Patent Description:
In the measurement for detecting a biological substance such as protein or DNA, if a minute amount of a substance to be detected can be detected with high sensitivity and quantitatively, it is possible to immediately grasp the patient's condition and perform treatment. Therefore, there is a demand for a detection method and a detection device for detecting weak light caused by a trace amount of a substance to be detected with high sensitivity and quantitatively. As one method for detecting a substance to be detected with high sensitivity, the surface plasmon resonance fluorescence analysis method (surface plasmon-field enhanced fluorescent spectroscopy: SPFS) is known (see, for example, Patent Document <NUM>).

In the SPFS, for example, a prism in which a metal film is disposed on a predetermined surface is used. Then, when the metal film is irradiated with excitation light from the excitation light irradiation unit at an angle at which surface plasmon resonance occurs via the prism, localized field light (enhanced electric field) can be generated on the surface of the metal film. Since the fluorescent substance that labels the substance to be detected captured on the metal film is excited by the localized field light, detecting the fluorescence emitted from the fluorescent substance allows the presence or amount of the substance to be detected to be detected.

<CIT> discloses a surface plasmon resonance fluorometry device and method, wherein a specimen is made to flow on a metal film on a prism, and excitation light is incident on the metal film through the prism. By changing the incident angle of the excitation light that enters the prism, the position of an irradiation spot on the metal film is adjusted and the intensity of light generated on the metal film is maximized. Different filters used when measuring plasmon scattered light and fluorescence reduce stray light incident on a detector, which receives light from a measurement area on the metal film including the irradiation spot.

In the SPFS, in order to efficiently excite plasmon resonance, a region corresponding to a region (reaction field) in which a substance to be detected on the metal film is captured is irradiated with excitation light α of substantially parallel light. In addition, in order to reduce the cost of the detection chip, improve the reaction efficiency, and reduce the amount of the specimen to reduce the burden on the subject, the reaction field is set in a limited region, and therefore, after being thinly beam-formed (beam size is reduced) using a pinhole or a slit, the excitation light α is applied to a region corresponding to the reaction field. However, as shown in <FIG>, thinning the excitation light α generates the diffracted light <NUM> in the beam passing through the pinhole or the slit, and the peripheral region of the region to be irradiated with the excitation light α of the metal film is irradiated with the diffracted light <NUM>.

As shown in <FIG>, when the diffracted light <NUM> reaches a structure around the metal film <NUM> of the detection chip <NUM>, for example, an adhesive layer <NUM> that adheres the prism <NUM> and the wall portion <NUM>, the diffracted light <NUM> is guided through the adhesive layer <NUM> and scattered to generate stray light <NUM>.

In addition, in the SPFS, in order to generate an enhanced electric field based on plasmon resonance, it is necessary to cause the excitation light α to be incident on the metal film <NUM> at a predetermined angle. When, in order to adjust the excitation light α to a predetermined incident angle with respect to the metal film <NUM>, the metal film <NUM> is irradiated with the excitation light α through the prism <NUM>, the plasmon scattered light β having the same wavelength as the excitation light α generated due to the surface plasmon resonance in the metal film <NUM> may be detected. At this time, when the above-described stray light <NUM> enters the detection range <NUM> of the detection unit, as shown in <FIG>, in addition to the plasmon scattered light β emitted from the region corresponding to the irradiation spot <NUM> of the excitation light α, the stray light <NUM> emitted from the stray light occurrence spot <NUM> is also detected. When not only the plasmon scattered light β but also the stray light <NUM> is detected as described above, it is difficult to appropriately adjust the incident angle of the excitation light α to an optimum angle (for example, an enhancement angle).

<FIG> is a graph showing the relationship between the incident angle of the excitation light α and the amount of received light of the detection unit when only the plasmon scattered light β emitted from the region corresponding to the irradiation spot <NUM> of the excitation light α is detected, and <FIG> is a graph showing the relationship between the incident angle of the excitation light α and the amount of received light of the detection unit when the stray light <NUM> emitted from the stray light occurrence spot <NUM> is also detected in addition to the plasmon scattered light β. By scanning the incident angle of the excitation light α, the irradiation position of the diffracted light <NUM> applied to the metal film <NUM> is changed, whereby the irregular stray light <NUM> is detected by the detection unit. Therefore, as is clear from <FIG>, in a case where the plasmon scattered light β and the stray light <NUM> are detected, noise occurs in the amount of received light, and it may be difficult to detect a peak. Therefore, when stray light is also detected, the plasmon scattered light β cannot be accurately detected, and the incident angle (for example, the enhancement angle) of the excitation light α cannot be accurately determined.

Here, in order for the detection unit not to detect stray light, it is conceivable to sufficiently separate a structure (for example, adhesive layer <NUM>) that causes stray light from the detection range <NUM> of the detection unit, for example, to make a detection chip larger. However, in this case, the cost increases due to the increase in the size of detection chip, the burden on the subject increases due to the need to increase the specimen amount, and the reaction efficiency of the reaction field decreases due to the increase in the flow path width. In addition, the detection unit may receive a large amount of autofluorescence generated from the prism <NUM>, and the measurement performance may be deteriorated. Therefore, it is not easy to largely separate the structure from the detection range <NUM> of the detection unit.

The present invention has been made in view of the above circumstances, and has an object to provide, in a detection device and a detection method for detecting the presence or amount of a substance to be detected using an enhanced electric field based on surface plasmon resonance, a detection device and a detection method which does not increase the cost of the detection chip, which does not deteriorate the measurement performance of the substance to be detected, and which includes a detection unit capable of accurately detecting plasmon scattered light β without detecting stray light derived from excitation light and accurately determining an incident angle of the excitation light.

The present invention is defined in claims <NUM> and <NUM>.

A detection device according to an embodiment of the present invention is a detection device configured to detect presence or an amount of a substance to be detected using an enhanced electric field based on surface plasmon resonance, the detection device including: a chip holder configured to hold a detection chip including a prism having an incident surface and a film formation surface, a metal film disposed on the film formation surface, and a capturing body fixed on the metal film and configured to capture a substance to be detected; a light source configured to irradiate an irradiation spot on the metal film of the detection chip held by the chip holder with excitation light through the prism; a detection unit arranged to face the metal film of the detection chip held by the chip holder, the detection unit configured to detect light emitted from the metal film and a region on the metal film when the light source irradiates the metal film with the excitation light; and a detection range control unit configured to control a detection range of the detection unit.

The detection range control unit is configured to: determine a first detection range of the detection unit when the detection unit detects plasmon scattered light emitted from the metal film; and determine a second detection range of the detection unit when the detection unit detects fluorescence emitted from a fluorescent substance that labels the substance to be detected captured by the capturing body, and wherein the first detection range is different from the second detection range in a position or a size, and is controlled such that, when plasmon scattered light is detected, a stray light occurrence spot generated by the excitation light does not fall within the first detection range, but the irradiation spot of excitation light falls within the first detection range.

A detection method according to an embodiment of the present invention is a detection method for detecting presence or an amount of a substance to be detected using an enhanced electric field based on surface plasmon resonance, the detection method including: preparing a detection chip including: a prism having an incident surface and a film formation surface, a metal film disposed on the film formation surface, and a capturing body fixed on the metal film and configured to capture a substance to be detected; irradiating an irradiation spot on the metal film with excitation light through the prism and detecting plasmon scattered light emitted from the metal film within a first detection range; and irradiating the metal film with the excitation light through the prism and detecting fluorescence emitted from a fluorescent substance that labels the substance to be detected captured by the capturing body within a second detection range.

The first detection range of the plasmon scattered light when the plasmon scattered light is detected is different from the second detection range of the fluorescence when the fluorescence is detected in a position or a size, and is controlled such that, when plasmon scattered light is detected , a stray light occurrence spot generated by the excitation light does not fall within the first detection range, but the irradiation spot of excitation light falls within the first detection range.

According to the present invention, in the detection device and the detection method for detecting the presence or the amount of the substance to be detected using the enhanced electric field based on the surface plasmon resonance, the plasmon scattered light β can be accurately detected without the detection unit detecting stray light derived from the excitation light, and the incident angle of the excitation light can be accurately determined.

<FIG> is a diagram showing a detection device <NUM> according to the first embodiment of the present invention.

The detection device <NUM> includes: a light source <NUM> for irradiating the detection chip <NUM> with the excitation light α, a first lens <NUM>, a second lens <NUM>, an excitation light cut filter <NUM>, a detection unit <NUM> for detecting light (plasmon scattered light β or fluorescence γ) emitted from the detection chip <NUM>, a chip holder <NUM>, a chip holder moving means <NUM>, and a control unit <NUM>. The detection device <NUM> is used together with the detection chip <NUM>. Thus, the detection chip <NUM> will be described first, and then each component of the detection device <NUM> will be described.

As shown in <FIG>, the detection chip <NUM> includes a prism <NUM> having an incident surface <NUM>, a film formation surface <NUM>, and an emitting surface <NUM>, a metal film <NUM> disposed on the film formation surface <NUM> of the prism <NUM>, and a flow path lid <NUM> disposed on the metal film <NUM>.

The prism <NUM> is made of a member transparent to the excitation light α. The prism <NUM> has an incident surface <NUM>, a film formation surface <NUM> on which a metal film <NUM> is formed, and an emitting surface <NUM>. The incident surface <NUM> allows the excitation light α from the light source <NUM> to be incident on the inside of the prism <NUM>. A metal film <NUM> is formed on the film formation surface <NUM>. The excitation light α incident on the inside of the prism <NUM> is reflected by the metal film <NUM>. More specifically, the excitation light α is reflected at the interface (film formation surface <NUM>) between the prism <NUM> and the metal film <NUM>. The emitting surface <NUM> emits the excitation light α reflected by the metal film <NUM> to outside the prism <NUM>. The shape of the prism <NUM> is not particularly limited. In the present exemplary embodiment, the shape of the prism <NUM> is a column body having a trapezoidal bottom. A surface corresponding to one base of the trapezoid is the film formation surface <NUM>, a surface corresponding to one leg is the incident surface <NUM>, and a surface corresponding to the other leg is the emitting surface <NUM>. The trapezoid serving as the bottom surface is preferably a substantially isosceles trapezoid. Thus, the incident surface <NUM> and the emitting surface <NUM> are substantially symmetrical, and the S-polarized component of the excitation light α is less likely to stay by being totally reflected in the prism <NUM>. It should be noted that since only the P-polarized component of the excitation light contributes to plasmon resonance, the S-polarized component is reflected by the metal film <NUM>. In addition, the incident surface <NUM> is formed so that the excitation light α does not return to the light source <NUM>. This is because, if the excitation light α returns to the light source <NUM> being a laser diode, for example, the excited state of the laser diode is disturbed, and the wavelength and power of the excitation light α fluctuate. Therefore, the angle of the incident surface <NUM> is set so that the excitation light α is not perpendicularly incident on the incident surface <NUM> in the scanning range centered on the ideal enhancement angle. For example, the angle between the incident surface <NUM> and the film formation surface <NUM> and the angle between the film formation surface <NUM> and the emitting surface <NUM> are both about <NUM>°. Examples of the material of the prism <NUM> include resin and glass. The material of the prism <NUM> is preferably a resin having a refractive index of <NUM> to <NUM> and small birefringence.

The metal film <NUM> is formed on the film formation surface <NUM> of the prism <NUM>. Providing the metal film <NUM> allows interactions between photons of the excitation light α incident on the film formation surface <NUM> under a total reflection condition and free electrons in the metal film <NUM> (surface plasmon resonance; SPR) to occur, and an enhanced electric field (localized field light) to be generated on the surface of the metal film <NUM>. The material of the metal film <NUM> is not particularly limited as long as it is a metal that causes surface plasmon resonance. Examples of the material of the metal film <NUM> include gold, silver, copper, aluminum, and alloys thereof. Among them, the metal constituting the metal film <NUM> is preferably gold from the viewpoint of suppressing non-specific adsorption of a substance in a specimen. In the present embodiment, a metal constituting the metal film <NUM> is gold. A method for forming the metal film <NUM> is not particularly limited. Examples of the method for forming the metal film <NUM> include sputtering, vapor deposition, and plating. The thickness of the metal film <NUM> is not particularly limited, but is preferably within the range of <NUM> to <NUM>.

In addition, although not particularly shown, a capturing body for capturing a substance to be detected is immobilized on a surface of the metal film <NUM> not facing the prism <NUM>. By immobilizing the capturing body, the substance to be detected can be selectively detected. In the present embodiment, the capturing body is uniformly immobilized in a predetermined region on the metal film <NUM>. The kind of the capturing body is not particularly limited as long as the capturing body 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 flow path lid <NUM> is disposed on a surface not facing the prism <NUM> of the metal film <NUM> across the flow path <NUM>. In the present embodiment, the flow path lid <NUM> is joined to the film formation surface <NUM> or the metal film <NUM> via the wall portion <NUM> and the adhesive layer <NUM>. The wall portion <NUM> defines a storage portion for storing liquid on the metal film <NUM>. In the present embodiment, the storage portion is the flow path <NUM>, but may be a well. In the present embodiment, the wall portion <NUM> includes at least a first side wall <NUM> and a second side wall <NUM> facing each other, and the second side wall <NUM> is farther away from the light source <NUM> than the first side wall <NUM>. It should be noted that in the present embodiment, the wall portion <NUM> has a light shielding function, but the adhesive layer <NUM> does not have a light shielding function. Specifically, the flow path lid <NUM> is joined to the metal film <NUM> or the prism <NUM> by, for example, adhesion with a double-sided tape or an adhesive, laser welding, ultrasonic welding, pressure bonding using a clamp member, or the like.

When the metal film <NUM> is formed only on a part of the film formation surface <NUM> of the prism <NUM>, the flow path lid <NUM> may be disposed on the film formation surface <NUM> across the flow path <NUM>. The flow path lid <NUM> forms, together with the metal film <NUM>, the wall portion <NUM>, and the adhesive layer <NUM>, a flow path <NUM> through which liquids such as a specimen, a fluorescent labeling liquid, and a cleaning liquid flow. The capture substance is exposed in the flow path <NUM>. Each of both ends of the flow path <NUM> is connected to an injection port and a discharge port (both are not shown) formed on the upper surface of the flow path lid <NUM>. When the liquids are injected into the flow path <NUM>, these liquids come into contact with the capture substance in the flow path <NUM>.

The flow path lid <NUM> is made of a material transparent to light (plasmon scattered light β and fluorescence γ) emitted from a surface not facing the prism <NUM> of the metal film <NUM> and its vicinity. Examples of the material of the flow path lid <NUM> include resin. If these rays of light can be guided to the detection unit <NUM>, a part of the flow path lid <NUM> may be made of an opaque material.

As shown in <FIG>, the excitation light α guided to the prism <NUM> enters the prism <NUM> from the incident surface <NUM>. The excitation light α incident into the prism <NUM> is incident on the interface (film formation surface <NUM>) between the prism <NUM> and the metal film <NUM> so as to have a total reflection angle (angle at which surface plasmon resonance occurs). The reflected light from the interface is emitted from the emitting surface <NUM> to outside the prism <NUM>. On the other hand, incidence of the excitation light α on the interface at an angle at which the surface plasmon resonance occurs emits the plasmon scattered light β and/or the fluorescence γ from the metal film <NUM> and the vicinity thereof toward the detection unit <NUM>.

Next, each component of the detection device <NUM> will be described. As described above, the detection device <NUM> includes the light source <NUM>, the first lens <NUM>, the second lens <NUM>, the excitation light cut filter <NUM>, the detection unit <NUM>, the chip holder <NUM>, the chip holder moving means <NUM>, and the control unit <NUM>.

The light source <NUM> emits the excitation light α. The position and orientation of the light source <NUM> are adjusted by the control unit <NUM>. Thus, the incident angle of the excitation light α with respect to the interface (film formation surface <NUM>) between the prism <NUM> and the metal film <NUM> is adjusted. When the metal film <NUM> is irradiated with the excitation light α, the plasmon scattered light β having the same wavelength as the excitation light α, the fluorescence γ emitted from the fluorescent substance, and the like are emitted upward from the surface not facing the prism <NUM> of the metal film <NUM> and the vicinity thereof. In addition, the excitation light α is reflected by the interface between the prism <NUM> and the metal film <NUM>, and is emitted from the emitting surface <NUM> to outside the prism <NUM>.

In the present embodiment, the light source <NUM> is a laser diode (hereinafter abbreviated as "LD"), and emits excitation light α (single mode laser light) toward the incident surface <NUM> of the detection chip <NUM>. More specifically, the light source <NUM> emits only the P waves with respect to the interface toward the incident surface <NUM> so that the excitation light α has a total reflection angle with respect to the interface (film formation surface <NUM>) between the prism <NUM> and the metal film <NUM> of the detection chip <NUM>. The excitation light α is preferably substantially parallel light.

It should be noted that the type of the light source <NUM> is not particularly limited, and does not need to be an LD. Examples of the light source <NUM> include a light emitting diode, a mercury lamp, and other laser light sources. When the light emitted from the light source <NUM> is not a beam, the light emitted from the light source <NUM> is preferably converted into a beam by a lens, a mirror, a pinhole, a slit, or the like. In the present embodiment, the beam size of the light emitted from the light source <NUM> is controlled by the slit. The beam size of the excitation light α is preferably controlled to <NUM> to <NUM> on the metal film surface. In addition, When the light emitted from the light source <NUM> is not monochromatic light, the light emitted from the light source <NUM> is preferably converted into monochromatic light by a diffraction grating or the like. Furthermore, when the light emitted from the light source <NUM> is not linearly polarized light, the light emitted from the light source <NUM> is preferably converted into linearly polarized light by a polarizer or the like.

The first lens <NUM> forms an image of the plasmon scattered light β or the fluorescence γ emitted from on the metal film <NUM> on the light receiving unit of the detection unit <NUM>. The first lens <NUM> is, for example, a condenser lens, and condenses light emitted from on the metal film <NUM>. The second lens <NUM> is, for example, an image forming lens, and forms an image of the light condensed by the first lens <NUM> on the light receiving unit of the detection unit <NUM>. The optical paths between the two lenses are substantially parallel optical paths.

The excitation light cut filter <NUM> blocks the plasmon scattered light β, stray light derived from the excitation light α, and the like, and transmits the fluorescence γ, thereby preventing light other than the wavelength of the fluorescence γ from reaching the detection unit <NUM>. That is, the excitation light cut filter <NUM> removes a noise component from the light emitted from the metal film <NUM>, and enables the detection unit <NUM> to detect the fluorescence γ at a high S/N ratio. In <FIG> showing the present embodiment, the excitation light cut filter <NUM> is disposed between the first lens <NUM> and the second lens <NUM>, but when the enhancement angle is determined, the excitation light cut filter <NUM> is removed from the optical path so that the plasmon scattered light β can be detected.

The detection unit <NUM> is disposed so as to face a surface not facing the prism <NUM> of the metal film <NUM> of the detection chip <NUM>. The detection unit <NUM> receives light (plasmon scattered light β or fluorescence γ) emitted from on the metal film <NUM>. The light receiving unit of the detection unit <NUM> includes, for example, an image sensor, a photomultiplier tube, a photodiode, or the like. The first lens <NUM>, the excitation light cut filter <NUM>, the second lens <NUM>, and the detection unit <NUM> are arranged in this order from the metal film <NUM> side so as to face the surface of the metal film <NUM>.

The detection chip <NUM> is set on the chip holder <NUM>. The chip holder <NUM> is not particularly limited as long as the detection chip <NUM> can be set. The chip holder <NUM> has a shape that can set the detection chip <NUM> and does not hinder the optical path of the excitation light α, the reflected light, the fluorescence γ, and the like. For example, the chip holder <NUM> is provided with an opening through which these rays of light pass.

The chip holder moving means <NUM> moves the chip holder <NUM>. The chip holder moving means <NUM> moves, for example, the chip holder <NUM> in one direction and the opposite direction. The chip holder moving means <NUM> is, for example, a motor or the like.

The control unit <NUM> centrally controls the light source <NUM>, the excitation light cut filter <NUM>, the detection unit <NUM>, and the chip holder moving means <NUM>. Specifically, the control unit <NUM> controls the position and orientation of the light source <NUM>, and sets the incident angle of the excitation light α with respect to the metal film <NUM> to a predetermined angle. In addition, when determining the enhancement angle, the control unit <NUM> removes the excitation light cut filter <NUM> from on the optical path so that the plasmon scattered light β reaches the detection unit <NUM>. In addition, when receiving the fluorescence γ, the control unit <NUM> disposes the excitation light cut filter <NUM> on the optical path so that light having the same wavelength as the excitation light α (plasmon scattered light β, stray light derived from the excitation light α, or the like) does not reach the detection unit <NUM>. In addition, the control unit (detection range control unit) <NUM> controls the chip holder moving means <NUM> to move the chip holder <NUM> and change the detection range of the detection unit <NUM>. The control unit <NUM> is, for example, a computer that executes software.

The detection device <NUM> may include a shape information acquisition unit <NUM> that acquires shape information on the detection chip <NUM>. The control unit (detection range control unit) <NUM> can determine the detection range <NUM> when the detection unit <NUM> detects the plasmon scattered light β based on the shape information on the detection chip <NUM> acquired by the shape information acquisition unit <NUM>.

The above-described shape information on the detection chip is, for example, information indicating a positional relationship between the prism <NUM> and the first side wall <NUM>. For example, at the time of manufacturing the detection chip <NUM>, this positional relationship can be put in a barcode or the like attached to the detection chip <NUM>. The shape information acquisition unit <NUM> is, for example, a barcode reader, and can acquire the shape information by reading the barcode. Based on the acquired shape information, the control unit (detection range control unit) <NUM> can determine the detection range <NUM> of the detection unit <NUM>. Thus, stray light can be removed without being affected by the manufacturing variations of the detection chip <NUM>, and the enhancement angle can be accurately measured.

The detection device <NUM> may include a positional information detection unit <NUM> that detects positional information of the detection chip <NUM>. The positional information of the detection chip <NUM> may be information including the position of at least one of the first side wall <NUM> and the second side wall <NUM> of the detection chip <NUM>. The positional information detection unit <NUM> is, for example, a camera, and can acquire positional information on at least one of the first side wall <NUM> and the second side wall <NUM> using the camera. Based on the acquired positional information on the detection chip, the control unit (detection range control unit) <NUM> can determine the detection range <NUM> of the detection unit <NUM> necessary for stray light removal.

<FIG> is a flowchart showing a detection method according to an embodiment of the present invention using the detection device <NUM>.

First, preparation for measurement is made. Specifically, the detection chip <NUM> is set in the chip holder <NUM> of the detection device <NUM> (step S10). In addition, when a humectant is present in the flow path <NUM> of the detection chip <NUM>, the inside of the flow path <NUM> is cleaned to remove the humectant so that the capture substance can appropriately capture the substance to be detected.

Next, the plasmon scattered light β is detected (step S20). Specifically, the chip holder <NUM> is moved from the position where the detection chip <NUM> is set so that a predetermined position of the metal film <NUM> (film formation surface <NUM>) can be irradiated with the excitation light α. The incident angle of the excitation light α with respect to the metal film <NUM> (film formation surface <NUM>) is scanned while a predetermined position of the metal film <NUM> (film formation surface <NUM>) is irradiated with the excitation light α, the plasmon scattered light β is detected with the detection unit <NUM>, and the optimum incident angle (enhancement angle) of the excitation light α for generating an enhanced electric field based on plasmon resonance is determined (step S30). This is performed by the control unit <NUM> controlling the light source <NUM> to scan the incident angle of the excitation light α with respect to the metal film <NUM> (film formation surface <NUM>) while irradiating a predetermined position of the metal film <NUM> (film formation surface <NUM>) with the excitation light α. At this time, as shown in <FIG> described below, the control unit <NUM> moves the chip holder <NUM> so that the end portion of the first side wall <NUM> on a side of the storage portion does not enter the detection range <NUM> of the detection unit <NUM>. When the end portion of the first side wall <NUM> on the side of the storage portion enters the detection range <NUM>, the control unit <NUM> controls the chip holder moving means <NUM> to move the chip holder <NUM> so that the end portion of the first side wall <NUM> does not enter the detection range <NUM>. In addition, the control unit <NUM> controls the excitation light cut filter <NUM> so that the excitation light cut filter <NUM> is not present on the optical path, and controls the detection unit <NUM> so that the detection unit <NUM> detects the plasmon scattered light β from on the metal film <NUM> (the surface of the metal film <NUM> and the vicinity thereof. The plasmon scattered light β from the on metal film <NUM> (the surface of the metal film <NUM> and the vicinity thereof) reaches the detection unit <NUM> via the first lens <NUM> and the second lens <NUM>.

<FIG> is a schematic diagram showing the detection range <NUM> of the detection unit <NUM> and the position of the light source <NUM> when the optimum incident angle (enhancement angle) of the excitation light α is determined. In this figure, a state when the detection chip <NUM> and the light source <NUM> are viewed from the detection unit <NUM> side is shown. As shown in <FIG> and <FIG>, the stray light derived from the excitation light α is guided toward the traveling direction side of the excitation light α through the adhesive layer <NUM> positioned on the first side wall <NUM> on the light source <NUM> side, and enters the flow path <NUM> from the end portion of the adhesive layer <NUM>. That is, the stray light occurrence spot <NUM> is likely to occur at the end portion of the first side wall <NUM> on the side of the storage portion, and large stray light is generated. However, in the present embodiment, when plasmon scattered light is detected, the detection chip <NUM> (chip holder <NUM>) is moved so that the end portion of the first side wall <NUM> on the side of the storage portion does not enter the detection range <NUM> of the detection unit <NUM> and the region corresponding to the irradiation spot <NUM> of the excitation light α enters the detection range <NUM> of the detection unit <NUM>. Therefore, the stray light occurrence spot <NUM> does not fall within the detection range <NUM>, but the irradiation spot <NUM> of the excitation light α falls within the detection range <NUM>. Therefore, the control unit <NUM> can obtain accurate data on the relationship between the incident angle of the excitation light α and the intensity of the plasmon scattered light β while preventing the influence of stray light derived from the excitation light α.

In addition, as shown in <FIG>, since the stray light occurrence spot <NUM> may also occur at the end portion of the second side wall <NUM> on a side opposite to the storage portion, it is more preferable that the end portion does not fall within the detection range <NUM>.

It should be noted that regarding the facing direction between the first side wall <NUM> and the second side wall <NUM>, the ratio (S/W) of the length (S) of the irradiation spot of the excitation light on the metal film <NUM> to the distance (W) between the first side wall <NUM> and the second side wall <NUM> is preferably more than <NUM> and less than <NUM>, and more preferably more than <NUM> and less than <NUM>.

As described above, when the ratio (S/W) exceeds <NUM>, the size of the irradiation spot <NUM> of the excitation light α becomes an appropriate size, and the light amount of the fluorescence caused by the amount of the substance to be detected becomes a sufficient amount, so that the detection accuracy of the substance to be detected becomes favorable. More specifically, the substance to be detected is captured in the reaction field (region where the capturing body is immobilized) in the flow path. When the size of the irradiation spot <NUM> of the excitation light α is appropriate for the reaction field, a fluorescent substance for labeling the captured substance to be detected can be appropriately excited, and the light amount of fluorescence sufficient for securing detection accuracy can be obtained.

As described above, when the ratio (S/W) is less than <NUM>, the irradiation spot of the excitation light and the place where the stray light occurs can be sufficiently separated from each other, and the operation for excluding the place where the stray light occurs from the detection range <NUM> becomes easy.

Then, the control unit <NUM> analyzes the data and determines the incident angle (enhancement angle) of the excitation light at which the intensity of the plasmon scattered light β is maximized. It should be noted that the enhancement angle is basically determined by the material and shape of the prism <NUM>, the thickness of the metal film <NUM>, the refractive index of the liquid in the flow path <NUM>, and the like, but slightly fluctuates due to various factors such as the type and amount of the substance in the flow path <NUM>, and the shape error of the prism <NUM>. Therefore, it is preferable to determine the enhancement angle each time detection is performed. The enhancement angle is determined on the order of about <NUM>°.

Next, the chip holder <NUM> is moved (step S40). Specifically, the control unit <NUM> controls the chip holder moving means <NUM> to cause the chip holder moving means <NUM> to move the chip holder <NUM>, so that the irradiation spot <NUM> of the excitation light α is positioned near the center of the detection range <NUM> as shown in <FIG>.

As shown in <FIG>, the end portion of the first side wall <NUM> on the side of the storage portion falls within the detection range <NUM>, and the stray light occurring here has the same wavelength as the excitation light α. As will be described below, in the measurement of the optical blank value and the measurement of the specimen signal, since the excitation light cut filter <NUM> is inserted to measure the signal, the stray light <NUM> having the same wavelength as the excitation light α is not detected by the detection unit <NUM> and does not affect the measurement performance. Furthermore, by moving the detection range so that the irradiation spot <NUM> of the excitation light α falls within the vicinity of the center of the detection range <NUM>, even if the positional relationship between the irradiation spot <NUM> of the excitation light α and the detection range <NUM> varies due to setting variations of the detection chip or the like, the fluorescence from the reaction field (region where the capturing body is immobilized) on the metal film <NUM> can be efficiently and reproducibly detected without the irradiation spot <NUM> of the excitation light α falling outside the detection range, and the detection accuracy (accuracy, accuracy of absolute value) of the substance to be detected can be improved.

Here, comparing <FIG> with <FIG>, the detection range <NUM> of the detection unit <NUM> when the detection unit <NUM> detects scattered light may be farther from the light source <NUM> than the detection range <NUM> of the detection unit <NUM> when the detection unit <NUM> detects fluorescence. In addition, the irradiation spot <NUM> (irradiation position of excitation light) of the excitation light α on the metal film <NUM> when the detection unit <NUM> detects plasmon scattered light β may be farther from the light source <NUM> than the irradiation spot <NUM> (irradiation position of excitation light) (see <FIG>) of the excitation light α on the metal film <NUM> when the detection unit <NUM> detects fluorescence.

It should be noted that, in steps S20 and S40, moving the chip holder <NUM> relatively moves the position of the detection range <NUM> of the detection unit <NUM> with respect to the detection chip <NUM>, but moving the detection unit <NUM> may relatively move the detection range <NUM> of the detection unit <NUM> with respect to the detection chip <NUM>.

<FIG> is a graph showing a relative movement distance of the detection range <NUM> of the detection unit <NUM> with respect to the irradiation spot of the excitation light α when the detection chip <NUM> is moved and when the detection unit <NUM> is moved. When the detection unit <NUM> is moved without the chip holder <NUM> (detection chip <NUM>) being moved, the position of the irradiation spot of the excitation light α does not change, but the position of the detection range <NUM> of the detection unit <NUM> is moved by the amount of movement of the detection unit <NUM>. Therefore, the relative movement distance of the detection range <NUM> of the detection unit <NUM> with respect to the irradiation spot of the excitation light α becomes longer by the same distance as the movement distance of the detection unit <NUM>. On the other hand, when the chip holder <NUM> (detection chip <NUM>) is moved without the detection unit <NUM> being moved, the position of the detection range <NUM> of the detection unit <NUM> is moved by the amount of movement of the chip holder <NUM> (detection chip <NUM>), but the position of the irradiation spot of the excitation light α is also moved. Therefore, the relative movement distance of the detection range <NUM> of the detection unit <NUM> with respect to the irradiation spot of the excitation light α is shorter than the distance by which the chip holder <NUM> (detection chip <NUM>) is moved. As described above, when the chip holder <NUM> (detection chip <NUM>) is moved, the relative position of the detection range <NUM> of the detection unit <NUM> with respect to the irradiation spot of the excitation light α is less likely to change, which is preferable from the viewpoint of the detection accuracy.

Next, the incident angle of the excitation light α with respect to the metal film <NUM> (film formation surface <NUM>) is set to the enhancement angle determined in step S30 (step S50). Specifically, the control unit <NUM> controls the light source <NUM> to set the incident angle of the excitation light α with respect to the metal film <NUM> (film formation surface <NUM>) to the enhancement angle. In the subsequent steps, the incident angle of the excitation light α with respect to the metal film <NUM> (film formation surface <NUM>) remains at the enhancement angle.

Next, the substance to be detected in the specimen is reacted with the capture substance (primary reaction, step S60). Specifically, the specimen is injected into the flow path <NUM>, and the specimen and the capture substance are brought into contact with each other. When the substance to be detected is present in the specimen, at least a part of the substance to be detected is captured by the capture substance. Thereafter, the inside of the flow path <NUM> is cleaned with a buffer solution or the like to remove a substance not captured by the capture substance. The type of specimen is not particularly limited. Examples of the specimen include body fluids such as blood, serum, plasma, urine, nasal fluid, saliva, and semen, and diluents thereof.

Next, an optical blank value is measured (step S70). Specifically, the control unit <NUM> controls the light source <NUM> so that the light source <NUM> emits the excitation light α. At the same time, the control unit <NUM> controls the detection unit <NUM> so that the detection unit <NUM> detects light having the same wavelength as fluorescence γ. In addition, at this time, the control unit <NUM> moves the excitation light cut filter <NUM> so that the excitation light cut filter <NUM> is present on the optical path. Thus, since the excitation light cut filter <NUM> does not transmit the plasmon scattered light β and the stray light derived from the excitation light α, only light having the same wavelength as the fluorescence γ is detected by the detection unit <NUM>. The obtained detection value is recorded as an optical blank value.

Next, the substance to be detected captured by the capture substance is labeled with a fluorescent substance (secondary reaction, step S80). Specifically, a fluorescent labeling liquid is injected into the flow path <NUM>. The fluorescent labeling liquid is, for example, a buffer solution containing an antibody (secondary antibody) labeled with a fluorescent substance. When the fluorescent labeling liquid is injected into the flow path <NUM>, the fluorescent labeling liquid comes into contact with the substance to be detected, and the substance to be detected is labeled with the fluorescent substance. Thereafter, the inside of the flow path <NUM> is cleaned with a buffer solution or the like and free fluorescent substances and the like are removed.

Next, the metal film <NUM> (film formation surface <NUM>) is irradiated with the excitation light α, and fluorescence γ emitted from the fluorescent substance on the metal film <NUM> (the surface of the metal film <NUM> and the vicinity thereof) is detected (specimen signal measurement, step S90). Specifically, the control unit <NUM> controls the light source <NUM> so that the light source <NUM> emits the excitation light α. At the same time, the control unit <NUM> controls the detection unit <NUM> so that the detection unit <NUM> detects fluorescence γ emitted from on the metal film <NUM> (metal film <NUM> and its vicinity). In addition, also at this time, the control unit <NUM> moves the excitation light cut filter <NUM> so that the excitation light cut filter <NUM> is present on the optical path. Thus, since the excitation light cut filter <NUM> does not transmit the plasmon scattered light β and the stray light derived from the excitation light α, only the fluorescence γ is detected by the detection unit <NUM>.

Next, the control unit <NUM> subtracts the optical blank value detected in step S70 from the obtained detection value (signal value) to calculate a difference signal value corresponding to the amount of the substance to be detected. From this difference signal value, information on the presence or absence or amount of the substance to be detected in the specimen can be obtained.

It should be noted that when the detection device <NUM> includes the shape information acquisition unit <NUM>, a step of acquiring the shape information on the detection chip <NUM> may be performed between step S10 and step S20. In this case, the control unit (detection range control unit) <NUM> determines the detection range of the detection unit <NUM> when the detection unit <NUM> detects the plasmon scattered light β based on the acquired shape information on the detection chip <NUM>.

In addition, when the detection device <NUM> includes the positional information detection unit <NUM>, a step of detecting the positional information on the detection chip may be performed between step S10 and step S20. In this case, the control unit (detection range control unit) <NUM> determines the detection range of the detection unit <NUM> when the detection unit <NUM> detects the plasmon scattered light β based on the acquired positional information on the detection chip <NUM>.

In the detection device and the detection method according to the present embodiment, the position of the light receiving range is different between the light receiving range at the time of detecting the plasmon scattered light β in order to determine the enhancement angle and the light receiving range at the time of detecting the fluorescence in order to detect the presence or the amount of the substance to be detected. Thus, when the plasmon scattered light β is detected in order to determine the enhancement angle, stray light derived from (diffracted light of) the excitation light is less likely to fall within the light receiving range of the detection unit, and the enhancement angle can be accurately determined. In addition, when the presence or the amount of the substance to be detected is detected, the maximum amount of fluorescence can be received, and the detection accuracy of the substance to be detected is improved.

<FIG> is a diagram showing a detection device <NUM> according to the second embodiment of the present invention. Means for changing the light receiving range <NUM> of the detection unit <NUM> is different between the detection device <NUM> according to the first embodiment and the detection device <NUM> according to the second embodiment. Regarding the detection device <NUM>, the same components as those of the detection device <NUM> are denoted by the same reference numerals, and the description thereof will be omitted.

In the present embodiment, the detection device <NUM> further includes a diaphragm <NUM> arranged between the detection chip <NUM> and the detection unit <NUM>. The diaphragm <NUM> adjusts the detection range <NUM> when the detection unit <NUM> detects light emitted from the metal film <NUM> and the region on the metal film <NUM>. Specifically, under the control of the control unit (detection range control unit) <NUM>, the diaphragm <NUM> narrows the light receiving range of the detection unit <NUM> when the detection unit <NUM> detects the plasmon scattered light β in order to determine the enhancement angle, and widens the light receiving range of the detection unit <NUM> when the detection unit <NUM> detects fluorescence in order to detect the presence or amount of the substance to be detected. In the present embodiment, the diaphragm <NUM> is disposed between the second lens <NUM> and the detection unit <NUM>, but the position of the diaphragm <NUM> is not particularly limited as long as the diaphragm <NUM> can exhibit the above function. In particular, the surface of the diaphragm <NUM> and the surface of the metal film <NUM> are desirably optically conjugated surfaces.

<FIG> is a schematic diagram showing the detection range <NUM> of the detection unit <NUM> and the position of the light source <NUM> when the optimum incident angle (enhancement angle) of the excitation light α is determined. In this figure, a state when the detection chip <NUM> and the light source <NUM> are viewed from the detection unit <NUM> side is shown. As shown in <FIG>, in the present embodiment, when the plasmon scattered light β is detected, the light receiving range of the detection unit <NUM> is narrowed so that the end portion (stray light occurrence spot <NUM>) of the first side wall <NUM> on the side of the storage portion does not fall within the detection range <NUM> of the detection unit <NUM>, and the region corresponding to the irradiation spot <NUM> of the excitation light α falls within the detection range <NUM> of the detection unit <NUM>. Therefore, the stray light occurrence spot <NUM> does not fall within the detection range <NUM>, but the irradiation spot <NUM> of the excitation light α falls within the detection range <NUM>. Therefore, the control unit <NUM> can obtain accurate data on the relationship between the incident angle of the excitation light α and the intensity of the plasmon scattered light β while preventing the influence of stray light derived from the excitation light α.

<FIG> is a schematic diagram showing the detection range <NUM> of the detection unit <NUM> and the position of the light source <NUM> when the fluorescence is measured. As shown in <FIG>, by expanding the detection range <NUM> of the detection unit <NUM> so that the end portions of the first side wall <NUM> and the second side wall <NUM> on the side of the storage portion fall within both sides of the detection range <NUM> and the irradiation spot <NUM> of the excitation light α falls within the vicinity of the center of the detection range <NUM>, it is possible to efficiently and reproducibly detect the fluorescence from the reaction field (region where the capturing body is immobilized) on the metal film <NUM> and to improve the detection accuracy (accuracy, accuracy of absolute value) of the substance to be detected. Since the stray light occurring here has the same wavelength as the excitation light α, in the measurement of the optical blank value measured by inserting the excitation light cut filter <NUM> and the measurement of the specimen signal, the stray light <NUM> is not detected by the detection unit <NUM> and does not affect the measurement performance. Furthermore, since the irradiation spot <NUM> of the excitation light α falls within the vicinity of the center of the detection range <NUM>, even if the irradiation spot <NUM> of the excitation light α and the detection range <NUM> varies due to setting variations of the detection chip <NUM> or the like, the fluorescence from the reaction field (region where the capturing body is immobilized) on the metal film <NUM> can be efficiently and reproducibly detected without the irradiation spot <NUM> of the excitation light α falling outside the detection range <NUM>, and the detection accuracy (accuracy, accuracy of absolute value) of the substance to be detected can be improved.

As is clear from <FIG>, the detection range <NUM> when the plasmon scattered light β is detected and the detection range <NUM> of the fluorescence when the fluorescence is detected have different sizes, and the detection range <NUM> of the detection unit <NUM> when the plasmon scattered light β is detected is smaller than the detection range <NUM> when the detection unit <NUM> detects the fluorescence.

As in the first embodiment, in the detection device and the detection method according to the present embodiment, the size of the light receiving range is different between the light receiving range at the time of detecting the plasmon scattered light in order to determine the enhancement angle and the light receiving range at the time of detecting the fluorescence in order to detect the presence or the amount of the substance to be detected. Thus, when the plasmon scattered light is detected in order to determine the enhancement angle, stray light derived from (diffracted light of) the excitation light is less likely to fall within the light receiving range of the detection unit, and the enhancement angle can be accurately determined. In addition, when the presence or the amount of the substance to be detected is detected, the maximum amount of fluorescence can be received, and the detection accuracy of the substance to be detected is improved.

The present application claims priority based on <CIT>.

The detection device and the detection method according to the present invention can more accurately determine the enhancement angle and improve the detection accuracy of the substance to be detected, and thus are useful for, for example, clinical examinations and the like.

Claim 1:
A detection device (<NUM>) configured to detect presence or an amount of a substance to be detected using an enhanced electric field based on surface plasmon resonance, the detection device (<NUM>) comprising:
a chip holder (<NUM>) configured to hold a detection chip (<NUM>) including a prism (<NUM>) having an incident surface (<NUM>) and a film formation surface (<NUM>),
a metal film (<NUM>) disposed on the film formation surface (<NUM>), and a capturing body fixed on the metal film (<NUM>) and configured to capture a substance to be detected;
a light source (<NUM>) configured to irradiate an irradiation spot (<NUM>) on the metal film (<NUM>) of the detection chip (<NUM>) held by the chip holder (<NUM>) with excitation light (α) through the prism (<NUM>);
a detection unit (<NUM>) arranged to face the metal film (<NUM>) of the detection chip (<NUM>) held by the chip holder (<NUM>), the detection unit (<NUM>) configured to detect light (β, γ) emitted from the metal film (<NUM>) and a region on the metal film (<NUM>) when the light source (<NUM>) irradiates the metal film (<NUM>) with the excitation light; and
a detection range control unit (<NUM>) configured to control a detection range (<NUM>) of the detection unit (<NUM>),
wherein the detection range control unit (<NUM>) is configured to
determine a first detection range of the detection unit when the detection unit (<NUM>) detects plasmon scattered light (β) emitted from the metal film (<NUM>); and
determine a second detection range of the detection unit when the detection unit (<NUM>) detects fluorescence (γ) emitted from a fluorescent substance that labels the substance to be detected captured by the capturing body;
characterized in that
the first detection range is different from the second detection range in a position or a size, and is controlled such that, when plasmon scattered light (β) is detected, a stray light occurrence spot (<NUM>) generated by the excitation light (α) does not fall within the first detection range, but the irradiation spot (<NUM>) of excitation light falls within the first detection range