WO2018100780A1

WO2018100780A1 - Target substance detection device and target substance detection method

Info

Publication number: WO2018100780A1
Application number: PCT/JP2017/023038
Authority: WO
Inventors: 雅人安浦; 藤巻　真; 裕樹芦葉; 島　隆之
Original assignee: 国立研究開発法人産業技術総合研究所
Priority date: 2016-11-30
Filing date: 2017-06-22
Publication date: 2018-06-07
Also published as: JPWO2018100780A1; JP7028455B2

Abstract

[Problem] To detect a target substance efficiently and with a high degree of accuracy. [Solution] This target substance detection device 1 comprises: a liquid sample introduction plate 2 formed from a light-transparent plate and which, when a liquid sample containing a target substance and magnetic particles is introduced onto the surface, allows transmitted light shone from the rear side to propagate as propagated light above the surface; a rear-side light-shining unit 3 which can shine the light from the rear side; a first magnetic field application unit 4 which is positioned on the surface side and which moves the aggregates in the liquid sample which has been introduced to the surface in a direction away from the liquid sample introduction plate 2 by applying a magnetic field; and an optical signal detection unit 5 which can detect signal changes in the optical signal before and after the application of the magnetic field by the first magnetic field application unit 4. The optical signal detection unit 5 can detect, from among optical signals and as signals to which signal changes apply, only optical signals on the basis of aggregates obtained by two or more magnetic particles bonding to a single target substance.

Description

Target substance detection apparatus and target substance detection method

The present invention relates to a target substance detection apparatus and a target substance detection method for detecting a target substance by using a change in an optical signal when a target substance existing in a liquid sample is changed using a magnetic field.

In recent years, methods for detecting and quantifying minute substances present in a solution, in particular, biological substances such as DNA, RNA, proteins, viruses, and bacteria have been developed. Examples of the method include surface plasmon resonance (SPR) immunoassay, total reflection illumination fluorescence microscope (TIRFM), surface plasmon resonance excitation enhanced fluorescence spectroscopy (SPFS), and the like.

The surface plasmon resonance immunoassay method combines a specific selectivity of an antigen-antibody reaction with a surface plasmon resonance sensor which is a highly sensitive refractometer, and an antigen in an enhanced electric field generated on the gold thin film surface of the total reflection surface. -Antibody binding can be detected and quantified in real time with high accuracy (see Non-Patent Document 1).

The total reflection illumination fluorescent microscope totally reflects incident light at the interface between a sample and a cover glass or a slide glass, and uses the evanescent field generated thereby as excitation light to perform fluorescence observation with less background light that becomes noise. Technology (see Patent Document 1). This technique is a technique capable of realizing super-resolution, and enables single molecule observation.

The surface plasmon resonance excitation-enhanced fluorescence spectroscopy uses an optical arrangement called a Kretschmann arrangement to cause a total reflection of incident light at the interface between the gold thin film layer on the glass surface in contact with the prism and the liquid sample, on the gold thin film. It is characterized by exciting surface plasmon resonance and forming an enhanced electric field on the gold thin film surface. This is a technology that uses fluorescence enhanced by surface plasmon resonance in the vicinity of the surface of the gold thin film as excitation light to excite fluorescent molecules in the enhanced electric field to generate strong fluorescence and perform fluorescence observation with little background light (patent) Reference 2).

In addition, as a method of obtaining an enhanced electric field by causing an electric field enhancement by such total reflection of light, there are known methods as described in Non-Patent Documents 2 to 8, for example. The inventor installs a detection plate in which a silicon layer and a SiO ₂ layer are laminated in this order on a silica glass substrate on a trapezoidal prism made of silica glass, and transmits light under the condition of total reflection on the surface of the detection plate through the prism. In Non-Patent Document 2, a method for obtaining an enhanced electric field by irradiating the light was reported.

Non-Patent Document 3 discloses a method for obtaining an enhanced electric field by generating surface plasmon resonance using a Kretschmann arrangement. Non-Patent Document 4 discloses a method of obtaining an enhanced electric field by causing light to be incident on a prism in a Kretschmann arrangement using a dove prism to generate surface plasmon resonance. Non-Patent Document 5 and Non-Patent Document 6 disclose a method of obtaining an enhanced electric field using a resonant mirror. In Non-Patent Document 7, a metal layer and a transparent dielectric layer are laminated on a prism in this order to form a structure called a leaky mode sensor, and light is irradiated through the prism to form a surface of the dielectric layer. Discloses a method for obtaining an enhanced electric field. In Non-Patent Document 8, a metal layer is formed on a prism, and two types of transparent dielectric layers having different refractive indexes are laminated on each of them to provide an enhanced electric field stronger than the leaky mode sensor structure. A method of obtaining is disclosed.

Further, Patent Documents 3 and 4 disclose a method in which a prism shape for generating surface plasmon resonance is imparted to the flow path, and surface plasmon resonance is generated on the bottom or side surface of the flow path to obtain an enhanced electric field.

According to the prior art literature search related to the present application, a method using magnetic particles for labeling is known in order to promote adsorption or proximity of a target substance to a detection surface and realize measurement in a short time. (Patent Documents 5 and 6). In Patent Documents 5 and 6, a conjugate of a magnetic label, a photoresponsive labeling substance, and a target substance is attracted to a local area by applying a magnetic field, and only a predetermined area including the local area is irradiated with excitation light. Thus, the detection is performed by eliminating the signal of the photoresponsive label that does not form a conjugate of the target substance and the magnetic label.

As a method for detecting and quantifying the biological substance, a method using propagation light as detection light has also been proposed. Examples of such methods include fluorescence immunoassay (FIA method), enzyme-linked immunosorbent method (ELISA method), and the like.
In the FIA method, a fluorescent dye is bound using an antibody that specifically binds to a target substance such as a specific bacterium or virus, and the target substance is detected and quantified by observing the emission of the fluorescent dye with a fluorescence microscope or the like. It is a technique.
In the ELISA method, the target substance is immobilized on a detection plate using an antigen-antibody reaction, then an enzyme-labeled antibody is bound, a substrate that develops color with the enzyme is added, and the target substance is determined from the change in color. Is detected and quantified.
Both methods are widely used as established biologically relevant substance detection methods, but these methods require multiple reaction steps and repeated washing steps, and require a lot of time and labor to obtain measurement results. There is a problem that is needed. There is also a need for further improvement in detection sensitivity.

As a method for improving the detection sensitivity in the detection of the target substance using the biological substance-related detection method, a measurement method using magnetic particles has been proposed. For example, a detection method in which a conjugate containing the target substance and the magnetic particles is concentrated on the bottom surface side of a liquid sample container, and is immobilized on the bottom surface of the container by an antigen-antibody reaction between the antibody disposed on the bottom surface of the container and the conjugate. Is disclosed (see Patent Document 7).
However, in the measurement method using such magnetic particles, although the detection sensitivity can be improved by the concentration effect of collecting the conjugate at the detection position by a magnetic field, the contaminants floating at the detection position of the concentration destination, the liquid The contaminants adsorbed on the bottom surface of the sample container, scratches on the bottom surface of the liquid sample container, noise signal due to fluctuations in the light source output of detection light used for detection, and an optical signal based on the combined body Since these cannot be distinguished, there is a problem that detection accuracy is low. Such a problem becomes more apparent when the minute substance is detected.
Further, in order to eliminate the noise signal based on the contaminants adsorbed on the bottom surface of the liquid sample container, it is necessary to perform a cleaning process to remove the contaminants for each detection, and the detection efficiency is still low. is there.

JP 2002-236258 A International Publication No. 2015/194663 JP 2013-24606 A JP 2010-145408 A JP 2011-33454 A JP 2005-77338 A Japanese Patent Laid-Open No. 4-102062

The inventor performs the detection of a target substance that eliminates a noise signal based on scratches on the surface of the detection plate, adsorption on the surface or impurities existing on the surface, fluctuation of a light source output, etc. We are investigating the detection of the target substance by binding magnetic particles to the target substance and moving the conjugate by applying a magnetic field.
Specifically, the following methods are being considered.

First, the target substance detection apparatus which concerns on examination is demonstrated, referring FIG. In addition, FIG. 1 is explanatory drawing of the target substance detection apparatus which concerns on examination.
As shown in FIG. 1, a target substance detection apparatus 1 is configured according to a known transmission microscope, and includes a liquid sample introduction plate 2, a light irradiation unit 3, a first magnetic field application unit 4, an imaging device 5a, and It is comprised with the optical signal detection part 5 comprised with the objective lens 5b. Note that the imaging device 5a is constituted by, for example, a known CCD image sensor or the like, and can acquire a two-dimensional image.

Liquid sample introduction plate 2, the transmitted light T _L of the light L liquid sample containing the magnetic particles forming the coupling member and the target substance and the target substance is irradiated from the back side while being introduced onto the surface It is formed of a translucent plate that can propagate as propagating light above the surface. Further, the liquid sample introduction plate 2 itself constitutes a liquid sample holding unit, and after the liquid sample is introduced onto the surface, a cover glass or the like is disposed so as to cover the liquid sample, thereby the liquid sample introduction plate 2 Hold.
The light irradiation unit 3 is configured as a back side light irradiation unit that can irradiate the light L from the back side of the liquid sample introduction plate 2.
The first magnetic field application unit 4 is arranged on the surface side of the liquid sample introduction plate 2 and applies a magnetic field to the combination in the liquid sample introduced on the surface of the liquid sample introduction plate 2. Is configured to move away from the liquid sample introduction plate 2. Here, the first magnetic field applying unit 4 is formed by an annular electromagnet which through-holes are formed in the center, the optical signal is the through hole based on the transmitted light T _L of the light L irradiated from the light irradiation section 3 Through the optical signal detection unit 5, the detection is possible.
The optical signal detection unit 5 is arranged on the surface side of the liquid sample introduction plate 2 and can detect a signal change of the optical signal based on the propagation light before and after application of the magnetic field by the first magnetic field application unit 4. The

In the target substance detection device 1 configured as described above, first, the liquid sample is introduced and held on the surface of the liquid sample introduction plate 2 (liquid sample introduction and holding step).
Next, after the conjugate floating in the liquid layer of the liquid sample is gravity settled on the surface of the liquid sample introduction plate 2, the light L is irradiated from the back side of the liquid sample introduction plate 2 (light irradiation step). ), The objective lens 5b is adjusted so that the surface or the vicinity thereof is within the imageable range, and an optical signal on the surface is acquired by the imaging device 5a (optical signal detection step). Here, the imageable range refers to a range in which an optical signal at the focal depth and in the vicinity thereof can be acquired.

FIG. 2 schematically shows a state on the surface of the liquid sample introduction plate 2 in the observation field observed by the imaging device 5a at this time.
As shown in FIG. 2, on the surface of the liquid sample introduction plate 2 in the observation field, for example, the light transmitted through the liquid sample that propagates above the surface of the liquid sample introduction plate 2 is transmitted. Four optical signals a to d that can be distinguished from the background signal are observed based on a contrast difference with the signal (background signal). In FIG. 2, the optical signals a and d are observed as light spots, and the optical signals b and c are observed as dark spots.

Further, FIG. 3 shows a state when the substance a ′ for generating the optical signal a and the substance b ′ for generating the optical signal b at this time are viewed from the side surface of the liquid sample introduction plate 2. 3 is a cross-sectional view taken along line AA in FIG. Further, an arrow B in FIG. 3 indicates an imageable range where an optical signal can be acquired.
As shown in FIG. 3, the substance a ′ and the substance b ′ are in a state of being gravity settled on the surface of the liquid sample introduction plate 2.

Next, the electromagnet of the first magnetic field application unit 4 is excited to apply the magnetic field to the combined body in the liquid sample introduced onto the surface of the liquid sample introduction plate 2 by applying a magnetic field. And the combined body is moved in a direction away from the liquid sample introduction plate 2 (a combined body changing step).
Next, an optical signal on the surface of the liquid sample introduction plate 2 after the combined body is moved away from the liquid sample introduction plate 2 while maintaining the imageable range and the observation field is acquired by the imaging device 5a. (Optical signal detection step).

FIG. 4 schematically shows a state on the surface of the liquid sample introduction plate 2 in the observation field observed by the imaging device 5a after the combined body changing step.
As can be understood from a comparison between FIG. 2 showing the state before the combined body changing step and FIG. 4 showing the state after the combined body changing step, the optical signals a and b are before and after the combined body changing step. The optical signal changes, and the optical signals c and d do not change before and after the combined body changing process.
From this, it is understood that the substances a ′ and b ′ that generate the optical signals a and b are the combined body including the magnetic particles attracted to the first magnetic field application unit 4 and include the target substance. .
On the other hand, the optical signals c and d, which are not confirmed before and after the combined body changing step, are scratches on the surface of the liquid sample introduction plate 2, adsorbed on the surface or impurities existing on the surface, light source It can be seen that this is a noise signal such as output fluctuation.

FIG. 5 shows a state in which the substance a ′ that generates the optical signal a and the substance b ′ that generates the optical signal b after the conjugate changing step are viewed from the side surface of the liquid sample introduction plate 2. FIG. 5 is a cross-sectional view taken along line AA in FIG. Further, an arrow B in FIG. 5 indicates an imageable range where an optical signal can be acquired.
As shown in FIG. 5, the substance a ′ and the substance b ′ are moved in a direction away from the liquid sample introduction plate 2 by application of the magnetic field by the first magnetic field application unit 4.

In the optical signal a, the size of the light spot is observed large before and after the conjugate variation process (see FIG. 4). This is based on the depth of focus in a state where the surface of the liquid sample introduction plate 2 is in focus before the conjugate variation step, although the substance a ′ exists within the imageable range of the optical signal detector 5. Since it is off, the size of the light spot is observed large (see FIG. 5).
On the other hand, it is confirmed that the optical signal b disappears after the conjugate variation step (see FIG. 4). This is because the substance b ′ has moved out of the imageable range of the optical signal detector 5 (see FIG. 5).

As described above, in the target substance detection device 1, the optical signal based on the target substance is detected on the surface of the liquid sample introduction plate 2, scratches on the surface, impurities present on the surface or impurities on the surface, and light source output. The target substance can be detected with high accuracy because it can be clearly distinguished from a noise signal such as fluctuation. In addition, even when the contaminants are adsorbed on the surface of the liquid sample introduction plate 2, detection can be performed while ignoring the presence of the impurities, and therefore the cleaning process for the liquid sample introduction plate 2 is not necessarily performed for each detection. Efficient detection can be performed without having to do so.

By the way, the conjugate used for detection in the target substance detection apparatus 1 is a combination of the target substance and the magnetic particles.
When the target substance does not generate optical signals a and b that can be distinguished from the background signal illustrated in FIG. 4, the magnetic particles may generate an optical signal that can be distinguished from the background signal. preferable.
In addition to this, as a technique for discriminating the conjugate as the optical signals a and b, a technique of binding a labeling substance that generates an optical signal that can be distinguished from the background signal to the target substance can be considered. Since the step of binding the labeling substance is necessary, preparation for detection becomes complicated.

Therefore, once again, the function of detecting the target substance using the target substance detection device 1 and generating the optical signal that can be distinguished from the background signal of the label substance on the magnetic particles (M) is provided. Let's think about the case where it is also used.
As shown in FIG. 4, the target substance detection device 1 processes an optical signal that changes before and after application of a magnetic field from the first magnetic field application unit 4 as an optical signal based on the target substance.
Here, when the magnetic particle (M) has a function (O) for generating an optical signal distinguishable from the background signal (the magnetic particle in this case is referred to as MO), the first magnetic field application unit 4 The generation source of the optical signal that changes before and after the application of the magnetic field is considered as follows.

First, as shown in FIG. 6, the case where one magnetic particle MO couple | bonds with one target substance T can be considered.
Moreover, as shown in FIG. 7, the case where two or more magnetic particles MO couple | bond with one target substance T can be considered. In the illustrated example, a case where two magnetic particles MO are bonded to one target substance T is representatively shown.
Moreover, as shown in FIG. 8, the case of the magnetic particle MO simple substance can be considered. In particular, the liquid sample is preferably prepared by increasing the content of the magnetic particles MO with respect to the content of the target material T in order to prevent detection of the target material T from being detected. It is assumed that the substance T and unbonded magnetic particles MO alone are included.

Here, since the magnetic particle MO alone is not bonded to the target substance T, the optical signal based on the magnetic particle MO shown in FIG. 8 is a noise signal.
Further, this noise signal cannot be distinguished from the optical signal based on the combined body when one magnetic particle MO is bonded to one target substance T shown in FIG.

On the other hand, when two or more magnetic particles MO are bonded to one target substance T shown in FIG. 7, the optical signal based on the combined body is an optical signal derived from one magnetic particle MO shown in FIGS. Can be distinguished.
That is, an optical signal derived from two or more magnetic particles MO has a larger signal size than an optical signal derived from one magnetic particle MO or is different from an optical signal derived from one magnetic particle MO. Since it has signal strength, it can be distinguished from an optical signal derived from one magnetic particle MO.
Therefore, in detecting an optical signal, if only an optical signal based on the combined body in which two or more magnetic particles MO are bonded to one target substance T is detected as a signal change target, the presence of the noise signal is present. It is possible to perform detection with higher accuracy while ignoring.

Here, the target substance detection device using the propagating light has been described as an example. However, in the target substance detection device using the enhanced electric field (near-field light), the magnetic particle MO may be used. The same situation occurs.

The present invention provides a target substance detection apparatus and a target substance detection method capable of solving the above-mentioned problems in the prior art and capable of detecting a target substance with high accuracy when an optical signal generated from a magnetic particle is used for detection. Objective.

The present invention is based on the above knowledge, and means for solving the above problems are as follows. That is,
<1> A liquid sample containing magnetic particles having a target substance and a conjugate with the target substance and having photoresponsiveness is introduced onto the surface and transmits transmitted light that is irradiated from the back side or the surface side. A translucent plate capable of propagating to a surface opposite to the side irradiated with the light as light, and the reflected light of the light irradiated from the surface side while the liquid sample is introduced onto the surface A reflecting plate capable of propagating above the surface as propagating light, an introducing plate on which the liquid sample is introduced onto the surface, and the liquid sample are introduced onto the surface and irradiated on the surface under total reflection conditions. A liquid sample introduction plate formed by any of the detection plates capable of generating near-field light on the surface by the light to be emitted, and the liquid sample can be held on the surface of the liquid sample introduction plate Liquid sample holder to be When the liquid sample introduction plate is formed of the translucent plate, a back side light irradiating unit that is capable of irradiating the light from the back side of the liquid sample introduction plate, and the liquid sample introduction plate is the translucent plate And the liquid sample introduction plate is formed by the introduction plate, and the liquid sample introduction plate can be irradiated with the light from the surface side of the liquid sample introduction plate. Sometimes, the liquid sample held on the liquid sample introduction plate can be irradiated with the light from the side surface side of the liquid sample introduction plate, and the liquid sample introduction plate serves as the detection plate. A light irradiating part formed by any of the total reflected light irradiating parts capable of irradiating the surface with total reflection conditions on the surface, and the surface side of the liquid sample introduction plate or The liquid sample introduced while being arranged on the side surface side A direction having a vector component in a direction parallel to an in-plane direction of the surface of the liquid sample introduction plate by applying a magnetic field to the combined body in the liquid sample introduced on the surface of the liquid sample, and from the liquid sample introduction plate The first magnetic field applying unit that moves in any direction of the moving away direction or changes the posture of the combined body and the rear surface side of the liquid sample introduction plate, and on the surface of the liquid sample introduction plate The combined body in the liquid sample introduced into the liquid sample can be attracted onto the surface of the liquid sample introduction plate by applying a magnetic field, and the surface of the surface of the liquid sample introduction plate in a state where the magnetic field is applied A magnetic field application unit formed by one of the second magnetic field application units capable of moving in a direction having a vector component in a direction parallel to the inward direction, the surface side of the liquid sample introduction plate, and the back side The propagating light or the near-field light, which is arranged on the side or the side surface side and is in the front-rear relationship between before and after application of the magnetic field by the first magnetic field application unit and before and after movement of the second magnetic field application unit An optical signal detection unit capable of detecting a signal change of the optical signal based on the optical signal, wherein the optical signal detection unit emits the propagation light or the near-field light among the optical signals. It is possible to detect only an optical signal generated from the magnetic particles when received and based on the conjugate in which two or more magnetic particles are bound to one target substance as a signal change target. A target substance detection device.
<2> When the magnetic field application unit is formed by the first magnetic field application unit, the combined body in the liquid sample that is further arranged on the back side of the liquid sample introduction plate and introduced into the liquid sample introduction plate <3> The target substance detection device according to <1>, further including a third magnetic field application unit that can be drawn onto the surface of the liquid sample introduction plate by application of.
<3> The target substance detection device according to any one of <1> to <2>, wherein the optical signal detection unit can acquire the state of the detection region on the surface of the liquid sample introduction plate as a two-dimensional image.
<4> The target substance detection device according to any one of <1> to <3>, wherein the surface of the liquid sample introduction plate is surface-treated with an adsorption inhibitor that suppresses adsorption of the conjugate.
<5> A liquid sample containing a target substance and a magnetic particle having a photoresponsiveness and forming a conjugate with the target substance is introduced onto the surface and transmits light transmitted from the back side or the front side. A translucent plate capable of propagating to the side opposite to the side irradiated with the light as light, and propagating reflected light of the light irradiated from the surface side while the liquid sample is introduced onto the surface A reflecting plate capable of propagating as light above the surface, an introducing plate into which the liquid sample is introduced onto the surface, and the liquid sample are introduced onto the surface and irradiated on the surface under total reflection conditions A liquid sample introduction plate formed by one of detection plates capable of generating near-field light on the surface by light is disposed, and the liquid sample can be held on the surface of the liquid sample introduction plate For the liquid sample holder A liquid sample introduction and holding step for introducing and holding the liquid sample on the surface of the liquid sample introduction plate; and from the back side of the liquid sample introduction plate when the liquid sample introduction plate is formed by the translucent plate A back side light irradiation step for irradiating the light, and a surface for irradiating the light from the surface side of the liquid sample introduction plate when the liquid sample introduction plate is formed of either the light transmitting plate or the reflection plate Side light irradiation step, when the liquid sample introduction plate is formed by the introduction plate, the liquid sample held on the liquid sample introduction plate is irradiated from the side surface side of the liquid sample introduction plate A light irradiation step that is one of a side light irradiation step and a total reflection light irradiation step of irradiating the surface with total reflection conditions on the surface when the liquid sample introduction plate is formed by the detection plate; The table of the liquid sample introduction plate A direction having a vector component parallel to an in-plane direction of the surface of the liquid sample introduction plate and a direction away from the liquid sample introduction plate by applying a magnetic field to the conjugate in the liquid sample introduced above. The liquid sample is moved by a first combined body changing step of moving in any direction or changing the posture of the combined body and application of a magnetic field from a magnetic field applying unit disposed on the back side of the liquid sample introduction plate. The combined body in the liquid sample introduced onto the surface of the introduction plate is drawn onto the surface of the liquid sample introduction plate and the magnetic field application unit is configured to apply the magnetic field in a state where the magnetic field is applied. Move in a direction having a vector component in a direction parallel to the in-plane direction of the surface, and move the combined body following the movement of the magnetic field applying unit, or change the posture of the combined body Any one of the combination variation step that is one of the two combination variation steps, before and after application of the magnetic field by the first combination variation step, and before and after movement of the magnetic field application unit by the second combination variation step An optical signal detection step of detecting a signal change of the optical signal based on the propagating light or the near-field light in the context, wherein the optical signal detection step includes the combination of the optical signals, Only an optical signal generated from the magnetic particle when irradiated with propagating light or the near-field light and based on the combined body in which two or more magnetic particles are bonded to one target substance is changed in signal. A method for detecting a target substance, wherein the target substance is detected as a target of the target.
<6> The target substance detection method according to <5>, wherein the magnetic particles are particles that generate scattered light upon irradiation with propagating light or near-field light.
<7> The target substance detection method according to <5>, wherein the magnetic particles are spherical particles having a diameter of 50 nm to 6,500 nm.
<8> The target substance detection method according to <5>, wherein the magnetic particles contain a fluorescent dye.
<9> When the optical signal detection step is a step of detecting a signal change of an optical signal based on propagating light, the magnetic particles include a light absorbing material that generates light absorption upon irradiation with the propagating light. > The target substance detection method described in>.
<10> When the conjugate variation step is the first conjugate variation step, after the liquid sample introduction and holding step and before the conjugate variation step, all or all of the conjugates in the liquid sample are applied by applying an attracting magnetic field. The target substance detection method according to any one of <5> to <9>, wherein a conjugate drawing step of drawing a part once onto the surface of the liquid sample introduction plate is performed.

According to the present invention, there are provided a target substance detection apparatus and a target substance detection method capable of solving the above-described problems in the prior art and capable of detecting a target substance with high accuracy when an optical signal generated from magnetic particles is used for detection. be able to.

It is explanatory drawing of the target substance detection apparatus which concerns on examination. It is the figure which showed typically the mode on the surface of the liquid sample introduction plate in the observation visual field observed with an imaging device. FIG. 3 is a sectional view taken along line AA in FIG. 2. It is the figure (1) which showed typically the mode on the surface of the liquid sample introducing | transducing board in the observation visual field observed with an imaging device after a coupling body change process. FIG. 5 is a sectional view taken along line AA in FIG. 4. It is a figure which shows a mode that one magnetic particle MO couple | bonded with respect to one target substance. It is a figure which shows a mode that two magnetic particle MO couple | bonded with respect to one target substance. It is explanatory drawing which shows the magnetic particle MO simple substance. It is a figure which shows a mode that three magnetic particles MO couple | bonded with respect to one target substance T. FIG. It is a figure which shows the coupling | bonding particle | grains of the magnetic particle which does not have photoresponsiveness, and a photoresponsive substance. It is explanatory drawing of the target substance detection apparatus which concerns on a 1st modification. It is explanatory drawing of the target substance detection apparatus which concerns on a 2nd modification. It is the figure (2) which showed typically the mode on the surface of the liquid sample introducing | transducing board in the observation visual field observed with an imaging device after a conjugate | bonded_body fluctuation | variation process. FIG. 14 is a sectional view taken along line AA in FIG. 13. It is explanatory drawing of the target substance detection apparatus which concerns on 2nd Embodiment. It is the figure (3) which showed typically the mode on the surface of the liquid sample introducing | transducing board in the observation visual field observed with an imaging device after a coupling body change process. It is the figure which showed typically the mode on the surface of the liquid sample introducing | transducing board in the observation visual field observed with an imaging device before a coupling body change process. It is the figure (4) which showed typically the mode on the surface of the liquid sample introducing | transducing board in the observation visual field observed with an imaging device after a conjugate | bonded_body fluctuation | variation process. It is the sectional view on the AA line in FIG. It is the sectional view on the AA line in FIG. It is explanatory drawing of the target substance detection apparatus which concerns on 3rd Embodiment. It is explanatory drawing of the target substance detection apparatus which concerns on the modification of the target substance detection apparatus which concerns on 3rd Embodiment. It is explanatory drawing of the target substance detection apparatus which concerns on 4th Embodiment. It is explanatory drawing of the target substance detection apparatus which concerns on 5th Embodiment. It is a figure (1) which shows the mode on the said surface of a liquid sample introduction plate before a coupling body change process. It is a figure (1) which shows the mode on the said surface of a liquid sample introduction plate after a conjugate | bonded_body fluctuation | variation process. It is explanatory drawing of the target substance detection apparatus which concerns on 6th Embodiment. It is a figure (2) which shows the mode on the said surface of a liquid sample introduction plate before a conjugate | bonded_body change process. It is a figure (2) which shows the mode on the said surface of a liquid sample introduction plate after a conjugate | bonded_body fluctuation | variation process. It is an image which shows the mode on the surface of the liquid sample introduction plate observed in the imaging device in the exposure time of 5 seconds before the combined body changing step. It is an image which shows the mode on the surface of the liquid sample introducing | transducing board observed by exposure time 0.5 second in the imaging device before a conjugate | bonded_body change process. It is an image which shows the mode on the surface of the liquid sample introducing | transducing board observed with the exposure time 0.5 second in the imaging device after the conjugate | bonded_body change process.

(Target substance detection device)
The target substance detection apparatus of the present invention includes a liquid sample holding unit, a light irradiation unit, a magnetic field application unit, and an optical signal detection unit, and includes other units as necessary.

<Liquid sample holder>
The liquid sample holding part is a part in which a liquid sample introduction plate is arranged and a liquid sample is held on the surface of the liquid sample introduction plate.

-Liquid sample-
The liquid sample includes a target substance and magnetic particles that form a conjugate with the target substance and have photoresponsiveness.
Specific examples of the liquid sample include, for example, solid samples such as blood, saliva, urine, liquid chemicals, environmental water, water and sewage, beverages, food homogenization solutions, wipes, and powders dissolved in a solvent such as water. Examples thereof include a solution, a gas phase concentrated liquid in which gas and fine particles in the gas phase are collected. Specific examples of the target substance include DNA, RNA, protein, virus, fungus, and contaminant.

As described above, the magnetic particles have photoresponsiveness. Here, “light responsiveness” refers to the property of generating an optical signal that can be detected by the optical signal detection unit upon receiving irradiation of propagating light or near-field light.
The magnetic particles are not particularly limited as long as they have such properties, can be appropriately selected according to the purpose, and known magnetic beads can be used.

For example, when the magnetic particle is irradiated with the propagating light or the near-field light and generates scattered light as an optical signal that can be detected by the optical signal detection unit, the magnetic particle may be an organic polymer. Known magnetic beads that generate the scattered light upon irradiation with light such as ferrite-based particles whose surface is modified can be used.
The magnetic particles at this time are not particularly limited and may be appropriately selected depending on the intended purpose. However, spherical particles having a diameter of 50 nm to 6,500 nm are preferable.
With respect to the upper limit of the diameter, the optical signal based on one magnetic particle is based on the combination including three or more magnetic particles rather than the optical signal based on the combination including two magnetic particles. The optical signal is a significantly distinguishable optical signal. Therefore, for example, as shown in FIG. 9, the diameter is preferably limited so that three or more magnetic particles MO having photoresponsiveness can be bonded to one target substance T. Here, the size of the target substance T is generally about 20 nm to 1,000 nm, although it depends on the type, in order to bind three photoresponsive magnetic particles MO to one target substance T. For this, it is necessary that the diameter of the magnetic particle MO having photoresponsiveness is within about 6.5 times the diameter of the target substance T. Therefore, the upper limit of the diameter is preferably 6,500 nm. Since the target substance T can be used even when the target substance T has a size of 150 nm or less, which is difficult to observe in the visible light wavelength region, such as a virus, the diameter is more preferably 1,000 nm or less. FIG. 9 is a diagram showing a state where three magnetic particles MO having photoresponsiveness are bonded to one target substance T. FIG.
In addition, regarding the lower limit of the diameter, since a light source mainly in the visible light wavelength range (400 nm to 700 nm) is used, a conjugate of two or more magnetic particles MO having photoresponsiveness to one target substance is bound. Since the size is preferably not less than one quarter of the light source wavelength, the diameter is desirably 50 nm or more.
The spherical particles include not only true spheres but also irregular spherical particles such as elliptical spheres. The diameter of the irregular spherical particle corresponds to the maximum diameter of the particle. Further, the magnetic particles MO having photoresponsiveness may be constituted by a combined body including magnetic particles M having no photoresponsiveness and a photoresponsive substance O as shown in FIG.

In addition, when the magnetic particle is irradiated with the propagating light or the near-field light and generates fluorescence as an optical signal whose signal change can be detected by the optical signal detection unit, examples of the magnetic particle include a fluorescent dye. Known magnetic beads containing can be used.

Further, when the magnetic particles receive irradiation of propagating light and generate optical absorption as an optical signal whose signal change can be detected by the optical signal detector (discriminated from the optical signal of the transmitted light with respect to the liquid sample. In the case of generating a dark spot signal due to light absorption as a possible optical signal), the magnetic particles may be, for example, known magnetic beads enclosing a plurality of magnetic cores, or known magnetic beads and known light-absorbing substances. Binding particles (for example, see FIG. 10 showing a binding particle of a magnetic particle having no photoresponsiveness and a photoresponsive substance).
In addition, as said light absorption substance, a gold nanoparticle etc. can be used, for example.

The method for binding the target substance and the magnetic particles is not particularly limited and can be appropriately selected according to the purpose. Examples thereof include physical adsorption, antigen-antibody reaction, DNA hybridization, biotin-avidin binding, A known binding method such as a chelate bond or an amino bond can be used. Examples of the binding method by physical adsorption include a method of binding the target substance and the magnetic particles using an electrostatic binding force such as hydrogen bonding.

When the bonding method using physical adsorption is used, there is an advantage that the magnetic particles can be easily implemented without prior treatment. However, the magnetic particles do not specifically adsorb only to the target substance, and may bind to impurities other than the target substance contained in the liquid sample. Therefore, as a method of binding the target substance and the magnetic particles, the magnetic particles are subjected to prior treatment, the antigen-antibody reaction, the DNA hybridization, the biotin-avidin bond, the chelate bond, It is preferable that the target substance and the magnetic particles are specifically bound by a binding method such as an amino bond.

In addition, as a method of binding the magnetic beads and the light absorbing material when the magnetic particle is a binding particle with the light absorbing material, the method described as the method of binding the target material and the magnetic particle, A similar method can be mentioned.

-Liquid sample introduction plate-
The liquid sample introduction plate is disposed on the surface side opposite to the side irradiated with the light as the propagating light transmitted from the rear surface side or the front surface side when the liquid sample is introduced onto the surface. A translucent plate capable of propagating, a reflective plate capable of propagating reflected light of light irradiated from the surface side as the propagating light as the propagating light, and the liquid sample. The near-field light can be generated on the surface by the introduction plate introduced on the surface, and the liquid sample introduced on the surface and light irradiated on the surface under total reflection conditions It is formed with any of the detection plates.
The propagating light is generally light that does not include near-field light that shows abrupt attenuation at a position that is a distance of several hundred nm to several μm from the generation source. It means that near-field light is not included, and means light that does not show abrupt attenuation at a position separated from the surface of the liquid sample introduction plate by a distance of several hundred nm to several μm. The near-field light means light that exhibits abrupt attenuation at a position separated from the surface of the liquid sample introduction plate by a distance of several hundred nm to several μm.

There is no restriction | limiting in particular as said light transmission board, According to the objective, it can select suitably, For example, well-known light transmission boards, such as a glass plate used for the observation stage of a well-known transmission microscope, a plastic plate, are used. Can be used.
The reflecting plate is not particularly limited and may be appropriately selected depending on the purpose. For example, a known plate such as a glass plate, a plastic plate, or a metal plate used for an observation stage of a known episcopic microscope is used. A reflector can be used.
The introduction plate is not particularly limited and may be appropriately selected according to the purpose. For example, the introduction plate includes the light transmission plate and the reflection plate, and is a known plate for introducing other liquid samples. A shaped member can be used.
The detection plate is not particularly limited and may be appropriately selected depending on the purpose. A known detection plate such as a known surface plasmon resonance sensor or a known optical waveguide mode sensor is used. be able to.

There is no restriction | limiting in particular as said liquid sample introduction board, Although it can select suitably according to the objective, It is preferable that the said surface is surface-treated with the adsorption inhibitor which suppresses adsorption | suction of the said conjugate | bonded_body. When such a surface treatment is performed, it is possible to suppress the combined body from being adsorbed on the surface of the liquid sample introduction plate, and to assist the variation by the magnetic field application unit.
There is no restriction | limiting in particular as said adsorption inhibitor, According to the kind of substance which comprises the said conjugate | bonded_body, it can select from a well-known adsorption inhibitor suitably.
For example, when the target substance is the protein, a known blocking method that suppresses adsorption of the protein can be selected as the surface treatment technique. The blocking method is not particularly limited, and examples thereof include a method using polyethylene glycol, a method using ethanolamine, and a method using skim milk.

There is no restriction | limiting in particular as a structure of the said liquid sample holding | maintenance part, According to the objective, it can select suitably, For example, you may be comprised with the said liquid sample introduction plate itself, and the said liquid sample is cover glass etc. The plate-like translucent member and the liquid sample introduction plate may be sandwiched to hold the liquid layer of the liquid sample on the surface of the liquid sample introduction plate.
In addition, the liquid sample holder may be configured as a bowl-shaped liquid cell whose bottom surface is formed of the liquid sample introduction plate.
In addition, as the liquid sample holding unit, a region on the surface of one liquid sample introduction plate may be fractionated into a plurality of channels.

Moreover, it is preferable that a flow path capable of feeding liquid is formed between the outside and the space on the surface of the liquid sample introduction plate as the liquid sample holding unit.
That is, according to the target substance detection device, since the target substance can be detected ignoring the presence of the contaminants adsorbed on the liquid sample introduction plate, the cleaning process of the liquid sample introduction plate is sequentially performed. Therefore, when the flow path is formed in the liquid sample holder, the liquid sample is simply exchanged through introduction and discharge of the liquid sample through the flow path. The next detection can proceed, and the detection operation can be made more efficient.
In the present specification, the “cleaning process” means a process of removing the contaminants adsorbed on the surface of the liquid sample introduction plate by a physical polishing process, a peeling process using chemicals, or a dissolution process. , It does not include a process of rinsing with water when changing the liquid sample.

<Light irradiation part>
The light irradiation unit is formed of any one of a back surface side light irradiation unit, a front surface side light irradiation unit, a side surface side light irradiation unit, and a total reflection light irradiation unit.

The back surface side light irradiating unit can irradiate the light from the back surface of the liquid sample introducing plate when the liquid sample introducing plate is formed of the light transmitting plate.
There is no restriction | limiting in particular as a structure of the said back surface side light irradiation part, According to the objective, it can select suitably, For example, it can comprise similarly to the well-known light irradiation part used for a well-known transmission microscope. .

The surface-side light irradiating unit can irradiate the light from the surface side of the liquid sample introducing plate when the liquid sample introducing plate is formed of either the light transmitting plate or the reflecting plate.
There is no restriction | limiting in particular as a structure of the said surface side light irradiation part, According to the objective, it can select suitably, For example, when forming with the said reflecting plate, the well-known light irradiation used for a well-known episcopic microscope In the case of being formed of the light transmitting plate, it can be configured in the same manner as a known light irradiation unit used for a known transmission microscope.

The side-side light irradiating unit is configured so that the liquid sample is introduced from the side surface side of the liquid sample introduction plate with respect to the liquid sample held on the liquid sample introduction plate when the liquid sample introduction plate is formed by the introduction plate. The light can be irradiated in a direction parallel to the in-plane direction of the surface of the sample introduction plate.
There is no restriction | limiting in particular as a structure of the said side surface side light irradiation part, According to the objective, it can select suitably, For example, it can comprise similarly to a well-known light irradiation part.

The total reflection light irradiation unit can irradiate the light with the total reflection condition on the surface when the liquid sample introduction plate is formed by the detection plate. The total reflection on the surface is not particularly limited in the incident direction of the light as long as the total reflection condition can be satisfied on the surface of the detection plate. For example, by making the detection plate surface into a waveguide structure, a grating formed on the front surface side, the back surface side, or the side surface side of the detection plate, or arranged on the front surface side, the back surface side, or the side surface side. The light can be introduced into the waveguide structure from the surface side through the prism, and the total reflection condition on the surface of the detection plate can be satisfied by utilizing the total reflection in the waveguide structure. The prism may be formed as a partial structure of the detection plate.
There is no restriction | limiting in particular as a structure of the said total reflection light irradiation part, According to the objective, it can select suitably, It is the same as the well-known light irradiation part used for a well-known surface plasmon resonance sensor or a well-known optical waveguide mode sensor. Can be configured.

In addition, as a light source in the back surface side light irradiation unit, the front surface side light irradiation unit, the side surface side light irradiation unit and the total reflection light irradiation unit, there is no particular limitation, can be appropriately selected according to the purpose, A known light emitting device such as a lamp, an LED device, or a laser light irradiation device can be used.
Further, the back side light irradiation unit, the front side light irradiation unit, and the total reflection light irradiation unit are not particularly limited with respect to optical elements other than the light source, and include a known optical microscope, a known surface plasmon resonance sensor, A known optical element used in a known optical waveguide mode sensor can be appropriately adopted depending on the purpose.

<Magnetic field application unit>
The magnetic field application unit is formed by one of a first magnetic field application unit and a second magnetic field application unit. Each of the first magnetic field application unit and the second magnetic field application unit has a role of moving the combined body introduced on the surface of the liquid sample introducing unit and a posture of the combined body. The target substance detection device uses the variation of the conjugate for detection of the target substance.
“Fluctuation” means movement of the combined body and change in posture of the combined body.

-First magnetic field application unit-
The first magnetic field application unit is disposed on the surface side or the side surface side of the liquid sample introduction plate and magnetically applies the combination in the liquid sample introduced onto the surface of the liquid sample introduction plate. Is moved in either a direction having a vector component parallel to an in-plane direction of the surface of the liquid sample introduction plate and a direction away from the liquid sample introduction plate, or the posture of the combined body is changed. It is a member to change.
The first magnetic field application unit is not particularly limited as long as it is such a member, and can be appropriately selected according to the purpose. For example, the first magnetic field application unit can be configured using a known electromagnet and permanent magnet. In the case of using the permanent magnet, for example, the permanent magnet is held on a moving member, and the magnetic field by the permanent magnet and the magnetic field by the permanent magnet are in the proximity state where the magnetic field by the permanent magnet extends on the surface of the liquid sample introduction plate. It is possible to control the movement between the liquid sample introduction plate and the separated state that does not reach the surface, and to turn on and off the application state of the magnetic field on the surface of the liquid sample introduction plate. In addition, for example, a known magnetic shield member is controlled to open and close in an open state in which the magnetic field is applied to the surface of the liquid sample introduction plate and a shield state in which the magnetic field is not applied to the surface of the liquid sample introduction plate. The application state of the magnetic field to the surface of the liquid sample introduction plate can be turned on and off. When the electromagnet is used, on / off control of the application state of the magnetic field to the surface of the liquid sample introduction plate can be performed through excitation and demagnetization of the electromagnet.
The first magnetic field application unit is not particularly limited, but has a through-hole, an incomplete ring shape such as a U shape, or a plurality of members arranged in an annular or incomplete ring shape. It is preferable that it is the structure comprised. When the first magnetic field application unit is formed in this way, the surface side of the liquid sample introduction plate through the through hole or the inside of the ring or the incomplete ring when the surface side light irradiation unit is used. The light propagation from the surface of the liquid sample introduction plate is propagated in any case of the front side light irradiation unit, the back side light irradiation unit, and the total reflection light irradiation unit. An optical signal based on light can be detected by the optical signal detection unit through the inside of the through hole or the annular member. The members arranged in an annular shape are not particularly limited as long as they do not obstruct the light irradiation or the optical path of the optical signal, and may be those that can individually control the application state of the magnetic field.

-Second magnetic field application unit-
The second magnetic field application unit is arranged on the back side of the liquid sample introduction plate and applies the combined body in the liquid sample introduced onto the surface of the liquid sample introduction plate by applying a magnetic field. The liquid sample introduction plate can be drawn toward the surface side and can be moved in a direction having a vector component in a direction parallel to the in-plane direction of the surface of the liquid sample introduction plate with the magnetic field applied. It is a member.
The second magnetic field application unit is not particularly limited as long as it is such a member, and can be appropriately selected according to the purpose. For example, the second magnetic field application unit can be configured using a known electromagnet and permanent magnet. For example, the electromagnet or the permanent magnet is held on a slide member, and the light is irradiated from the light irradiation unit on the front surface side or the back surface side of the liquid sample introduction plate or the total reflection light irradiation unit (detection) The electromagnet or the permanent magnet is directed toward a direction having a vector component in a direction parallel to an in-plane direction of the surface of the liquid sample introduction plate, and an initial state in which the electromagnet or the permanent magnet is positioned in the vicinity of the region) It can be configured by performing movement control between the moved state. When the electromagnet is used, the electromagnet is continuously or intermittently excited during the movement control. Further, the intensity of excitation may be changed during the movement control.
Also, a configuration in which a plurality of the electromagnets or permanent magnets are arranged, and the movement control is performed by holding the electromagnets or the permanent magnets on the slide member by controlling the application state of the magnetic field in each member. The same effect can be obtained.
The second magnetic field application unit is not particularly limited, but has a through-hole formed therein, an incomplete ring shape such as a U shape, or a plurality of members arranged in a ring shape or an incomplete ring shape. It is preferable that it is the structure comprised. When the second magnetic field application unit is formed in this way, light irradiation from the back side of the liquid sample introduction plate is performed through the through hole or the inside of the annular or incomplete ring in the back side light irradiation unit. It becomes possible. The members arranged in an annular shape are not particularly limited as long as they do not obstruct the light irradiation or the optical path of the optical signal, and may be those that can individually control the application state of the magnetic field.

<Optical signal detector>
The optical signal detection unit is disposed on the front surface side, the back surface side, or the side surface side of the liquid sample introduction plate, and before and after application of the magnetic field by the first magnetic field application unit, and the second magnetic field application unit. It is possible to detect a signal change of an optical signal based on the propagating light or the near-field light in any context before and after the movement.

The optical signal detector is not particularly limited and may be appropriately selected depending on the purpose. A known optical element such as a photodiode or a photomultiplier tube or a known optical element such as an objective lens may be used. Can be configured.
Further, the optical signal detection unit is not particularly limited, but it is preferable that the state of the detection region on the surface of the liquid sample introduction plate can be acquired as a two-dimensional image. If the two-dimensional image can be acquired, position information and size information of the optical signal in the two-dimensional image appearing as a light spot or a dark spot can be easily acquired, and the two-dimensional image before and after the combined body is moved. Comparing images, whether the optical signal is information related to the combined body, or the combined body such as scratches on the surface of the liquid sample introduction plate, the foreign matter, fluctuation of the light source output, etc. It is possible to clearly distinguish whether the information is not involved. In order to enable acquisition of such a two-dimensional image, an imaging device may be selected as the optical signal detection unit.
There is no restriction | limiting in particular as said imaging device, According to the objective, it can select suitably, For example, well-known image sensors, such as a CCD image sensor and a CMOS image sensor, can be used.
In addition, as a method for detecting the optical signal by the optical signal detection unit, the optical signal detection unit is out of the imageable range and the near-field light generation region (several hundred nm from the surface of the liquid sample introduction plate). In order to prevent detection leakage of the conjugate existing outside (a region several μm above), a method of performing detection once the conjugate is disposed on or near the surface of the liquid sample introduction plate is provided. preferable.
Examples of detecting the target substance include detection of the presence or absence of the target substance, detection of the amount of the target substance (quantitative measurement), real-time observation of the presence state of the target substance, and the like.

Further, the optical signal detection unit is generated from the magnetic particles when the combined body receives the propagating light or the near-field light out of the propagating light or the optical signal based on the near-field light. Only an optical signal based on the conjugate in which two or more magnetic particles are bound to the target substance can be detected as a signal change target.
That is, in the present invention, when an optical signal generated from the magnetic particles is detected as a signal change target, only the combined body in which two or more magnetic particles are bound to one target substance is set as a signal change target. The core of the technology is to eliminate the noise signal based on the single magnetic particle shown in FIG. 8 by not detecting the optical signal generated from one magnetic particle and subjecting it to signal change.
There is no restriction | limiting in particular as a structure of the said optical signal detection part which performs such a detection, According to the objective, it can select suitably, For example, one said target substance rather than the optical signal which arises from one said magnetic particle By using the fact that the optical signal generated from the combined body in which two or more magnetic particles are coupled to each other has a larger signal size, a size larger than the signal size of the optical signal generated from one magnetic particle is set as a threshold value. In this configuration, only the optical signal exceeding the threshold is detected, or the optical signal generated from the combined body in which two or more magnetic particles are bound to one target substance rather than the optical signal generated from one magnetic particle. Using the fact that the signal intensity is stronger, a configuration for detecting only an optical signal equal to or higher than the threshold by setting a signal intensity stronger than the signal intensity of the optical signal generated from one magnetic particle as a threshold, It includes be like configuration with the configuration of both these. As the signal intensity, when detecting an optical signal as a light spot, a higher signal is used as a strong signal, and when detecting an optical signal as a dark spot, a lower signal is used as a strong signal.
Further, in place of the configuration based on the setting of the threshold value, the optical signal detection unit adjusts the light source output in the light irradiation unit, the exposure time of the optical signal detection unit, and the like, so that the optical signal detection unit has one magnetic particle. It is good also as a structure which does not detect the optical signal resulting from this as a signal below a detection limit.
Moreover, as information such as the signal size, signal intensity, and detection limit of an optical signal generated from one magnetic particle necessary for adopting these configurations, for example, only the magnetic particles are dispersed in water or the like in advance. This can be grasped by detecting the sample.

Next, detection in the optical signal detection unit accompanying the movement of the combined body by the magnetic field application unit will be described.
The optical signal based on the propagating light detected by the optical signal detection unit includes an upper part of the surface of the liquid sample introduction plate in the same manner as the optical signal acquired by the known transmission microscope or the epi-illumination microscope. Optical signal 1 of transmitted light or reflected light of the propagating light propagating to the liquid sample and generated when the combined light in the liquid sample is irradiated with the propagating light and can be distinguished from the optical signal 1 An optical signal 2, an optical signal 3 that can be distinguished from the optical signal 1, and generated on the surface of the liquid sample introduction plate. There are optical signals 4 generated when the propagating light is irradiated on scratches and the foreign matter adsorbed on the surface. The optical signal also includes a noise signal caused by fluctuations in the light source output.
If the optical signals 2 to 4 and the noise signal cannot be distinguished except for the optical signal 1 processed as a background signal, the detection accuracy is lowered.
However, in the target substance detection device, the conjugate is changed based on the magnetic field application unit formed by the first magnetic field application unit and the second magnetic field application unit of the conjugate, and the fluctuation is Since it is detected as a signal change of the optical signal based on the propagating light, the optical signal 2, the optical signals 3 and 4, and the noise signal can be clearly distinguished.
That is, the optical signals 3 and 4 and the noise signal are optical signals that do not change before and after the application of the magnetic field by the first magnetic field application unit and before and after the movement of the second magnetic field application unit. Is an optical signal that changes before and after the application of the magnetic field by the first magnetic field application unit and before and after the movement of the second magnetic field application unit because it originates from the combined body including the magnetic particles. By detecting a signal change of the optical signal based on light, it is possible to detect the conjugate, and thus the target substance constituting the conjugate, with high accuracy.

Here, as an aspect of the optical signal 2 which is noted as the changing optical signal, various aspects can be taken according to the type of the magnetic particles and the type of the optical system of the target substance detection device. That is, as the optical signal 2, emitted light such as scattered light, reflected light, phase difference, transmitted light based on differential interference, fluorescence of the magnetic particles, phosphorescence, etc. emitted when the magnetic particles are irradiated with the propagating light. And an optical signal based on light absorption of the magnetic particles. In addition, when the transmitted light based on the phase difference and the differential interference is detected as an optical signal 2, each of the liquid sample holding unit, the light irradiation unit, and the optical signal detection unit may be a known phase contrast microscope, The optical system in the differential interference microscope is configured.
Examples of the change of the optical signal 2 include intensity increase / decrease, phase change, position movement, shape rotation, defocus, and appearance / disappearance.

Further, the optical signal based on the near-field light detected by the optical signal detection unit includes, in the liquid sample, the same as the optical signal acquired by a known surface plasmon resonance sensor or a known optical waveguide mode sensor. The optical signal 5 generated when the near-field light is irradiated to the combined body, the optical signal 6 generated when the contaminant in the liquid sample is irradiated with the near-field light, and the liquid There are scratches on the surface of the sample introduction plate, optical signals 7 generated when the near-field light is irradiated on the contaminants adsorbed on the surface, and the like. The optical signal also includes a noise signal caused by fluctuations in the light source output.
That is, when the near-field light is used, similarly to the case where the propagation light is used, if the optical signals 5 to 7 and the noise signal cannot be distinguished, the detection sensitivity is lowered.
However, in the target substance detection device, the conjugate is changed based on the magnetic field application unit formed by the first magnetic field application unit and the second magnetic field application unit of the conjugate, and the change is Since it is detected as a signal change of the optical signal based on near-field light, the optical signal 5, the optical signals 6 and 7, and the noise signal can be clearly distinguished.
That is, the optical signals 6 and 7 and the noise signal are optical signals that do not change before and after the application of the magnetic field by the first magnetic field application unit and before and after the movement of the second magnetic field application unit, whereas the optical signal 5 Is an optical signal that changes before and after the application of the magnetic field by the first magnetic field application unit and before and after the movement of the second magnetic field application unit because it is caused by the combined body including the magnetic particles. By detecting a signal change of the optical signal based on the field light, it is possible to detect the conjugate, and thus the target substance constituting the conjugate, with high accuracy.

Here, as the aspect of the optical signal 5 that is focused as the changing optical signal, various aspects can be taken according to the type of the magnetic particles and the type of the optical system of the target substance detection device. That is, as the optical signal 5, there are optical signals based on scattered light, emission of fluorescence of the conjugate, etc. emitted when the magnetic particles are irradiated with the near-field light, and light absorption of the magnetic particles. Can be mentioned.
In addition, examples of changes in the optical signal 5 include intensity increase / decrease, position movement, shape rotation, and appearance / disappearance.

<Other parts>
There is no restriction | limiting in particular as said other part, According to the objective, it can select suitably, For example, a 3rd magnetic field application part, a well-known transmission microscope, a well-known episcopic microscope, a well-known surface plasmon resonance sensor An arbitrary part used for a known optical waveguide mode sensor or the like can be mentioned.

-Third magnetic field application unit-
The third magnetic field application unit is further disposed on the back side of the liquid sample introduction plate and is disposed on the liquid sample introduction plate when the magnetic field application unit is formed by the first magnetic field application unit. It is a portion that can draw the combined body in the introduced liquid sample onto the surface of the liquid sample introduction plate by applying a magnetic field.

When the magnetic field application unit is formed by the second magnetic field application unit, the combined body in the liquid sample is attracted onto the surface of the liquid sample introduction plate by the application of the magnetic field. Therefore, by detecting the optical signal by the optical signal detection unit while focusing on the surface of the liquid sample introduction plate or in the vicinity thereof, the fluctuation state of the combined body attracted on the surface is detected. be able to.
However, when the magnetic field application unit is formed by the first magnetic field application unit, the optical signal detection unit performs the detection of the optical signal by focusing on the surface of the liquid sample introduction plate or the vicinity thereof. For example, immediately after the liquid sample is introduced into the liquid sample introduction plate, the conjugate does not necessarily attract the surface of the liquid sample introduction plate. It is in a state of floating in the liquid layer. When the combined body in the floating state exists outside the imageable range where the optical signal can be detected by the optical signal detection unit or outside the near-field light generation region, the combined body is not detected. Become.
Therefore, when the optical signal is detected by the optical signal detection unit while focusing on the surface of the liquid sample introduction plate or the vicinity thereof, the liquid sample is introduced into the liquid sample introduction plate, and then the coupling is performed. It is necessary to wait for the body to gravity settle on the surface of the liquid sample introduction plate, and it takes time to prepare for detection. In particular, when the specific gravity of the conjugate is small, a longer time is required.
Therefore, by applying the magnetic field by the third magnetic field applying unit, the conjugate floating in the liquid layer of the liquid sample is drawn to the surface side of the liquid sample introduction plate, thereby shortening the detection preparation time. And more efficient detection can be performed.

There is no restriction | limiting in particular as said 3rd magnetic field application part, According to the objective, it can select suitably, For example, it can comprise using a well-known electromagnet and a permanent magnet.
The third magnetic field application unit is configured to prevent the combination from being moved by the first magnetic field application unit after the combination is attracted to the surface side of the liquid sample introduction plate. On-off control is required to stop the application state of the magnetic field that attracts. In this regard, when the permanent magnet is used, for example, the permanent magnet is held on a moving member, and the magnetic field by the permanent magnet and the magnetic field by the permanent magnet are in close proximity to the liquid layer of the liquid sample. It is possible to control the movement of the sample introduction plate between a separated state that does not reach the liquid layer of the liquid sample and to turn on and off the application state of the magnetic field. When the electromagnet is used, on / off control of the application state of the magnetic field can be performed through excitation and demagnetization of the electromagnet. Further, for example, using a known magnetic shield member, an open state in which the magnetic field attracted to the surface is applied to the combined body and a shielded state in which the magnetic field attracted to the surface is not applied to the combined body. And the application state of the magnetic field can be turned on and off.
The third magnetic field application unit is not particularly limited, but has a through-hole formed therein, an incomplete ring shape such as a U shape, or a plurality of members arranged in a ring shape or an incomplete ring shape. It is preferable that it is the structure comprised. When the third magnetic field application unit is formed in this way, light irradiation from the back surface side of the liquid sample introduction plate through the through hole or the inside of the annular or incomplete ring in the back surface side light irradiation unit. Is possible. The members arranged in an annular shape are not particularly limited as long as they do not obstruct the light irradiation or the optical path of the optical signal, and may be those that can individually control the application state of the magnetic field.
Further, when the third magnetic field application unit is provided, the conjugate can be concentrated in the detection region on the surface of the liquid sample introduction plate, and the target substance can be detected with higher accuracy. It can be carried out.

(Target substance detection method)
The target substance detection method of the present invention includes a liquid sample introduction / holding step, a light irradiation step, a conjugate variation step, and an optical signal detection step, and includes other steps as necessary.

<Liquid sample introduction holding process>
In the liquid sample introduction / holding step, a liquid sample containing a target substance and magnetic particles forming a conjugate with the target substance is introduced onto the surface, and transmitted light of light irradiated from the back side or the front side is propagated light. A translucent plate capable of propagating to the side opposite to the side irradiated with the light, and the reflected light of the light irradiated from the surface side as the liquid sample is introduced onto the surface A reflector capable of propagating above the surface, an introduction plate into which the liquid sample is introduced onto the surface, and the liquid sample is introduced onto the surface and irradiated to the surface under total reflection conditions. A liquid sample introduction plate formed by any of the detection plates capable of generating near-field light on the surface by the light to be emitted, and the liquid sample can be held on the surface of the liquid sample introduction plate Liquid sample holder Against a step of holding introducing the liquid sample on the surface of the liquid sample introducing plate.
As the liquid sample, the liquid sample described for the target substance detection device can be used.
Further, as the liquid sample introduction plate, the liquid sample introduction plate described for the target substance detection device can be used.

The liquid sample is prepared by mixing the magnetic particles as a pre-process of the liquid sample introduction and holding step. That is, generally, the magnetic particles are dispersed and stored in a solution or stored in a powder form, and are added to the liquid sample and mixed at the time of use.
The mixing method of the liquid sample is not particularly limited and may be appropriately selected depending on the purpose. For example, (1) the liquid sample without the magnetic particles added is held in the liquid sample holding unit. (2) After the magnetic particles are held in the liquid sample holder, the liquid sample in which the magnetic particles have not been added is added to the liquid sample. (3) adding the magnetic particles to the liquid sample to which the magnetic particles have not been added before introducing the liquid sample into the liquid sample holding unit; Examples of the method include a method of mixing (pre-mixing method).
In particular, according to the pre-mixing method of (3), the magnetic particles in the mixing container and the combined body containing the magnetic particles are collected by the magnet through the mixing container, and these are prevented from flowing down by the magnet. In addition, by separating a part of the liquid mixture, contamination of the liquid sample introduced into the liquid sample holding unit can be suppressed and the introduced into the liquid sample holding unit In a liquid sample, the conjugate can be concentrated. As a result, it is possible to carry out detection with higher accuracy than when the methods (1) and (2) are applied.
In this step, the liquid sample may be prepared by mixing the target substance that has been solidified by drying or the like with a solution in which the magnetic particles, the labeling substance, and the weight substance are dispersed. Good.

In addition, it is important to prepare the liquid sample so as to contain the magnetic particles in a content number that is twice or more the content number of the target substance.
That is, an optical signal based on the combined body in which one magnetic particle is bonded to one target substance shown in FIG. 6 is an undetected optical signal, which leads to detection omission of the target substance.
Accordingly, by preparing the liquid sample so as to contain the magnetic particles at a content number more than twice the content number of the target substance, one magnetic substance for one target substance shown in FIG. In addition to reducing the number of the conjugates to which the particles are bound, and increasing the number of the conjugates to which two or more of the magnetic particles are bound to one target substance shown in FIG. It is preferable to suppress detection omission of the target substance.
In addition, since the magnetic particles may bind three or more to one target substance, the liquid sample has a magnetic content of three times or more the content of the target substance. It is good also as preparing so that it may contain the particle | grains, and also containing the said magnetic particle more than the content number which brings about saturation to the coupling | bonding with the said target material with respect to the content number of the said target material. .

Note that the number of the target substances present in the liquid sample is unknown at the stage before detection, but in view of the number of the target substances assumed from empirical rules, an excessive amount of the magnetic particles is introduced. Two or more magnetic particles can be bound to one target substance. In addition, the magnetic particles are introduced into the liquid sample in a plurality of times, and detection is performed each time the liquid sample is introduced, and the combined body in which two or more magnetic particles are bound to one target substance. By stopping the introduction of the magnetic particles when the number of generated optical signals is saturated, two or more magnetic particles can be bound to one target substance without waste.

<Light irradiation process>
The light irradiation step includes a back side light irradiation step of irradiating the light from the back side of the liquid sample introduction plate when the liquid sample introduction plate is formed of the translucent plate, and the liquid sample introduction plate is A surface-side light irradiating step of irradiating the light from the surface side of the liquid sample introduction plate when formed by either a light-transmitting plate or the reflection plate; and the liquid sample introduction plate is formed by the introduction plate A side surface that irradiates the liquid sample held on the liquid sample introduction plate from the side surface side of the liquid sample introduction plate in a direction parallel to the in-plane direction of the surface of the liquid sample introduction plate. It is one of a side light irradiation step and a total reflection light irradiation step of irradiating the light with total reflection conditions on the surface when the liquid sample introduction plate is formed by the detection plate.
In addition, as the said back surface side light irradiation process, it can implement by the said back surface side light irradiation part demonstrated in the said target substance detection apparatus.
Further, the surface side light irradiation step can be performed by the surface side light irradiation unit described in the target substance detection apparatus.
Further, the side surface side light irradiation step can be performed by the side surface side light irradiation unit described in the target substance detection device.
The total reflected light irradiation step can be performed by the total reflected light illumination unit described in the target substance detection device.

<Conjugate change process>
In the combined body changing step, the combined body in the liquid sample introduced onto the surface of the liquid sample introduction plate is moved in a direction parallel to an in-plane direction of the surface of the liquid sample introduction plate by applying a magnetic field. A first combined body changing step for moving in a direction having a vector component and a direction away from the liquid sample introducing plate or changing the posture of the combined body, and the back side of the liquid sample introducing plate The combined body in the liquid sample introduced onto the surface of the liquid sample introduction plate is attracted onto the surface of the liquid sample introduction plate by applying a magnetic field from a magnetic field application unit arranged and the magnetic field is applied In this state, the magnetic field application unit is moved in a direction having a vector component parallel to the in-plane direction of the surface of the liquid sample introduction plate, and the coupling is performed by following the movement of the magnetic field application unit. Which is the one step of the second coupling member variation step of changing the posture of or the conjugate move.
In addition, the first conjugate variation step can be performed by the first magnetic field application unit described in the target substance detection device.
In addition, the second conjugate variation step can be performed by the second magnetic field application unit described in the target substance detection device.
In addition, the detection accuracy can be improved by repeatedly performing the first conjugate variation step and the second conjugate variation step while sandwiching the optical signal detection step. At this time, in the target substance detection device having both the first magnetic field applying unit and the second magnetic field applying unit, the first combined body changing step and the second combined body changing step are interwoven. Can be implemented.
In the combined body changing step, the same effect can be obtained by moving the liquid sample introduction plate in a direction having a vector component parallel to the in-plane direction of the surface of the liquid sample introduction plate when a magnetic field is applied. May be.

<Optical signal detection process>
The optical signal detecting step includes the propagating light or Detecting a signal change of an optical signal based on the near-field light.
In addition, the optical signal detection step is generated from the magnetic particles when the combined body receives the propagating light or the near-field light out of the propagating light or the optical signal based on the near-field light. This is a step of detecting only an optical signal based on the conjugate in which two or more magnetic particles are bound to a target substance as a signal change target.
The optical signal detection step can be performed by the optical signal detection unit described in the target substance detection device.

<Other processes>
There is no restriction | limiting in particular as said other process, According to the objective, it can select suitably, For example, a conjugate | zygote drawing process can be mentioned.

-Assembly drawing process-
In the conjugate pulling step, when the conjugate changing step is the first conjugate changing step, further after the liquid sample introduction and holding step and before the conjugate changing step, by applying a drawing magnetic field, This is a step of drawing all or part of the combined body in the liquid sample once onto the surface of the liquid sample introduction plate.

When the conjugate variation step is performed by the first conjugate variation step, the optical signal is detected by focusing on the surface of the liquid sample introduction plate or the vicinity thereof in the optical signal detection step. For example, immediately after the liquid sample is introduced into the liquid sample introduction plate, the conjugate does not necessarily attract the surface of the liquid sample introduction plate. It is in a state of floating in the liquid layer. When the combined body in a floating state exists outside the imageable range where the optical signal can be detected in the optical signal detection step or outside the near-field light generation region, the combined body is not detected. Become.
Therefore, when the detection of the optical signal by the optical signal detection step is performed focusing on the surface of the liquid sample introduction plate or the vicinity thereof, the liquid sample is introduced into the liquid sample introduction plate, and then the coupling is performed. It is necessary to wait for the body to gravity settle on the surface of the liquid sample introduction plate, and it takes time to prepare for detection. In particular, when the specific gravity of the conjugate is small, a longer time is required.
Therefore, when the conjugate variation step is the first conjugate variation step, the conjugate pulling step is further performed to shorten detection preparation time and perform more efficient detection. preferable.
The conjugate drawing step can be performed by the third magnetic field application unit described in the target substance detection device.

In the case of performing the combined body drawing step, and in the case of performing the first combined body variation step by moving the combined body in a direction away from the liquid sample introduction plate, there is no particular limitation, After the liquid sample introduction / holding step, it is preferable to repeat the conjugate drawing step, the conjugate variation step, and the optical signal detection step in this order a plurality of times (alternating magnetic field application).
By applying the alternating magnetic field, the optical signals originating from the same combined body are repeatedly detected, so that the detection accuracy can be improved. Furthermore, it is also possible to amplify the optical signal by periodically applying the alternating magnetic field and applying a known lock-in amplifier to the frequency of the optical signal caused by the same combination. Thus, the detection sensitivity can be improved.

Hereinafter, embodiments of the present invention will be described more specifically with reference to the drawings.

[First Embodiment]
The target substance detection apparatus according to the first embodiment of the present invention is configured according to the target substance detection apparatus related to the examination described above with reference to FIG. 1, and the optical signal detection unit 5 includes the optical signal, When the combined body is irradiated with the propagating light or the near-field light, the combined body is formed from the magnetic particles, and two or more magnetic particles are bonded to one target substance (FIG. 7). Only an optical signal based on the reference) can be detected as a signal change target, and an optical signal based on one magnetic particle (see FIGS. 6 and 8) is not detected as a signal change target.

Here, the optical signal detection unit 5 is configured such that, for example, an optical signal generated from the combined body in which two or more magnetic particles are bonded to one target substance is more signal than an optical signal generated from one magnetic particle. A configuration in which only a light signal that is equal to or larger than the threshold is detected by setting a size larger than the signal size of an optical signal generated from one of the magnetic particles by using the large size, or generated from one of the magnetic particles Utilizing the fact that the optical signal generated from the combined body in which two or more magnetic particles are bound to one target substance is stronger than the optical signal, the signal of the optical signal generated from one magnetic particle. By configuring a signal intensity stronger than the intensity as a threshold value, a configuration in which only an optical signal equal to or higher than the threshold value is detected, or a configuration including both configurations is configured.

Alternatively, in place of the configuration by setting the threshold value, the optical signal detection unit 5 adjusts the light source output in the light irradiation unit 3 and the exposure time of the optical signal detection unit 5 so that the optical signal detection unit 5 has one magnetic particle. In this configuration, the optical signal generated from the signal is not detected as a signal below the detection limit.

According to such a target substance detection device according to the first embodiment, only an optical signal based on the combined body (see FIG. 7) in which two or more magnetic particles are bound to one target substance is changed in signal. If the target substance is detected, the target substance can be detected with higher accuracy while ignoring the presence of the single magnetic particle (see FIG. 8).
In the following, in the optical signal detector, two of the optical signals are generated from the magnetic particles when the combined body is irradiated with the propagating light or the near-field light, and two for one target substance. Only the optical signal based on the combined body (see FIG. 7) to which the magnetic particles are combined can be detected as a signal change target, and the optical signal based on one magnetic particle (see FIGS. 6 and 8) is signaled. Based on the premise that it is not detected as a change target, other modifications and embodiments in which high-precision detection by such an optical signal detection unit can be performed will be described.
Moreover, in the following description, since the target substance detection device according to the first embodiment is configured according to the target substance detection device according to the study shown in FIG. The target substance detection apparatus according to the first embodiment will be described.

First, a target substance detection device according to a first modification of the target substance detection device 1 according to the first embodiment will be described with reference to FIG. In addition, FIG. 11 is explanatory drawing of the target substance detection apparatus which concerns on a 1st modification.
As shown in FIG. 11, the target substance detection device 1 </ b> A according to the first modification is configured by further arranging a third magnetic field application unit 6 with respect to the target substance detection device 1 according to the first embodiment. Is done. In addition, since other than this is the same as that of the target substance detection apparatus which concerns on 1st Embodiment, description is abbreviate | omitted.

The third magnetic field application unit 6 is arranged on the back side of the liquid sample introduction plate 2 and the combined body in the liquid sample introduced into the liquid sample introduction plate 2 is applied to the liquid sample introduction plate 2 by applying a magnetic field. It can be drawn on the surface, and here is formed of an annular electromagnet having a through hole, and the light irradiation unit 3 can irradiate light from the back side of the liquid sample introduction plate 2 through the through hole.

In the target substance detection device 1A configured as described above, as in the case of using the target substance detection device 1 according to the first embodiment, the liquid sample floats in the liquid layer after the liquid sample introduction and holding step. Without waiting for the conjugate to gravity settle on the surface of the liquid sample introduction plate 2, after the liquid sample introduction / holding step and before the conjugate variation step, the third magnetic field application unit 6 draws By applying a magnetic field, all or part of the combined body in the liquid sample can be once pulled onto the surface of the liquid sample introduction plate 2 (a combined body drawing step).
Therefore, according to the target substance detection apparatus 1A, in addition to the advantages of the target substance detection apparatus 1 according to the first embodiment, the time required for detection can be shortened, and the target substance can be detected more efficiently. It can be carried out.

Next, a target substance detection device according to a second modification of the target substance detection device 1 according to the first embodiment will be described with reference to FIG. In addition, FIG. 12 is explanatory drawing of the target substance detection apparatus which concerns on a 2nd modification.
As shown in FIG. 12, in the target substance detection device 1B according to the second modification, instead of the first magnetic field application unit 4 in the target substance detection device according to the first embodiment, a first magnetic field application unit. 7 is arranged. In addition, since other than this is the same as that of the target substance detection apparatus 1 which concerns on 1st Embodiment, description is abbreviate | omitted.

The first magnetic field application unit 7 is configured by an electromagnet, and is detected on the surface of the liquid sample introduction plate 2 (received by the light irradiation unit 3 on the back surface side, and propagates above the surface). The region in the liquid sample introduction plate 2 is disposed obliquely above the surface of the liquid sample introduction plate 2 and is applied to the surface of the liquid sample introduction plate 2 by applying a magnetic field. It is moved in a direction having a vector component in a direction parallel to the in-plane direction (first combined body changing step).

FIG. 13 schematically shows the state on the surface of the liquid sample introduction plate 2 in the observation field of view observed by the imaging device 5a after the first combined body changing step performed using the first magnetic field application unit 7. Shown in The state before the first combined body changing step is the same as in FIG.

As can be understood from a comparison between FIG. 2 showing the state before the first combined body changing step and FIG. 13 showing the state after the first combined body changing step, the optical signals a and b are The optical signal changes before and after the first combined body changing step, and the optical signals c and d do not change before and after the first combined body changing step.
Therefore, according to the target substance detection device 1B, similarly to the target substance detection device 1 according to the first embodiment, the substances a ′ and b ′ that generate the optical signals a and b include the target substance, and the optical signal It can be determined that c and d are noise signals such as scratches on the surface of the liquid sample introduction plate 2, adsorption on the surface or impurities existing on the surface, fluctuations in the light source output, and the like.

After the first combined body changing step performed using the first magnetic field application unit 7, the substance a ′ that generates the optical signal a and the substance b ′ that generates the optical signal b are separated from the side surface of the liquid sample introduction plate 2. FIG. 14 shows the state when viewed from above. 14 is a cross-sectional view taken along line AA in FIG. Further, an arrow B in FIG. 14 indicates an imageable range where an optical signal can be acquired.
As shown in FIG. 14, the substance a ′ and the substance b ′ are each in a direction parallel to the in-plane direction of the surface of the liquid sample introduction plate 2 due to the magnetic field attracted obliquely from above by the first magnetic field application unit 7. Move in a direction having vector components x ₁ and x ₂ and vector components y ₁ and y ₂ in a direction away from the liquid sample introduction plate 2.
Therefore, in the target substance detection apparatus 1B, the target substance detection apparatus 1 according to the first embodiment that moves the substance a ′ and the substance b ′ only in the direction away from the liquid sample introduction plate 2, and the first conjugate fluctuation The situation after the process is different.

This difference leads to a reduction in the detection burden of the target substance.
That is, when comparing the case where the detection of the target substance based on the optical signals a and b is attempted while comparing FIG. 4 and FIG. 13, the optical signal b is the result of the disappearance of the optical signal in both figures. However, for the optical signal a, in the case shown in FIG. 4, only the detection of the target substance based on the size change is possible and the detection of the target substance based on the movement cannot be performed, whereas in the case shown in FIG. The case shown in FIG. 13 is easier to detect the target substance in that the target substance can be detected based on movement in addition to the detection of the target substance based on the change.
Therefore, the target substance detection apparatus 1B can detect the target substance with higher accuracy.

[Second Embodiment]
Next, a target substance detection apparatus according to a second embodiment of the present invention will be described with reference to FIG. FIG. 15 is an explanatory diagram of the target substance detection device according to the second embodiment.
As shown in FIG. 15, the target substance detection device 10 according to the second embodiment is configured according to a known transmission microscope, and includes a liquid sample introduction plate 12, a light irradiation unit 13, and a second magnetic field application. Unit 18 and an optical signal detection unit 15 including an imaging device 15a and an objective lens 15b.
The liquid sample introduction plate 12, the light irradiation unit 13, and the optical signal detection unit 15 are the same as the liquid sample introduction plate 2, the light irradiation unit 3, and the optical signal detection unit 5 in the target substance detection apparatus 1 according to the first embodiment. The target substance detection device 10 according to the second embodiment can be configured, and the target according to the first embodiment is provided in that a second magnetic field application unit 18 is provided instead of the first magnetic field application unit 4. It differs from the substance detection apparatus 1. Hereinafter, differences will be described.

The second magnetic field application unit 18 is disposed on the back surface side of the liquid sample introduction plate 12, and the combined body in the liquid sample introduced onto the surface of the liquid sample introduction plate 12 is liquidated by applying a magnetic field. It can be drawn onto the surface of the sample introduction plate 12 and can be moved in a direction having a vector component in a direction parallel to the in-plane direction of the surface of the liquid sample introduction plate 12 with the magnetic field applied. . Here, the second magnetic field applying unit 18 is formed out with sliding member for sliding said permanent magnet and the permanent magnet of annular through holes are formed in the direction of the X ₁ or X ₂ (not shown), The light irradiation unit 13 can irradiate light from the back side of the liquid sample introduction plate 12 through the through hole.
The combined sample is moved by using the second magnetic field application unit 18 as a magnetic field application unit, and the liquid sample introduced onto the surface of the liquid sample introduction plate 12 by application of the magnetic field from the second magnetic field application unit 18. The combined body in the liquid sample introduction plate 12 is attracted to the surface of the liquid sample introduction plate 12 and the second magnetic field application unit 18 is applied in a direction parallel to the in-plane direction of the surface of the liquid sample introduction plate 12 with the magnetic field applied. The movement is performed in a direction having a vector component, and the combined body is moved following the movement of the second magnetic field applying unit 18 (second combined body changing step). Moreover, when the 2nd magnetic field application part 18 is comprised by the some member arrange | positioned cyclically | annularly, the 2nd conjugate | bonded_body fluctuation | variation process is performed without using a slide moving member by controlling a magnetic field application state for every member. It is also possible to perform.
When the second magnetic field applying unit 18 is used, in the second combined body changing step, all or a part of the combined body in the liquid sample is applied on the surface of the liquid sample introducing plate 12 by applying the magnetic field. Therefore, after the liquid sample introduction and holding step, there is no need to wait for the conjugate floating in the liquid layer of the liquid sample to gravity settle on the surface of the liquid sample introduction plate 12.

FIG. 16 schematically shows the state on the surface of the liquid sample introduction plate 12 in the observation field observed by the imaging device 15a after the second combined body changing step. The state before the second combined body changing step is the same as FIG. In addition, FIG. 17 schematically shows a state on the surface before the second combined body changing step when anisotropy can be confirmed in the shape of the optical signal. In addition, FIG. 18 schematically shows the state on the surface after the second combined body changing step in this case.
As can be understood from the comparison between FIG. 2 and FIG. 16 showing the state after the second combined body variation process, or the comparison between FIG. 17 and FIG. 18, the optical signals a and b are The optical signal changes before and after the second conjugate variation step, and the optical signals c and d do not change before and after the second conjugate variation step.
Therefore, according to the target substance detection device 10, the substances a ′ and b ′ that generate the optical signals a and b include the target substance, and the optical signals c and d are scratches on the surface of the liquid sample introduction plate 12, It can be determined that the signal is a noise signal such as a foreign matter adsorbed on the surface or a contaminant present on the surface, or a fluctuation in light source output.

FIGS. 19 and 20 show the state when the substance a ′ for generating the optical signal a and the substance b ′ for generating the optical signal b are viewed from the side surface of the liquid sample introduction plate 12 after the second combined body changing step. Shown in 19 is a cross-sectional view taken along line AA in FIG. 16, and FIG. 20 is a cross-sectional view taken along line AA in FIG. In addition, the arrow B in FIG. 19, FIG. 20 has shown the imaging possible range which can acquire an optical signal.
As shown in FIG. 19, the substance a ′ and the substance b ′ are attracted onto the surface of the liquid sample introduction plate 12 by application of the magnetic field from the second magnetic field application unit 18, and then applied with the second magnetic field. Based on the movement (direction X ₁ or X _{2 in} FIG. 15) of the liquid sample introduction plate 12 of the unit 18 in the direction having a vector component parallel to the in-plane direction of the surface, the second magnetic field application unit 18 Is moved or rotated in a direction parallel to the in-plane direction of the surface of the liquid sample introduction plate 12.
16 and 19 show an example in which the substance a ′ and the substance b ′ move within the observation field of view, but the second magnetic field application unit 18 is arranged in the in-plane direction of the surface of the liquid sample introduction plate 12. When moving in a direction having a vector component in a parallel direction and parallel to the direction of any one side that regulates the rectangular observation field, the distance in the observation field is longer than the length of the one side. The substance a ′ and the substance b ′ can be moved out of the observation field, and highly accurate detection based on the disappearance of the optical signals a and b can be performed.
In the target substance detection apparatus according to the first and second embodiments, the optical system is irradiated with light from the back side of the liquid

sample introduction plates

2 and 12 according to the configuration of a known upright microscope, and the surface The optical signal based on the propagating light transmitted to the side is detected by the

optical signal detectors

5 and 15, and light is irradiated from the surface side of the liquid sample introduction plate according to the configuration of a known inverted microscope. And the said optical signal based on the said propagation light permeate | transmitted to the said back surface side is good also as detecting with the said optical signal detection part distribute | arranged to the said back surface side.

[Third Embodiment]
Next, a target substance detection apparatus according to a third embodiment of the present invention will be described with reference to FIG. In addition, FIG. 21 is explanatory drawing of the target substance detection apparatus which concerns on 3rd Embodiment.
As shown in FIG. 21, the target substance detection apparatus 20 is configured according to a known epi-illumination microscope, and includes a liquid sample introduction plate 22, a light irradiation unit 23, a first magnetic field application unit 24, an imaging device 25a, The optical signal detection unit 25 includes an objective lens 25b and a half mirror (such as a dichroic mirror) 25c. Note that the imaging device 25a is configured with, for example, a known CCD image sensor or the like, and can acquire a two-dimensional image. The half mirror 25c is also used as an optical element of the light irradiation unit 23 for introducing irradiation light onto the surface of the liquid sample introduction plate 22 by reflection.

Liquid sample introduction plate 22, the liquid sample is formed by the propagation possible reflector to said surface upward reflected light R _L as the propagation of the light L emitted from the front surface side while being introduced onto the surface The In addition, the liquid sample introduction plate 22 itself constitutes the liquid sample holding unit, and after the liquid sample is introduced onto the surface, a cover glass or the like is disposed so as to cover the liquid sample, whereby the liquid is introduced. Hold the sample.
The light irradiation unit 23 is configured as a surface side irradiation unit that can irradiate the light L from the surface side of the liquid sample introduction plate 22 by the reflected light from the half mirror 25c.
The first magnetic field application unit 24 is arranged on the surface side of the liquid sample introduction plate 22 and applies a magnetic field to the conjugate in the liquid sample introduced onto the surface of the liquid sample introduction plate 22. Is configured to move away from the liquid sample introduction plate 22. Here, the first magnetic field application unit 24 is formed of an annular electromagnet having a through hole formed in the center, and the light L emitted from the light irradiation unit 23 is applied to the liquid sample introduction plate 22 through the through hole. optical signal based on the reflected light R _L of the light L while being capable radiation is detectable by the optical signal detection unit 25 through the through hole.
The optical signal detection unit 25 is arranged on the surface side of the liquid sample introduction plate 22 and can detect a signal change of the optical signal based on the propagation light before and after application of the magnetic field by the first magnetic field application unit 24. The
Note that the liquid sample introduction plate 22, the light irradiation unit 23, and the optical signal detection unit 25 (the imaging device 25a, the objective lens 25b, and the half mirror 25c) can be configured according to a known episcopic microscope.

In the target substance detection device 20 configured as described above, first, the liquid sample is introduced and held on the surface of the liquid sample introduction plate 22 (liquid sample introduction and holding step).
Next, after the conjugate floating in the liquid layer of the liquid sample is gravity settled on the surface of the liquid sample introduction plate 22, the light L emitted from the light irradiation unit 23 is liquidated via the half mirror 25c. The surface of the sample introduction plate 22 is irradiated (light irradiation step), the irradiation objective lens 25b is adjusted to place the surface or the vicinity thereof within an imageable range, and the imaging device 25a emits the light L on the surface. An optical signal based on the reflected light _RL is acquired (optical signal detection step).

Next, the electromagnet of the first magnetic field application unit 24 is excited to apply the combined body in the liquid sample introduced onto the surface of the liquid sample introduction plate 22 by applying a magnetic field. And the combined body is moved in a direction away from the liquid sample introduction plate 22 (first combined body changing step).
Next, an optical signal on the surface of the liquid sample introduction plate 22 after the combined body is moved in a direction away from the liquid sample introduction plate 22 while maintaining the imageable range and the observation field of view is acquired by the imaging device 25a. (Optical signal detection step).

In the target substance detection device 20 configured in this way, the optical signals before and after the first conjugate fluctuation process in the optical signal detection process are obtained as shown in FIGS. 2 and 4, and light based on the target substance is obtained. The signal can be detected by clearly distinguishing it from noise signals such as scratches on the surface of the liquid sample introduction plate 22, adsorption on the surface or impurities present on the surface, and fluctuations in the light source output.
Therefore, according to the target substance detection device 20, the target substance can be detected with high accuracy. Further, even when the contaminants are adsorbed on the surface of the liquid sample introduction plate 22, since the detection can be performed while ignoring the presence thereof, the cleaning process for the liquid sample introduction plate 22 is not necessarily performed for each detection. Efficient detection can be performed without having to do so. Further, an optical signal generated based on various phenomena such as the scattered light, the reflected light, the fluorescence, and the light absorption can be handled as an identification signal, and can be expected to be used in a wide range of fields. In addition, as a mode of change of the optical signal, it is possible to use a phenomenon of disappearing in addition to defocusing, so that the change of the optical signal can be clearly captured.

Next, a target substance detection device according to a modification of the target substance detection device according to the third embodiment will be described with reference to FIG. In addition, FIG. 22 is explanatory drawing of the target substance detection apparatus which concerns on the modification of the target substance detection apparatus which concerns on 3rd Embodiment.
As shown in FIG. 22, in the target substance detection device 20A according to the modified example, a third magnetic field application unit 26 is further arranged with respect to the target substance detection device 20 according to the third embodiment. Instead of the magnetic field application unit 24, a first magnetic field application unit 27 is provided. In addition, since other than this is the same as that of the target substance detection apparatus 20 which concerns on 3rd Embodiment, description is abbreviate | omitted.

The third magnetic field application unit 26 is formed of an electromagnet, and is disposed on the back side of the liquid sample introduction plate 22, and the combination in the liquid sample introduced into the liquid sample introduction plate 22 is liquidated by applying a magnetic field. It can be drawn onto the surface of the sample introduction plate 22.
According to the third magnetic field application unit 26, as in the case where the target substance detection device 20 is used, after the liquid sample introduction and holding step, the combined body floating in the liquid layer of the liquid sample is a liquid sample introduction plate. Without waiting for gravity sedimentation on the surface of 22, after the liquid sample introduction and holding step, and before the combined body changing step, the application of the attracting magnetic field in the third magnetic field applying unit 26 causes the All or a part of the combined body can be once drawn on the surface of the liquid sample introduction plate 22 (a combined body drawing step).
Therefore, according to the target substance detection device 20A, in addition to the advantages of the target substance detection device 20, the time required for detection can be shortened and the target substance can be detected more efficiently.

The first magnetic field application unit 27 is composed of an electromagnet, and a detection region on the surface of the liquid sample introduction plate 22 (received by the light irradiation unit 23 on the surface side, and above the surface). The region of the liquid sample introduction plate 2 is arranged obliquely above the propagation light generation region) and introduced into the liquid sample introduced on the surface of the liquid sample introduction plate 22 by applying a magnetic field. It is moved in a direction having a vector component in a direction parallel to the in-plane direction of the surface (first combined body changing step).
When the first magnetic field application unit 27 is used instead of the first magnetic field application unit 24, optical signals before and after the first combined body changing step in the optical signal detection step are obtained as shown in FIGS. In addition to the detection of the target substance based on the change in size of the optical signal a shown in FIG. 13, the target substance can be detected based on the movement of the optical signal a, and the target substance can be detected with higher accuracy. Can be detected.

[Fourth Embodiment]
Next, a target substance detection apparatus according to a fourth embodiment of the present invention will be described with reference to FIG. FIG. 23 is an explanatory diagram of the target substance detection device according to the fourth embodiment.
As shown in FIG. 23, the target substance detection device 30 according to the fourth embodiment is configured according to a known epi-illumination microscope, and includes a liquid sample introduction plate 32, a light irradiation unit 33, and a second magnetic field application. And an optical signal detection unit 35 including an imaging device 35a, an objective lens 35b, and a half mirror 35c.
The liquid sample introduction plate 32, the light irradiation unit 33, and the optical signal detection unit 35 are the same as the liquid sample introduction plate 22, the light irradiation unit 23, and the optical signal detection unit 25 in the target substance detection device 20 according to the third embodiment. The target substance detection device 30 according to the fourth embodiment can be configured and the target according to the third embodiment in that a second magnetic field application unit 38 is provided instead of the first magnetic field application unit 24. It differs from the substance detection device 20. Hereinafter, differences will be described.

The second magnetic field application unit 38 is arranged on the back surface side of the liquid sample introduction plate 32 and applies the liquid to the combined body in the liquid sample introduced onto the surface of the liquid sample introduction plate 32 by applying a magnetic field. It can be drawn onto the surface of the sample introduction plate 32 and can be moved in a direction having a vector component in a direction parallel to the in-plane direction of the surface of the liquid sample introduction plate 32 with the magnetic field applied. . Here, the second magnetic field applying unit 38 is formed out with sliding member for sliding said permanent magnet and the permanent magnet in the direction of the X ₁ or X ₂ (not shown).
The liquid sample introduced onto the surface of the liquid sample introduction plate 32 by the application of the magnetic field from the second magnetic field application unit 38, using the second magnetic field application unit 38 as a magnetic field application unit. The combined body in the liquid sample introduction plate 32 is attracted to the surface of the liquid sample introduction plate 32 and the second magnetic field application unit 38 is applied in a direction parallel to the in-plane direction of the surface of the liquid sample introduction plate 32 with the magnetic field applied. The movement is performed in a direction having a vector component, and the combined body is moved following the movement of the second magnetic field applying unit 38 (second combined body changing step). Moreover, when the 2nd magnetic field application part 38 is comprised by the some member arrange | positioned cyclically | annularly, it is the 2nd conjugate | bonded_body fluctuation | variation process, without using a slide moving member by controlling a magnetic field application state for every member. It is also possible to perform.
When the second magnetic field application unit 38 is used, in the second combined body changing step, all or a part of the combined body in the liquid sample is applied on the surface of the liquid sample introduction plate 32 by applying the magnetic field. Therefore, it is not necessary to wait for the combined substance floating in the liquid layer of the liquid sample to gravity settle on the surface of the liquid sample introduction plate 2 after the liquid sample introduction and holding step.

In the target substance detection device 30 configured as described above, the optical signals before and after the second conjugate fluctuation process in the optical signal detection process are obtained as shown in FIGS. 2 and 16, and light based on the target substance is obtained. The signal can be detected by clearly distinguishing it from noise signals such as scratches on the surface of the liquid sample introduction plate 32, adsorption on the surface or impurities existing on the surface, and fluctuations in the light source output.
FIG. 16 shows an example in which the substance a ′ and the substance b ′ move within the observation field. However, the second magnetic field application unit 38 is parallel to the in-plane direction of the surface of the liquid sample introduction plate 32. When the object a ′ in the observation field is moved in a direction having a vector component of the direction and moving in a direction parallel to the direction of any one side defining the rectangular observation field, the distance a is longer than the length of the one side. And the substance b ′ can be moved out of the observation field, and highly accurate detection based on the disappearance of the optical signals a and b can be performed.

In addition, examples of the optical signal based on the combined body are shown in FIGS. 2, 4, 13, 16, and 18 to explain that the optical signal is caused by the scattered light, the reflected light, the fluorescence, and the like. However, this is for the convenience of drawing display, and the optical signal may be an optical signal resulting from the transmitted light due to the phase difference, the differential interference, or the like.
In addition, examples of changes in the optical signal based on the combined body are shown in FIGS. 4, 13, 16, and 18, and the positional movement, the defocus, the disappearance, and the rotation (the posture change of the combined body). As described above, as the mode of change of the optical signal, increase / decrease in intensity (intensity decrease based on the defocus, etc.), phase change (phase change after position movement), appearance (position movement from outside observation field) Can also be mentioned.

[Fifth Embodiment]
Next, a target substance detection apparatus according to a fifth embodiment of the present invention will be described with reference to FIG. FIG. 24 is an explanatory diagram of a target substance detection device according to the fifth embodiment.
As shown in FIG. 24, the target substance detection device 40 is configured according to a known surface plasmon resonance sensor, and includes a liquid sample introduction plate 42, a light irradiation unit including a light source 43a and an optical prism 43b, a first A magnetic field application unit 47 and an optical signal detection unit 45 (imaging device) are included. Note that the imaging device is composed of a known CCD image sensor or the like, for example, and can acquire a two-dimensional image.

The liquid sample introduction plate 42 is capable of generating near-field light above the surface when the liquid sample E is introduced onto the surface and irradiated with the light L irradiated on the surface under total reflection conditions. It is formed with a detection plate. Further, the liquid sample introduction plate 42 itself constitutes the liquid sample holding unit, and after the liquid sample E is introduced onto the surface, the cover glass G is disposed so as to cover the liquid sample. A liquid sample E is held.
The light irradiation unit is configured as a total reflection light irradiation unit that can irradiate the surface of the liquid sample introduction plate 42 with the light L from the light source 43a through the optical prism 43b under total reflection conditions. . Note that the total reflection light irradiation unit introduces light L emitted from the light source 43a under total reflection conditions onto the surface of the liquid sample introduction plate 42 via a grating, for example, instead of the optical prism 43b. It can also be configured.
The first magnetic field application unit 47 receives the detection region on the surface of the liquid sample introduction plate 42 (irradiation of the light L by the light irradiation unit on the back surface side, and generates the near-field light on the surface. In the in-plane direction of the surface of the liquid sample introduction plate 42 by applying a magnetic field to the combined body in the liquid sample disposed on the surface of the liquid sample introduction plate 42. It is comprised so that it may move in the direction with a vector component of the direction parallel to.

In the target substance detection device 40 configured as described above, first, the liquid sample is introduced and held on the surface of the liquid sample introduction plate 42 (liquid sample introduction and holding step).
Next, after the conjugate floating in the liquid layer of the liquid sample is gravity settled on the surface of the liquid sample introduction plate 42, the light L irradiated from the light source 43a is introduced into the liquid sample via the optical prism 43b. The surface of the plate 42 is irradiated under total reflection conditions (light irradiation step), and the optical signal S based on the near-field light on the surface is acquired by the optical signal detection unit 45 (optical signal detection step).

Next, the electromagnet of the first magnetic field application unit 47 is excited and the combined body in the liquid sample introduced onto the surface of the liquid sample introduction plate 42 is applied to the first magnetic field application unit 47 by applying a magnetic field. The combined body is moved in a direction having a vector component parallel to the in-plane direction of the surface of the liquid sample introduction plate 42 by applying a magnetic field (first combined body changing step).
Next, the optical signal on the surface of the liquid sample introduction plate 42 after the combined body is moved while maintaining the observation visual field is acquired by the optical signal detection unit 45 (optical signal detection step).

In the target substance detection device 40 configured as described above, the optical signals before and after the first conjugate fluctuation process in the optical signal detection process are obtained as shown in FIGS. 25 and 26, and the optical signal based on the target substance is obtained. e and g can be detected by clearly distinguishing them from noise signals f such as scratches on the surface of the liquid sample introduction plate 42, adsorption on the surface or impurities existing on the surface, fluctuations in the light source output, and the like. . FIG. 25 is a diagram showing a state on the surface of the liquid sample introduction plate 42 before the conjugate variation step, and FIG. 26 is a situation on the surface of the liquid sample introduction plate 42 after the conjugate variation step. FIG.
As shown in FIGS. 25 and 26, the optical signal obtained by using the near-field light has a dark field as a background due to the attenuation of the near-field light. Based on this, the target substance is detected. Although not shown, the appearance of an optical signal based on movement from outside the observation field of view can also be a detection target.
According to the target substance detection device 40, the target substance can be detected with high accuracy. In addition, even when the contaminants are adsorbed on the surface of the liquid sample introduction plate 42, it is possible to perform detection while ignoring the presence of the contaminants. Efficient detection can be performed without having to do so. Further, an optical signal generated based on the phenomenon such as the scattered light and the fluorescence can be handled as an identification signal. In addition to the position movement, the appearance / disappearance / rotation of the shape can also be used as the mode of change of the optical signal, so that the change of the optical signal can be clearly captured.

[Sixth Embodiment]
Next, a target substance detection apparatus according to a sixth embodiment of the present invention will be described with reference to FIG. FIG. 27 is an explanatory diagram of the target substance detection device according to the sixth embodiment.
As shown in FIG. 27, the target substance detection device 50 according to the sixth embodiment is configured according to a known surface plasmon resonance sensor, and includes a liquid sample introduction plate 52, a light source 53a, and an optical prism 53b. The light irradiation unit, the second magnetic field application unit 58, and the optical signal detection unit 55 are configured.
The liquid sample introduction plate 52, the light irradiation unit, and the optical signal detection unit 55 are the same as the liquid sample introduction plate 42, the light irradiation unit, and the optical signal detection unit 45 in the target substance detection device 40 according to the fifth embodiment. The target substance detection device 50 according to the sixth embodiment can be configured, and the target according to the fifth embodiment is provided in that a second magnetic field application unit 58 is provided instead of the first magnetic field application unit 47. This differs from the substance detection device 40. Hereinafter, differences will be described.

The second magnetic field application unit 58 is arranged on the back surface side of the liquid sample introduction plate 52 and applies the liquid to the combination in the liquid sample introduced onto the surface of the liquid sample introduction plate 52 by applying a magnetic field. It can be drawn onto the surface of the sample introduction plate 52 and can be moved in a direction having a vector component in a direction parallel to the in-plane direction of the surface of the liquid sample introduction plate 52 with the magnetic field applied. . Here, the second magnetic field applying unit 58 is formed out with sliding member for sliding said permanent magnet and the permanent magnet in the direction of the X ₁ or X ₂ (not shown).
The fluctuation of the combined body is caused by the liquid sample introduced onto the surface of the liquid sample introduction plate 52 by application of the magnetic field from the second magnetic field application unit 58 using the second magnetic field application unit 58 as a magnetic field application unit. The combined body in the liquid sample introduction plate 52 is attracted to the surface of the liquid sample introduction plate 52 and the second magnetic field application unit 58 is applied in a direction parallel to the in-plane direction of the surface of the liquid sample introduction plate 52 with the magnetic field applied. This is performed by moving in the direction having the vector component and moving the combined body according to the movement of the second magnetic field applying unit 58 or changing the posture of the combined body (second combined body changing step). . Moreover, when the 2nd magnetic field application part 18 is comprised by the some member arrange | positioned cyclically | annularly, the 2nd conjugate | bonded_body fluctuation | variation process is performed without using a slide moving member by controlling a magnetic field application state for every member. It is also possible to perform.
When the second magnetic field applying unit 58 is used, in the second combined body changing step, all or a part of the combined body in the liquid sample is applied on the surface of the liquid sample introducing plate 52 by applying the magnetic field. Therefore, it is not necessary to wait for the combined substance floating in the liquid layer of the liquid sample to gravity settle on the surface of the liquid sample introduction plate 52 after the liquid sample introduction and holding step.

In the target substance detection device 50 configured as described above, optical signals before and after the second conjugate fluctuation process in the optical signal detection process are obtained as shown in FIGS. 28 and 29, and the optical signal based on the target substance is obtained. h can be detected by clearly distinguishing it from a noise signal i such as a scratch on the surface of the liquid sample introduction plate 52, adsorption on the surface or impurities existing on the surface, and fluctuations in the light source output. FIG. 28 is a diagram showing a state on the surface of the liquid sample introduction plate 52 before the conjugate changing step, and FIG. 29 is a situation on the surface of the liquid sample introducing plate 52 after the conjugate changing step. FIG.

The target substance detection devices according to the fifth and sixth embodiments are configured according to the configuration of the surface plasmon resonance sensor, but as a modification of these embodiments, liquid

sample introduction plates

42 and 52 are used. As the detection plate used in the optical waveguide mode sensor and the optical system as the optical system used in the optical waveguide mode sensor, the target substance based on the movement of the conjugate is similar to these embodiments. Detection can be performed. In addition, an optical system that uses near-field light generated by total reflection for illumination, such as an optical system of a known total reflection microscope, can also be used.

In addition, the example shown by the said embodiment etc. was described in order to make invention easy to understand, and is not limited to this form.

In the above-described embodiments, the liquid sample introduction plate is configured to use the translucent plate, the reflection plate, and the detection plate. However, the liquid sample introduction plate may be configured to use the introduction plate. In this case, the side light irradiation unit is employed as the light irradiation unit, and in the optical signal detection unit disposed on the front surface side or the back surface side of the liquid sample introduction plate, scattered light from the combined body, It can be set as the structure which detects reflected light etc. Alternatively, in the optical signal detection unit disposed on the side of the liquid sample introduction plate (the side opposite to the side on which the side light irradiation unit of the liquid sample introduction plate is disposed), It can be set as the structure which detects light absorption, transmitted light, etc.

A target substance detection device according to Example 1 was produced according to the configuration of the target substance detection device 40 according to the fifth embodiment shown in FIG. Below, for convenience of explanation, each component of the target substance detection device according to the first embodiment will be described with the same reference numerals as those used for the description of the target substance detection device 40.
Specifically, as the liquid sample introduction plate 42, a planar waveguide chip in which a Si layer having a thickness of 36 nm and a SiO ₂ layer having a thickness of 368 nm are laminated in this order on a SiO ₂ substrate having a thickness of 0.725 mm is used. It was. The light source 43a is connected to a red LED light source (Thorlabs, model number M625F2) with an optical fiber having a core diameter of 600 μm with a collimating lens attached to the emission end, and a 650 nm short-pass filter and a polarization filter are arranged at the tip of the emission end. What was done was used. A prism 43b made of SiO ₂ glass is optically adhered to the back surface of the liquid sample introduction plate 42, and light from the light source 43a is incident on the surface of the liquid sample introduction plate 42 at an incident angle of 67.6 °. did.

The detection method of this example corresponds to the fifth embodiment. Norovirus virus-like particles were selected as the target substance. Further, as the magnetic particles, particles obtained by binding an anti-norovirus antibody to fluorescent magnetically labeled beads having a diameter of 200 nm (manufactured by Tamagawa Seiki Co., Ltd., FF beads Cy5 Streptavidin, model number TAB8851N2170) were used.
After the fluorescent magnetically labeled beads are mixed with the solution containing the target substance to prepare a mixed solution (liquid sample), 100 μL of the mixed solution is 2 mm thick having a through hole with a diameter of 8 mm on the liquid sample introduction plate 42. Were introduced into the liquid sample holder formed by installing the silicon rubber sheet.
After introducing the mixed solution, a cover glass G was arranged, the liquid sample holding part was covered, and the magnetic particles were gravity settled on the surface of the liquid sample introduction plate 42.
After gravity settling, a near field was formed on the surface of the liquid sample introduction plate 42 by irradiation with the incident light, and an optical signal was measured.
The optical signal was observed with a cooled CCD camera (BITRAN, model number BU-59LIR) through a 650 nm long pass filter using an optical microscope equipped with a 5 × objective lens. That is, a CCD camera provided with a 5 × objective lens and a 650 nm long pass filter is used as the optical signal detection unit 45 (imaging device). The magnetic particles include a fluorescent dye that emits fluorescence in a wavelength range having a peak at about 660 nm to 670 nm when receiving light from the light source 43a, and the imaging device only observes fluorescence from the magnetic particles. be able to.

FIG. 30 shows an image observed with an exposure time of 5 seconds before the conjugate changing step. In FIG. 30, the background is black and the optical signal detection position is white. Moreover, the visual field of the figure is approximately 2.5 mm × 2.0 mm. In the figure, several tens to hundreds of light spots can be confirmed in the entire visual field.
Next, FIG. 31 shows an image observed at an exposure time of 0.5 seconds before the conjugate changing step. The field of view is the same as that of FIG. By shortening the exposure time, it becomes impossible to observe a light spot having only one magnetic particle, and only a light spot having a plurality of the magnetic particles at the same position is observed. Thus, by controlling the exposure time, it is possible to distinguish between a light spot formed by the magnetic particle alone and a light spot formed by a plurality of the magnetic particles.
Furthermore, observation is performed after applying a magnetic field having a vector component in a direction parallel to the surface of the surface of the liquid sample introduction plate 42 using a permanent magnet as the first magnetic field applying unit 47 (after the combined body changing step). The resulting image is shown in FIG. The field of view is the same as that shown in FIGS. The solid circle and the dotted circle in FIGS. 31 and 32 are added for the convenience of explanation.
When the magnetic particles are moved from the surface of the liquid sample introduction plate 42 by the application of the magnetic field, three of the light spots in FIG. 31 observed by the CCD camera before application of the magnetic field (the solid circle in the figure). It can be seen that it has disappeared in FIG. 32 (it does not exist in the circle surrounded by a dotted line in the figure).
Here, the extincted light spot is due to the fact that the combined body of the target substance and the magnetic particles is moved away from the surface of the liquid sample introduction plate 42 or moved in the form shown in FIG. 14 by the application of the magnetic field. . The disappearance of the light spot observed in this example is considered to be due to the fact that the combined body at the position of the light spot has moved out of the near field range or out of the observation field.
31 and 32 are considered to have caused non-specific adsorption on the surface of the liquid sample introduction plate 42, and a plurality of the magnetic particles are targeted at the same location. Even when non-specific adsorption occurs without using a substance, it is possible to distinguish it from the conjugate through a target substance by using the movement of a light spot by applying a magnetic field as in this embodiment.

In this way, the detection of the target substance that suppresses false detection is performed using the fact that only the conjugate can be detected by taking the difference between the control of the exposure time and the optical signal before and after the application of the magnetic field. it can.

1, 1A, 1B, 10, 20, 20A, 30, 40, 50 Target

substance detection device

2, 12, 22, 32, 42, 52 Liquid

sample introduction plate

3, 13, 23, 33

Light irradiation unit

4, 7, 24, 27, 47 First magnetic

field application unit

5a, 15a, 25a,

35a Imaging device

5b, 15b, 25b,

35b Objective lens

25c,

35c Half mirror

5, 15, 25, 35, 45, 55 Optical signal detection unit 6 , 26 Third magnetic

field applying unit

18, 38, 58 Second magnetic

field applying unit

43a,

53a Light source

43b, 53b Optical prism L light T _L transmitted light R _L reflected light a to i optical signals a ′, b ′ substance X ₁ , X ₂ direction x ₁ , x ₂ , y ₁ , y ₂ vector component G cover glass E liquid sample S optical signal

Claims

A liquid sample containing a target substance and a magnetic particle that forms a conjugate with the target substance and has photoresponsiveness is introduced onto the surface, and transmitted light of light irradiated from the back side or the surface side is used as propagating light. A translucent plate capable of propagating to the surface opposite to the side irradiated with light, and the reflected light of the light irradiated from the surface side as the liquid sample is introduced onto the surface as the propagating light A reflection plate capable of propagating above the surface, an introduction plate into which the liquid sample is introduced onto the surface, and light that is introduced onto the surface and irradiated on the surface under total reflection conditions. A liquid in which a liquid sample introduction plate formed by one of detection plates capable of generating near-field light is disposed on the surface, and the liquid sample can be held on the surface of the liquid sample introduction plate A sample holder,
When the liquid sample introduction plate is formed of the translucent plate, a back side light irradiating unit that is capable of irradiating the light from the back side of the liquid sample introduction plate, and the liquid sample introduction plate is the translucent plate And the liquid sample introduction plate is formed by the introduction plate, and the liquid sample introduction plate can be irradiated with the light from the surface side of the liquid sample introduction plate. Sometimes, the liquid sample held on the liquid sample introduction plate can be irradiated with the light from the side surface side of the liquid sample introduction plate, and the liquid sample introduction plate serves as the detection plate. A light irradiating part formed by any of the total reflection light irradiating parts capable of irradiating the light with total reflection conditions on the surface when formed with
The liquid sample introduction plate is arranged on the surface side or side surface side of the liquid sample introduction plate and introduced into the liquid sample introduction plate on the surface of the liquid sample introduction plate by applying a magnetic field to the liquid sample introduction plate. A first magnetic field applying unit that moves in any direction of a vector component in a direction parallel to the in-plane direction of the surface and a direction away from the liquid sample introduction plate, or changes the posture of the combined body; The combined body in the liquid sample disposed on the back side of the liquid sample introduction plate and introduced onto the surface of the liquid sample introduction plate is placed on the surface of the liquid sample introduction plate by applying a magnetic field. A second magnetic field mark that can be drawn and movable in a direction having a vector component in a direction parallel to the in-plane direction of the surface of the liquid sample introduction plate with the magnetic field applied. And a magnetic field applying section which is formed in either part,
Either before or after application of the magnetic field by the first magnetic field application unit and before or after movement of the second magnetic field application unit while being arranged on the front surface side, the back surface side or the side surface side of the liquid sample introduction plate An optical signal detector capable of detecting a signal change of an optical signal based on the propagating light or the near-field light in a context, and
The optical signal detection unit is generated from the magnetic particles when the combined body is irradiated with the propagating light or the near-field light in the optical signal, and two or more for one target substance. Only the optical signal based on the said conjugate | bonded_body with which the said magnetic particle couple | bonded can be detected as an object of signal change, The target substance detection apparatus characterized by the above-mentioned.
When the magnetic field application unit is formed by the first magnetic field application unit, the combined body in the liquid sample that is further disposed on the back side of the liquid sample introduction plate and introduced into the liquid sample introduction plate is applied by applying a magnetic field. The target substance detection device according to claim 1, further comprising a third magnetic field application unit that can be drawn onto the surface of the liquid sample introduction plate.
3. The target substance detection device according to claim 1, wherein the optical signal detection unit can acquire the state of the detection region on the surface of the liquid sample introduction plate as a two-dimensional image.
4. The target substance detection device according to claim 1, wherein the surface of the liquid sample introduction plate is surface-treated with an adsorption inhibitor that suppresses adsorption of the conjugate.
A liquid sample containing a target substance and a magnetic particle that forms a conjugate with the target substance and has photoresponsiveness is introduced onto the surface, and transmitted light of light irradiated from the back side or the surface side is used as propagating light. A translucent plate capable of propagating to the surface opposite to the side irradiated with light, and the liquid sample is introduced onto the surface and reflected light of light irradiated from the surface side is used as the propagating light. A reflection plate capable of propagating above the surface, an introduction plate into which the liquid sample is introduced onto the surface, and light that is introduced onto the surface and irradiated on the surface under total reflection conditions. A liquid sample in which a liquid sample introduction plate formed by one of detection plates capable of generating near-field light is arranged on the surface, and the liquid sample can be held on the surface of the liquid sample introduction plate The liquid with respect to the holding part A liquid sample introduction holding step of holding introducing the liquid sample on the surface of the sample introduction plate,
When the liquid sample introduction plate is formed of the translucent plate, a back side light irradiation step of irradiating the light from the back side of the liquid sample introduction plate, the liquid sample introduction plate includes the translucent plate and the reflection A surface-side light irradiation step of irradiating the light from the surface side of the liquid sample introduction plate when formed by any of the plates; the liquid sample introduction when the liquid sample introduction plate is formed by the introduction plate A side-side light irradiation step of irradiating the liquid sample held on the plate from the side surface side of the liquid sample introduction plate and the surface when the liquid sample introduction plate is formed by the detection plate On the other hand, a light irradiation step that is one of the total reflection light irradiation steps of irradiating the light under total reflection conditions;
A direction having a vector component in a direction parallel to an in-plane direction of the surface of the liquid sample introduction plate by applying a magnetic field to the combined body in the liquid sample introduced onto the surface of the liquid sample introduction plate; From the first combined body changing step for moving in any direction away from the liquid sample introducing plate or changing the posture of the combined body, and from the magnetic field applying unit disposed on the back side of the liquid sample introducing plate The magnetic field application unit is configured to draw the combined body in the liquid sample introduced onto the surface of the liquid sample introduction plate by applying the magnetic field of the liquid sample and draw the combined body on the surface of the liquid sample introduction plate and apply the magnetic field. Is moved in a direction having a vector component in a direction parallel to the in-plane direction of the surface of the liquid sample introduction plate, and the combined body is moved in accordance with the movement of the magnetic field application unit. A conjugate fluctuation step is either a second conjugate fluctuation step of changing the posture of the conjugate,
Light based on the propagating light or the near-field light in any one of the before-and-after relations before and after the application of the magnetic field by the first combined body changing step and before and after the movement of the magnetic field applying unit by the second combined body changing step An optical signal detection step of detecting a signal change of the signal,
The optical signal detection step is generated from the magnetic particles when the combined body is irradiated with the propagating light or the near-field light in the optical signal, and two or more for one target substance. A method for detecting a target substance, the method comprising detecting only an optical signal based on the conjugate with which the magnetic particles are bound as a signal change target.
The target substance detection method according to claim 5, wherein the magnetic particles are particles that generate scattered light upon irradiation with propagating light or near-field light.
6. The target substance detection method according to claim 5, wherein the magnetic particles are spherical particles having a diameter of 50 nm to 6,500 nm.
The target substance detection method according to claim 5, wherein the magnetic particles contain a fluorescent dye.
6. The optical particle detection method according to claim 5, wherein when the optical signal detection step is a step of detecting a signal change of the optical signal based on the propagation light, the magnetic particles include a light absorbing material that generates light absorption upon receiving the propagation light. Target substance detection method.
When the conjugate variation step is the first conjugate variation step, all or part of the conjugate in the liquid sample is further applied by applying an attracting magnetic field after the liquid sample introduction and holding step and before the conjugate variation step. The target substance detection method according to any one of claims 5 to 9, wherein a conjugate drawing step of drawing the liquid sample once onto the surface of the liquid sample introduction plate is performed.