JP2015059898A - Immunoassay analysis method and device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an immunoassay analysis method and device in which excess time for switching does not occur even when a detection range of a light reception sensor is switched.SOLUTION: A detection range of a light reception sensor is changed by changing control voltage of the light reception sensor, in an SPFS device. Before washing processing before signal measurement is completed (S210, 211), steps from prior measurement for determination of the detection range to setting of a proper detection range (S411-413) are completed. Before actual measurement, the detection range is optimized, and after washing of a channel after a fluorescent label, the actual measurement is immediately made possible.

Description

  The present invention relates to an immunoassay analysis method and an immunoassay analyzer, and more particularly, to a surface plasmon resonance fluorescence analyzer and a surface plasmon for detecting a substance to be detected contained in a sample liquid using surface plasmon resonance (SPR). The present invention relates to an immunoassay analysis method and an immunoassay analyzer suitable for a resonance fluorescence analysis method.

  In a measurement for detecting a biological substance such as protein or DNA, if a small amount of a substance to be detected can be detected quickly, it is possible to immediately grasp the patient's condition and perform treatment. Therefore, there is a need for an analysis method and an analysis apparatus that can detect faint light caused by a small amount of a substance to be detected quickly and with high sensitivity. As one method for detecting the target substance with high sensitivity, a surface plasmon resonance fluorescence analysis (Surface Plasmon-field enhanced Fluorescence Spectroscopy: SPFS) method is known.

  The SPFS method uses a prism in which a metal film made of gold or silver is formed on a predetermined surface. Then, when the metal film is irradiated with excitation light through the prism at an angle at which surface plasmon resonance occurs, localized field light can be generated on the surface of the metal film. The detected substance captured on the metal film or the fluorescent substance that labels the detected substance is selectively excited by the localized field light, and the fluorescence emitted from the excited detected substance or the fluorescent substance is detected. Thus, the presence of the substance to be detected and the amount thereof can be detected.

  In order to detect faint light with high quantitativeness, it is indispensable to use a highly sensitive light receiving sensor. As the light receiving sensor, a photomultiplier (PMT) and an avalanche photodiode (APD) are used.

  The light receiving sensor can detect, for example, from a very small amount of a substance to be detected to a sufficient amount of a substance to be detected by switching the range of the measurement light quantity (hereinafter also referred to as “detection range”). There are a mechanical method and an electrical method for switching the detection range. The mechanical method is a method in which an ND (attenuation) filter is put in and out of the optical path of light incident on the light receiving sensor, thereby reducing the amount of light incident on the light receiving sensor and switching the detection range ( For example, see Patent Document 1). The electrical method is a method of changing the control voltage of the light receiving sensor, whereby the detection range of the light receiving sensor is electrically changed and the detection range is switched (for example, see Non-Patent Document 1).

International Publication No. 2012/172987

Hamamatsu Photonics Co., Ltd. Editorial Board, “Photomultiplier Tubes-Fundamentals and Applications”, 3rd edition, Hamamatsu Photonics, Inc., August 1, 2005, p. 154-166

  The detection range is usually determined based on the detected light amount once the light amount of the fluorescence from the fluorescent substance labeling the substance to be detected is detected once by the light receiving sensor. Then, an optimal detection range is selected based on the determination result. Then, the detection range of the light receiving sensor is switched to the optimum detection range by any one of the methods described above. Thereafter, the light amount of the fluorescence related to the substance to be detected is detected by the light receiving sensor having the optimum detection range.

  When the detection range is switched by the above method, it is necessary to further switch the detection range after the target substance is fluorescently labeled. The time for switching the detection range is the time for the ND filter to advance and retreat with respect to the optical path in the mechanical method. In the electrical method described above, it is the time from when the detection range of the light receiving sensor is changed until the light receiving sensor is electrically stabilized. In either method, for example, about 10 seconds are required to switch the detection range.

  However, when the substance to be detected is labeled with a fluorescent substance by a biochemical reaction, the measurement accuracy or reproducibility of the fluorescence may decrease depending on the timing of measuring the fluorescence after labeling. This is because the fluorescent labeling of a substance to be detected by a biochemical reaction usually uses the interaction between the substance to be detected and the fluorescent substance, and the substance to be detected is detected after the fluorescent labeling of the substance to be detected is completed. This is because dissociation of the fluorescent substance with time occurs. For this reason, in the immunoassay analysis method, it is desired to measure the fluorescence as quickly as possible after fluorescent labeling of the substance to be detected.

  An object of the present invention is to provide an immunoassay analysis method and an immunoassay analyzer that do not cause an excess time for switching even if the detection range of a light receiving sensor is switched.

  The present invention irradiates excitation light toward an analysis chip in which a target substance labeled with a fluorescent substance is immobilized in a flow path, and detects fluorescence from the excited fluorescent substance with a light receiving sensor. In the immunoassay analysis method for measuring the amount of the substance to be detected, the substance to be detected is supplied to the flow path of the analysis chip together with the fluorescent substance, and the substance to be detected labeled with the fluorescent substance is supplied to the flow. When performing the fluorescent substance washing step of washing the flow path so as to remove excess fluorescent substance in the flow path after the first immobilization process of immobilizing in the path, the first immobilization process Before the fluorescent substance cleaning step is performed, the excitation light is irradiated toward the analysis chip, and the amount of first emission light emitted from the analysis chip by the excitation light irradiation or For irradiation of the excitation light A first light quantity detection step for detecting the light quantity of the first total reflected light reflected by the analysis chip, or a second immobilization step for immobilizing the substance to be detected in the flow path of the analysis chip A first washing step for washing the flow path after the labeling, and then supplying the fluorescent substance to the flow path and labeling the target substance immobilized in the flow path with the fluorescent substance When performing the second washing step of washing the flow path so as to remove excess fluorescent substance in the flow path after the process, from after the second immobilization process to before the labeling process During this time, the excitation light is irradiated toward the analysis chip, and the amount of second emission light emitted from the analysis chip by the irradiation of the excitation light or reflected by the analysis chip by the irradiation of the excitation light. Second to detect the amount of second total reflected light A quantity detection step is performed, or during the period from the labeling step until the second washing step is performed, the excitation light is irradiated toward the analysis chip, and the analysis is performed by irradiation with the excitation light. Performing a third light quantity detection step of detecting a light quantity of the third emitted light emitted from the chip or a third total reflected light reflected by the analysis chip by irradiation of the excitation light, and the amount of the substance to be detected is determined. Before irradiating the excitation light toward the analysis chip to detect the fluorescence from the fluorescent material for measurement, the first light amount detection step, the second light amount detection step, and the third light amount detection Based on the amount of light detected by at least one of the steps, one detection range is selected from a plurality of detection ranges of the light receiving sensor, and switching to the selected detection range is performed. Then, in order to measure the amount of the substance to be detected, an immunoassay analysis method is provided in which the fluorescence from the fluorescent substance is detected by the light receiving sensor whose detection range is switched.

Further, the present invention is to label the substance to be detected fixed in the flow path of the analysis chip with a fluorescent substance, and when irradiating excitation light from a light source toward the portion where the substance to be detected in the flow path is fixed, In an immunoassay analyzer for detecting the fluorescence from the excited fluorescent substance with a light receiving sensor and measuring the amount of the detected substance, the excitation light is irradiated toward the part where the detected substance is fixed. And a control for setting a detection range of the first light receiving sensor for detecting at least the light amount of the fluorescent light, and a first light receiving sensor for detecting the light amount of the fluorescent light generated in the flow path. And an immunoassay analyzer that satisfies the following (A) or (B).
(A) The control unit is configured so that the flow path is washed after the substance to be detected is labeled with the fluorescent substance or before the washing (that is, after the process of fixing the substance to be detected is completed, Emission emitted from the channel by irradiating the excitation light toward the portion where the substance to be detected is fixed while the excess fluorescent substance in the channel is being cleaned and removed (until cleaning is completed) One detection range of the first light receiving sensor when detecting the light amount of the fluorescence is selected from a plurality of detection ranges from the light amount detected by the first light receiving sensor of the light, and the flow is at the latest. By the time the cleaning of the road is completed, the switching of the detection range of the first light receiving sensor to one detection range is completed.
(B) A second light receiving sensor for detecting the amount of total reflected light on the analysis chip generated by irradiation of the excitation light from the light source toward the portion where the substance to be detected is fixed is further provided. Then, the control unit fixes the detected substance, before the flow path is cleaned, or while the flow path is being cleaned (that is, after completion of the process of fixing the detected substance, The total reflected light generated by irradiating the excitation light toward the portion where the substance to be detected is fixed while the excessive fluorescent substance in the path is being washed away (until washing is completed), One detection range of the first light receiving sensor for detecting the light amount of the fluorescence is selected from a plurality of detection ranges from the light amount detected by the second light receiving sensor, and the flow path is washed at the latest. Until , The detection range of the first light receiving sensor to complete the switch to one of the detection range.

  According to the present invention, when the cleaning operation of the flow path after the detection target substance is labeled with the fluorescent substance is completed, the detection range of the light receiving sensor can be switched to the optimum detection range. Therefore, according to the present invention, the detection range of the light receiving sensor can be switched to, for example, an optimal detection range, and the time for the switching does not exceed the time required for preparation for measurement. As a result, since the amount of the substance to be detected can be measured immediately after washing the channel, the influence of dissociation between the substance to be detected and the fluorescent substance can be reduced in the immunoassay analysis. Therefore, according to the present invention, the amount of the substance to be detected can be detected quickly and with high quantitativeness regardless of the amount of the substance to be detected.

It is a figure which shows typically the structure of the immunoassay analyzer (SPFS apparatus) which concerns on 1st embodiment of this invention. It is a flowchart which shows the main operation | movement procedures of the said SPFS apparatus. It is a figure which shows roughly the relationship between the washing | cleaning operation time (washing | cleaning frequency) of the flow path in the said SPFS apparatus, and the density | concentration of the fluorescent substance in a flow path. It is a flowchart which shows the procedure of the 1st example of the determination of the detection range of the light reception sensor in the said SPFS apparatus, and the setting method. It is a figure which shows typically the structure of the immunoassay analyzer (SPFS apparatus) which concerns on 2nd embodiment of this invention. It is a flowchart which shows the procedure of the 2nd example of the determination of the detection range of the light reception sensor in the said SPFS apparatus, and the setting method. It is a figure which shows typically the structure of the immunoassay analyzer which concerns on 3rd embodiment of this invention.

  The immunoassay analysis method according to the present invention irradiates an analysis chip with excitation light, and detects the amount of fluorescence generated by excitation of a fluorescent substance that labels the detection target substance arranged on the analysis chip by a light receiving sensor. Measure the amount of the detected substance. Examples of the immunoassay analysis method include surface plasmon resonance fluorescence analysis (SPFS). Hereinafter, an embodiment using the SPFS method will be described as an embodiment of the present invention. First, a surface plasmon resonance fluorescence analyzer (hereinafter also referred to as “SPFS apparatus”) according to an embodiment of the present invention will be described.

  FIG. 1 is a schematic diagram showing a configuration of an SPFS apparatus 100 according to an embodiment of the present invention. The SPFS device 100 makes the excitation light incident on the metal film on the dielectric prism at an angle at which surface plasmon resonance occurs, thereby causing localized field light (generally “enhanced evanescent wave” or “ (Also called “enhanced electric field”). The concentration or amount of the target substance is detected by selectively exciting the fluorescent substance that labels the target substance placed on the metal film by the localized field light and detecting the amount of fluorescence emitted from the fluorescent substance. Is detected.

  As shown in FIG. 1, the SPFS apparatus 100 includes an excitation optical system unit 110, a light receiving optical system unit 120, a liquid feeding unit 130, and a control unit 160. The SPFS device 100 is used in a state in which the analysis chip 10 is mounted on a chip holder (not shown) for measurement of a substance to be detected. Therefore, the analysis chip 10 will be described first, and then each component of the SPFS device 100 will be described.

  As shown in FIG. 1, the analysis chip 10 includes a prism 20 having an incident surface 21, a film formation surface 22 and an emission surface 23, a metal film 30 formed on the film formation surface 22, and a film formation surface 22 or a metal. And a flow path lid 40 disposed on the membrane 30. Usually, the analysis chip 10 is replaced for each analysis. The analysis chip 10 is preferably a structure in which each piece has a length of several mm to several cm, but is a smaller structure or a larger structure not included in the category of “chip”. Also good.

  The prism 20 is made of a dielectric that is transparent to the excitation light α. The prism 20 has an incident surface 21, a film forming surface 22, and an exit surface 23. The incident surface 21 allows the excitation light α from the excitation optical system unit 110 to enter the prism 20. A metal film 30 is formed on the film formation surface 22. The excitation light α incident on the inside of the prism 20 is reflected by the metal film 30. More specifically, the light is reflected at the interface (deposition surface 22) between the prism 20 and the metal film 30. The emission surface 23 emits the excitation light α reflected by the metal film 30 to the outside of the prism 20.

  The shape of the prism 20 is not particularly limited. In the present embodiment, the shape of the prism 20 is a column having a trapezoidal bottom surface. The surface corresponding to one base of the trapezoid is the film formation surface 22, the surface corresponding to one leg is the incident surface 21, and the surface corresponding to the other leg is the emission surface 23. The trapezoid serving as the bottom surface is preferably an isosceles trapezoid. Thereby, the entrance surface 21 and the exit surface 23 are symmetric, and the S wave component of the excitation light α is less likely to stay in the prism 20.

  The incident surface 21 is formed so that the excitation light α does not return to the excitation optical system unit 110. This is because when the excitation light α returns to the laser diode that is the excitation light source, the excitation state of the laser diode is disturbed, and the wavelength and output of the excitation light α change. Therefore, the angle of the incident surface 21 is set so that the excitation light α does not enter the incident surface 21 perpendicularly in the scanning range centered on the ideal enhancement angle. For example, the angle between the incident surface 21 and the film formation surface 22 and the angle between the film formation surface 22 and the emission surface 23 are both about 80 °.

  It should be noted that the resonance angle (and the enhancement angle in the vicinity thereof) is substantially determined by the design of the analysis chip 10. Design factors include prism refractive index, metal refractive index, metal film thickness, metal extinction coefficient, excitation wavelength, and the like. The resonance angle and the enhancement angle are shifted according to the amount of the substance to be detected fixed to the metal film, but the amount is less than a few degrees.

  The prism 20 has a number of birefringence characteristics. Examples of the material of the prism 20 include resin and glass. The material of the prism 20 is preferably a resin having a refractive index of 1.4 to 1.6 and a small birefringence.

  The metal film 30 is formed on the film formation surface 22 of the prism 20. As a result, an interaction, that is, surface plasmon resonance occurs between the photon of the excitation light α incident on the film formation surface 22 under the total reflection condition and the free electrons in the metal film 30, and is locally on the surface of the metal film 30. In-situ light can be generated.

  The material of the metal film 30 is not particularly limited as long as it is a metal that causes surface plasmon resonance. Examples of the material of the metal film 30 include gold, silver, copper, aluminum, and alloys thereof. In the present embodiment, the metal film 30 is a gold thin film. The method for forming the metal film 30 is not particularly limited. Examples of the method for forming the metal film 30 include sputtering, vapor deposition, and plating. The thickness of the metal film 30 is not particularly limited, but is preferably in the range of 30 to 70 nm.

  Although not shown in FIG. 1, a capturing body for capturing a substance to be detected may be fixed to the surface of the metal film 30 that does not face the prism 20 (the surface of the metal film 30). By fixing the capturing body, it becomes possible to selectively detect the substance to be detected. In the present embodiment, the capturing body is uniformly fixed to a predetermined region on the metal film 30. The type of capturing body is not particularly limited as long as it can capture the substance to be detected. For example, the capturing body is an antibody specific to the substance to be detected or a fragment thereof.

  The channel lid 40 is disposed on the surface of the metal film 30 that does not face the prism 20 with the channel interposed therebetween. When the metal film 30 is formed only on a part of the film formation surface 22 of the prism 20, the flow path lid 40 may be disposed on the film formation surface 22 with the flow path interposed therebetween. The channel lid 40 forms a channel through which liquid flows together with the metal film 30 (and the prism 20). Examples of the liquid include a sample solution and a chemical solution that are solutions of the substance to be detected, and examples of the chemical solution include a solution of a fluorescent material and a cleaning solution. The capturing body is exposed in a flow path (not shown). Both ends of the channel are connected to an inlet and an outlet (not shown) formed on the upper surface of the channel lid 40, respectively. When the liquid is injected into the flow path, the liquid comes into contact with the capturing body in the flow path.

  The channel lid 40 is made of a material that is transparent to light (plasmon scattered light and fluorescence) emitted from the metal film 30. An example of the material of the flow path lid 40 includes a resin. The flow path cover 40 only needs to be able to guide these lights to the light receiving optical system unit 120. For example, a part of the channel lid 40 may be made of an opaque material as long as at least the surface from which the fluorescence from the fluorescent substance labeled with the substance to be detected is extracted is optically transparent. The flow path lid 40 is bonded to the metal film 30 or the prism 20 by, for example, adhesion using a double-sided tape or an adhesive, laser welding, ultrasonic welding, or pressure bonding using a clamp member.

  As described above, the analysis chip 10 includes the prism 20, the metal film 30 disposed on one surface of the prism 20, and the flow path in which the surface of the metal film 30 is exposed. In the analysis chip 10 configured as described above, a fluorescent substance is supplied into the flow path. Then, the analysis chip having the detection target substance supplied on the metal film and labeled with the fluorescent substance is installed and held in a predetermined posture on the chip holder. In the present embodiment, the detection range of the light receiving sensor 127 is adjusted in parallel with the labeling of the substance to be detected by the fluorescent substance. The adjustment of the detection range will be described in detail later.

  The excitation optical system unit 110 includes a configuration for emitting the excitation light α toward the flow path of the prism 20 and a configuration for scanning the incident angle of the excitation light α on the metal film 30. The excitation optical system unit 110 includes, for example, a light source unit 111, an angle adjustment mechanism 112, and a light source control unit 113. Here, the “excitation light” is light that directly or indirectly excites a fluorescent substance that labels the substance to be detected. If the “excitation light” is light that directly excites the fluorescent substance, it is irradiated directly on the target substance, and if it is light that indirectly excites the fluorescent substance, it is a place where excitation of the fluorescent substance occurs in the analysis chip. Is irradiated. For example, the excitation light α is light that generates localized field light on the surface of the metal film 30, and is light that indirectly excites the fluorescent substance by the localized field light. The excitation light α is irradiated to the metal film 30 through the prism at an angle at which surface plasmon resonance occurs.

  The light source unit 111 emits the collimated excitation light α having a constant wavelength and light amount so that the shape of the irradiation spot on the surface of the metal film 30 is substantially circular. The light source unit 111 includes, for example, a light source for excitation light, a beam shaping optical system, an APC mechanism, and a temperature adjustment mechanism (all not shown). The light source of the excitation light α is, for example, a laser diode (hereinafter also referred to as “LD”).

  The LD emits excitation light α (single mode laser light) toward the incident surface 21 of the analysis chip 10 held by the chip holder. More specifically, the light source unit 111 emits only the P wave for the metal film 30 toward the incident surface 21 at an angle at which the excitation light α causes surface plasmon resonance with respect to the metal film 30 of the analysis chip 10.

  The light source unit 111 includes a light source for irradiating the excitation light α toward the portion of the analysis chip in which the detected substance is fixed (hereinafter also referred to as “detected substance fixing portion”). As described above, the excitation light α may be applied to a part of the analysis chip other than the detected substance fixing portion in the flow path as long as the fluorescent substance that labels the detected substance is excited. In the present embodiment, the light source irradiates the excitation light α on the interface between the prism 20 and the metal film 30. The type of the light source is not particularly limited as long as the excitation light α can be emitted, and may not be LD. Examples of light sources include light emitting diodes, mercury lamps, and other laser light sources. When the light emitted from the light source is not a beam, the light emitted from the light source is converted into a beam by a lens, a mirror, a slit, or the like. When the light emitted from the light source is not monochromatic light, the light emitted from the light source is converted into monochromatic light by a diffraction grating or the like. Furthermore, when the light emitted from the light source is not linearly polarized light, the light emitted from the light source is converted into linearly polarized light by a polarizer or the like.

  The beam shaping optical system in the light source unit 111 includes, for example, a collimator, a band pass filter, a linear polarization filter, a half-wave plate, a slit, and a zoom unit. The beam shaping optical system may be configured by all of the above-described components or may be configured by a part. The collimator collimates the excitation light α emitted from the LD. The emitted light of the LD is already flat in the emitted state, and the polarization direction is substantially biased to one side. Even when collimated, the LD is held so as to be incident from the short axis side so that the irradiation surface is circular at the position irradiated under the total reflection condition (shallow angle).

  The band pass filter turns the excitation light α from the light source unit 111 into a narrow band light having only the center wavelength. This is because the excitation light α from the light source unit 111 has a slight wavelength distribution width. The linear polarization filter converts the excitation light α from the light source unit 111 into completely linearly polarized light. The half-wave plate adjusts the polarization direction of the excitation light α so that the P-wave component is incident on the metal film 30. The slit and zoom means adjust the beam diameter, contour shape, and the like of the excitation light α so that the shape of the irradiation spot on the surface of the metal film 30 becomes a circle of a predetermined size.

  The APC mechanism in the light source unit 111 detects the amount of light branched from the collimated excitation light α with a photodiode (not shown) or the like, and uses a regression circuit so that the amount of excitation light α is constant. The output is controlled to be constant by controlling the input energy.

  The temperature adjustment mechanism in the light source unit 111 is, for example, a heater or a Peltier element. The wavelength and energy of the emitted light from the LD vary with temperature. Therefore, the wavelength is controlled to be constant by keeping the temperature of the LD constant by the temperature adjusting mechanism.

  The angle adjustment mechanism 112 scans and adjusts the incident angle of the excitation light α to the metal film 30 (the interface between the prism 20 and the metal film 30 (film formation surface 22)). In order to irradiate the excitation light α at a predetermined incident angle to a predetermined position of the metal film 30 (deposition surface 22) via the prism 20, the angle adjustment mechanism 112 uses the optical axis of the excitation light α and the chip holder. Rotate relatively.

  For example, the angle adjustment mechanism 112 rotates the light source unit 111 around an axis (axis perpendicular to the paper surface of FIG. 1) orthogonal to the optical axis of the excitation light α. At this time, the position of the rotation axis is set so that the incident position of the excitation light α on the metal film 30 (deposition surface 22) hardly moves even when the incident angle is scanned.

  The light source unit 111 is rotated about the axis orthogonal to the incident optical axis (one-axis rotation) to scan the incident angle of the excitation light α, so that the prism 20 is irradiated with the excitation light α while changing the incident angle. . The position of the rotation axis is set so that the incident position of the excitation light α on the metal film 30 (deposition surface 22) of the analysis chip 10 is hardly shifted even when the rotation for scanning the incident angle is performed. To do. The position of the rotation center is set near the intersection of the optical axes of the two excitation lights α at both ends of the scanning range of the incident angle (between the incident position of the excitation light α on the film formation surface 22 and the incident surface 21). Thus, the deviation of the incident position of the excitation light α can be minimized.

  Among the incident angles of the excitation light α, the angle at which the amount of plasmon scattered light is maximized by the scanning is the enhancement angle. By setting the incident angle of the excitation light α to the enhancement angle or an angle in the vicinity thereof, it becomes possible to measure fluorescence with high intensity. The basic excitation light incident conditions are determined by the prism material, shape, metal film thickness, refractive index of fluid in the flow path, etc. attached to the analysis chip, but the material and amount of the fluorescent substance in the flow path, the prism side A slight fluctuation of the condition occurs due to the error of. For this reason, it is preferable to obtain an optimal enhancement angle for each measurement. The enhancement angle is obtained up to the first decimal place, for example.

  The light source control unit 113 operates and controls the above-described various devices that constitute the light source unit 111, and controls emission of emitted light from the light source unit 111. The light source control unit 113 includes, for example, a known computer or microcomputer including an arithmetic device, a control device, a storage device, an input device, and an output device.

  Light such as plasmon scattered light or fluorescence generated by irradiation of the excitation light α onto the metal film 30 (deposition surface 22) is incident on the light receiving optical system unit 120. The light receiving optical system unit 120 includes, for example, a light receiving unit 121, a position switching mechanism 122, and a sensor control unit 123.

  The light receiving unit 121 is disposed in the normal direction of the metal film (deposition surface 22) of the analysis chip 10. The light receiving unit 121 includes, for example, a first lens 124, an optical filter 125, a second lens 126, and a light receiving sensor 127.

  The first lens 124 is a condensing lens, for example, and condenses light emitted from the metal film 30. The second lens 126 is, for example, an imaging lens, and re-images the light collected by the first lens 124 on the light receiving surface of the light receiving sensor 127. Between both lenses, an optical path is formed in which light travels substantially parallel along the optical axis. The optical filter 125 is disposed between both lenses.

  The optical filter 125 guides only the fluorescence component to the light receiving sensor 127 and removes the excitation light component (plasmon scattered light) in order to detect the fluorescence component with a high S / N ratio. Examples of the optical filter 125 include an excitation light reflection filter, a short wavelength cut filter, and a band pass filter. The optical filter 125 is, for example, a filter made of a multilayer film that removes a predetermined light component by reflecting it, but may be a colored glass filter that generally removes the predetermined light component by absorbing it. .

  The light receiving sensor 127 has high sensitivity capable of detecting weak fluorescence from a very small amount of a substance to be detected. The light receiving sensor 127 is a light receiving sensor for detecting the amount of fluorescence. The light receiving sensor 127 is, for example, a head-on type photomultiplier tube (PMT).

  The position switching mechanism 122 switches the position of the optical filter 125 to a position in the optical path of the light receiving unit 121 or a position deviated from the optical path. The position switching mechanism 122 is, for example, a known mechanism that causes the optical filter 125 to advance and retreat from the optical path by a rotational motion, such as a rotation drive unit and a turntable or rack and pinion having the optical filter 125. It is comprised by.

  The sensor control unit 123 controls the detection of the light amount by the light receiving sensor 127 and the management and change of the detection range of the light receiving sensor 127. The sensor control unit 123 includes, for example, a known computer or microcomputer including an arithmetic device, a control device, a storage device, an input device, and an output device.

  In addition, the light receiving unit 121 determines the incident area when the light generated on the surface side of the metal film 30 is incident on the light receiving surface of the light receiving sensor 127 when the excitation light α is incident on the back surface of the metal film 30. It is comprised so that it may become smaller.

  The liquid feeding unit 130 supplies a sample liquid or a chemical liquid to the analysis chip 10. The liquid feeding unit 130 includes, for example, a chemical liquid chip 131, a syringe pump 132, and a liquid feeding pump drive mechanism 133.

  The chemical solution chip 131 is a container that stores a sample solution and a chemical solution. A plurality of chemical liquid chips 131 are usually arranged according to the types of sample liquid and chemical liquid.

  The syringe pump 132 includes a syringe 134 and a plunger 135 that can reciprocate inside the syringe 134. By the reciprocating motion of the plunger 135, the sample liquid or the chemical liquid is sucked and discharged quantitatively.

  If the syringe 134 is replaceable, the syringe 134 need not be cleaned. For this reason, it is preferable from the viewpoint of suppressing the influence of impurities and the like. If the syringe 134 is not configured to be replaceable, the syringe 134 can be used without being replaced by further having a configuration for cleaning the inside of the syringe 134. For this reason, it is preferable from a viewpoint of suppressing consumption of the syringe 134.

  The liquid feed pump drive mechanism 133 is configured by, for example, a drive device for the plunger 135 and a moving device for the syringe pump 132.

  The drive device of the plunger 135 is a device for reciprocating the plunger 135 and includes, for example, a stepping motor. The driving device including the stepping motor is preferable from the viewpoint of managing the liquid feeding amount and the liquid feeding speed of the syringe pump 132 uniformly and managing the residual liquid amount of the analysis chip 10 constant.

  The moving device of the syringe pump 132 is a device that freely moves the syringe pump 132 in two directions, for example, an axial direction (for example, a vertical direction) of the syringe 134 and a direction crossing the axial direction (for example, a horizontal direction). . The moving device of the syringe pump 132 is configured by, for example, a robot arm, a two-axis stage, or a turntable that can move up and down.

  The liquid feeding unit 130 further includes a device that detects the position of the tip of the syringe 134, the relative height between the syringe 134 and the analysis chip 10 is adjusted to be constant, and the remaining liquid amount in the analysis chip 10 Is preferable from the viewpoint of maintaining a constant value.

  The liquid feeding unit 130 sucks liquids such as various chemical solutions from the chemical solution chip 131, moves to the analysis chip 10, and supplies the chemical solution to the analysis chip 10. At this time, by moving the plunger 135 in the direction of advancement and retreat with respect to the analysis chip 10, for example, the vertical direction, the liquid reciprocates in the flow path in the analysis chip 10, and the liquid in the flow path Stir. From the viewpoint of performing the above operation, the liquid insertion portion in the analysis chip 10 that stores the liquid to be supplied to the analysis chip 10 is protected by a multilayer film, and the syringe 134 is used for supplying the liquid. It is preferable that the analysis chip 10 and the syringe 134 are configured so as to seal the liquid insertion portion when penetrating the multilayer film.

  By the above operation, the concentration of the liquid is made uniform, and the immobilization reaction of the substance to be detected at a predetermined position of the analysis chip 10 is promoted. The liquid after the reaction is again sucked by the syringe pump 132 and discharged to the chemical liquid chip 131 and the like. By repeating the above operation, reaction with various chemical solutions, washing, and the like are performed, and the detection target substance is arranged at a predetermined position of the analysis chip 10, and the detection target substance is labeled with the fluorescent substance.

  The control unit 160 drives and controls the angle adjustment mechanism 112, the light source control unit 113, the position switching mechanism 122, the sensor control unit 123, and the liquid feed pump drive mechanism 133. The control unit 160 starts setting the detection range of the light receiving sensor 127 and executes various operations for control. The control unit 160 includes, for example, a known computer or microcomputer including an arithmetic device, a control device, a storage device, an input device, and an output device.

  In the present embodiment, the SPFS device 100 irradiates the metal film 30 disposed on one surface of the prism 20 with the excitation light α via the prism 20 and is detected on the metal film 30. The fluorescent substance for labeling is selectively excited, the fluorescence emitted from the fluorescent substance is detected, and the amount of the substance to be detected is measured. In the present embodiment, the SPFS device 100 determines the detection range of the light receiving sensor 127 and switches the detection range of the light receiving sensor 127 to an appropriate range before the cleaning operation before the measurement ends. Complete. Based on an example of sequence control in the SPFS apparatus 100, implementation of the SPFS method by the SPFS apparatus 100 will be described.

  First, as illustrated in FIG. 2, the control unit 160 performs the following operation on the analysis chip 10 held in the chip holder.

  A humectant may be applied in the flow path of the analysis chip 10. For example, in the case where a solid phase film that captures a target substance is disposed on the metal film 30 of the analysis chip 10, the substrate specificity for capturing the target substance in the solid phase film is fixed for a long period of time. In general, a moisturizing agent is applied to the solid phase film so as not to decrease during the period. The solid phase film is, for example, a film containing the above-described capturing body. When the solid phase film is disposed on the metal film 30, the control unit 160 causes the liquid feeding unit 130 to wash the humectant in the flow path of the analysis chip 10 (step 201). By this washing, the high substrate specificity for capturing the substance to be detected in the analysis chip 10 is restored.

  Next, the control unit 160 causes the liquid feeding unit 130 to supply the sample liquid from the chemical liquid chip 131 into the flow path of the analysis chip 10 (step 202). Thereby, the substance to be detected is captured in the flow path (solid phase film on the surface of the metal film 30) by the immune reaction (primary reaction). Next, the controller 160 causes the liquid feeding unit 130 to remove excess sample liquid from the flow path, and to clean the flow path with an appropriate cleaning liquid such as a pH buffer solution (step 203).

  Next, when the optical filter 125 such as an excitation light reflection filter is disposed in the light receiving unit 121, the control unit 160 causes the position switching mechanism 122 to retract the optical filter 125 from the optical path in the light receiving unit 121 (step 204). ).

  Further, the control unit 160 causes the excitation optical system unit 110 and the light receiving optical system unit 120 to cooperate to measure the enhancement angle (step 205), and causes the excitation optical system unit 110 to obtain a position and orientation at which the enhancement angle is obtained. The light source unit 111 is arranged (step 206). Next, the control unit 160 causes the light receiving optical system unit 120 to place the optical filter 125 in the optical path in the light receiving unit 121 (step 207). When a neutral density filter is disposed in the light receiving optical system unit 120, the control unit 160 retracts the neutral density filter from the optical path as necessary.

  Then, the control unit 160 records the output value (optical blank value) of the light receiving sensor 127 with the amount of light detected by the light receiving sensor 127 when the optical filter 125 is interposed (step 208).

  Next, the control unit 160 causes the liquid feeding unit 130 to supply a chemical solution containing a fluorescent substance from the chemical solution chip 131 into the flow channel of the analysis chip 10, and into the flow channel by an antigen labeling reaction (secondary reaction). The fluorescent substance is bound to the fixed substance to be detected (step 209).

  In this way, the substance to be detected is labeled with the fluorescent substance, and fluorescence having a light amount corresponding to the amount of the substance to be detected is obtained. After the labeling reaction, the excess fluorescent substance emits light regardless of the amount of the substance to be detected, resulting in noise. Therefore, the control unit 160 causes the liquid feeding unit 130 to remove excess fluorescent material in the flow path and clean the flow path. The controller 160 determines and sets the detection range of the light receiving sensor 127 in parallel with the cleaning. Each of the two operations, that is, the washing operation, the determination, and the setting operation in parallel will be described.

[Cleaning operation]
As shown in FIG. 3, the controller 160 causes the liquid feeding unit 130 to perform the cleaning operation in a plurality of times, for example, three times. The control unit 160 causes the liquid feeding unit 130 to suck and remove the chemical solution in the flow path, supply the cleaning liquid to the flow path, and perform the first cleaning to suck and remove from the flow path (step 210). ). Next, the control unit 160 causes the liquid feeding unit 130 to supply the cleaning liquid to the flow path, and perform the second cleaning to suck and remove from the flow path. Further, the control unit 160 causes the liquid feeding unit 130 to perform the third cleaning by the same operation as the second cleaning (step 211).

[Judgment and setting operations]
The control unit 160 adjusts the detection range of the light receiving sensor 127 as shown in FIG. 4 while performing the above-described cleaning operation. For example, after supplying the cleaning liquid in the second cleaning (during cleaning), the control unit 160 causes the excitation light α to be incident on the analysis chip 10 at the above-described enhancement angle to the excitation optical system unit 110 and enter the light receiving optical system unit 120. The amount of fluorescence is detected (step 411). After the detection, the control unit 160 performs suction and removal of the cleaning liquid in the second cleaning of the analysis chip 10, and then performs the third cleaning.

  While cleaning the analysis chip 10, the control unit 160 compares the output value obtained by detecting the light amount in step 411 with the current detection range of the light receiving sensor 127, for example, the number of digits of the output value. The optimum detection range is selected from the preset detection ranges in accordance with (Step 412). Next, the control unit 160 causes the light receiving optical system unit 120 to change the control voltage of the light receiving sensor 127 as necessary, and switches the detection range of the light receiving sensor 127 to the selected optimum detection range (step 413). The sensitivity of the light receiving sensor 127 whose detection range has been switched is stable before the end of the third cleaning at the latest.

  Immediately after completion of the third cleaning, the control unit 160 causes the excitation optical system unit 110 to emit the excitation light α toward the analysis chip 10 and causes the light reception optical system unit 120 to detect the amount of fluorescence (see FIG. Step 212). The light quantity of the fluorescence is selected by the above determination and setting operation, and is detected by the light receiving sensor 127 of the optimum detection range that has been switched.

  In the measurement of fluorescence, the excitation light α guided to the prism 20 enters the prism 20 from the incident surface 21 and irradiates the metal film 30. Then, plasmon scattered light and fluorescence generated on the surface of the metal film 30 by surface plasmon resonance are emitted to the light receiving unit 121.

The plasmon scattered light is reflected or absorbed by the optical filter 125, and the fluorescence is incident on the light receiving sensor 127. The output value (fluorescence signal) (S) related to the amount of fluorescence of the light receiving sensor 127 is output from the light receiving sensor 127 and stored in the control unit 160 or the sensor control unit 123. From the fluorescence signal (S), in step 208 The difference obtained by subtracting the measured optical blank value (oB) is calculated, and the fluorescence intensity (ΔS) correlated with the amount of the substance to be detected is obtained (see the following formula). Then, the amount of the substance to be detected is obtained. The above calculation may be performed by the control unit 160 or may be performed by the sensor control unit 123.
ΔS = S−oB

  As described above, the SPFS apparatus 100 performs the SPFS method.

  The SPFS apparatus 100 can quickly measure the amount of a substance to be detected with high quantitativeness. This is because the optimization of the detection range of the light receiving sensor 127 is completed during the cleaning of the flow path after labeling the substance to be detected. The flow path cleaning and detection range optimization will be further described below. In the following description, it is assumed that 95% of the liquid in the flow path is removed in each of the three washing operations.

  In the flow path after labeling, all of the fluorescent material remaining after labeling remains. Assuming that the amount of the remaining fluorescent material in the channel is “100%”, the amount of the fluorescent material in the channel during the first cleaning (step 210) is “5%” from 100 × 0.05. " The amount of the fluorescent substance in the flow path during the second cleaning is “0.25%” from 100 × 0.05 × 0.05, and the amount of the fluorescent substance in the flow path during the third cleaning is It becomes “0.0125%” from 100 × 0.05 × 0.05 × 0.05 (FIG. 3).

  In the SPFS method, the fluorescent material on the surface of the metal film 30 is selectively excited. For this reason, basically, the fluorescent substance bound to the detected substance trapped on the surface of the metal film 30 is preferentially excited. Nevertheless, some of the fluorescent material present in the channel is also excited. Therefore, if the concentration of the fluorescent substance in the channel is high, the fluorescent substance in the channel affects the measured value.

  For this reason, even if the amount of fluorescent light is detected during cleaning of the flow path, the obtained output value is not accurate as the measured value of the substance to be detected. However, if the flow path has been washed to some extent, it is sufficiently accurate if it is about the order (number of digits) of the amount of fluorescent light in this measurement (measurement of the substance to be detected) from the output value by the amount of fluorescent light in the flow path. Can be determined. For example, the amount of the fluorescent substance in this measurement is about 5 pg to 50 ng, and each of the plurality of detection ranges in which the light receiving sensor 127 is set has a range of 2 to 3 digits in terms of the concentration of the fluorescent substance. And

  The amount of the fluorescent material is “5%” in the flow path during the first cleaning. Therefore, the amount of the fluorescent substance in the channel during the first cleaning is too large compared to the quantity of the fluorescent substance in this measurement. For this reason, even if the amount of fluorescent light in the flow path is detected during the first cleaning, the output value obtained is too larger than the output value based on the amount of fluorescent light to be detected in this measurement. Therefore, it is not suitable as a material for determining the detection range of the light receiving sensor 127 for the main measurement. However, the amount of the fluorescent substance in the flow path during the second cleaning is “0.25%”. Further, the amount of the fluorescent substance in the flow path during the third cleaning is “0.0125%”. Thus, the amount of the fluorescent substance in the flow path decreases exponentially according to the number of times of cleaning. Therefore, if the amount of fluorescent light in the flow path is detected during the second cleaning, more preferably during the third cleaning, the influence of the fluorescent substance remaining in the flow path is sufficiently small. An output value of the light receiving sensor 127 suitable as a determination material is obtained.

  As described above, optimization of the detection range requires a predetermined waiting time for moving the optical filter, stabilizing the control voltage of the light receiving sensor, and the like. However, the waiting time is approximately 10 seconds, and can be predicted in advance. Therefore, if the drain time required to suck and remove the liquid from the flow path in the third washing is shorter than the waiting time, as described above, the detection range is not changed during the second washing. The amount of light in the flow path for determination is detected. When the drainage time in the third cleaning is longer than the standby time, light irradiation for determination of the detection range can be performed during the third cleaning. In addition, when it is not necessary to switch the detection range of the light receiving sensor 127, the switching of the detection range is omitted, and the subsequent standby time is unnecessary.

  As is clear from the above description, the SPFS device 100 selects the optimum detection range of the light receiving sensor 127 during the cleaning of the flow path after the fluorescent labeling of the detection target substance, and the cleaning of the flow path is completed. Complete the switch to the optimal detection range selected in. For this reason, in the SPFS apparatus 100, it is possible to detect the light quantity of the fluorescence in the main measurement immediately after the cleaning of the flow path with an optimal detection range. Therefore, even if the detection range of the light receiving sensor 127 is switched to the optimal detection range, the SPFS device 100 can perform immunoassay analysis without causing an operation time excess associated with the switching. As a result, the SPFS device 100 can reduce the influence of dissociation between the substance to be detected and the fluorescent substance, and can detect the amount of the substance to be detected quickly and with high quantitativeness regardless of the amount of the substance to be detected. can do.

  Further, when the SPFS device 100 determines the detection range of the light receiving sensor 127, the SPFS device 100 selects the optimum detection range of the light receiving sensor 127 based on the amount of fluorescence of the flow path during the second cleaning. Therefore, the detection range of the light receiving sensor 127 can be optimized with high accuracy.

  Further, the SPFS device 100 determines the detection range of the light receiving sensor 127 based on the amount of fluorescent light in the flow path being cleaned. The fluorescence is the same kind of light as that measured in the main measurement. Therefore, it is possible to further increase the accuracy of determination of the detection range.

  Note that the SPFS apparatus 100 can determine the detection range based on the detection result of light other than the fluorescence. For example, the enhancement angle measured in the measurement of the enhancement angle (step 205) is compared with the known enhancement angle value (reference enhancement angle) of the analysis chip 10 before fixing the detection target substance. Determine the shift amount of the enhancement angle. The shift amount indirectly reflects the amount of the detection target substance fixed on the surface of the metal film 30. For this reason, the amount of the substance to be detected can be estimated from the shift amount. Therefore, the control unit 160, the enhancement angle shift value obtained from the comparison between the measurement value of the enhancement angle and the reference enhancement angle, the relationship between the abundance of the detection target substance obtained in advance and the shift value, In addition, the optimum detection range of the light receiving sensor 127 in this measurement can be determined from the relationship between the abundance of the detected substance to be detected in advance and the relationship between the amount of fluorescence to be detected.

  In the present invention, it is also possible to determine the detection range of the light receiving sensor 127 based on the measured value of the resonance angle instead of the enhancement angle. An SPFS device capable of measuring the resonance angle is shown in FIG. As shown in FIG. 5, the SPFS device 500 is configured in the same way as the SPFS device 100 described above except that it further includes a photodiode 527. In the photodiode 527, the excitation light α emitted from the light source unit 111 is the analysis chip 10. For example, the surface of the metal film 30 on the opposite side to the detected substance fixing portion in the flow path (metal film 30 is a second light receiving sensor for receiving the totally reflected light that is totally reflected by the back surface of 30. The excitation light α is irradiated to a position in the analysis chip 10 where the detected substance fixing portion in the flow path generates the localized field light. At this time, among the components of the excitation light α, the localized field light is generated. The component of light that does not contribute is totally reflected. For example, the photodiode 527 is disposed so as to rotate relative to the analysis chip 10 around an axis orthogonal to the optical axis of the total reflected light (an axis perpendicular to the paper surface of FIG. 5). ing.

  As shown in FIG. 6, the measurement of the resonance angle is performed in parallel with, for example, the operation from the measurement of the enhancement angle in step 205 to the third cleaning of the flow path in step 211. The control unit 160 scans the incident angle of the incident light from the light source unit 111 at the time of measuring the enhancement angle, and causes the photodiode 527 to detect the amount of the total reflected light totally reflected on the back surface of the metal film 30, and thereby the resonance angle. Is measured (step 611). The resonance angle is an incident angle of the incident light when the amount of the total reflected light detected by the photodiode 527 is the smallest.

  Then, the control unit 160 compares the measured resonance angle with the known resonance angle value (reference resonance angle) of the analysis chip 10 before fixing the substance to be detected, and determines the measured resonance angle. Find the shift amount. The shift amount also indirectly reflects the amount of the substance to be detected. For this reason, the amount of the detection target substance fixed on the surface of the metal film 30 can be estimated from the shift amount. Therefore, the control unit 160 determines the resonance angle shift value obtained from the comparison between the measured value of the resonance angle and the reference resonance angle, the relationship between the abundance of the detection target substance obtained in advance and the shift value, The optimum detection range in the main measurement of the light receiving sensor 127 is determined based on the relationship between the abundance of the target substance to be detected and the amount of fluorescence detected (step 612). The detection range 127 is set to the optimum detection range (step 613).

  For the reference enhancement angle and the reference resonance angle, for example, values specific to the analysis chip 10 measured at the time of manufacturing the analysis chip 10 can be used. However, the reference enhancement angle and the reference resonance angle may be values measured from the analysis chip 10 before fixing the detection target substance. Further, the correlation between the amount of the substance to be detected and the shift value of the enhancement angle or the resonance angle, the correlation between the amount of the substance to be detected and the amount of fluorescent light, etc. can be obtained from a calibration curve prepared in advance, for example. . Further, the detection range of the light receiving sensor 127 can be set based on any one of the fluorescence of the flow path of the analysis chip 10 being cleaned, the shift value of the enhancement angle, and the shift value of the resonance angle. However, it is also possible to perform based on two or more or all of these.

  In the SPFS devices 100 and 500, the detection range of the light receiving sensor 127 is set by changing the control voltage of the light receiving sensor 127. However, the detection range setting method of the light receiving sensor 127 may be another method. For example, the detection range of the light receiving sensor 127 can be changed by moving an ND filter as the optical filter 125 back and forth in the optical path of the light receiving unit 121. The change of the detection range by the ND filter may be performed in parallel with the change of the control voltage.

  The adjustment of the detection range of the light receiving sensor 127 described above can also be applied to immunoassay analyzers other than the SPFS devices 100 and 500. For example, the immunoassay analyzer 700 includes a light source unit 711 and a light receiving unit 727 as shown in FIG. The excitation light incident on the analysis chip 70 from the light source unit 711 is irradiated to the detected substance fixing portion in the flow path of the analysis chip 70, and the fluorescence-labeled detected substance fixed to the flow path of the analysis chip 70. Excites fluorescent material. Fluorescence generated by excitation enters the light receiving sensor 727 and is detected. The adjustment of the detection range of the light receiving sensor 727 by the fluorescence can be applied to such an immunoassay analyzer 700 as well.

  In addition, the operations of immobilizing the substance to be detected, fluorescent labeling of the substance to be detected by the fluorescent substance, washing the flow path, and detecting the light amount of the substance to be detected fixed part are not limited to the order described above. As long as the effects of the present invention can be obtained, it can be performed in various orders.

  For example, after the first immobilization step of supplying the detected substance together with the fluorescent substance to the flow path of the analysis chip and immobilizing the detected substance labeled with the fluorescent substance in the flow path It is possible to perform a fluorescent substance cleaning step of cleaning the flow path so as to remove excess fluorescent substance in the flow path. In this case, during the period from the first immobilization step until the fluorescent substance washing step is performed, the excitation light is irradiated toward the analysis chip, and the light amount of the first emitted light or the first A first light amount detection step for detecting the amount of total reflected light can be performed. The first emitted light is light emitted from the analysis chip by irradiation with the excitation light, and is, for example, the fluorescence or the plasmon scattered light. The first total reflected light is light reflected by the analysis chip by irradiation with the excitation light.

  Alternatively, after the second immobilization step of immobilizing the substance to be detected in the flow channel of the analysis chip, a first washing step of washing the flow channel is performed, and then the fluorescent material is supplied to the flow channel. A labeling step of labeling the substance to be detected immobilized in the flow path with the fluorescent material, and then washing the flow path to remove excess fluorescent material in the flow path. Two cleaning steps can be performed. In this case, between the second immobilization step and before the labeling step, the excitation light is irradiated toward the analysis chip, and the amount of the second emitted light or the second total reflected light is irradiated. A second light quantity detection step for detecting the light quantity can be performed. The second emitted light is light emitted from the analysis chip by irradiation with the excitation light, for example, the plasmon scattered light. The second total reflected light is light reflected by the analysis chip by irradiation with the excitation light.

  Alternatively, after the second immobilization step of immobilizing the substance to be detected in the flow channel of the analysis chip, a first washing step of washing the flow channel is performed, and then the fluorescent material is supplied to the flow channel. A labeling step of labeling the substance to be detected immobilized in the flow path with the fluorescent material, and then washing the flow path to remove excess fluorescent material in the flow path. Two cleaning steps can be performed. In this case, during the period from the labeling step to the time when the second washing step is performed, the excitation light is irradiated toward the analysis chip, and the amount of the third emitted light or the third total reflection is irradiated. A third light quantity detection step for detecting the quantity of light can be performed. The third emission light is light emitted from the analysis chip by irradiation of the excitation light, and is, for example, the fluorescence or the plasmon scattered light. The third total reflected light is light reflected by the analysis chip by irradiation with the excitation light.

  And before irradiating the said excitation light toward the said analysis chip in order to detect the said fluorescence from the said fluorescent substance for measuring the quantity of the said to-be-detected substance, said 1st light quantity detection process, said 2nd Based on the light amount detected by at least one of the light amount detection step and the third light amount detection step, one detection range is selected from the plurality of detection ranges of the light receiving sensor, and the selected detection range is selected. Switch to. Thereafter, in order to measure the amount of the substance to be detected, the fluorescence from the fluorescent substance is detected by the light receiving sensor whose detection range is switched.

  As is clear from the description of the above-described embodiment, in this embodiment, the flow path in the analysis chip is washed while the flow path of the analysis chip after the substance to be detected is labeled with the fluorescent substance. Before irradiating excitation light toward the detected substance fixing portion (in this embodiment, the film formation surface 22 of the prism 20) or before the detected substance is fixed and the flow path is washed. Irradiate excitation light toward the target substance fixing part. Then, the light amount generated from the flow path by the irradiation of the excitation light is detected by the first light receiving sensor (light receiving sensor 127), or the light amount of the total reflection light at the analysis chip by the irradiation of the excitation light is Detection is performed by a second light receiving sensor (photodiode 527). Then, based on the light amount of the light or total reflected light detected by the first light receiving sensor or the second light receiving sensor, an optimum detection range of the first light receiving sensor when detecting the light amount of the fluorescence is obtained. Select and complete the switching of the detection range of the first light receiving sensor to the optimum detection range by the time the cleaning of the flow path is completed at the latest. As described above, in the present embodiment, the optimization of the detection range from the wide dynamic range of the light receiving sensor is completed before the cleaning of the flow path after the fluorescent labeling of the detection target substance is completed.

For example, in this embodiment,
(A) supplying a substance to be detected together with the fluorescent substance to the flow path of the analysis chip, and immobilizing the substance to be detected labeled with the fluorescent substance in the flow path; Later, when performing the fluorescent substance cleaning process for cleaning the flow path so as to remove excess fluorescent substance in the flow path, the fluorescent substance cleaning process is performed after the first immobilization process. Until the analysis chip is irradiated with the excitation light, and the amount of first emission light emitted from the analysis chip by the excitation light irradiation or reflected by the analysis chip by the excitation light irradiation. Performing a first light quantity detection step for detecting the amount of the first total reflected light, or
(B1) After the second immobilization step of immobilizing the substance to be detected in the flow channel of the analysis chip, a first washing step of washing the flow channel is performed, and then the fluorescent material is supplied to the flow channel Then, a labeling step of labeling the target substance immobilized in the flow path with the fluorescent material is performed, and then the flow path is washed so as to remove excess fluorescent material in the flow path. When performing the second washing step, the excitation light is irradiated toward the analysis chip after the second immobilization step and before the labeling step, and the analysis is performed by irradiation with the excitation light. Performing a second light amount detection step of detecting the amount of second emitted light emitted from the chip or the amount of second total reflected light reflected by the analysis chip by irradiation of the excitation light,
(B2) Or, after the labeling step and before the second washing step is performed, the excitation light is irradiated toward the analysis chip and emitted from the analysis chip by the irradiation of the excitation light. Performing a third light quantity detection step of detecting the light quantity of the third emitted light or the third total reflected light reflected by the analysis chip by irradiation of the excitation light,
(C) before irradiating the excitation light toward the analysis chip in order to detect the fluorescence from the fluorescent material for measuring the amount of the substance to be detected, Based on the light quantity detected by at least one of the two light quantity detection process and the third light quantity detection process, one detection range is selected from the plurality of detection ranges of the light receiving sensor, and the selected detection is performed. Switch to the range,
(D) Thereafter, in order to measure the amount of the substance to be detected, the fluorescence from the fluorescent substance is detected by the light receiving sensor whose detection range is switched.

  For this reason, in the present embodiment, it is possible to detect the amount of fluorescence of the substance to be detected immediately after completion of the cleaning. Therefore, the influence in the measurement of the dissociation of the fluorescent substance from the substance to be detected is reduced. As a result, it is possible to measure a substance to be detected quickly and with high quantitativeness.

  In addition, it is more effective from the viewpoint of cost to change the detection range of the first light receiving sensor to the optimum detection range by changing the control voltage of the first light receiving sensor. That is, since the dynamic range can be electrically changed, the SPFS device can be downsized and manufactured at a lower cost than the installation and operation of physical means such as an ND filter. Is possible.

  In addition, this embodiment is particularly suitable for the SPFS method. In the SPFS method, the analysis chip includes a prism, a metal film disposed on one surface of the prism, and the flow path from which the surface of the metal film is exposed. Then, the excitation light is irradiated to the back surface of the metal film through the prism at an incident angle that generates surface plasmon resonance, and the localized field light generated on the surface of the metal film by the irradiation of the excitation light, The fluorescent substance is excited, and the amount of the fluorescence generated by the excitation is detected by the first light receiving sensor.

  In addition, after the substance to be detected is labeled with the fluorescent substance, while the excess fluorescent substance in the flow path is washed away, the excitation light is irradiated toward the detected substance fixing part, Detecting the light amount of the light generated from the flow path (for example, the first emission light, the second emission light, or the third emission light) by the first light receiving sensor is an output based on the amount of fluorescence. It is more effective from the viewpoint of determining the detection range more directly from the value and more appropriately determining and setting the detection range of the light receiving sensor.

  In addition, while changing the incident angle of the excitation light to the metal film on which the substance to be detected is fixed, the metal film is irradiated with excitation light through the prism, and the metal film is generated on the metal film. The enhancement angle is measured by detecting the amount of light (for example, the first emission light, the second emission light, or the third emission light) by the first light receiving sensor, and the reference enhancement angle of the analysis chip is measured. When the amount of shift of the measured enhancement angle with respect to is obtained, and the amount of fluorescence from the fluorescent substance labeled with the target substance is detected based on the amount of the target substance estimated from the shift amount In the case where the optimum detection range of the first light receiving sensor is selected, excitation light is irradiated to the detection target substance before being labeled with the fluorescent substance. For this reason, it is more effective from the viewpoint of suppressing the attenuation of the fluorescence accompanying the determination of the detection range.

  In addition, while changing the incident angle of the excitation light to the metal film on which the substance to be detected is fixed, the metal film is irradiated with excitation light through the prism, and the total reflection light in the metal film The resonance angle is measured by detecting the amount of light (for example, the first total reflection light, the second total reflection light, or the third total reflection light) by the second light receiving sensor, and the reference resonance of the analysis chip A shift amount of the measured resonance angle with respect to an angle is obtained, and based on the amount of the detected substance estimated from the shift amount, the amount of fluorescence from the fluorescent substance labeled with the detected substance is detected. Even when the optimal detection range of the first light receiving sensor is selected, the detection target substance before being labeled with the fluorescent substance is irradiated with the excitation light. For this reason, it is more effective from the viewpoint of suppressing the attenuation of the fluorescence accompanying the determination of the detection range. In addition, the resonance angle has a higher detectability of the amount of the detection target substance fixed to the metal film than the enhancement angle. Therefore, it is more effective from the viewpoint of determining the detection range of the light receiving sensor with higher accuracy than the enhancement angle.

  Further, when the reference enhancement angle or the reference resonance angle is a value measured at the time of manufacturing the analysis chip, it is not necessary to detect the output value of the light receiving sensor before the shift at the time of the determination. Therefore, it is more effective from the viewpoint of simplification and speeding up of the detection range and setting of the light receiving sensor.

As described above, the immunoassay analyzer includes a light source for irradiating excitation light toward a portion where the substance to be detected is fixed, and a first light source for detecting the amount of fluorescence generated in the flow path. And a control unit that sets a detection range of the first light receiving sensor when detecting at least the amount of fluorescence light, and satisfies the following (A) or (B).
(A) The control unit is configured such that the excitation light is directed toward a portion where the detected substance is fixed while the flow path is cleaned after the fluorescent substance is labeled with the detected substance or before the cleaning. One detection range of the first light receiving sensor when detecting the amount of fluorescent light from the light amount detected by the first light receiving sensor of the emitted light emitted from the flow path by irradiating The detection range is selected from a plurality of detection ranges, and the switching of the detection range of the first light receiving sensor to the one detection range is completed before the cleaning of the flow path is completed at the latest.
(B) A second light receiving sensor for detecting the amount of total reflected light at the analysis chip generated by irradiation of the excitation light from the light source toward the portion where the substance to be detected is fixed is further provided. Then, the control unit directs the excitation light toward a portion where the detection target substance is fixed before the flow path is cleaned or while the flow path is cleaned. One detection range of the first light receiving sensor when detecting the light amount of the fluorescence from the light amount detected by the second light receiving sensor of the total reflected light generated by irradiation is a plurality of detection ranges. The detection range of the first light receiving sensor is switched to one detection range until the cleaning of the flow path is completed at the latest.

  Therefore, according to the immunoassay analyzer, optimization of the detection range selected from the wide dynamic range of the light receiving sensor is completed before the washing operation is completed. For this reason, it is possible to detect the amount of fluorescent light immediately after cleaning. Therefore, the influence in the measurement of the dissociation of the fluorescent substance from the captured substance to be detected is reduced. As a result, it is possible to measure a substance to be detected quickly and with high quantitativeness.

  In addition, as described above, the irradiation of the excitation light for measuring the resonance angle to estimate the amount of the detection target substance is performed toward the flow path where the detection target substance is fixed before being fluorescently labeled. Although it is preferable, it is also possible to carry out toward the flow path during washing after the fluorescent labeling. That is, the estimation of the amount of the substance to be detected based on the resonance angle and the optimization of the detection range of the first light receiving sensor can be performed during the cleaning of the flow path after the fluorescent labeling.

  Further, in the SPFS devices 100 and 500, various processes such as cleaning of the analysis chip 10, supply of a chemical solution to the analysis chip 10, and irradiation of the excitation light α to the analysis chip 10 are performed at the irradiation position of the analysis chip 10. Done. Therefore, the SPFS devices 100 and 500 require more time for operations from cleaning to determination and setting than the SPFS device that transports the analysis chip 10 to the supply position of the chemical solution and the irradiation position at the transport stage, respectively. It can be shortened. On the other hand, the SPFS apparatuses 100 and 500 may have the above-described transfer stage as long as the effects of the present invention are obtained. In this case, the effect of shortening the above time is reduced, but it is possible to avoid physical interference between the light receiving optical system unit and the liquid feeding system unit by retracting the analysis chip from the irradiation position on the transfer stage. An effect of increasing the degree of freedom is further provided to the SPFS devices 100 and 500.

  The immunoassay analysis method and the immunoassay analysis apparatus according to the present invention can be suitably applied to a surface plasmon resonance fluorescence analysis apparatus and a surface plasmon resonance fluorescence analysis method, and can detect a substance to be detected with high reliability. For example, it is useful for clinical examinations.

DESCRIPTION OF SYMBOLS 10,70 Analysis chip 20 Prism 21 Incident surface 22 Film-forming surface 23 Output surface 30 Metal film 40 Channel cover 100, 500 SPFS apparatus 110 Excitation optical system unit 111,711 Light source unit 112 Angle adjustment mechanism 113 Light source control part 120 Light reception optical System unit 121, 727 Light receiving unit 122 Position switching mechanism 123 Sensor control unit 124 First lens 125 Optical filter 126 Second lens 127 Light receiving sensor 130 Liquid feeding unit 131 Chemical liquid chip 132 Syringe pump 133 Liquid feeding pump drive mechanism 134 Syringe 135 Plunger 160 Control Unit 527 Photodiode 700 Immunoassay Analyzer α Excitation Light

Claims (8)

The detection substance labeled with the fluorescent substance is irradiated with excitation light toward the analysis chip fixed in the flow path, and the fluorescence from the excited fluorescent substance is detected by the light receiving sensor, and the detection target is detected. In an immunoassay analysis method for measuring the amount of a substance,
After the first immobilization step of supplying the substance to be detected together with the fluorescent substance to the flow path of the analysis chip and immobilizing the substance to be detected labeled with the fluorescent substance in the flow path, When performing the fluorescent substance cleaning step of cleaning the flow path so as to remove excess fluorescent substance in the flow path,
After the first immobilization process and before the fluorescent substance cleaning process is performed, the analysis chip is irradiated with the excitation light, and is emitted from the analysis chip by the excitation light irradiation. Performing a first light quantity detection step of detecting a light quantity of one emitted light or a first total reflected light reflected by the analysis chip by irradiation of the excitation light, or
After the second immobilization step of immobilizing the substance to be detected in the flow channel of the analysis chip, a first washing step of washing the flow channel is performed, and then the fluorescent material is supplied to the flow channel to A second cleaning is performed in which a labeling step of labeling the target substance immobilized in the flow path with the fluorescent material is performed, and then the flow path is cleaned so as to remove excess fluorescent material in the flow path. When performing the process,
Between the second immobilization step and before the labeling step, the analysis chip is irradiated with the excitation light, and the second emission light emitted from the analysis chip by the excitation light irradiation. A second light quantity detection step for detecting the light quantity or the second total reflected light reflected by the analysis chip by irradiation of the excitation light is performed, or the second cleaning process is performed after the labeling process. Until the analysis chip is irradiated with the excitation light, and the amount of third emission light emitted from the analysis chip by irradiation of the excitation light or reflected by the analysis chip by irradiation of the excitation light. Performing a third light amount detection step for detecting the amount of the third totally reflected light,
Before irradiating the excitation light toward the analysis chip to detect the fluorescence from the fluorescent substance for measuring the amount of the substance to be detected, the first light quantity detection step, the second light quantity detection Selecting one detection range from a plurality of detection ranges of the light receiving sensor based on the amount of light detected by at least one of the step and the third light amount detection step, to the selected detection range Switch
Then, in order to measure the amount of the substance to be detected, the fluorescence from the fluorescent substance is detected by the light receiving sensor whose detection range is switched.
Immunoassay analysis method.   The immunoassay analysis method according to claim 1, wherein the detection range of the light receiving sensor is switched to an optimum detection range by changing a control voltage of the light receiving sensor. The analysis chip includes a prism having a metal film on one surface, and the flow path from which the surface of the metal film is exposed,
The immunoassay analysis method comprises:
Irradiating the metal film with the excitation light through the prism at an incident angle that generates surface plasmon resonance;
Exciting the fluorescent material by localized field light based on surface plasmon resonance,
The light receiving sensor detects the amount of fluorescence generated by the excitation,
A surface plasmon resonance fluorescence analysis method,
The immunoassay analysis method according to claim 1 or 2. After the substance to be detected is labeled with the fluorescent substance, the excitation light is irradiated toward the portion where the substance to be detected is fixed while the excessive fluorescent substance in the flow path is washed and removed. ,
Detecting the light quantity of at least one of the first emission light, the second emission light, and the third emission light by the light receiving sensor;
The immunoassay analysis method according to any one of claims 1 to 3. While irradiating the metal film with the excitation light through the prism while changing the incident angle of the excitation light to the metal film on which the detection target substance is fixed, the first generated on the metal film The light receiving sensor detects the light quantity of at least one of the emitted light, the second emitted light and the third emitted light, and measures the enhancement angle,
Obtaining a shift amount of the measured enhancement angle with respect to a reference enhancement angle of the analysis chip before the detection target substance is fixed;
Based on the amount of the substance to be detected estimated from the shift amount, a detection range of the light receiving sensor when detecting the amount of fluorescence from the fluorescent substance labeled with the substance to be detected is selected.
The immunoassay analysis method according to claim 3. While changing the incident angle of the excitation light to the metal film on which the detection target substance is fixed, the metal film is irradiated with excitation light through the prism, and the first total reflected light in the metal film , By measuring the amount of light of at least one of the second total reflected light and the third total reflected light by the light receiving sensor to measure the resonance angle,
Obtaining a shift amount of the measured resonance angle with respect to a reference resonance angle of the analysis chip before the detection target substance is fixed;
Based on the amount of the substance to be detected estimated from the shift amount, an optimum detection range of the light receiving sensor when detecting the amount of fluorescence from the fluorescent substance labeled with the substance to be detected is selected.
The immunoassay analysis method according to claim 3.   The immunoassay analysis method according to claim 5 or 6, wherein the reference enhancement angle or the reference resonance angle is a value measured when the analysis chip is manufactured. The fluorescent substance excited when a substance to be detected fixed in the flow path of the analysis chip is labeled with a fluorescent substance and excitation light is irradiated from a light source toward a portion where the substance to be detected is fixed in the flow path In an immunoassay analyzer for measuring the amount of the substance to be detected by detecting fluorescence from a light receiving sensor,
A light source for irradiating excitation light toward a portion where the substance to be detected is fixed;
A first light receiving sensor for detecting the amount of fluorescence generated in the flow path;
A control unit for setting a detection range of the first light receiving sensor when detecting at least the amount of fluorescence light,
Satisfy the following (A) or (B),
Immunoassay analyzer.
(A) The control unit may emit the excitation light toward a portion where the target substance is fixed while the flow path is cleaned after the target substance is labeled with the fluorescent substance or before the cleaning. One detection range of the first light receiving sensor when detecting the amount of fluorescent light from the light amount detected by the first light receiving sensor of the emitted light emitted from the flow path by irradiating The detection range is selected from a plurality of detection ranges, and the switching of the detection range of the first light receiving sensor to the one detection range is completed by the time the cleaning of the flow path is completed at the latest.
(B) A second light receiving sensor for detecting the amount of total reflected light on the analysis chip generated by irradiation of the excitation light from the light source toward the portion where the substance to be detected is fixed is further provided. And
The control unit irradiates the excitation light toward a portion where the target substance is fixed before or while the flow path is cleaned, where the target substance is fixed. One detection range of the first light receiving sensor when detecting the light amount of the fluorescence from the light amount detected by the second light receiving sensor of the total reflected light generated by And the switching of the detection range of the first light receiving sensor to one of the detection ranges is completed before the cleaning of the flow path is completed at the latest.

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