and the sample hold circuits 22a, 22b, 22c. And a controller 26 that controls the operation of the drive circuit 25 based on an instruction from the signal processing unit 20. The differential amplifier array 18, the driver 19, and the signal processing unit 20 are configured to perform the same processing in parallel on the inputs from the two photodiode arrays 17.

  The outputs of the photodiodes 17a, 17b, 17c,... Are input to the differential amplifiers 18a, 18b, 18c,. At this time, the outputs of two photodiodes adjacent to each other are input to a common differential amplifier. Therefore, it can be considered that the outputs of the differential amplifiers 18a, 18b, 18c,... Are obtained by differentiating the photodetection signals output from the plurality of photodiodes 17a, 17b, 17c,.

  The outputs of the differential amplifiers 18a, 18b, 18c... Are sampled and held at predetermined timings by the sample hold circuits 22a, 22b, 22c. The multiplexer 23 inputs the sampled and held outputs of the differential amplifiers 18a, 18b, 18c,... Into the A / D converter 24 in a predetermined order. The A / D converter 24 digitizes these outputs and inputs them to the signal processing unit 20.

  FIG. 11 illustrates the relationship between the light intensity for each incident angle θ of the light beam 13 totally reflected at the interface 50d (or 50e) and the outputs of the differential amplifiers 18a, 18b, 18c. Here, it is assumed that the relationship between the incident angle θ of the light beam 13 with respect to the interface 50d (or 50e) and the light intensity I is as shown in the graph of FIG.

Light incident at a specific incident angle θ _SP at the interface 50d (or 50e) excites surface plasmons at the interface between the metal film 55 and the buffer 72, and the reflected light intensity I sharply decreases for this light. That theta _SP is attenuated total reflection angle, the reflected light intensity I in the angle theta _SP takes a minimum value. This decrease in the reflected light intensity I is observed as a dark line in the reflected light, as indicated by D in FIG.

  11 (2) shows the direction in which the photodiodes 17a, 17b, 17c,... Are arranged in parallel. As described above, the positions of the photodiodes 17a, 17b, 17c,. It uniquely corresponds to the incident angle θ.

  The relationship between the positions of the photodiodes 17a, 17b, 17c..., That is, the incident angle θ, and the output I ′ (differential value of the reflected light intensity I) of the differential amplifiers 18a, 18b, 18c. As shown in FIG.

Based on the value of the differential value I ′ input from the A / D converter 24, the signal processing unit 20 selects the differential corresponding to the total reflection attenuation angle θ _SP from among the differential amplifiers 18a, 18b, 18c. After selecting the output that is closest to the value I ′ = 0 (becomes the differential amplifier 18d in the example of FIG. 11) and applying a predetermined correction process to the differential value I ′ that is output, The value is displayed on the display unit 21. In some cases, there may be a differential amplifier that outputs a differential value I ′ = 0. In this case, the differential amplifier is naturally selected.

Thereafter, the differential value I ′ output from one of the differential amplifiers selected as described above every time a predetermined time elapses is displayed on the display unit 21 after receiving a predetermined correction process. The differential value I'has a dielectric constant clogging material in contact with the metal film 55 of the measuring chip refractive index changes, the attenuated total reflection angle theta _SP is changed, the left and right curve shown in FIG. 11 (1) If it changes in a moving direction, it will move up and down accordingly. Therefore, by continuously measuring this differential value I ′ with the passage of time, the refractive index change of the buffer 72 (or the ligand 73) in contact with the metal film 55 can be examined.

  In particular, in the surface plasmon sensor 1, if the analyte contained in the buffer 72 is a specific substance that binds to the ligand 73 in the detection region, the refractive index of the ligand 73 changes according to the binding state between the ligand 73 and the analyte. Therefore, it is possible to detect whether the analyte is a specific substance that binds to the ligand 73 by continuing to measure the differential value I ′.

  Further, the surface plasmon sensor 1 has two regions, a detection region and a reference region, for performing the reference method, and the measurement of the two regions is performed at the same time. Since the error due to disturbance such as light source fluctuation can be calibrated, the measurement accuracy can be improved.

  Next, a second embodiment of the present invention will be described with reference to FIG. In FIG. 12, the same elements as those in FIG. 8 are denoted by the same reference numerals, and description thereof will be omitted unless particularly required. The sample supply apparatus according to the second embodiment is modified so that samples can be supplied simultaneously to a plurality of flow paths. FIG. 12 is an external view of this sample supply apparatus.

  The sample supply device 80 ′ includes a main body 81 ′ in which a plurality of sets of inlet pipette tip mounting portions 83 and outlet pipette tip mounting portions 84 are formed, and the main body 81 ′, and the inlet pipette tip mounting portion 83 The inlet piston (not shown) that sucks and discharges air from the inlet pipette tip mounting portion 83, the operation portion 82 for operating the inlet piston, and the inlet pipette tip mounting portion 83 A plurality of inlet pipette tips 90 and a plurality of outlet pipette tips 91 attached to the outlet pipette tip attaching portion 84.

  By using such a sample supply device 80 ', it is possible to supply samples to a plurality of flow paths simultaneously.

  The sample supply apparatus described in the first and second embodiments includes only an inlet piston that communicates with the inlet pipette tip mounting portion and sucks and discharges air from the inlet pipette tip mounting portion. Is further provided with an outlet piston that communicates with the outlet pipette tip mounting portion and sucks and discharges air from the outlet pipette tip mounting portion so that the inlet piston and the outlet piston can be controlled independently. May be.

  The pipette tip tip is inserted into the inlet or outlet of the flow channel member and the sample in the pipette tip is not injected into the flow channel, but as shown in FIG. The tip 90a 'and the tip 91a' of the outlet pipette tip 91 'are brought into close contact with the pipette engaging portion 52d' of the holding member 52 'on the channel member, respectively, and only the sample in the pipette tip is injected into the channel 60. It is good also as an aspect to do. Of course, the pipette tip and the flow path member may be in direct contact with each other.

  In addition, the sample collected by the outlet pipette tip is sent out from the outlet, and the fluid pushed out from the flow path to the inlet by the sent sample is collected by the inlet pipette tip, that is, from the outlet toward the inlet. The sample may be made to flow backward.

  The sample supply device can be used not only for a surface plasmon sensor measurement unit but also for various types such as a leakage mode sensor measurement unit.

The top view which shows schematic structure of the surface plasmon sensor corresponding to the sample supply apparatus of the 1st Embodiment of this invention Plan view of the measurement system of the above surface plasmon sensor Side view of the measurement system of the above surface plasmon sensor Perspective view of the measurement unit of the surface plasmon sensor Exploded perspective view of the measurement unit Top view of the above measurement unit VII-VII line sectional view in FIG. 1 is an external view of a sample supply device according to a first embodiment of the present invention. IX-IX sectional view in FIG. Block diagram showing the electrical configuration of the measurement system of the surface plasmon sensor Schematic showing the relationship between the light beam incident angle and the detected light intensity and the relationship between the light beam incident angle and the differential value of the light intensity detection signal in the measurement system of the surface plasmon sensor. External view of the sample supply device of the second embodiment of the present invention Sectional drawing which shows the other aspect of the sample supply apparatus of this invention

Explanation of symbols

10 Measuring unit
13 Light beam
14 Laser light source
15 Optical system
16 Collimator lens
17 Photodiode array
17a, 17b, 17c …… Photodiode
18 Differential amplifier array
18a, 18b, 18c ... Differential amplifier
19 Drivers
20 Signal processor
21 Display
22a, 22b, 22c ... Sample hold circuit
23 Multiplexer
24 A / D converter
25 Drive circuit
26 Controller
50 dielectric block
51 Channel member
52 Holding member
53 Double-sided tape
54 Evaporation prevention member
55 Metal film
56 Clad layer
57 Lightwave layer
60 channels
61 entrance
62 Supply channel
63 Measurement unit
64 Discharge channel
65 Exit
72 Liquid sample
73 Ligand
80 Sample feeder
81 body
82 Operation unit
83 Entrance pipette tip attachment
84 Exit pipette tip mounting part
90 Pipette tip for entrance
91 Pipette tip for outlet

Claims (6)

A dielectric block in which a thin film layer is formed on a smooth surface, and a flow path member in close contact with the thin film layer of the dielectric block, the flow path member being a measurement section facing the thin film layer, It is composed of a supply path from the inlet on the flow path member to one end of the measurement part, and a discharge path from the other end of the measurement part to the outlet on the flow path member, and from the inlet through the measurement part A sample supply method for supplying a sample to a measurement unit having a flow path through which the sample can flow to the outlet,
Attach an inlet pipette tip into which the sample is sucked,
Attach an outlet pipette tip to the outlet,
The sample sucked by the inlet pipette tip is sent out from the inlet, and the fluid pushed out from the flow path by the sample to the outlet is collected by the outlet pipette tip. Supply method.   2. The sample collected by the outlet pipette tip is sent out from the outlet, and the fluid pushed out from the flow path into the inlet by the sample is collected by the inlet pipette tip. The sample supply method described. A dielectric block in which a thin film layer is formed on a smooth surface, and a flow path member in close contact with the thin film layer of the dielectric block, the flow path member being a measurement section facing the thin film layer, It is composed of a supply path from the inlet on the flow path member to one end of the measurement part, and a discharge path from the other end of the measurement part to the outlet on the flow path member, and from the inlet through the measurement part A sample supply device for supplying a sample to a measurement unit having a flow path through which the sample can flow to the outlet,
An inlet pipette tip for attachment to the inlet;
An outlet pipette tip for mounting on the outlet;
An inlet pipette tip mounting portion to which the inlet pipette tip can be attached and detached; and
An outlet pipette tip mounting portion to which the outlet pipette tip can be attached and detached;
A sample supply device comprising: an inlet piston communicating with the inlet pipette tip mounting portion and for sucking and discharging air from the inlet pipette tip mounting portion.   The sample supply device according to claim 3, wherein the outlet pipette tip mounting portion includes a check valve that prevents a backflow of the sample that has entered from the outlet pipette tip side. An outlet piston that communicates with the outlet pipette tip mounting portion and sucks and discharges air from the outlet pipette tip mounting portion;
5. The sample supply device according to claim 3, wherein the inlet piston and the outlet piston can be controlled independently.   6. The sample according to claim 3, comprising a plurality of sets of the inlet pipette tip, the outlet pipette tip, the inlet pipette tip mounting portion, and the outlet pipette tip mounting portion. Feeding device.

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