Patent Application
20170122965
The present invention relates to a reaction method using a container including a reaction field to which a capturing body is fixed, and a detection method and a detection device for detecting a detection target substance using the container.
In a clinical test or the like, if a small amount of detection target substance such as a protein or DNA in a specimen can be detected quantitatively with a high sensitivity, a treatment can be performed by recognizing a patient's condition rapidly. Therefore, a method capable of detecting a detection target substance in a specimen quantitatively with a high sensitivity has been desired.
In recent years, in order to analyze a small amount of detection target substance, a micro total analysis system (p-TAS) has been used. In the micro total analysis system, analysis can be performed using small amounts of reagents and samples in a short time advantageously. Examples of the micro total analysis system include a microchannel system using a microchannel chip having a fine channel. A reaction field to which a capturing body for capturing a detection target substance is fixed is disposed in the channel of the microchannel chip. In this reaction field, an antigen-antibody reaction to bond a detection target substance included in a specimen supplied into the channel to the capturing body (hereinafter, also referred to as “primary reaction”) and the like are performed. This microchannel system detects a detection target substance captured by the capturing body.
However, the detection target substance can adhere also to an inner surface of the channel nonspecifically. The detection target substance adhering to the inner surface of the channel outside the reaction field is not detected. Therefore, a reliable detection result cannot be obtained disadvantageously. Therefore, as a means for preventing nonspecific adhesion of the detection target substance to the inner surface of the channel, it has been proposed to subject the inner surface of the channel to a blocking treatment (for example, refer to Patent Literature 1).
In a micro total analysis system described in Patent Literature 1, the blocking treatment is performed as a separate step from the primary reaction. Specifically, the inner surface of the channel is subjected to the blocking treatment. Thereafter, a blocking liquid is removed from the inside of the channel, and the inside of the channel is cleaned. Thereafter, a specimen including a detection target substance is introduced into the channel, and the primary reaction is performed in a reaction field. This prevents nonspecific adhesion of the detection target substance to the inner surface of the channel in the micro total analysis system described in Patent Literature 1.
Patent Literature 1: JP 2006-292472 A
When the inside of the channel is cleaned after the blocking treatment, detection time is increased due to increase in the number of steps. In addition, a blocking agent may be peeled off from the inner surface of the channel by cleaning, and an effect of the blocking treatment may be reduced. That is, there is room for improvement of the micro total analysis system described in Patent Literature 1 from viewpoints of reducing detection time and improving a detection accuracy.
A first object of the present invention is to provide a reaction method capable of bonding a capturing body to a reaction target substance (detection target substance) without reducing an effect of a blocking treatment to an inner surface of a channel (containing unit). A second object of the present invention is to provide a detection method and a detection device capable of detecting a detection target substance in a short time with a high accuracy without reducing an effect of a blocking treatment to an inner surface of a channel (containing unit) by using this reaction method.
In order to solve the above problems, a reaction method according to an embodiment of the present invention is a reaction method for bonding a capturing body to a reaction target substance in a container including a containing unit for containing a liquid and a reaction field which is exposed to the containing unit and to which the capturing body is fixed. The reaction method includes a step of subjecting the inside of the containing unit to a blocking treatment by moving a blocking liquid containing a blocking agent in the containing unit, and a step of causing the capturing body to capture the reaction target substance by supplying a liquid containing the reaction target substance into the containing unit. The step of causing the capturing body to capture the reaction target substance is performed following the step of performing the blocking treatment or simultaneously with the step of performing the blocking treatment.
In order to solve the above problems, a detection method according to an embodiment of the present invention is a detection method for detecting presence or the amount of a detection target substance in a specimen by utilizing surface plasmon resonance using a detection chip including a containing unit for containing a liquid and a metal film with a reaction field which is exposed to the containing unit and to which the capturing body is fixed. The detection method includes a step of subjecting the inside of the containing unit to a blocking treatment by moving a blocking liquid containing a blocking agent in the containing unit, a step of causing the capturing body to capture the detection target substance by supplying the specimen into the containing unit, and a step of detecting a light quantity of fluorescence emitted from the vicinity of a surface of the metal film on a side of the containing unit or a light quantity of light reflected by the metal film by irradiating the metal film with light such that surface plasmon resonance occurs on the metal film while the detection target substance is bonded to the capturing body. The step of causing the capturing body to capture the detection target substance is performed following the step of performing the blocking treatment or simultaneously with the step of performing the blocking treatment.
In order to solve the above problems, a detection device according to an embodiment of the present invention is a detection device for detecting presence or the amount of a detection target substance in a specimen by utilizing surface plasmon resonance using a detection chip including a containing unit for containing a liquid and a metal film with a reaction field which is exposed to the containing unit and to which the capturing body is fixed. The detection device includes a holder for holding the detection chip, a liquid feeding unit for supplying a liquid into the containing unit while the detection chip is held by the holder, a light irradiation unit for irradiating the metal film of the detection chip held by the holder with light such that surface plasmon resonance occurs on the metal film, and a light detection unit for detecting a light quantity of fluorescence emitted from the vicinity of a surface of the metal film on a side of the containing unit or a light quantity of light reflected by the metal film when the light irradiation unit irradiates the metal film with light. The liquid feeding unit performs a step of subjecting the inside of the containing unit to a blocking treatment by supplying a blocking liquid containing a blocking agent into the containing unit and moving the blocking liquid in the containing unit, and a step of causing the capturing body to capture the detection target substance by supplying the specimen into the containing unit. The liquid feeding unit performs the step of causing the capturing body to capture the detection target substance following the step of performing the blocking treatment or simultaneously with the step of performing the blocking treatment.
A reaction method according to the present invention makes it possible to bond a capturing body to a reaction target substance (detection target substance) efficiently in a short time without reducing an effect of a blocking treatment to an inner surface of a channel (containing unit). Therefore, a detection method and a detection device according to the present invention make it possible to detect presence or the amount of a detection target substance in a short time with a high accuracy.
Hereinafter, an embodiment of the present invention will be described in detail with reference to the drawings. Here, as a typical example of a detection device according to the present invention, an analyzing device (SPFS device) utilizing a surface plasmon-field enhanced fluorescence spectroscopy (hereinafter, abbreviated as “SPFS”) for detecting the amount of a detection target substance (reaction target substance) in a specimen (liquid containing a reaction target substance) will be described.
(Configurations of SPFS Device and Detection Chip)
The detection chip 10 includes a prism 20 having an incident surface 21, a film forming surface 22, and a light emitting surface 23, a metal film 30 formed on the film forming surface 22, and a channel lid 40 disposed on the metal film 30. As described below, the detection chip 10 (container) includes a channel 41 (containing unit) and a reaction field exposed to the channel 41. In the reaction field, capture of a detection target substance (reaction target substance) by a capturing body, labeling of the detection target substance by a fluorescence substance, or the like is performed. The detection chip 10 is usually replaced whenever analysis is performed. The detection chip 10 is a structure preferably having each length of several mm to several cm, but may be a smaller or larger structure not included in a category of the “chip”.
The prism 20 is formed of a dielectric transparent with respect to excitation light α. The prism 20 includes the incident surface 21, the film forming surface 22, and the light emitting surface 23. The incident surface 21 allows the excitation light α from the excitation light irradiation unit 110 to enter the prism 20. The metal film 30 is disposed on the film forming surface 22. The excitation light α which has entered the prism 20 is reflected on a back surface of the metal film 30 to become reflected light β. More specifically, the excitation light α is reflected on an interface (film forming surface 22) between the prism 20 and the metal film 30 to become the reflected light β. The light emitting surface 23 emits the reflected light β outside 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 columnar body having a trapezoidal bottom surface. A surface corresponding to one of bases of the trapezoid is the film forming surface 22, a surface corresponding to one of legs is the incident surface 21, and a surface corresponding to the other leg is the light emitting surface 23. The trapezoid as the bottom surface is preferably an isosceles trapezoid. This makes the incident surface 21 and the light emitting surface 23 symmetric to each other, and makes it difficult for an S-wave component of the excitation light α to stay in the prism 20.
The incident surface 21 is formed such that the excitation light α does not return to the excitation light irradiation unit 110. When a light source of the excitation light α is a laser diode (hereinafter, also referred to as “LD”), return of the excitation light α to the LD disturbs an excitation state of the LD, and causes a wavelength of the excitation light α or an output thereof to fluctuate. Therefore, an angle of the incident surface 21 is set such that the excitation light α is not vertically incident on the incident surface 21 in a scanning range around an ideal resonance angle or enhancement angle. Here, the “resonance angle” means an incident angle when a light quantity of the reflected light β emitted from the light emitting surface 23 is minimized in scanning for an incident angle of the excitation light α with respect to the metal film 30. The “enhancement angle” means an incident angle when a light quantity of scattered light having the same wavelength as the excitation light α emitted above the detection chip 10 (hereinafter, referred to as “plasmon scattered light”) δ is maximized in scanning for an incident angle of the excitation light α with respect to the metal film 30. In the present embodiment, each of an angle between the incident surface 21 and the film forming surface 22 and an angle between the film forming surface 22 and the light emitting surface 23 is about 80°.
Design of the detection chip 10 generally determines the resonance angle (and the enhancement angle extremely close thereto). Examples of a design element include a refractive index of the prism 20, a refractive index of the metal film 30, a film thickness of the metal film 30, an extinction coefficient of the metal film 30, and a wavelength of the excitation light α. The resonance angle and the enhanced angle are shifted by a detection target substance captured on the metal film 30, but the amount thereof is less than a few degrees.
The prism 20 has not a little birefringence characteristic. Examples of a material of the prism 20 include resin and glass. A 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 forming surface 22 of the prism 20. This can cause surface plasmon resonance (hereinafter, abbreviated as “SPR”) between a photon of the excitation light α incident on the film forming surface 22 under total reflection conditions and a free electron in the metal film 30, and can cause localized field light (generally, also referred to as “evanescent light” or “near-field light”) on a surface of the metal film 30. In the present embodiment, the metal film 30 is formed on the entire surface of the film forming surface 22.
A material of the metal film 30 is not particularly limited as long as being a metal capable of causing surface plasmon resonance. Examples of a material of the metal film 30 include gold, silver, copper, aluminum, and an alloy thereof. In the present embodiment, the metal film 30 is a gold thin film. A method for forming the metal film 30 is not particularly limited. Examples of a 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 from 30 to 70 nm.
A capturing body for capturing a detection target substance is fixed to a surface of the metal film 30 not facing the prism 20 (surface of the metal film 30), although not particularly illustrated. By fixing the capturing body, a detection target substance can be detected selectively. In the present embodiment, the capturing body is uniformly fixed in a predetermined region (reaction field) on the metal film 30. The kind of the capturing body is not particularly limited as long as the capturing body can capture a detection target substance. In the present embodiment, the capturing body is an antibody specifically bonded to a detection target substance or a fragment thereof. The capturing body is usually stored with a protective layer when the capturing body is not in use from a viewpoint of preventing denaturation caused by drying.
The channel lid 40 is disposed on the metal film 30. When the metal film 30 is formed only on a part of the film forming surface 22 of the prism 20, the channel lid 40 may be disposed on the film forming surface 22. In the present embodiment, the channel lid 40 is disposed on the metal film 30. A channel groove is formed on a back surface of the channel lid 40. The channel lid 40 forms a containing unit for containing a liquid together with the metal film 30 (and the prism 20). In the present embodiment, the containing unit is the channel 41 in which a liquid flows. A moving rate of a liquid in the channel 41 can be adjusted by a size of a cross-sectional area of the channel 41. A cross-sectional area of the channel 41 in a cross section perpendicular to a flowing direction of a liquid on a reaction field described below is not particularly limited, but for example, is preferably 0.01 mm2 or more and 100 mm2 or less. The cross-sectional area is more preferably 0.01 mm2 or more and 10 mm2 or less, and still more preferably 0.01 mm2 or more and 1 mm2 or less from a viewpoint of moving a liquid at a higher speed in the channel 41. The reaction field is disposed so as to be exposed to the channel 41 (containing unit). Examples of the liquid include a specimen containing a detection target substance (for example, blood, serum, plasma, urine, a nasal fluid, saliva, or semen), a blocking liquid containing a blocking agent, a labeling liquid containing a capturing body labeled with a fluorescent substance, a standard liquid, and a cleaning liquid. The capturing body fixed to the metal film 30 is exposed to the inside of the channel 41. Both ends of the channel 41 are connected to an inlet and an outlet (not illustrated) formed on an upper surface of the channel lid 40. When a liquid is supplied into the channel 41, the liquid comes into contact with the capturing body.
The channel lid 40 is preferably formed of a material transparent with respect to fluorescence γ and the plasmon scattered light δ emitted from an upper surface of the metal film 30. Examples of a material of the channel lid 40 include resin. When a portion to extract the fluorescent γ and the plasmon scattered light S to an outside is transparent with respect to the fluorescence γ and the plasmon scattered light δ, the other portions of the channel lid 40 may be formed of an opaque material. For example, the channel lid 40 is joined to the metal film 30 or the prism 20 by adhesion using a double-sided tape or an adhesive, laser welding, ultrasonic welding, or crimping using a clamp member.
The detection chip 10 may include a frame body having a through hole and a top plate stacked on the frame body in place of the channel lid 40. In this case, the channel 41 (containing unit) is formed by stacking the frame body and the top plate in this order on the prism 20 having the metal film 30 formed. A surface of the metal film 30 becomes a bottom surface of the channel 41. An inner surface of the through hole of the frame body becomes a side surface of the channel 41. One surface (inner surface) of the top plate becomes a top surface of the channel 41.
As illustrated in
Next, components of the SPFS device 100 will be described. As described above, the SPFS device 100 includes the excitation light irradiation unit 110, the reflected light detecting unit 120, the fluorescence detecting unit 130, the liquid feeding unit 140, the conveying unit 150, and the control unit 160.
The excitation light irradiation unit 110 irradiates the detection chip 10 held by the chip holder 152 with the excitation light α. When the fluorescence γ or the plasmon scattered light δ is measured, the excitation light irradiation unit 110 emits only P-wave with respect to the metal film 30 toward the incident surface 21 such that an incident angle thereof with respect to the metal film 30 becomes an angle to cause SPR. Here, the “excitation light” is light to excite a fluorescence substance directly or indirectly. For example, the excitation light α is light to generate localized field light to excite a fluorescence substance on a surface of the metal film 30 when the metal film 30 is irradiated with the excitation light α 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 adjusting 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 quantity such that an irradiation spot on a back surface of the metal film 30 has a substantially circular shape. For example, the light source unit 111 includes a light source of the excitation light α, a beam shaping optical system, an APC mechanism, and a temperature adjusting mechanism (none of them is illustrated).
The kind of a light source is not particularly limited, but is a laser diode (LD), for example. Examples of a light source further include a light emitting diode, a mercury lamp, and other laser light sources. When light emitted from a 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 light emitted from a 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. When light emitted from a 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.
For example, the beam shaping optical system includes a collimator, a bandpass filter, a linearly polarizing filter, a half-wave plate, a slit, and a zooming means. The beam shaping optical system may include all of these means, or may include a part thereof. The collimator collimates the excitation light α emitted from a light source. The bandpass filter changes the excitation light α emitted from a light source into narrow band light having only a center wavelength. This is because the excitation light α from a light source has a small wavelength distribution width. The linearly polarizing filter changes the excitation light α emitted from a light source into completely linearly polarized light. The half-wave plate adjusts a polarization direction of the excitation light α such that a P-wave component is incident on the metal film 30. The slit and zooming means adjust a beam diameter of the excitation light α and a contour shape thereof such that the irradiation spot on the back surface of the metal film 30 has a circular shape having a predetermined size.
The APC mechanism controls a light source such that an output of the light source is constant. More specifically, the APC mechanism detects a light quantity of light branching from the excitation light α with a photodiode (not illustrated) or the like. The APC mechanism controls an output of a light source constantly by controlling input energy in a regression circuit.
Examples of the temperature adjusting mechanism include a heater and a Peltier element. A wavelength and energy of light emitted from a light source may vary according to a temperature. Therefore, by keeping the temperature of a light source constantly by the temperature adjusting mechanism, the wavelength and energy of light emitted from a light source is controlled constantly.
The angle adjusting mechanism 112 adjusts an incident angle of the excitation light α with respect to the metal film 30 (an interface (film forming surface 22) between the prism 20 and the metal film 30). The angle adjusting mechanism 112 rotates an optical axis of the excitation light α and the chip holder 152 relatively in order to emit the excitation light α toward a predetermined position of the metal film 30 through the prism. 20 at a predetermined incident angle.
For example, the angle adjusting mechanism 112 rotates the light source unit 111 around an axis (axis perpendicular to paper face of
As described above, among incident angles of the excitation light α with respect to the metal film 30, an angle at which a light amount of the plasmon scattered light δ is maximum is an enhanced angle. By setting an incident angle of the excitation light α to the enhanced angle or an angle in the vicinity thereof, the fluorescence γ having a high intensity can be measured. Basic incident conditions of the excitation light α are determined by a material of the prism 20 of the detection chip 10 and a shape thereof, a film thickness of the metal film 30, a refractive index of a liquid in the channel 41, and the like. However, an optimum incident condition is varied slightly according to the kind of the fluorescence substance in the channel 41, the amount thereof, a shape error of the prism 20, and the like. Therefore, an optimum enhancement angle is preferably determined for each measurement.
The light source control unit 113 controls various devices included in the light source unit 111 to control emission of the excitation light α from the light source unit 111. For example, the light source control unit 113 is constituted by a known computer or microcomputer including a computing device, a control device, a storage device, an input device, and an output device.
The reflected light detecting unit 120 measures a light quantity of the reflected light β generated by irradiation to the detection chip 10 with the excitation light α for measurement of a resonance angle or the like. The reflected light detecting unit 120 includes a light receiving sensor 121, an angle adjustment mechanism 122, and a sensor control unit 123.
The light receiving sensor 121 is disposed at a position on which the reflected light β is incident, and measures a light quantity of the reflected light β. The kind of the light receiving sensor 121 is not particularly limited. For example, the light receiving sensor 121 is a photodiode (PD).
The angle adjustment mechanism 122 adjusts a position (angle) of the light receiving sensor 121 according to an incident angle of the excitation light α with respect to the metal film 30. The angle adjustment mechanism 122 rotates the light receiving sensor 121 and the chip holder 152 relatively such that the reflected light β is incident on the light receiving sensor 121.
The sensor control unit 123 controls detection of an output value of the light receiving sensor 121, management of a sensitivity of the light receiving sensor 121 with the detected output value, change of the sensitivity of the light receiving sensor 121 for obtaining a proper output value, and the like. For example, the sensor control unit 123 is constituted by a known computer or microcomputer including a computing device, a control device, a storage device, an input device, and an output device.
The fluorescence detecting unit 130 detects the fluorescence γ generated by irradiation to the metal film 30 with the excitation light α. In addition, the fluorescence detecting unit 130 detects the plasmon scattered light δ generated by irradiation to the metal film 30 with the excitation light α, if necessary. The fluorescence detecting unit 130 includes a light receiving unit 131, a position switching mechanism 132, and a sensor control unit 133.
The light receiving unit 131 is disposed in a normal direction of the metal film 30 of the detection chip 10. The light receiving unit 131 includes a first lens 134, an optical filter 135, a second lens 136, and a light receiving sensor 137.
For example, the first lens 134 is a condenser lens, and condenses light emitted from an upper surface of the metal film 30. For example, the second lens 136 is an imaging lens, and images the light condensed by the first lens 134 on a light receiving surface of the light receiving sensor 137. An optical path between the two lenses is substantially parallel. The optical filter 135 is disposed between the two lenses.
The optical filter 135 leads only a fluorescence component to the light receiving sensor 137, and removes an excitation light component (plasmon scattered light δ) in order to detect the fluorescence γ at a high S/N ratio. Examples of the optical filter 135 include an excitation light reflection filter, a short wavelength cut filter, and a bandpass filter. For example, the optical filter 135 is a filter including a multilayer film for reflecting a predetermined light component (light having a predetermined wavelength component) or a colored glass filter for absorbing a predetermined light component.
The light receiving sensor 137 detects the fluorescence γ and the plasmon scattered light δ. The light receiving sensor 137 has such a high sensitivity to be able to detect the weak fluorescence γ from a small amount of detection target substance. For example, the light receiving sensor 137 is a photomultiplier tube (PMT) or an avalanche photodiode (APD).
The position switching mechanism 132 switches a position of the optical filter 135 between a position on an optical path and a position outside the optical path in the light receiving unit 131. Specifically, the optical filter 135 is disposed on the optical path of the light receiving unit 131 when the light receiving sensor 137 detects the fluorescence γ, and the optical filter 135 is disposed outside the optical path in the light receiving unit 131 when the light receiving sensor 137 detects the plasmon scattered light δ.
The sensor control unit 133 controls detection of an output value of the light receiving sensor 137, management of a sensitivity of the light receiving sensor 137 with the detected output value, change of the sensitivity of the light receiving sensor 137 for obtaining a proper output value, and the like. For example, the sensor control unit 133 is constituted by a known computer or microcomputer including a computing device, a control device, a storage device, an input device, and an output device.
The liquid feeding unit 140 supplies a specimen, a blocking liquid containing a blocking agent, a labeling liquid, a standard liquid, a cleaning liquid, and the like into the channel 41 of the detection chip 10 held by the chip holder 152. The liquid feeding unit 140 includes a liquid chip 141, a syringe pump 142, and a liquid feeding pump drive mechanism 143.
The liquid chip 141 is a container for containing a liquid such as a specimen, a blocking liquid, a labeling liquid, a standard liquid, or a cleaning liquid. As the liquid chip 141, usually, a plurality of containers is disposed according to the kind of liquid, or a chip obtained by integrating a plurality of containers is disposed.
The syringe pump 142 is constituted by a syringe 144 and a plunger 145 capable of being reciprocated in the syringe 144. By the reciprocating motion of the plunger 145, suction of a liquid and ejection thereof are performed quantitatively. When the syringe 144 is replaceable, cleaning of the syringe 144 is not required. Therefore, the replaceable syringe 144 is preferable from a viewpoint of preventing contamination of impurities or the like. When the syringe 144 is not replaceable, by further adding a configuration for cleaning the inside of the syringe 144, the syringe 144 can be used without being replaced.
The liquid feeding pump drive mechanism 143 includes a device for driving the plunger 145 and a device for moving the syringe pump 142. The device for driving the syringe pump 142 is a device for reciprocating the plunger 145, and for example, includes a stepping motor. A driving device including a stepping motor can manage a liquid feeding amount of the syringe pump 142 or a liquid feeding rate thereof, and is therefore preferable from a viewpoint of managing a residual liquid amount of the detection chip 10. For example, the device for moving the syringe pump 142 freely moves the syringe pump 142 in two directions of an axial direction of the syringe 144 (for example, vertical direction) and a direction crossing the axial direction (for example, horizontal direction). For example, the device for moving the syringe pump 142 is constituted by a robot arm, a 2-axis stage, or a vertically movable turntable.
The liquid feeding unit 140 sucks various liquids from the liquid chip 141, and supplies the liquids into the channel 41 of the detection chip 10. At this time, by moving the plunger 145, a liquid is reciprocated in the channel 41 of the detection chip 10, and the liquid in the channel 41 is stirred. This can make the concentration distribution of the liquid uniform, or can accelerate a reaction in the channel 41 (for example, an antigen-antibody reaction), for example. An inlet of the detection chip 10 is preferably protected by a multilayer film, and the detection chip 10 and the syringe 144 are preferably configured so as to be able to seal the inlet when the syringe 144 penetrates through the multilayer film from a viewpoint of performing such an operation.
The liquid in the channel 41 is sucked by the syringe pump 142 again, and is discharged to the liquid chip 141 or the like. By repeating these operations, a reaction of various liquids, cleaning, and the like can be performed, and a detection target substance labeled with a fluorescent substance can be disposed in a reaction field in the channel 41.
The conveying unit 150 conveys the detection chip 10 to a measurement position or a liquid feeding position, and fixes the detection chip 10. Here, the “measurement position” is a position at which the reflected light detecting unit 120 or the fluorescence detecting unit 130 detects the reflected light β, the fluorescence γ, or the plasmon scattered light δ generated by irradiation to the detection chip 10 with the excitation light α by the excitation light irradiation unit 110. The “liquid feeding position” is a position at which the liquid feeding unit 140 supplies a liquid into the channel 41 of the detection chip 10 or removes the liquid in the channel 41 of the detection chip 10. The conveying unit 150 includes a conveying stage 151 and the chip holder 152. The chip holder 152 is fixed to the conveying stage 151, and holds the detection chip 10 detachably. The chip holder 152 has a shape capable of holding the detection chip 10 and not blocking an optical path of the excitation light α, the reflected light β, the fluorescence γ, or the plasmon scattered light δ. For example, the chip holder 152 includes an opening through which the excitation light α, the reflected light β, the fluorescence γ, and the plasmon scattered light δ pass. The conveying stage 151 moves the chip holder 152 in one direction and in the opposite direction thereto. The conveying stage 151 also has a shape not blocking an optical path of the excitation light α, the reflected light β, the fluorescence γ, or the plasmon scattered light δ. For example, the conveying stage 151 is driven by a stepping motor.
The control unit 160 controls the angle adjusting mechanism 112, the light source control unit 113, the angle adjustment mechanism 122, the sensor control unit 123, the position switching mechanism 132, the sensor control unit 133, the liquid feeding pump drive mechanism 143, and the conveying stage 151. For example, the control unit 160 is constituted by a known computer or microcomputer including a computing device, a control device, a storage device, an input device, and an output device.
(Detection Operation of SPFS Device)
Next, a detection operation of the SPFS device 100 according to the present embodiment (detection method according to an embodiment of the present invention) will be described. For example, the SPFS device 100 can operate according to either of the following two detection operations. Hereinafter, each of the detection operations will be described with reference to the drawings.
First, detection is prepared (step S10). Specifically, the detection chip 10 is installed in the chip holder 152 of the SPFS device 100.
Subsequently, a protective layer of a capturing body is removed, and an inner surface of the channel 41 is subjected to a blocking treatment (step S20). Specifically, the control unit 160 operates the conveying stage 151, and moves the detection chip 10 to the liquid feeding position. Thereafter, the control unit 160 operates the liquid feeding pump drive mechanism 143, and supplies a blocking liquid of the liquid chip 141 into the channel 41 of the detection chip 10. In the channel 41, the blocking liquid removes a protective layer of a capturing body, moves in the channel 41, and subjects an inner surface of the channel 41 to a blocking treatment. At this time, in order to make the concentration distribution of a blocking agent in the channel 41 more uniform and to accelerate the blocking treatment, the blocking liquid is moved in the channel 41. A method for moving the blocking liquid is not particularly limited. Examples of a method for moving the blocking liquid include repeating suction and ejection alternately with a syringe and irradiation with an ultrasonic wave, a microwave, or the like. A flow rate (moving rate) of the blocking liquid is not particularly limited. When the blocking treatment (step S20) and a primary reaction (step S30) are performed separately, the flow rate of the liquid (blocking liquid) in the channel 41 is preferably 1600 μL/min or more and 3300 μL/min or less. This can make the concentration distribution of a blocking agent in the channel 41 uniform, can accelerate the blocking treatment, and can reduce time required for the blocking treatment. In addition, the concentration of the blocking agent in the blocking liquid is preferably about from 5 to 8% by mass. The blocking agent having a higher concentration is more preferable from a viewpoint of accelerating the blocking treatment. However, the blocking agent having a too high concentration makes the viscosity of the blocking liquid high, and inhibits movement of the blocking liquid. On the other hand, the blocking agent having a too low concentration makes it impossible to perform the blocking treatment properly. Thereafter, the control unit 160 operates the liquid feeding pump drive mechanism 143, and removes the blocking liquid in the channel 41.
The kind of the blocking agent contained in the blocking liquid is not particularly limited as long as the blocking agent can suppress nonspecific bonding of a detection target substance to an inner surface of the channel 41. Examples of the kind of the blocking agent include a high molecular compound such as albumin, casein, skim milk, gelatin, or polyethylene glycol; and a low molecular compound such as a phospholipid, ethylenediamine, or acetonitrile. These blocking agents may be used singly or in combination of two or more kinds thereof.
Following the blocking treatment (step S20), a capturing body is caused to capture a detection target substance (primary reaction; step S30). Here, “following the blocking treatment” means that no other step (for example, a cleaning step or a storage step for a long time) is performed after the blocking treatment. Specifically, the control unit 160 operates the liquid feeding pump drive mechanism 143, and supplies a specimen in the liquid chip 141 into the channel 41. In the channel 41, the detection target substance is captured by the capturing body on the metal film 30 by an antigen-antibody reaction. Thereafter, the control unit 160 operates the liquid feeding pump drive mechanism 143, removes the specimen in the channel 41, and cleans the inside of the channel 41 at least once using a cleaning liquid (for example, a phosphate buffer).
Subsequently, the control unit 160 measures an optical blank value (step S40). Here, the “optical blank value” means a light quantity of background light emitted above the detection chip 10 in measurement of the fluorescence γ described below (step S60). Specifically, the control unit 160 operates the conveying stage 151, and moves the detection chip 10 to a detection position. Thereafter, the control unit 160 operates the excitation light irradiation unit 110 and the fluorescence detecting unit 130, irradiates a back surface of the metal film 30 corresponding to a region to which the capturing body is fixed with the excitation light α through the incident surface 21 so as to cause surface plasmon resonance, and records an output value (optical blank value) of the light receiving sensor 137. At this time, the control unit 160 operates the angle adjusting mechanism 112, and sets an incident angle of the excitation light α to an enhancement angle. In addition, the control unit 160 controls the position switching mechanism 132, and disposes the optical filter 135 in an optical path of the light receiving unit 131. A measured optical blank value is recorded in the control unit 160.
Subsequently, the control unit 160 labels the detection target substance captured on the metal film 30 with a fluorescence substance (secondary reaction; step S50). Specifically, the control unit 160 operates the conveying stage 151, and moves the detection chip 10 to the liquid feeding position. Thereafter, the control unit 160 operates the liquid feeding pump drive mechanism 143, and supplies a liquid labeled with the fluorescence substance and containing the capturing body (labeling liquid) into the channel 41 of the detection chip 10. In the channel 41, the detection target substance captured on the metal film 30 by an antigen-antibody reaction is labeled with the fluorescence substance. Thereafter, the control unit 160 operates the liquid feeding pump drive mechanism 143, removes the labeling liquid in the channel 41, and cleans the inside of the channel 41 using a cleaning liquid.
Subsequently, the control unit 160 measures ae light quantity of the fluorescence γ emitted from the vicinity of a surface of the metal film 30 on a side of the channel 41 (containing unit) to obtain a signal value indicating presence of the detection target substance in the specimen or the amount thereof (step S60). Specifically, the control unit 160 operates the conveying stage 151, and moves the detection chip 10 to a detection position. Thereafter, the control unit 160 operates the excitation light irradiation unit 110 and the fluorescence detecting unit 130, irradiates a back surface of the metal film 30 corresponding to a region to which the capturing body is fixed with the excitation light α through the incident surface 21 from a side of the prism 20 while the detection target substance is bonded to the capturing body and the specimen is not present on the metal film 30, and records an output value of the light receiving sensor 137. At this time, the control unit 160 operates the angle adjusting mechanism 112, and sets an incident angle of the excitation light α to an enhancement angle. In addition, the control unit 160 controls the position switching mechanism 132, and disposes the optical filter 135 in an optical path of the light receiving unit 131. The control unit 160 calculates a signal value indicating presence of the detection target substance in the specimen or the amount thereof by subtracting the optical blank value from a detection value. The signal value is converted into the amount of the detection target substance, the concentration thereof, or the like, if necessary. By the above procedures, presence of the detection target substance in the specimen or the amount thereof can be detected.
Next, a second detection operation of the SPFS device 100 will be described.
First, detection is prepared by installing the detection chip 10 in the chip holder 152 of the SPFS device 100 in a similar manner to the above operation procedure (step S10′).
Subsequently, a protective layer of a capturing body is removed, and the inside of the channel 41 is subjected to a blocking treatment and a primary reaction simultaneously (step S20′). Specifically, the control unit 160 operates the conveying stage 151, and moves the detection chip 10 to the liquid feeding position. Thereafter, the control unit 160 operates the liquid feeding pump drive mechanism 143, and supplies a mixed liquid of a blocking liquid of the liquid chip 141 and a specimen containing a detection target substance into the channel 41 of the detection chip 10. In the channel 41, the mixed liquid of the blocking liquid and the specimen moves in the channel 41, the protective layer of the capturing body is removed, and an inner surface of the channel 41 is subjected to a blocking treatment. In addition, the detection target substance is captured by the capturing body on the metal film 30 by an antigen-antibody reaction. Also in the present operation procedure, the liquid (mixed liquid of the blocking liquid and the specimen) in the channel 41 is moved in a similar manner to the first operation procedure in order to accelerate the blocking treatment. A flow rate of the liquid (mixed liquid of the blocking liquid and the specimen) in the channel 41 is not particularly limited, but is similar to that in the first operation procedure. On the other hand, for example, the concentration of the blocking agent in the liquid (mixed liquid of the blocking liquid and the specimen) in the channel 41 is preferably about from 2 to 3% by mass. When the blocking treatment and the primary reaction are performed simultaneously, the liquid moving in the channel 41 contains various proteins such as the detection target substance in addition to the blocking agent. Therefore, the viscosity of the liquid is higher than that in a case where the blocking treatment and the primary reaction are performed separately (the concentration of the blocking agent is from 5 to 8% by mass), and it may be difficult to move the liquid properly. Therefore, the concentration of the blocking agent contained in the liquid in the channel 41 is preferably reduced. Thereafter, the control unit 160 operates the liquid feeding pump drive mechanism 143, removes the liquid in the channel 41, and cleans the inside of the channel 41 at least once using a cleaning liquid (for example, a phosphate buffer).
Subsequently, an optical blank value is measured in a similar manner to the above operation procedure (step S30′), the secondary reaction is performed (step S40′), and then a signal value indicating presence of the detection target substance in the specimen or the amount thereof is obtained (step S50′). The control unit 160 calculates a signal value indicating presence of the detection target substance in the specimen or the amount thereof by subtracting the optical blank value from a detection value, and converts the signal value into the amount of the detection target substance, the concentration thereof, or the like, if necessary. By the above procedures, presence of the detection target substance in the specimen or the amount thereof can be detected.
(Effect)
In the SPFS device 100 according to the present embodiment, by moving the blocking liquid in the channel 41, the concentration of the blocking agent can be uniform. As a result, the blocking treatment is accelerated, and the blocking treatment can be performed in a short time. In addition, in the SPFS device 100 according to the present embodiment, the blocking liquid is moved, and therefore the blocking treatment can be performed properly in a short time regardless of the viscosity of the blocking liquid.
Conventionally, adjustment of the pH of the blocking liquid or the like has been performed from a viewpoint of accelerating the blocking treatment. However, when the pH of the blocking liquid is different from that of the specimen, the detection target substance in the specimen and the blocking agent may be denatured. Therefore, it is necessary to clean the inside of the channel 41 after the blocking treatment in order to remove the blocking liquid from the inside of the channel 41. However, as described above, an effect of the blocking treatment may be reduced by falling off of the blocking agent due to the cleaning liquid. Therefore, it may be impossible to detect the detection target substance with a high accuracy by a conventional detection method. In addition, by performing another step such as a cleaning step, the number of steps before detection of the detection target substance increases, and time required for detection is longer.
In contrast, in the present embodiment, by moving the blocking liquid, the blocking treatment is accelerated. Therefore, it is not necessary to adjust the pH of the blocking liquid. In addition, in the present embodiment, it is not necessary to perform another step such as cleaning the inside of the channel 41 between the blocking treatment and the primary reaction, and therefore there is no risk of falling off of the blocking agent in the other step. Therefore, the SPFS device 100 according to the present embodiment can detect presence of the detection target substance or the amount thereof in a short time with a high accuracy without reducing the effect of the blocking treatment.
In the above embodiment, the detection method and the detection device for bonding excited light to surface plasmon by totally reflecting the excitation light on an interface between the prism 20 and the metal film 30 have been described. However, in the detection method and the detection device according to the present invention, the excitation light may be bonded to the surface plasmon using a diffraction grating. In this case, in the SPFS device 100, it is not always necessary to dispose the metal film 30 on the prism 20, but the SPFS device 100 may include a diffraction grating in place thereof.
In the above embodiment, the detection method and the detection device using SPFS have been described. However, the detection method and the detection device according to the present invention are not limited to the detection method and the detection device using SPFS. For example, the detection method and the detection device according to the present invention may use an SPR method. In this case, the SPFS device 100 detects presence of the detection target substance or the amount thereof by measuring a light quantity of the reflected light β as a signal value without measuring a light quantity of the fluorescence γ. Therefore, measurement of the optical blank value (step S40, step S30′) and the secondary reaction (step S50, step S40′) are not required.
Hereinafter, the present invention will be described in detail with reference to Examples, but is not limited by these Examples.
In Example 1, an effect of moving a blocking liquid in a blocking treatment was examined.
1. Case where Primary Reaction is Performed Following Blocking Treatment
(1) Preparation of Detection Chip
A detection chip in which a reaction field on a metal film (region to which an anti-troponin antibody was fixed as a capturing body) was protected by a protective layer was prepared. A cross-sectional area of a channel on the reaction field was 0.46 mm2. The prepared detection chip was installed in a chip holder of an SPFS device.
(2) Preparation of Blocking Liquid
A blocking agent (bovine serum albumin (BSA)) was added to an acetate buffer such that a final concentration became 5% by mass to prepare a blocking liquid.
(3) Blocking Treatment
By performing reciprocating liquid feeding of a blocking liquid (100 μL) using a syringe pump in a liquid feeding unit for 35 seconds at a predetermined flow rate (1 to 3300 μL/min) in a channel of the detection chip, a blocking treatment was performed. Subsequently, the blocking liquid in the channel was removed using the syringe pump in the liquid feeding unit.
(4) Primary Reaction
Following the blocking treatment, by performing reciprocating liquid feeding of a diluted liquid of plasma (100 μL) obtained by mixing a phosphate buffer and plasma (specimen) containing troponin (detection target substance) such that a volume ratio thereof was 2:1 using the syringe pump in the liquid feeding unit for four minutes at a flow rate of 3300 μL/min in the channel, a primary reaction was performed.
(5) Confirmation of Effect of Blocking Treatment
After the primary reaction, the metal film was irradiated with light having a wavelength of 635 nm from a side of the prism. Subsequently, reflected light from the metal film was detected, and a reflectance was measured. A blocking ratio was calculated on the basis of a calibration curve between a reflectance and a blocking ratio created in advance and a measured reflectance. When the calibration curve was created, time for the blocking treatment was increased, or the concentration of the blocking agent was increased, and a blocking ratio when no change in the reflectance was observed was assumed to be 100%.
2. Case where Blocking Treatment and Primary Reaction are Performed Simultaneously
(1) Preparation of Detection Chip
The prepared detection chip was installed in the chip holder of the SPFS device in a similar manner to the above 1.(1).
(2) Preparation of Mixed Liquid of Blocking Liquid and Specimen
A blocking agent (BSA) was added to an acetate buffer to prepare a blocking liquid. The prepared blocking liquid and plasma were mixed such that a volume ratio thereof was 2:1 to prepare a mixed liquid of the blocking liquid and plasma. The final concentration of the blocking agent in the mixed liquid was 2% by mass.
(3) Blocking Treatment and Primary Reaction
By performing reciprocating liquid feeding of the mixed liquid of the blocking liquid and plasma (100 μL) using the syringe pump in the liquid feeding unit for one minute 28 seconds at a predetermined flow rate (1 to 3300 μL/min) in the channel, the blocking treatment and the primary reaction were performed. Subsequently, the mixed liquid in the channel was removed using the syringe pump in the liquid feeding unit.
(4) Confirmation of Effect of Blocking Treatment
An inner surface of the channel was subjected to the blocking treatment, and then a blocking ratio was calculated by a similar procedure to the above 1.(5).
3. Result
As illustrated in
In Example 2, a relationship between presence or absence of movement of a blocking liquid and a concentration of a blocking agent, and time required for the blocking treatment when the primary reaction was performed following the blocking treatment was examined.
(1) Preparation of Blocking Liquid
A blocking agent (BSA) was added to an acetate buffer such that a final concentration became 0% by mass, 1% by mass, 2% by mass, 5% by mass, or 8% by mass to prepare five kinds of blocking liquids.
(2) Preparation of Detection Chip
A plurality of detection chips in each of which a reaction field on a metal film (region to which an anti-troponin antibody was fixed as a capturing body) was protected by a protective layer was manufactured in a similar manner to Example 1. A cross-sectional area of a channel on the reaction field was 0.46 mm2. Some of the detection chips were subjected to the blocking treatment by supplying a blocking liquid (100 μL) containing a blocking agent at 1% by mass into the channel immediately after manufacturing and allowing the detection chips to stand for a predetermined time. Thereafter, the blocking liquid was removed from the inside of the channel, and was stored at room temperature and atmospheric pressure for one day or more.
(3) Blocking Treatment
Each of the detection chips which had not been subjected to the blocking treatment immediately after manufacturing was installed in the chip holder of the SPFS device. By performing reciprocating liquid feeding (stirring) of a blocking liquid (100 μL) containing a blocking agent at 0% by mass, 2% by mass, 5% by mass, or 8% by mass using the syringe pump in the liquid feeding unit for a predetermined time at a predetermined flow rate in the channel, the blocking treatment was performed. Subsequently, the blocking liquid in the channel was removed using the syringe pump in the liquid feeding unit.
(4) Primary Reaction
Following the blocking treatment, by performing reciprocating liquid feeding of a diluted liquid of plasma (100 μL) obtained by mixing a phosphate buffer and plasma (specimen) containing troponin (detection target substance) such that a volume ratio thereof was 2:1 for four minutes at a flow rate of 3300 μL/min in the channel, the primary reaction was performed. In addition, each of the detection chips which had been subjected to the blocking treatment immediately after manufacturing was installed in the chip holder of the SPFS device, and then was subjected to the primary reaction by a similar procedure.
(5) Determination of Blocking Time
After the primary reaction, a blocking ratio of an inner surface of the channel was calculated by a similar procedure to Example 1. For each condition of each blocking treatment, a lower limit value of treatment time when the blocking ratio was 90% or more (hereinafter, also referred to as “blocking time”) was determined.
Table 1 indicates a relationship between a condition of the blocking treatment and the blocking time. Under conditions A and C to E, the blocking treatment was performed immediately before the primary reaction. Under condition B, the blocking treatment was performed immediately after a detection chip was manufactured.
As indicated in Table 1, the blocking time in a case where the blocking liquid was not moved (condition B) was five hours. On the other hand, the blocking time in a case where the blocking liquid was moved (conditions C to E) was significantly shorter than that in the case where the blocking liquid was not moved (condition B). This result indicates that the blocking time can be largely reduced by moving the blocking liquid. In addition, the result indicates that the higher the concentration of the blocking agent is, the shorter the blocking time can be.
In Example 3, a relationship between presence or absence of movement of a blocking liquid and a concentration of a blocking agent, and a signal value when the primary reaction was performed following the blocking treatment was examined. As Comparative Examples, a case where another step was performed between the blocking treatment and the primary reaction was examined.
(1) Preparation of Detection Chip
A detection chip which had been subjected to the blocking treatment immediately after manufacturing and a detection chip which had not been subjected to the blocking treatment immediately after manufacturing were prepared by a similar procedure to Example 2 except that the detection chip was allowed to stand for 30 minutes in the blocking treatment immediately after manufacturing. These detection chips were stored at room temperature and atmospheric pressure for one day or more.
(2) Blocking Treatment
Each of the detection chips which had not been subjected to the blocking treatment immediately after manufacturing was installed in the chip holder of the SPFS device. Reciprocating liquid feeding of a blocking liquid (100 μL) containing a blocking agent at 0% by mass, 2% by mass, 5% by mass, or 8% by mass was performed using the syringe pump in the liquid feeding unit for 15 seconds at a flow rate of 3300 μL/min in the channel by a similar procedure to Example 2. Subsequently, the blocking liquid in the channel was removed using the syringe pump in the liquid feeding unit.
Table 2 indicates conditions of the blocking treatment. Under conditions F and H to J, the blocking treatment was performed immediately before the primary reaction. Under condition G, the blocking treatment was performed immediately after a detection chip was manufactured.
(3) Primary Reaction
Following the blocking treatment, by performing reciprocating liquid feeding of a diluted liquid of plasma (100 μL) obtained by mixing a phosphate buffer and plasma (specimen) such that a volume ratio thereof was 2:1 using a syringe pump in the liquid feeding unit for four minutes at a flow rate of 3300 μL/min in the channel, the primary reaction was performed. Subsequently, the diluted liquid in the channel was removed using the syringe pump in the liquid feeding unit. In addition, each of the detection chips which had been subjected to the blocking treatment immediately after manufacturing was installed in the chip holder of the SPFS device, and then was subjected to the primary reaction by a similar procedure.
In this experiment, as a specimen, plasma containing a detection target substance (troponin) and plasma containing no troponin were subjected to liquid feeding. Hereinafter, the primary reaction using a specimen containing a detection target substance is also referred to as a “positive reaction”, and the primary reaction using a specimen containing no detection target substance is also referred to as a “negative reaction”.
(4) Measurement of Optical Blank Value
After the primary reaction, the inside of the channel was cleaned by introducing a phosphate buffer into the channel. Thereafter, the metal film was irradiated with light having a wavelength of 635 nm from a side of the prism. Light emitted from the vicinity of a surface of the metal film on a side of the channel was detected to obtain an optical blank value.
(5) Secondary Reaction
Thereafter, by performing reciprocating liquid feeding of a solution (100 μL) containing an anti-troponin antibody labeled with a fluorescence dye using the syringe pump in the liquid feeding unit for one minute at a flow rate of 3300 μL/min in the channel, a secondary reaction was performed. In each of a case where the positive reaction was performed and a case where the negative reaction was performed, the secondary reaction was performed. Subsequently, the solution in the channel was removed using the syringe pump in the liquid feeding unit.
(6) Measurement of Signal Value Indicating Amount of Detection Target Substance
After the inside of the channel was cleaned, fluorescence emitted from the vicinity of a surface of the metal film on a side of the channel was detected in a similar manner to measurement of an optical blank value. A signal value indicating the amount of a detection target substance was obtained by subtracting the optical blank value from a detection value.
In
In
On the other hand, when results of the positive reactions under conditions F to J are compared with one another, the signal value is not reduced even when the concentration of the blocking agent is increased. This indicates that the concentration of the blocking agent has no influence on the amount of a detection target substance captured by a capturing body when the primary reaction is performed following the blocking treatment.
The above results indicate that time required for detecting a detection target substance (including time required for the blocking treatment) can be reduced, the SN ratio can be improved, and the amount of the detection target substance can be detected with a high accuracy by moving the blocking liquid and increasing the concentration of the blocking agent.
In Example 4, a relationship between a concentration of the blocking agent and a signal value when the blocking treatment and the primary reaction were performed simultaneously was examined.
(1) Preparation of Detection Chip
A detection chip in which a reaction field on a metal film (region to which an anti-troponin antibody was fixed as a capturing body) was protected by a protective layer was installed in the chip holder of the SPFS device in a similar manner to Example 1. A cross-sectional area of a channel on the reaction field was 0.46 mm2.
(2) Preparation of Mixed Liquid of Blocking Liquid and Specimen
A blocking agent (BSA) was added to an acetate buffer to prepare a blocking liquid. The prepared blocking liquid and plasma (specimen) were mixed such that a volume ratio thereof was 2:1 to prepare six kinds of mixed liquids of the blocking liquid and plasma. The final concentrations of the blocking agent in the mixed liquids were 0% by mass, 1% by mass, 2% by mass, 3% by mass, 4% by mass, and 5% by mass. At this time, by using plasma containing a detection target substance (troponin) and plasma containing no troponin as a specimen, 12 kinds of mixed liquids were prepared.
(3) Blocking Treatment and Primary Reaction
Reciprocating liquid feeding of a mixed liquid (100 μL) was performed using the syringe pump in the liquid feeding unit for four minutes at a flow rate of 3300 μL/min in the channel. Subsequently, the mixed liquid in the channel was removed using the syringe pump in the liquid feeding unit.
(4) Measurement of Signal Value Indicating Amount of Detection Target Substance
Measurement of an optical blank value and the secondary reaction were performed by a similar procedure to Example 2 to obtain a signal value indicating the amount of a detection target substance.
Also in
When results of the negative reactions are compared with one another, the signal value (noise) at a concentration of the blocking agent of 0% by mass is the highest, and there is a tendency that the higher the concentration of the blocking agent is, the lower the signal value (noise) is. This result indicates that a higher concentration of the blocking agent makes an effect of the blocking treatment higher, and can suppress nonspecific bonding in the channel.
On the other hand, when results of the positive reactions are compared with one another, in a case where the concentration of the blocking agent is higher than 3% by mass, the higher the concentration of the blocking agent is, the lower the signal value is. This result suggests that the concentration of the blocking agent higher than 3% by mass reduces the amount of a detection target substance bonded to a capturing body. It is considered that the primary reaction tends to be inhibited because the concentration of the blocking agent higher than 3% by mass increases the viscosity of the mixed liquid and suppresses movement of a detection target substance.
The above results indicate that the concentration of the blocking agent in the mixed liquid is preferably 3% by mass or less in order to prevent the primary reaction from being inhibited when the blocking treatment and the primary reaction are performed simultaneously.
This application claims priority based on Japanese Patent Application No. 2014-170438 filed on Aug. 25, 2014. Contents described in the application specification and drawings are all incorporated herein by reference.
The method and device for detecting a detection target substance using the reaction method according to the present invention can detect the detection target substance with a high reliability, and therefore are useful for examination of a disease, for example.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2014-170438 | Aug 2014 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2015/070150 | 7/14/2015 | WO | 00 |
