JPWO2017057136A1 - Surface plasmon excitation enhanced fluorescence spectrometry method and measurement kit - Google Patents

Abstract

The present invention provides a method for measuring the amount of a substance to be measured with high sensitivity in SPFS, and a measurement kit used in the method.
In the surface plasmon excitation enhanced fluorescence spectroscopic measurement method of the present invention, a specimen is provided on a measurement chip including a metal film and a first capturing body fixed on the metal film, and the substance to be measured is contained in the specimen Binding the first capturing body to the first capturing body, binding the second capturing body labeled with a fluorescent material to the substance to be measured bound to the first capturing body, and the surface of the metal film. Irradiating with light so as to generate plasmon resonance, and detecting fluorescence emitted from the fluorescent material excited by the localized field light based on the surface plasmon resonance. At this time, the number of the fluorescent substances bonded to one second capturing body is set to 8-22.

Description

  The present invention relates to a surface plasmon excitation enhanced fluorescence spectroscopic measurement method for measuring the amount of a substance to be measured in a specimen, and a measurement kit used therefor.

  Conventionally, many measuring methods using an antigen-antibody reaction are known for measuring substances in living bodies. For example, in a fluorescence immunoassay (hereinafter also referred to as “FIA”), a measurement substance (antigen) is reacted with a labeling substance containing a fluorescent substance. Thereafter, the substance to be measured labeled with the labeling substance is irradiated with excitation light, and the fluorescence emitted from the fluorescent substance is detected. Then, the amount of the substance to be measured is specified from the detected fluorescence intensity or the like. Also, among FIA, as a method capable of detecting a substance to be measured with particularly high sensitivity, a surface plasmon-field enhanced fluorescence spectroscopy (hereinafter also referred to as “SPFS”) is known. (For example, Patent Document 1).

  In SPFS, a first capturing body (for example, a primary antibody) that can specifically bind to a substance to be measured is fixed on a metal film to form a reaction field for capturing the substance to be measured. When a specimen (for example, blood) containing a substance to be measured is provided in this reaction field, the substance to be measured is bound to the first capturing body. Next, when a second capture body (for example, a secondary antibody) labeled with a fluorescent substance is provided to the reaction field, the second capture body is bound to the substance to be measured that is bound to the first capture body. That is, the substance to be measured is indirectly labeled with a fluorescent substance. When the metal film is irradiated with excitation light in this state, the fluorescent material is excited by the localized field light enhanced by surface plasmon resonance (hereinafter also referred to as “SPR”), and emits fluorescence. The amount of the substance to be measured can be measured by detecting the fluorescence emitted by the fluorescent substance.

JP 2013-88221 A

  In general FIA, it is known that the number of fluorescent substances bound per labeling substance (hereinafter also referred to as “labeling rate”) affects the fluorescence detection sensitivity. Usually, when the labeling rate is increased too much, background noise increases or concentration quenching is likely to occur, and the fluorescence detectability decreases. Therefore, in FIA, it is preferable from the viewpoint of fluorescence detection sensitivity and the like that the labeling rate is within the range recommended by the reagent manufacturer. Against this background, also in SPFS, which is a type of FIA, the number of fluorescent substances that bind to each second capture body is generally within the range recommended by the reagent manufacturer.

  In recent years, SPFS is required to measure the presence or absence and the amount of a substance to be measured with higher sensitivity. Accordingly, an object of the present invention is to provide a method for detecting the amount of a substance to be measured with high sensitivity in SPFS, and a measurement kit for use in the method.

  As a result of intensive studies by the present inventors, in SPFS, the number of fluorescent substances detected by binding the second capturing body to more than the number recommended by the reagent manufacturer, specifically, 8 to 22 fluorescent substances. It was found that the signal becomes higher while noise is difficult to increase. That is, it has been found that by setting the labeling rate of the fluorescent substance within the above range, the signal / noise ratio to be detected can be greatly increased, and the substance to be measured can be measured with high sensitivity, leading to the present invention.

  That is, the surface plasmon excitation enhanced fluorescence spectroscopic measurement method according to an embodiment of the present invention includes a metal film and a first recognition site that is fixed on the metal film and specifically binds to a substance to be measured. A sample is provided to a measurement chip including the first capture body, and the target substance contained in the specimen is bound to the first capture body; and the target substance bound to the first capture body is provided. A step of binding a second capturing body having a recognition site for specifically binding to a site different from the site binding to the first capturing body of the substance to be measured and labeled with a fluorescent material And irradiating the metal film with light so as to generate surface plasmon resonance, and detecting fluorescence emitted from the fluorescent material excited by localized field light based on the surface plasmon resonance. At this time, the number of the fluorescent substances bonded per one second capturing body is set to 8-22.

  In addition, a measurement kit according to an embodiment of the present invention includes a prism made of a dielectric, a metal film disposed on one surface of the prism, and fixed on the metal film, and is specific to a substance to be measured. A first capture body having a recognition site for binding to a measurement chip, and for binding specifically to a site different from the site of the substance to be measured that binds to the first capture body A second capturing body having a recognition site and labeled with a fluorescent substance. At this time, the number of the fluorescent substances bonded per one second capturing body is set to 8-22.

  According to the surface plasmon resonance measurement method or measurement kit of the present invention, the signal / noise ratio of the detected fluorescence can be increased. Therefore, the presence / absence and amount of the substance to be measured can be measured with high sensitivity.

FIG. 1 is a schematic diagram showing the configuration of the SPFS apparatus. FIG. 2 is a flowchart illustrating an example of an operation procedure of the SPFS apparatus when performing the measurement method according to the embodiment of the present invention. FIG. 3 is a perspective view of a metal film including a diffraction grating. FIG. 4 is a graph showing the relationship between the number of fluorescent substances (labeling rate) that binds to each second capture body, the signal value, the noise value, and the signal / noise ratio in SPFS.

  An embodiment of the present invention will be described below with reference to the drawings. In the following, the measurement chip and apparatus used in the present embodiment will be described first, and then the measurement method using the apparatus and measurement chip will be described.

(Measurement chip and device)
FIG. 1 is a schematic diagram showing a configuration of an SPFS apparatus 100 used in an embodiment of the present invention. As illustrated in FIG. 1, the SPFS apparatus 100 includes an excitation light irradiation unit 110, a fluorescence detection unit 120, a liquid feeding unit 130, a transport unit 140, and a control unit 150. In the SPFS apparatus 100, the measurement chip 10 is mounted on the chip holder 142 of the transport unit 140, and the excitation light α is irradiated so that surface plasmon resonance is generated on the metal film 30 of the measurement chip 10, and based on the surface plasmon resonance. Generates localized field light. Then, the presence or amount of the substance to be measured in the specimen is measured by exciting the fluorescent substance existing on the metal film 30 with the local field light and detecting the fluorescence β emitted from the fluorescent substance.

  The measurement chip 10 is arranged on the prism 20 having the incident surface 21, the film formation surface 22, and the emission surface 23, the metal film 30 formed on the film formation surface 22, and the film formation surface 22 or the metal film 30. And a flow path lid 40. The channel lid 40 has a channel groove on the surface facing the metal film 30, and a space surrounded by the film formation surface 22, the metal film 30, and the channel lid 40 contains a liquid (for example, a specimen). It becomes the accommodating part 41 for doing. The measurement chip 10 is usually replaced for each measurement. The measuring chip 10 is preferably a structure having a length of several mm to several cm for each piece, but is a smaller structure or a larger structure not included in the category of “chip”. Also good.

  The prism 20 included in the measurement chip 10 is made of a dielectric that is transparent to the excitation light α, and has an incident surface 21, a film formation surface 22, and an output surface 23. The incident surface 21 is a surface for causing the excitation light α from the excitation light irradiation unit 110 to enter the prism 20. Further, the metal film 30 is disposed on the film formation surface 22, and the excitation light α incident on the inside of the prism 20 is reflected on the back surface of the metal film 30, more specifically, between the prism 20 and the metal film 30. Reflected at the interface (deposition surface 22). On the other hand, the emission surface 23 is a surface for emitting the reflected light reflected by the film formation surface 22 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 light irradiation unit 110. When the light source of the excitation light α is a laser diode (hereinafter also referred to as “LD”), when the excitation light α returns to the LD, the excitation state of the LD 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 a scanning range centered on an ideal resonance angle or enhancement angle. Here, the “resonance angle” means an incident angle when the amount of reflected light emitted from the emission surface 23 is minimized when the incident angle of the excitation light α with respect to the metal film 30 is scanned. The “enhancement angle” refers to scattered light having the same wavelength as the excitation light α emitted above the measurement chip 10 when the incident angle of the excitation light α with respect to the metal film 30 is scanned (hereinafter referred to as “plasmon scattered light”). It means the incident angle when the amount of γ is maximized. In the present embodiment, 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 °.

  The design of the measuring chip 10 largely determines the resonance angle (and the enhancement angle near the pole). The design factors are the refractive index of the prism 20, the refractive index of the metal film 30, the film thickness of the metal film 30, the extinction coefficient of the metal film 30, the wavelength of the excitation light α, and the like. The resonance angle and the enhancement angle are shifted by the measured substance captured on the metal film 30 via the first capturing body, but the amount is less than several 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 disposed on the film formation surface 22 of the prism 20. As a result, an interaction (SPR) occurs between the photon of the excitation light α incident on the film formation surface 22 under total reflection conditions and the free electrons in the metal film 30, and a localized field is formed on the surface of the metal film 30. Light (generally referred to as “evanescent light” or “near-field light”) is generated.

  The material of the metal film 30 is not particularly limited as long as it is a metal that can cause surface plasmon resonance. Examples of the material of the metal film 30 include gold, silver, copper, aluminum, and alloys thereof. 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, in the present embodiment, the first capturing body is fixed to the surface of the metal film 30 that does not face the prism 20 (the surface of the metal film 30). The first capturing body is a substance having a recognition site for specifically binding to the substance to be measured in the specimen, and the first capturing body is exposed in the accommodating portion 41. When the specimen is provided in the storage unit 41, the first capturing body and the substance to be measured come into contact with each other, whereby the first capturing body and the substance to be measured are selectively combined. That is, the substance to be measured can be disposed on the metal film 30 via the first capturing body.

  The kind of the 1st capture body currently fixed on the metal film 30 will not be restrict | limited especially if it has the recognition site | part for couple | bonding specifically with a to-be-measured substance. Examples of the first capturing body include a biomolecule specific to the substance to be measured (for example, a monoclonal antibody, a polyclonal antibody, a nucleic acid aptamer, etc.), or a fragment thereof. The method for fixing the first capturing body to the metal film 30 is not particularly limited, and may be any of physical adsorption, chemical bonding (amide coupling, reaction between Au and thiol, silane coupling) and the like.

  In addition, the flow path lid 40 has a flow path groove and has a shape capable of forming a space (accommodating portion 41) for accommodating a liquid by the wall surface and upper surface of the flow path groove and the metal film 30. The shape is not particularly limited. Further, the shape and size of the accommodating portion 41 formed between the channel lid 40 and the metal film 30 are not limited. The accommodating portion 41 may be a space for temporarily storing liquid, such as a well, but is preferably a flow path for allowing the accommodating portion 41 to flow the liquid from the viewpoint of measurement efficiency and the like. When the accommodating part 41 is a flow path, both ends or one end part of the accommodating part 41 may be connected to an inlet and an outlet (not shown) formed on the upper surface of the channel lid 40.

  In the channel lid 40, a portion for taking out the fluorescence β and plasmon scattered light γ emitted from the metal film 30 to the outside is formed of a material transparent to these lights. The entire channel lid 40 may be formed of a material transparent to the light, or a part thereof may be formed of a material opaque to the light. Examples of the material transparent to the fluorescent β and the plasmon scattered light γ include a resin. The channel lid 40 is joined 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, pressure bonding using a clamp member, or the like.

  Next, components other than the measurement chip of the SPFS device 100 will be described. As described above, the SPFS apparatus 100 includes the excitation light irradiation unit 110, the fluorescence detection unit 120, the liquid feeding unit 130, the transport unit 140, and the control unit 150.

  The excitation light irradiation unit 110 irradiates the measurement chip 10 held by the chip holder 142 with excitation light α. When measuring the fluorescence β or the plasmon scattered light γ, the excitation light irradiation unit 110 emits only the P wave with respect to the metal film 30 toward the incident surface 21 so that the incident angle with respect to the metal film 30 is an angle that causes SPR. To do. Here, the “excitation light” is light that directly or indirectly excites the fluorescent material. For example, the excitation light α is light that generates localized field light on the surface of the metal film 30 that excites the fluorescent material when the metal film 30 is irradiated through the prism 20 at an angle at which SPR occurs. The excitation light irradiation unit 110 includes a light source unit 111, an angle adjustment mechanism 112, and a light source control unit 113.

  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 back surface of the metal film 30 is substantially circular. The light source unit 111 includes, for example, a light source of excitation light α, a beam shaping optical system, an APC mechanism, and a temperature adjustment mechanism (all not shown).

  The type of the light source is not particularly limited, and a laser diode (LD) is an example. Other 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 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 include all of these or a part thereof. The collimator collimates the excitation light α emitted from the light source. The band-pass filter turns the excitation light α emitted from the light source into narrowband light having only the center wavelength. This is because the excitation light α from the light source has a slight wavelength distribution width. The linear polarization filter turns the excitation light α emitted from the light source 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 back surface of the metal film 30 is a circle having a predetermined size.

  The APC mechanism controls the light source so that the output of the light source is constant. More specifically, the APC mechanism detects the amount of light branched from the excitation light α with a photodiode (not shown) or the like. The APC mechanism controls the input energy by a regression circuit, thereby controlling the output of the light source to be constant.

  The temperature adjustment mechanism is, for example, a heater or a Peltier element. The wavelength and energy of the light emitted from the light source may vary depending on the temperature. For this reason, the wavelength and energy of the light emitted from the light source are controlled to be constant by keeping the temperature of the light source constant by the temperature adjusting mechanism.

  The angle adjustment mechanism 112 adjusts the incident angle of the excitation light α with respect to the metal film 30 (the interface between the prism 20 and the metal film 30 (film formation surface 22)). The angle adjusting mechanism 112 relatively irradiates the optical axis of the excitation light α and the chip holder 142 in order to irradiate the excitation light α at a predetermined incident angle toward a predetermined position of the metal film 30 via the prism 20. Rotate.

  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 position of the irradiation spot on the metal film 30 hardly changes even when the incident angle is scanned. By setting the position of the center of rotation 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 irradiation position on the film forming surface 22 and the incident surface 21), the irradiation position Can be minimized.

  As described above, among the incident angles of the excitation light α to the metal film 30, the angle at which the amount of plasmon scattered light γ is maximum 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 the high-intensity fluorescence β. The basic incident condition of the excitation light α is determined by the material and shape of the prism 20 of the measurement chip 10, the film thickness of the metal film 30, the refractive index of the liquid in the container 41, etc., but the fluorescent substance in the container 41 The optimum incident condition varies slightly depending on the type and amount of the light and the shape error of the prism 20. For this reason, it is preferable to obtain an optimal enhancement angle for each measurement.

  The light source control unit 113 controls various devices included in the light source unit 111 to control the emission of the excitation light α 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.

  The fluorescence detection unit 120 detects the fluorescence β generated by irradiating the metal film 30 with the excitation light α. Further, as necessary, the fluorescence detection unit 120 also detects plasmon scattered light γ generated by irradiation of the excitation light α to the metal film 30. The fluorescence detection unit 120 includes a light receiving unit 121, a position switching mechanism 122, and a sensor control unit 123.

  The light receiving unit 121 is arranged in the normal direction of the metal film 30 of the measurement chip 10. The light receiving unit 121 includes a first lens 124, an optical filter 125, a second lens 126, and a light receiving sensor 127.

  The first lens 124 is, for example, a condensing lens, and condenses light emitted from the metal film 30. The second lens 126 is an imaging lens, for example, and forms an image of the light collected by the first lens 124 on the light receiving surface of the light receiving sensor 127. The optical path between both lenses is a substantially parallel optical path. 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 γ) in order to detect the fluorescence β with a high S (signal) / N (noise) 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 including a multilayer film that reflects a predetermined light component, or a color glass filter that absorbs a predetermined light component.

  The light receiving sensor 127 detects fluorescence β and plasmon scattered light γ. The light receiving sensor 127 has a high sensitivity capable of detecting weak fluorescence β from a minute amount of a substance to be measured. The light receiving sensor 127 is, for example, a photomultiplier tube (PMT) or an avalanche photodiode (APD).

  The position switching mechanism 122 switches the position of the optical filter 125 on or off the optical path in the light receiving unit 121. Specifically, when the light receiving sensor 127 detects the fluorescence β, the optical filter 125 is disposed on the optical path of the light receiving unit 121, and when the light receiving sensor 127 detects the plasmon scattered light γ, the optical filter 125 is moved to the light receiving unit 121. Placed outside the optical path.

  The sensor control unit 123 controls detection of an output value of the light receiving sensor 127, management of sensitivity of the light receiving sensor 127 based on the detected output value, change of sensitivity of the light receiving sensor 127 for obtaining an appropriate output value, and the like. 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.

  The liquid feeding unit 130 supplies various liquids into the accommodating portion 41 of the measuring chip 10 held by the chip holder 142. In the present embodiment, for example, a specimen, a cleaning liquid, a labeling liquid containing a second capturing body labeled with a fluorescent substance (hereinafter also referred to as “labeling liquid”), and the like are supplied to the storage unit 41. The liquid feeding unit 130 includes a liquid chip 131, a syringe pump 132, and a liquid feeding pump driving mechanism 133.

  The liquid chip 131 is a container for storing a liquid such as a specimen, a cleaning liquid, or a labeling liquid. As the liquid chip 131, a plurality of containers are usually arranged according to the type of liquid, or a chip in which a plurality of containers are integrated is arranged.

  The syringe pump 132 in the liquid feeding unit 130 includes a syringe 134 and a plunger 135 that can reciprocate inside the syringe 134. By the reciprocating motion of the plunger 135, the 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 preventing contamination of impurities. In the case where the syringe 134 is not configured to be replaceable, it is possible to use the syringe 134 without replacing it by adding a configuration for cleaning the inside of the syringe 134.

  The liquid feed pump drive mechanism 133 includes a drive device for the plunger 135 and a moving device for the syringe pump 132. The drive device of the syringe pump 132 is a device for reciprocating the plunger 135, and includes, for example, a stepping motor. A driving device including a stepping motor is preferable from the viewpoint of managing the amount of liquid remaining in the measurement chip 10 because the amount of liquid fed and the liquid feeding speed of the syringe pump 132 can be managed. For example, the moving device of the syringe pump 132 freely moves the syringe pump 132 in two directions, that is, an axial direction (for example, a vertical direction) of the syringe 134 and a direction (for example, a horizontal direction) crossing the axial 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 sucks various liquids from the liquid chip 131 and supplies the liquids into the accommodating portion 41 of the measuring chip 10. At this time, by moving the plunger 135, the liquid can reciprocate in the housing part 41 of the measurement chip 10, and the liquid in the housing part 41 can be stirred. Thereby, the concentration of the liquid provided in the container 41 can be made uniform, and the reaction (for example, antigen-antibody reaction) in the container 41 can be promoted. From the viewpoint of performing such operations, the measurement chip 10 and the syringe 134 are protected so as to be sealed when the injection port of the measurement chip 10 penetrates the multilayer film. It is preferable to be configured.

  The liquid in the storage unit 1 is again sucked by the syringe pump 132 and discharged to the liquid chip 131 and the like. By repeating these operations, the inside of the container 41 is washed, in the container 41, the first capturing body reacts with the substance to be measured, or the second capture that is labeled with the substance to be measured and the fluorescent substance. You can react with the body.

  The transport unit 140 transports and fixes the measurement chip 10 to the measurement position or the liquid feeding position. Here, the “measurement position” is a position where the excitation light irradiation unit 110 irradiates the measurement chip 10 with the excitation light α, and the fluorescence detection unit 120 detects the fluorescence β or the plasmon scattered light γ generated accordingly. Further, the “liquid feeding position” is a position where the liquid feeding unit 130 supplies the liquid into the housing part 41 of the measuring chip 10 or removes the liquid from the housing part 41 of the measuring chip 10. The transport unit 140 includes a transport stage 141 and a chip holder 142. The chip holder 142 is fixed to the transfer stage 141, and holds the measurement chip 10 in a detachable manner. The shape of the chip holder 142 is a shape that can hold the measurement chip 10 and does not obstruct the optical paths of the excitation light α, the fluorescence β, and the plasmon scattered light γ. For example, the chip holder 142 is provided with an opening through which excitation light α, fluorescence β, and plasmon scattered light γ pass. The transfer stage 141 moves the chip holder 142 in one direction and in the opposite direction. The transport stage 141 also has a shape that does not interfere with the optical paths of the excitation light α, fluorescence β, and plasmon scattered light γ. The transport stage 141 is driven by, for example, a stepping motor.

  The control unit 150 controls the angle adjustment mechanism 112, the light source control unit 113, the position switching mechanism 122, the sensor control unit 123, the liquid feed pump drive mechanism 133, and the transport stage 141. The control unit 150 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.

(Measuring method)
Next, a measurement method according to this embodiment will be described. FIG. 2 is a flowchart illustrating an example of an operation procedure of the SPFS apparatus 100 when performing the measurement method of the embodiment.

  First, preparation for measurement is performed (step S10). Specifically, the measurement chip 10 described above is installed in the chip holder 142 of the SPSF apparatus 100. Further, when a moisturizing agent is present in the housing part 41 of the measurement chip 10, the inside of the housing part 41 is washed to remove the moisturizing agent.

  Next, the incident angle of the excitation light α with respect to the metal film 30 (deposition surface 22) of the measurement chip 10 is set to an enhancement angle (step S20). Specifically, the control unit 150 controls the transfer stage 141 to move the measurement chip 10 from the installation position to the measurement position. Thereafter, the control unit 150 controls the light source control unit 113 and the angle adjustment unit 112 to irradiate the excitation light α from the light source unit 111 to a predetermined position of the metal film 30 (deposition surface 22). The incident angle of the excitation light α with respect to 30 (deposition surface 22) is scanned. At this time, the control unit 150 controls the position switching mechanism 122 to move the optical filter 125 out of the optical path of the light receiving unit 121. At the same time, the control unit 150 controls the sensor control unit 123 so that the light receiving sensor 127 detects the plasmon scattered light γ. The control unit 150 obtains data including the relationship between the incident angle of the excitation light α and the intensity of the plasmon scattered light γ. Then, the control unit 150 analyzes the data and determines an incident angle (enhancement angle) that maximizes the intensity of the plasmon scattered light γ. Finally, the control unit 150 controls the angle adjustment unit 112 to set the incident angle of the excitation light α with respect to the metal film 30 (deposition surface 22) as an enhancement angle.

  Here, the enhancement angle is determined by the material and shape of the prism 20, the thickness of the metal film 30, the refractive index of the liquid in the storage portion 41, etc., but the type and amount of the liquid in the storage portion 41, the shape of the prism 20. It varies slightly due to various factors such as errors. For this reason, it is preferable to determine the enhancement angle every time measurement is performed. The enhancement angle is determined on the order of about 0.1 °.

  Next, the specimen is provided to the container 41 of the measurement chip 10 and the substance to be measured contained in the specimen is specifically bound to the first capturing body fixed on the metal film 30 in the measurement chip 10 ( Primary reaction (step S30)). Note that after the substance to be measured is bound, a buffer solution or the like is provided in the container 41, and the inside of the container 41 is washed to remove free substance to be measured.

  Here, the types of the specimen and the substance to be measured provided to the storage unit 41 in the present embodiment are not particularly limited. Examples of the specimen include body fluids such as blood, serum, plasma, urine, nasal fluid, saliva, semen, and diluted solutions thereof. Examples of substances to be measured contained in these specimens include nucleic acids (such as DNA and RNA), proteins (such as polypeptides and oligopeptides), amino acids, carbohydrates, lipids, and modified molecules thereof.

  After the first reaction, the optical blank value is measured (S40). Specifically, the control unit 150 controls the transfer stage 141 to move the measurement chip 10 from the installation position to the measurement position. Thereafter, the control unit 150 controls the light source control unit 113 to emit the excitation light α from the light source unit 111 at an enhancement angle toward the metal film 30 (deposition surface 22). At the same time, the control unit 150 controls the sensor control unit 123 to detect the amount of light by the light receiving sensor 127, and records this as a blank value.

  Subsequently, the second capturing body labeled with the fluorescent material is bound to the substance to be measured bound to the first capturing body on the metal film 30 (secondary reaction (step S50)). Specifically, the control unit 150 controls the transfer stage 141 to move the measurement chip 10 from the measurement position to the liquid feeding position. Thereafter, the control unit 150 controls the liquid feed pump drive mechanism 133 to provide the labeling liquid including the second capturing body in the liquid chip 131 in the storage unit 41. Here, the second capturing body is a substance that specifically binds to a site of the substance to be measured that is different from the site to which the first capturing body specifically binds. In addition, a fluorescent substance is bound to the second capturing body. Therefore, when the labeling solution is provided to the storage unit 41, the second capturing body specifically binds to the substance to be measured that is bound to the first capturing body, and the substance to be measured is indirectly labeled with the fluorescent substance. Is done. After the substance to be measured is labeled with a fluorescent substance, a buffer solution or the like is provided in the container 41, and the container 41 is washed to remove the free second capturing body and the like.

  Here, the second capturing body may be any substance that specifically binds to a site different from the site where the first capturing body specifically binds to the substance to be measured, and is specific to the substance to be measured. It may be a biomolecule (for example, a monoclonal antibody, a polyclonal antibody, a nucleic acid aptamer, etc.), or a fragment thereof. The second capturing body may be composed of one molecule or a complex in which two or more molecules are complexed.

  The fluorescent substance bound to the second capturing body is not particularly limited as long as it is a substance that emits fluorescence by being excited by the localized field light generated on the surface of the metal film. A fluorescent substance containing a rhodamine skeleton). Specific examples of the fluorescent material include fluorescent dyes manufactured by Biotium (for example, CF Dye (trade name) 350, 405M, 532, 543, 555, 568, 594, 642, 650, 667, 663, 681, 680). , 755, 770) and the like.

  In addition, the number of fluorescent substances bound to one second capturing body is 8 to 22, and preferably 13 to 16. “The number of fluorescent substances bonded to one second capturing body” means the average number of fluorescent substances bonded to each of the plurality of second capturing bodies provided in the accommodating portion 41. Value. Therefore, the labeling solution provided in the storage unit 41 may include a part of the second capturing body in which the fluorescent substance is less than 8 or more than 22 bound.

  Here, the amount of the fluorescent substance bound to one second capturing body is determined by the charging ratio of the second capturing body and the fluorescent substance when the fluorescent substance is bound to the second capturing body, the reaction Although it can be adjusted depending on the time and temperature at the time of reaction, it is difficult to increase the binding amount of the fluorescent substance beyond a certain level only by the reaction time and temperature at the time of reaction. Therefore, when increasing the binding amount of the fluorescent substance, it is preferable to adjust by the charging ratio. In addition, the number (average value) of fluorescent substances bonded to each second capturing body provided to the storage unit 41 can be measured by the following method. Using an absorptiometer, the absorbance of the second capture body to which the fluorescent substance is bound is measured, and the amount of the second capture body and the amount of the fluorescent substance are calculated. And the ratio of the quantity of the fluorescent substance with respect to the quantity of the 2nd supplement is calculated, and let the said value be the number of fluorescent substances.

  Next, with the substance to be measured labeled with a fluorescent substance disposed on the bottom surface (metal film 30) of the accommodating portion 41 via the first capturing body, the excitation light α is increased with the enhancement angle through the prism 20. The film 30 (deposition surface 22) is irradiated. And the fluorescence value from the fluorescent substance which labels a to-be-measured substance is measured (measurement process (process S60)). Specifically, the control unit 150 controls the transfer stage 141 to move the measurement chip 10 from the liquid feeding position to the measurement position. Thereafter, the control unit 150 controls the light source control unit 113 to emit the excitation light α from the light source unit 111 toward the metal film 30 (film formation surface 22). At the same time, the control unit 150 controls the sensor control unit 123 to detect the amount of light having the same wavelength as the fluorescence β by the light receiving sensor 127.

  Finally, a signal value indicating the presence or amount of the substance to be measured is calculated (step S70). The fluorescence value mainly includes a fluorescent component (signal value) derived from a fluorescent substance that labels the substance to be measured, and an optical blank value. Therefore, the control unit 150 can calculate a signal value correlated with the amount of the substance to be measured by subtracting the optical blank value obtained in step S40 from the fluorescence value obtained in step S60. Then, it is converted into the amount and concentration of the substance to be measured using a calibration curve prepared in advance.

(effect)
As described above, in general SPFS, the labeling rate is set within the range recommended by the fluorescent reagent manufacturer (usually 7 or less). On the other hand, when the labeling rate is 8 to 22 as in the present embodiment, the detected signal value becomes very high. On the other hand, the detected noise hardly changes compared to the case where the labeling rate is 7 or less. Therefore, when SPFS is performed, the S (signal) / N (noise) ratio to be detected can be greatly increased from a conventionally obtained value, and the amount of a substance to be measured can be specified with high sensitivity. Become.

  Further, in the SPFS of the present embodiment, a sufficiently high signal value can be obtained even if the amount of excitation light irradiated at the time of fluorescence detection is reduced. On the other hand, noise can be reduced as a whole by reducing the amount of excitation light. Therefore, according to the present embodiment, SPFS can be performed with a smaller amount of excitation light than in the past, and at that time, the amount of the substance to be measured can be specified with high sensitivity.

  Further, according to the present embodiment, a sufficiently high signal value can be obtained even if the amount of the fluorescent substance provided to the reaction field is reduced. Therefore, according to the present embodiment, the amount of the substance to be measured can be specified with high sensitivity with a small number of second capturing bodies, and the cost can be suppressed.

  Note that the measurement chip 10 used in the present embodiment and the second capturing body labeled with a fluorescent substance can be combined to form a measurement kit. In the measurement kit, the second capturing body labeled with a fluorescent substance may be in the form of powder or liquid.

[Other embodiments]
In the above-described embodiment, the prism coupling (PC) -SPFS (measuring method) and the measuring apparatus for coupling (coupling) photons and surface plasmons using the prism 20 on which the metal film 30 is formed have been described. . However, the measurement method and the measurement chip according to the present invention are not limited to this form. FIG. 3 is a perspective view of the metal film 30a including the diffraction grating. In the measuring method and measuring apparatus according to the present invention, as shown in FIG. 3, a measuring chip having a metal film 30a including a diffraction grating may be used. Also in this case, photons and surface plasmons can be combined to emit plasmon scattered light γ from the metal film 30a. In this case, the prism 20 is not necessary. Moreover, the excitation light irradiation unit 110 is disposed on the metal film 30a side of the measurement chip, and irradiates the excitation light α toward the diffraction grating in the fluorescence β detection step and the plasmon scattered light γ detection step.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail with reference to an Example, this invention is not limited by these Examples.

[Example 1]
(1) Preparation of measurement chip A chip having a metal film disposed on one surface of a prism was prepared. And the 1st capture body (anti-troponin antibody) was fixed to the metal film of the said prism. And the flow-path lid | cover was mounted on the metal film, and it was set as the measurement chip. The measurement chip was placed in the chip holder of the SPFS apparatus.

(2) Primary reaction 150 μL of a specimen containing troponin as a substance to be measured was prepared (specimen diluent 0.05% Tween-containing PBS pH 7.4, 3% BSA, substance to be measured having a concentration of 10 to 15 ng / mL). The sample is then reciprocated into the measurement chip housing (flow channel), whereby the substance to be measured in the sample reacts specifically with the first capturing body fixed in the flow channel (primary). Reaction).
Next, the specimen in the flow channel was removed, and the flow channel was washed with a washing solution (PBS pH 7.4 containing 0.05% Tween). Thereafter, a measurement solution (0.05% Tween, 5% BSA-containing PBS pH 7.4) was placed in the flow path, and excitation light having a wavelength of 660 nm was irradiated from the prism side to the metal film at an enhancement angle. Then, light emitted from the reaction field was detected to obtain an optical blank value.

(3) Preparation of second capture body labeled with fluorescent substance Concentration of 1 mg / mL of second capture body (antitroponin antibody that binds to a site different from the site to which the first complement of troponin binds) 100 μL of the solution (1 × PBS, pH 7.4) and 10 μL of 1M sodium bicarbonate (pH 8.3) were mixed. On the other hand, 1 μmol of a fluorescent substance CF660R having a rhodamine skeleton (manufactured by Biotium) was dissolved in 250 μL of dehydrated dimethyl sulfoxide (DMSO). Thereafter, 110 μL of the solution containing the second capturing body and 2.8 μL of the solution containing the fluorescent substance were mixed and reacted at room temperature for 1 hour. After the reaction, unreacted fluorescent material was removed by ultrafiltration. When the concentration of the second supplement of the reactant and the concentration of the fluorescent substance were confirmed with an absorptiometer and the amount of the fluorescent substance bound was calculated, the average fluorescent substance was 9.2 per second capture body. It was united. In addition, the manufacturer recommended value of the fluorescent material CF660R (manufactured by Biotium) is 4 to 7.

(4) Calculation of secondary reaction and signal value Thereafter, 40 μL of a labeling solution containing a second capturing body labeled with a fluorescent substance prepared as described above (labeled antibody diluent PBS pH 7.4, 1% BSA, (Measurement substance concentration: 4 μg / mL) is sent back and forth in the flow path, and the second capture body is bound to the measurement target substance disposed in the flow path via the first capture body (secondary reaction). )
After the secondary reaction, the labeling solution in the channel was removed, and the inside of the channel was washed with the same washing solution as described above. Thereafter, in the same manner as the measurement of the optical blank value, the measurement solution was put in the flow path, and the excitation light was irradiated at the enhancement angle to detect the fluorescence intensity emitted from the reaction field. Then, the optical blank value was subtracted from the fluorescence detection value, and the value was used as the signal value.

[Examples 2 and 3 and Comparative Examples 1 to 6]
The signal value was the same as in Example 1 except that the reaction equivalent ratio between the second capture body and the fluorescent substance was changed and the average number of fluorescent substances to be bound to the second capture body was changed as shown in Table 1. Was calculated. In addition, about each signal value, the signal value obtained by the comparative example 1 was made into 100%, and the signal value of each Example and the comparative example was evaluated by relative intensity (%) with respect to the comparative example 1. The relative intensity is shown in Table 1.

[Reference example]
On the other hand, in order to confirm the noise generated in each Example and Comparative Example, and the signal / noise ratio, Examples 1-3, except that a buffer solution not containing troponin was sent instead of the sample containing troponin. And the optical blank value and fluorescence intensity were measured similarly to Comparative Examples 1-6. And the value which subtracted the optical blank value from the detected fluorescence intensity was made into the noise value. The noise value is shown in Table 1. Further, the noise value corresponding to Comparative Example 1 was set to 100%, and the noise value corresponding to each Example and Comparative Example was expressed in relative intensity (%). These values are also shown in Table 1.
Then, the ratio (S / N ratio = signal value intensity / noise value intensity) between the signal value calculated in each example and comparative example and the noise value calculated above was calculated. Furthermore, the S / N ratio of Comparative Example 1 was set to 100%, and the S / N ratios of Examples and Comparative Examples were expressed as relative strength (%). These values are also shown in Table 1.

[result]
A graph showing the relationship between the number of fluorescent substances bound to the second capturing body (labeling rate), signal value (relative value), noise value (relative value), and signal / noise ratio (relative value), As shown in FIG. As shown in Table 1 and FIG. 4, when the number of fluorescent substances bound per second capturing body is less than 8 (Comparative Examples 1 to 5), the relative intensity of the signal value is 200%. On the other hand, when the number of fluorescent substances was 8 to 22, the relative intensity of the signal value exceeded 200% (Examples 1 to 3). Moreover, in these Examples 1-3, S / N ratio was 150 or more, and the high value was shown compared with the comparative example 1. That is, it can be seen that by setting the number of fluorescent substances in the above range, fluorescence can be detected (measured substance amount) with high sensitivity. On the other hand, when the amount of the fluorescent substance that binds to each second capturing body exceeds 22, the relative intensity of the signal value decreases, and the S / N ratio also decreases compared to Comparative Example 1 (Comparison Example 6). It is presumed that concentration quenching is likely to occur because the distance between the fluorescent substances bound to the second capturing body is short.

  As can be seen from the above results, the amount of the substance to be measured can be accurately measured with high sensitivity by employing the embodiment of the present invention.

  This application claims the priority based on Japanese Patent Application No. 2015-191380 of an application on September 29, 2015. The contents described in these application specifications and drawings are all incorporated herein.

  According to the measurement method and measurement kit for a substance to be measured according to the present invention, the substance to be measured can be measured with high sensitivity, and thus it is useful for, for example, examination of diseases.

DESCRIPTION OF SYMBOLS 10 Measurement chip | tip 20 Prism 21 Incidence surface 22 Deposition surface 23 Output surface 30 Metal film 40 Flow path cover 41 Storage part 100 SPFS apparatus 110 Excitation light irradiation unit 111 Light source unit 112 Angle adjustment mechanism 113 Light source control part 120 Fluorescence detection unit 121 Light reception Unit 122 Position switching mechanism 123 Sensor control unit 124 First lens 125 Optical filter 126 Second lens 127 Light receiving sensor 130 Liquid feed unit 131 Liquid chip 132 Syringe pump 133 Liquid feed pump drive mechanism 134 Syringe 135 Plunger 140 Transport unit 141 Transport stage 142 Chip holder 150 Control unit α Excitation light β Fluorescence γ Plasmon scattered light

Claims (7)

A sample is provided on a measurement chip that includes a metal film and a first capturing body that is fixed on the metal film and has a recognition site for specifically binding to the substance to be measured. Binding a measurement substance to the first capturing body;
The substance to be measured bound to the first capturing body has a recognition site for specifically binding to a site different from the site to bind to the first capturing body of the material to be measured and is fluorescent. Binding a second capturer labeled with a substance;
Irradiating the metal film with light so that surface plasmon resonance is generated, and detecting fluorescence emitted by the fluorescent material excited by localized field light based on the surface plasmon resonance;
A surface plasmon excitation enhanced fluorescence spectroscopy method comprising:
The number of the fluorescent substance bound per one second capturing body is 8-22,
Surface plasmon excitation enhanced fluorescence spectroscopy measurement method.   The surface plasmon excitation enhanced fluorescence spectroscopic measurement method according to claim 1, wherein the number of the fluorescent substances bonded per one second capturing body is 13 to 16. The fluorescent material includes a rhodamine skeleton,
The method of measuring surface plasmon excitation enhanced fluorescence spectroscopy according to claim 1 or 2. The measurement chip includes a prism, the metal film formed on one surface of the prism, and the first capturing body.
The surface plasmon excitation enhanced fluorescence spectroscopy measurement method according to any one of claims 1 to 3. The measurement chip includes a support, the metal film including a diffraction grating disposed on the support, and the first capturing body.
The surface plasmon excitation enhanced fluorescence spectroscopy measurement method according to any one of claims 1 to 3. A prism made of a dielectric, a metal film disposed on one surface of the prism, a first capturing body fixed on the metal film and having a recognition site for specifically binding to a substance to be measured; And a second capture having a recognition site for specifically binding to a site different from the site of binding to the first capture body of the substance to be measured and labeled with a fluorescent material body,
A measurement kit comprising:
The number of the fluorescent substance bound per one second capturing body is 8-22,
Measurement kit. A support, a metal film including a diffraction grating disposed on the support, a first capturing body having a recognition site fixed on the metal film and specifically binding to a substance to be measured; And a second capture having a recognition site for specifically binding to a site different from the site binding to the first capture body of the substance to be measured and labeled with a fluorescent material body,
A measurement kit comprising:
The number of the fluorescent substance bound per one second capturing body is 8-22,
Measurement kit.

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