JP7028455B2 - Target substance detection device and target substance detection method - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Law "Institute of Electrical Engineers of Japan Material Sensor / Micromachine Division General Study Group" (CD-ROM), Chemical Sensor Study Group pp. 49-52, Institute of Electrical Engineers of Japan

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

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

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

The total internal reflection fluorescence microscope completely reflects incident light at the interface between the sample and the cover glass or slide glass, and uses the resulting evanescent field as excitation light to perform fluorescence observation with less background light that causes noise. It is a technique (see Patent Document 1). The 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 the Klechmann arrangement, which is formed on the gold thin film by total internal 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. It is characterized by exciting surface plasmon resonance and forming an enhanced electric field on the surface of the gold thin film. It is a technology to excite fluorescent molecules existing in the enhanced electric field by using the light enhanced near the surface of the gold thin film by surface plasmon resonance as excitation light to generate strong fluorescence and perform fluorescence observation with less background light (patented). See Document 2).

Further, as a method of causing an electric field enhancement by such total reflection of light and obtaining an enhanced electric field, for example, there is a known method as described in Non-Patent Documents 2 to 8. The present 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 emits light under total reflection conditions on the surface of the detection plate via the prism. In Non-Patent Document 2, a method of irradiating with an enhanced electric field to obtain an enhanced electric field was reported.

Non-Patent Document 3 discloses a method of generating a surface plasmon resonance using a Kletchmann arrangement to obtain an enhanced electric field. Non-Patent Document 4 discloses a method of obtaining an enhanced electric field by injecting light into a prism in a Kletchman arrangement using a dove prism to generate surface plasmon resonance. Non-Patent Document 5 and Non-Patent Document 6 disclose a method for obtaining an enhanced electric field using a resonant mirror. In Non-Patent Document 7, a metal layer and a transparent dielectric layer are laminated in this order on a prism to form a structure called a leaky mode sensor, and light is irradiated through the prism to irradiate the surface of the dielectric layer. Discloses a method of obtaining an enhanced electric field in. 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 the metal layer one by one to obtain an enhanced electric field stronger than that of a leaky mode sensor structure. The 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 provided to the flow path and surface plasmon resonance is generated on the bottom surface or the 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 to 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 region by applying a magnetic field, and excitation light is applied only to a predetermined region including this local region. This is a technique for performing detection excluding the signal of the photoresponsive label that does not form a bond between the target substance and the magnetic label.

Further, as a method for detecting and quantifying the biological substance, a method using propagating light as detection light has also been proposed. For example, as such a method, a fluorescence immunoassay method (FIA method), an enzyme-linked immunosorbent assay method (ELISA method), and the like can be mentioned.
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 method.
Further, in the ELISA method, the target substance is immobilized on a detection plate using an antigen-antibody reaction, an enzyme-labeled antibody is bound to the detection plate, a substrate that develops a color by the enzyme is added, and the target substance is changed from the color change. Is detected and quantified.
Both methods are widely used as established bio-related substance detection methods, but these methods require multiple reaction steps and repeated cleaning steps, and it takes a lot of time and effort to obtain measurement results. There is a necessary problem. Further, further improvement in detection sensitivity is required.

As a method for improving the detection sensitivity in the detection of the target substance using the bio-related substance 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 aggregated on the bottom surface side of the liquid sample container and fixed to the bottom surface of the container by an antigen-antibody reaction between the antibody arranged 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 and the liquid floating at the detection position of the concentration destination can be improved. Noise signals caused by the contaminants adsorbed on the bottom surface of the sample container, scratches on the bottom surface of the liquid sample container, fluctuations in the light source output of the detection light used for detection, and optical signals based on the conjugate. There is a problem that the detection accuracy is low because it is not possible to distinguish between the two. 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, a cleaning process for removing the contaminants is required for each detection, and there is still a problem that the detection efficiency is low. be.

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

D. R. Shankaran et al. Sensors and Actuators B, Vol. 121 (2007) 158-177 M. Fujimaki et al. Optics Express, Vol. 23 (2015) pp.10925 -10937 C. Nylander et al. Sensors and Actuators, Vol. 3 (1982/83) pp. 79 --88 O.R.Bolduc et al. Talanta, Vol. 77 (2009) pp. 1680 --1687 R. Cush et al. Biosensors and Bioelectronics, Vol. 8 (1993) pp. 347 --353 P.E. Buckle et al. Biosensors and Bioelectronics, Vol. 8 (1993) pp. 355 --363 R.P. Podgorsek et al. Sensors and Actuators B, Vol. 38 --39 (1997) pp. 349 --352 S. Hayashi et al. Applied Physics Express, Vol. 8, 022201 (2015)

In order to detect the target substance excluding the noise signal due to the scratches on the surface of the detection plate, the contaminants adsorbed on the surface or existing on the surface, the fluctuation of the light source output, etc., the present inventor of the present invention. We are studying the detection of the target substance by binding magnetic particles to the target substance and moving the bonded body by applying a magnetic field.
Specifically, we are considering the following methods.

First, the target substance detection device according to the study will be described with reference to FIG. Note that FIG. 1 is an explanatory diagram of the target substance detection device according to the study.
As shown in FIG. 1, the target substance detection device 1 is configured according to a known transmission type microscope, and includes a liquid sample introduction plate 2, a light irradiation unit 3, a first magnetic field application unit 4, an image pickup device 5a, and an image pickup device 5a. It is composed of an optical signal detection unit 5 composed of an objective lens 5b. The image pickup device 5a is composed of, for example, a known CCD image sensor or the like, and can acquire a two-dimensional image.

In the liquid sample introduction plate 2, the transmitted light TL of the light _L emitted from the back surface side while the liquid sample containing the target substance and the magnetic particles forming the bond with the target substance is introduced onto the front surface. It is formed of a translucent plate that can propagate above the surface as propagating light. Further, the liquid sample introduction plate 2 constitutes a liquid sample holding portion by itself, and after the liquid sample is introduced onto the surface, a cover glass or the like is arranged so as to cover the liquid sample, whereby the liquid sample is arranged. To hold.
The light irradiation unit 3 is configured as a back surface side light irradiation unit capable of irradiating light L from the back surface side of the liquid sample introduction plate 2.
Further, 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 combined body 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 application unit 4 is formed of an annular electromagnet having a through hole formed in the center, and an optical signal based on the transmitted light TL of the light _L emitted from the light irradiation unit 3 is the through hole. It can be detected by the optical signal detection unit 5 through the light signal detection unit 5.
The optical signal detection unit 5 is arranged on the surface side of the liquid sample introduction plate 2 and can detect the signal change of the optical signal based on the propagated light before and after the application of the magnetic field by the first magnetic field application unit 4. To.

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 has gravity settled on the surface of the liquid sample introduction plate 2, light L is irradiated from the back surface side of the liquid sample introduction plate 2 (light irradiation step). ), The objective lens 5b is adjusted to put the surface or its vicinity within the imageable range, and the image pickup device 5a acquires an optical signal on the surface (optical signal detection step). Here, the imageable range refers to a range in which an optical signal can be acquired at the depth of focus and its vicinity.

FIG. 2 schematically shows the state on the surface of the liquid sample introduction plate 2 in the observation field of view observed by the image pickup 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 of the transmitted light propagated above the surface of the liquid sample introduction plate 2 with respect to the liquid sample. Four optical signals a to d that can be distinguished from the background signal are observed by the contrast difference with the signal (background signal). FIG. 2 shows that the optical signals a and d are observed as light spots, and that the optical signals b and c are observed as dark spots.

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

Next, by applying a magnetic field to the conjugate in the liquid sample introduced onto the surface of the liquid sample introduction plate 2 by exciting the electromagnet of the first magnetic field application unit 4, the first magnetic field application unit 4 The conjugate is moved toward the liquid sample introduction plate 2 in a direction away from the liquid sample introduction plate 2 (combined variable step).
Next, the image pickup device 5a acquires an optical signal on the surface of the liquid sample introduction plate 2 after moving the conjugate in a direction away from the liquid sample introduction plate 2 while maintaining the imageable range and the observation field of view. (Optical signal detection step).

FIG. 4 schematically shows the state on the surface of the liquid sample introduction plate 2 in the observation field of view observed by the image pickup device 5a after the bond fluctuation step.
As can be understood through comparison between FIG. 2 showing the state before the bond change step and FIG. 4 showing the state after the bond change step, the optical signals a and b are before and after the bond change step. The optical signal changes, and the optical signals c and d do not change before and after the conjugate change step.
From this, it can be seen that the substances a'and b'that generate the optical signals a and b are the conjugate containing 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 to change before and after the conjugate fluctuation step, are scratches on the surface of the liquid sample introduction plate 2, adsorbed on the surface or contaminants existing on the surface, and a light source. It can be seen that it is a noise signal such as output fluctuation.

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

In the optical signal a, the size of the light spot is observed to be large before and after the conjugate fluctuation step (see FIG. 4). This is because although the substance a'is within the imageable range of the optical signal detection unit 5, it is from the depth of focus in the state where the surface of the liquid sample introduction plate 2 is in focus before the bond fluctuation step. Since it deviates, the size of the light spot is observed to be large (see FIG. 5).
On the other hand, it is confirmed that the optical signal b disappears after the conjugate change step (see FIG. 4). This is because the substance b'has moved out of the imageable range of the optical signal detection unit 5 (see FIG. 5).

As described above, in the target substance detection device 1, the optical signal based on the target substance is adsorbed on the surface of the liquid sample introduction plate 2, the contaminants existing on the surface, and the output of the light source. Since it can be detected by clearly distinguishing it from noise signals such as fluctuations, 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 2, detection can be performed ignoring the presence thereof, so that the cleaning treatment for the liquid sample introduction plate 2 is not necessarily performed for each detection. It is not necessary to perform it, and efficient detection can be performed.

By the way, the conjugate used for detection by the target substance detection device 1 is a bond between the target substance and the magnetic particles.
When the target substance does not generate optical signals a and b distinguishable from the background signal exemplified in FIG. 4, the magnetic particles may generate an optical signal distinguishable from the background signal. preferable.
In addition to this, as a method for identifying the conjugate as optical signals a and b, a method for binding a labeling substance that generates an optical signal distinguishable from the background signal to the target substance can be considered, but the target substance can be used. Since the step of binding the labeling substance is required, the preparation for detection becomes complicated.

Therefore, once again, the detection of the target substance using the target substance detection device 1 is provided with a function (O) of generating an optical signal that can be distinguished from the background signal of the labeling substance in the magnetic particles (M). 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 the application of the magnetic field from the first magnetic field application unit 4 as an optical signal based on the target substance.
Here, when the magnetic particles (M) have a function (O) of generating an optical signal that can be distinguished from the background signal (the magnetic particles in this case are referred to as MO), the first magnetic field application unit 4 The source of the optical signal that changes before and after the application of the magnetic field of is considered as follows.

First, as shown in FIG. 6, it is conceivable that one magnetic particle MO is bound to one target substance T.
Further, as shown in FIG. 7, it is conceivable that two or more magnetic particles MO are bonded to one target substance T. In the illustrated example, a case where two magnetic particles MO are bonded to one target substance T is shown as a representative.
Further, as shown in FIG. 8, the case where the magnetic particle MO is a single substance can be considered. In particular, in the liquid sample, in order to prevent the detection omission of the target substance T, it is preferable to prepare the liquid sample by increasing the content of the magnetic particles MO with respect to the content of the target substance T. It is assumed that the magnetic particle MO alone, which is unbonded to the substance T, is contained.

Here, since the magnetic particle MO alone is not bound to the target substance T, the optical signal based on the magnetic particle MO alone shown in FIG. 8 becomes 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 bound to one target substance T shown in FIG.

On the other hand, when two or more magnetic particles MO are bound to one target substance T shown in FIG. 7, the optical signal based on the combined body is the optical signal derived from one magnetic particle MO shown in FIGS. 6 and 8. Can be distinguished.
That is, an optical signal derived from two or more magnetic particle MOs 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 a signal strength, it can be distinguished from an optical signal derived from one magnetic particle MO.
Therefore, in the detection of the optical signal, if only the optical signal based on the conjugate in which two or more magnetic particles MO are bonded to one target substance T is detected as the target of the signal change, the presence of the noise signal. It is possible to perform more accurate detection ignoring.

Here, the target substance detection device using the propagated light has been described as an example, but the magnetic particle MO may also be used in the target substance detection device using the enhanced electric field (near field light). The situation is similar.

The present invention solves the above-mentioned problems in the prior art, and provides a target substance detection device and a target substance detection method capable of detecting a target substance with high accuracy when an optical signal generated from magnetic particles is used for detection. The purpose.

The present invention is based on the above findings, and the means for solving the above-mentioned problems are as follows. That is,
<1> A liquid sample containing a target substance and magnetic particles that form a bond with the target substance and have photoresponsiveness is introduced onto the front surface, and light is emitted from either the back surface side or the front surface side. A translucent plate capable of propagating to the surface side opposite to the side to which the light is irradiated, the light to which the liquid sample is introduced onto the surface and irradiated from the surface side. A reflector capable of propagating the reflected light of the above surface as the propagating light, an introduction plate into which the liquid sample is introduced onto the surface, and an introduction plate into which the liquid sample is introduced onto the surface and with respect to the surface. A liquid sample introduction plate formed by any of the detection plates capable of generating near-field light by the light irradiated under the total reflection condition is arranged, and the liquid sample is the liquid sample introduction plate of the liquid sample introduction plate. A liquid sample holding portion that can be held on the front surface and a back surface that can irradiate the light from the back surface side of the liquid sample introduction plate when the liquid sample introduction plate is formed by the translucent plate. Surface side light irradiation that enables irradiation of the light from the surface side of the liquid sample introduction plate when the side light irradiation unit and the liquid sample introduction plate are formed by either the light transmission plate or the reflection plate. The liquid sample held on the liquid sample introduction plate when the liquid sample introduction plate is formed by the introduction plate can be irradiated with the light from the side surface side of the liquid sample introduction plate. Formed by either the side surface side light irradiation unit or the total reflected light irradiation unit capable of irradiating the surface with the light under the total reflection conditions when the liquid sample introduction plate is formed by the detection plate. The liquid sample is arranged on the back surface side of the liquid sample introduction plate and the combined body in the liquid sample introduced on the surface of the liquid sample introduction plate by applying a magnetic field. A magnetic field that can be attracted onto the surface of the introduction plate and can move 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 in a state where the magnetic field is applied. The propagating portion and the propagating light and the near-field light before and after the movement of the application portion and the magnetic field application portion are arranged on any of the front surface side, the back surface side, and the side surface side of the liquid sample introduction plate. There is an optical signal detection unit that can detect the signal change of the optical signal based on any of the above lights and can acquire the state of the detection region on the surface of the liquid sample introduction plate as a two-dimensional image. Then, among the optical signals, the optical signal detection unit is the light of either the propagating light or the proximity field light of the coupled body. It is possible to detect only an optical signal generated from the magnetic particles when irradiated with the above magnetic particles and based on the conjugate in which two or more of the magnetic particles are bound to one target substance as a target of signal change. The surface of the liquid sample introduction plate is surface-treated with an adsorption inhibitor that suppresses the adsorption of the bonded body, and the combined body in which the magnetic field application portion exists within the image forming range of the two-dimensional image is described . A target substance detection device characterized in that it can be moved to any position within the image-forming range and outside the image-forming range by following the movement of the magnetic field application unit .
<2> Light emitted from either the back surface side or the front surface side while a liquid sample containing the target substance and magnetic particles forming a conjugate with the target substance and having photoresponsiveness is introduced onto the front surface. A translucent plate that can propagate the transmitted light to the surface side opposite to the side to which the light is irradiated, the light that the liquid sample is introduced onto the surface and is irradiated from the surface side. A reflector capable of propagating the reflected light as the propagating light above the surface, an introduction plate into which the liquid sample is introduced onto the surface, and an introduction plate into which the liquid sample is introduced onto the surface and with respect to the surface. A liquid sample introduction plate formed by any of the detection plates capable of generating near-field light by the light irradiated under the total reflection condition is arranged, and the liquid sample is the liquid sample introduction plate. The liquid sample introduction and holding step of introducing and holding the liquid sample on the surface of the liquid sample introduction plate with respect to the liquid sample holding portion that can be held on the surface, and the liquid sample introduction plate is the translucent plate. A backside light irradiation step of irradiating the light from the back surface side of the liquid sample introduction plate when formed, and when the liquid sample introduction plate is formed by either the translucent plate or the reflector. A surface-side light irradiation step of irradiating the light from the surface side of the liquid sample introduction plate, for the liquid sample held on the liquid sample introduction plate when the liquid sample introduction plate is formed by the introduction plate. In the side light irradiation step of irradiating the light from the side surface side of the liquid sample introduction plate, and when the liquid sample introduction plate is formed by the detection plate, the light is emitted to the surface under all reflection conditions. On the surface of the liquid sample introduction plate by the light irradiation step, which is one of the steps of the total reflected light irradiation step of irradiating, and the application of a magnetic field from the magnetic field application portion arranged on the back surface side of the liquid sample introduction plate. The combined body in the liquid sample introduced into the liquid sample introduction plate is attracted onto the surface of the liquid sample introduction plate, and the magnetic field application portion is aligned with the in-plane direction of the surface of the liquid sample introduction plate in a state where the magnetic field is applied. The front by the composite moving step of moving in a direction having a vector component in a parallel direction and the optical signal detection unit capable of acquiring the state of the detection region on the surface of the liquid sample introduction plate as a two-dimensional image. An optical signal detection step of detecting a signal change of an optical signal based on either the propagating light or the near-field light before and after the movement of the magnetic field application portion by the coupling moving step based on the two-dimensional image. The optical signal detection step includes, among the optical signals, the connection. The conjugate is generated from the magnetic particles when the coalescence is irradiated with either the propagating light or the near-field light, and two or more of the magnetic particles are bound to one target substance. In the step of detecting only the optical signal based on the above as the target of the signal change, the surface of the liquid sample introduction plate is surface-treated with an adsorption inhibitor that suppresses the adsorption of the conjugate , and the conjugate movement step is the step. This is a step of following the movement of the magnetic field application portion to move the conjugate existing in the imageable range of the two-dimensional image to either the position within the imageable range or outside the imageable range. A method for detecting a target substance.
<3> The target substance detection method according to <2>, wherein the magnetic particles are particles that generate scattered light by being irradiated with either propagating light or near-field light.
<4> The target substance detection method according to <2>, wherein the magnetic particles are spherical particles having a diameter of 50 nm to 6,500 nm.
<5> The target substance detection method according to <2> above, wherein the magnetic particles contain a fluorescent dye.
<6> 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 are irradiated with the propagating light and contain a light absorbing substance that causes light absorption. > The target substance detection method.

INDUSTRIAL APPLICABILITY According to the present invention, there is provided a target substance detection device and a target substance detection method that can solve the above-mentioned problems in the prior art and can detect 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 study. It is a figure which schematically showed the state on the surface of the liquid sample introduction plate in the observation field of view observed by the image pickup device. FIG. 2 is a cross-sectional view taken along the line AA in FIG. FIG. 1 is a diagram schematically showing the state on the surface of the liquid sample introduction plate in the observation field of view observed by the imaging device after the combined body change step. FIG. 4 is a cross-sectional view taken along the line AA in FIG. It is a figure which shows the state that one magnetic particle MO is bonded to one target substance T. It is a figure which shows the appearance that two magnetic particles MO are bonded to one target substance T. It is explanatory drawing which shows the magnetic particle MO simple substance. It is a figure which shows the appearance that three magnetic particles MO are bonded to one target substance T. It is a figure which shows the bonded particle of a magnetic particle which does not have a photoresponsiveness, and a photoresponsive substance. It is explanatory drawing of the target substance detection apparatus which concerns on 1st modification. It is explanatory drawing of the target substance detection apparatus which concerns on the 2nd modification. FIG. 2 is a diagram schematically showing the state on the surface of the liquid sample introduction plate in the observation field of view observed by the imaging device after the combined body change step. 13 is a cross-sectional view taken along the line AA in FIG. It is explanatory drawing of the target substance detection apparatus which concerns on 2nd Embodiment. FIG. 3 is a diagram (3) schematically showing the state on the surface of the liquid sample introduction plate in the observation field of view observed by the imaging device after the combined body change step. It is a figure which schematically showed the state on the surface of the liquid sample introduction plate in the observation field of view observed by the image pickup device before the combined body change process. It is a figure (4) which shows typically the state on the surface of the liquid sample introduction plate in the observation field of view observed by the image pickup device after the combined body change process. 16 is a cross-sectional view taken along the line AA in FIG. FIG. 18 is a cross-sectional view taken along the line AA 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 state on the surface of the liquid sample introduction plate before the combined body change process. It is a figure (1) which shows the state on the surface of the liquid sample introduction plate after the combined body change step. It is explanatory drawing of the target substance detection apparatus which concerns on 6th Embodiment. It is a figure (2) which shows the state on the surface of the liquid sample introduction plate before the combined body change process. It is a figure (2) which shows the state on the surface of the liquid sample introduction plate after the combined body change step. It is an image which shows the state on the surface of the liquid sample introduction plate observed in the image pickup apparatus with an exposure time of 5 seconds before the combined body change process. It is an image which shows the state on the surface of the liquid sample introduction plate observed in the image pickup apparatus with an exposure time of 0.5 second before the combined body change process. It is an image which shows the state on the surface of the liquid sample introduction plate observed in the image pickup device with an exposure time of 0.5 second after a combined body change step.

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

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

-Liquid sample-
The liquid sample contains a target substance and magnetic particles that form a bond with the target substance and have photoresponsiveness.
As the specific liquid sample, for example, a solid sample such as blood, saliva, urine, liquid chemicals, environmental water, water and sewage, beverage, food homogenized solution, swab, powder, etc. was dissolved in a solvent such as water. Examples thereof include a solution and a gas phase concentrate that collects gas and fine particles in the gas phase. Specific examples of the target substance include DNA, RNA, protein, virus, fungus, pollutant and the like.

As described above, the magnetic particles have photoresponsiveness. Here, the "optical responsiveness" refers to a property of generating an optical signal capable of detecting a signal change by the optical signal detection unit by being irradiated with propagating light or near-field light.
The magnetic particles are not particularly limited as long as they have such properties, and can be appropriately selected depending on the intended purpose, and known magnetic beads can be used.

For example, when the magnetic particles are irradiated with the propagating light or the near-field light to generate scattered light as an optical signal whose signal change can be detected by the optical signal detection unit, the magnetic particles are organic polymers. Known magnetic beads that generate the scattered light by being irradiated with light such as ferrite 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, but spherical particles having a diameter of 50 nm to 6,500 nm are preferable.
With respect to the upper limit of the diameter, for an optical signal based on one magnetic particle, it is based on the conjugate containing three or more of the magnetic particles rather than an optical signal based on the conjugate containing two magnetic particles. The optical signal is considered to be a significantly identifiable optical signal. Therefore, for example, as shown in FIG. 9, it is preferable that the diameter is limited so that three or more magnetic particles MO having photoresponsiveness can be bound 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, and the magnetic particles MO having three photoresponsiveness are bound to one target substance T. The diameter of the photoresponsive magnetic particle MO needs to be 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 optically in the visible light wavelength region such as a virus, the diameter is more preferably 1,000 nm or less. Note that FIG. 9 is a diagram showing a state in which three magnetic particles MO having photoresponsiveness are bound to one target substance T.
Further, regarding the lower limit of the diameter, since a light source in the visible light wavelength range (400 nm to 700 nm) is mainly used, a conjugate in which two or more magnetic particles MO having photoresponsiveness to one target substance are bonded. Since it is desirable that the size is one-fourth or more of the wavelength of the light source, it is desirable that the diameter is 50 nm or more.
The spherical particles include not only true spherical particles but also distorted spherical particles such as elliptical spherical particles. The maximum diameter of the distorted spherical particles corresponds to the diameter of the particles. Further, the magnetic particle MO having photoresponsiveness may be composed of a conjugate containing the magnetic particles M having no photoresponsiveness and the photoresponsive substance O as shown in FIG.

Further, when the magnetic particles are irradiated with the propagating light or the near-field light to generate fluorescence as an optical signal capable of detecting a signal change by the optical signal detection unit, the magnetic particles may be, for example, a fluorescent dye. Known magnetic beads containing the above can be used.

Further, when the magnetic particles are irradiated with the propagating light and generate light absorption as an optical signal whose signal change can be detected by the optical signal detection unit (identified from the optical signal of the transmitted light of the propagating light with respect to the liquid sample). (When a dark spot signal is generated by light absorption as a possible light signal), the magnetic particles include, for example, known magnetic beads containing a plurality of magnetic cores, known magnetic beads, and known light absorbing substances. Bonded particles (see, for example, FIG. 10 showing bonded particles of a magnetic particle having no photoresponsiveness and a photoresponsive substance) can be mentioned.
As the light absorbing substance, for example, gold nanoparticles or the like can be used.

The method for binding the target substance to the magnetic particles is not particularly limited and may be appropriately selected depending on the intended purpose. For example, physical adsorption, antigen-antibody reaction, DNA hybridization, biotin-avidin binding, etc. Known binding methods such as chelate binding and amino binding can be used. Examples of the bonding method by physical adsorption include a method of bonding the target substance and the magnetic particles by utilizing an electrostatic bonding force such as a hydrogen bond.

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

The method for binding the magnetic beads and the light-absorbing substance when the magnetic particles are bound to the light-absorbing substance is the method described as the method for binding the target substance and the magnetic particles. A similar method can be mentioned.

-Liquid sample introduction plate-
In the liquid sample introduction plate, the transmitted light of the light emitted from the back surface side or the front surface side as the liquid sample is introduced onto the front surface is used as the propagating light on the surface side opposite to the side on which the light is irradiated. A transmissive plate capable of propagating, a reflecting plate capable of propagating the liquid sample above the surface using the reflected light of light emitted from the surface side as the propagating light, the liquid sample. Can generate the near-field light on the surface by the introduction plate introduced onto the surface and the light that is introduced onto the surface and irradiates the surface under all reflection conditions. It is formed by any of the detection plates.
The propagating light is generally a light that does not include near-field light that exhibits abrupt attenuation at a position separated from the source by a distance of several hundred nm to several μm. It means that it does not contain near-field light, and means light that does not show abrupt attenuation at a position within a distance of several hundred nm to several μm from the surface of the liquid sample introduction plate. Further, the near-field light means light showing a rapid 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 translucent plate is not particularly limited and may be appropriately selected depending on the intended purpose. For example, a known transmissive plate such as a glass plate or a plastic plate used for an observation stage of a known transmissive microscope may be used. Can be used.
The reflector is not particularly limited and may be appropriately selected depending on the intended purpose. For example, a known glass plate, plastic plate, metal plate or the like used for an observation stage of a known epi-illumination microscope is known. A reflector can be used.
The introduction plate is not particularly limited and may be appropriately selected depending on the intended purpose. For example, the introduction plate includes the light-transmitting 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 intended purpose. A known detection plate such as a known surface plasmon resonance sensor or a detection plate used in a known optical waveguide mode sensor is used. be able to.

The liquid sample introduction plate is not particularly limited and may be appropriately selected depending on the intended purpose, but it is preferable that the surface is surface-treated with an adsorption inhibitor that suppresses the adsorption of the conjugate. When such a surface treatment is applied, it is possible to suppress the adsorbed body from being adsorbed on the surface of the liquid sample introduction plate, and it is possible to assist the fluctuation due to the magnetic field application portion.
The adsorption inhibitor is not particularly limited and may be appropriately selected from known adsorption inhibitors depending on the type of the substance constituting the conjugate.
For example, as the surface treatment method, when the target substance is the protein, a known blocking method that suppresses the adsorption of the protein can be selected. 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.

The structure of the liquid sample holding portion is not particularly limited and may be appropriately selected depending on the intended purpose. For example, the liquid sample introduction plate itself may be used, or the liquid sample may be covered with a cover glass or the like. The liquid sample may be sandwiched between the plate-shaped translucent member and the liquid sample introduction plate, and the liquid layer of the liquid sample may be held on the surface of the liquid sample introduction plate.
Further, as the structure of the liquid sample holding portion, the bottom surface may be composed of a box-shaped liquid cell having the liquid sample introduction plate.
As the liquid sample holding portion, the region on the surface of one liquid sample introduction plate may be divided into a plurality of regions to form a multi-channel.

Further, as the liquid sample holding portion, it is preferable that a flow path capable of sending liquid is formed between the outside and the space on the surface of the liquid sample introduction plate.
That is, since the target substance detection device can detect the target substance 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. Since the next detection can be performed without any need, when the flow path is formed in the liquid sample holding portion, the liquid sample is simply replaced through the introduction and discharge of the liquid sample through the flow path. The next detection can be advanced, and the detection operation can be further made more efficient.
In the present specification, the "cleaning treatment" means a treatment for removing the contaminants adsorbed on the surface of the liquid sample introduction plate by a physical polishing treatment, a peeling treatment using a chemical, or a dissolution treatment. Does not include the process of rinsing with water when exchanging the liquid sample.

<Light irradiation part>
The light irradiation unit is formed by 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 reflected light irradiation unit.

The back surface side light irradiation unit is capable of irradiating the light from the back surface of the liquid sample introduction plate when the liquid sample introduction plate is formed of the translucent plate.
The configuration of the back surface side light irradiation unit is not particularly limited and may be appropriately selected depending on the intended purpose. For example, the configuration may be the same as that of a known light irradiation unit used in a known transmission electron microscope. ..

The surface-side light irradiation unit is capable of irradiating the light from the surface side of the liquid sample introduction plate when the liquid sample introduction plate is formed by either the light-transmitting plate or the reflector.
The configuration of the surface-side light irradiation unit is not particularly limited and may be appropriately selected depending on the intended purpose. For example, when formed by the reflector, known light irradiation used in a known epi-illumination microscope is used. It can be configured in the same manner as the unit, and when it is formed of the translucent plate, it can be configured in the same manner as the known light irradiation unit used in a known transmission type microscope.

The side surface side light irradiation unit is the liquid 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.
The configuration of the side surface side light irradiation unit is not particularly limited and may be appropriately selected depending on the intended purpose. For example, the configuration may be the same as that of a known light irradiation unit.

The total reflection light irradiation unit is capable of irradiating the surface with the light under total reflection conditions 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 forming the surface of the detection plate into a waveguide structure, the grating is formed on the front surface side, the back surface side, or the side surface side of the detection plate, or is 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 on the surface of the detection plate can be satisfied by utilizing the total reflection in the waveguide structure. Further, the prism may be formed as a partial structure of the detection plate.
The configuration of the total reflected light irradiation unit is not particularly limited and may be appropriately selected depending on the intended purpose, and is the same as the known light irradiation unit used in a known surface plasmon resonance sensor or a known optical waveguide mode sensor. Can be configured in.

The 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 reflected light irradiation unit is not particularly limited and may be appropriately selected according to the purpose. A light source such as a known lamp, LED device, or laser light irradiation device can be used.
Further, the back surface side light irradiation unit, the front surface side light irradiation unit, and the total reflected light irradiation unit are not particularly limited in terms of optical elements other than the light source, such as a known optical microscope, a known surface plasmon resonance sensor, and the like. A known optical element used in a known optical waveguide mode sensor can be appropriately adopted and configured according to the purpose.

<Magnetic field application part>
The magnetic field application section is formed by either a first magnetic field application section or a second magnetic field application section. Both the magnetic field application part of the first magnetic field application part and the second magnetic field application part play a role of moving the bond introduced on the surface of the liquid sample introduction part and the posture of the bond. The target substance detection device has a role of changing, and the fluctuation of the conjugate is used for detecting the target substance.
The “variation” means the movement of the combined body and the change in the posture of the combined body.

-First magnetic field application part-
The first magnetic field application portion is arranged on the front surface side or the side surface side of the liquid sample introduction plate, and the combined body in the liquid sample introduced on the surface of the liquid sample introduction plate is magnetically charged. Is applied to move the liquid sample introduction plate in either the direction having a vector component in a direction parallel to the in-plane direction of the surface of the liquid sample introduction plate or the direction away from the liquid sample introduction plate, or the posture of the conjugate. 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 depending on the intended purpose. For example, a known electromagnet and a permanent magnet can be used. When the permanent magnet is used, for example, the permanent magnet is held in a moving member, and the magnetic field generated by the permanent magnet is in close proximity to the surface of the liquid sample introduction plate and the magnetic field generated by the permanent magnet is described. The movement can be controlled between the liquid sample introduction plate and the separated state that does not reach the surface, and the magnetic field applied state to the surface of the liquid sample introduction plate can be turned on and off. Further, for example, the opening / closing control of a known magnetic shield member is performed in an open state in which the magnetic field is applied on the surface of the liquid sample introduction plate and a shield state in which the magnetic field is not applied on the surface of the liquid sample introduction plate. Then, the state in which the magnetic field is applied to the surface of the liquid sample introduction plate can be turned on and off. Further, when the electromagnet is used, on-off control of the application state of the magnetic field on the surface of the liquid sample introduction plate can be performed through excitation and demagnetization of the electromagnet.
The first magnetic field application portion is not particularly limited, but a through hole is formed, an incomplete annular shape such as a U-shape, or a plurality of members are arranged in an annular shape or an incomplete annular shape. It is preferable that the configuration is as follows. When the first magnetic field application portion is formed in this way, the surface side of the liquid sample introduction plate is passed through the through hole or the inside of the annular to the incomplete annular when the surface side light irradiation portion is used. In addition to being able to irradiate light from the surface, the propagation of the front surface side light irradiation unit, the back surface side light irradiation unit, and the total reflected light irradiation unit is propagated above the surface of the liquid sample introduction plate. An optical signal based on light can be detected by the optical signal detection unit through the inside of the through hole or the member arranged in the annular shape. The members arranged in the annular shape are not particularly limited as long as they are arranged so as not to obstruct the optical path of the light irradiation or the optical signal, and may be individually capable of controlling the applied state of the magnetic field.

-Second magnetic field application part-
The second magnetic field application portion is arranged on the back surface side of the liquid sample introduction plate, and the combined body in the liquid sample introduced on the front surface of the liquid sample introduction plate is subjected to the application of a magnetic field. It is possible to attract the liquid sample introduction plate to the surface side and to move 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 in a state where the magnetic field is 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 depending on the intended purpose. For example, a known electromagnet and a permanent magnet can be used. For example, a region (detection) in which the electromagnet or the permanent magnet is held on the slide member and the light is emitted from the light irradiation unit on the front surface side or the back surface side of the liquid sample introduction plate or the total reflected light irradiation unit. The electromagnet or the permanent magnet is oriented toward the initial state in which the electromagnet or the permanent magnet is positioned in the vicinity of the region) and the direction having the vector component in the direction parallel to the in-plane direction of the surface of the liquid sample introduction plate. It can be configured by controlling the movement between the moved state and the moved state. When the electromagnet is used, it is in a state of being continuously or intermittently excited during the movement control. Further, the intensity of excitation may be changed during the movement control.
Further, by arranging a plurality of the electromagnets or permanent magnets and controlling the applied state of the magnetic field in each member, the electromagnets or the permanent magnets are held on the slide member to control the movement. The same effect can be obtained.
The second magnetic field application portion is not particularly limited, but a through hole is formed, an incomplete annular shape such as a U-shape, or a plurality of members are arranged in an annular shape or an incomplete annular shape. It is preferable that the configuration is as follows. When the second magnetic field application portion 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 annular in the back surface side light irradiation portion is performed. It will be possible. The members arranged in the annular shape are not particularly limited as long as they are arranged so as not to obstruct the optical path of the light irradiation or the optical signal, and may be individually capable of controlling the applied state of the magnetic field.

<Optical signal detection unit>
The optical signal detection unit is arranged on the front surface side, the back surface side, or the side surface side of the liquid sample introduction plate, and before and after the 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 of the front-back relations before and after the movement of the light.

The optical signal detection unit is not particularly limited and may be appropriately selected depending on the intended purpose. A known optical detector such as a photodiode or a photomultiplier tube or a known optical element such as an objective lens is used. Can be configured.
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, the 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 movement of the conjugate can be easily acquired. By comparing the images with each other, whether the optical signal is information related to the conjugate, or to the conjugate such as scratches on the surface of the liquid sample introduction plate, impurities, fluctuation of 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.
The image pickup device is not particularly limited and may be appropriately selected depending on the intended purpose. For example, a known image sensor such as a CCD image sensor or a CMOS image sensor can be used.
As a method of detecting the optical signal by the optical signal detection unit, the region outside the imageable range of the optical signal detection unit and the region where the near-field light is generated (several hundred nm from the surface of the liquid sample introduction plate). In order to prevent detection omission of the conjugate existing outside the region (up to several μm above), a method of once arranging the conjugate on or near the surface of the liquid sample introduction plate and then performing detection is performed. preferable.
Further, the detection of the target substance includes detection of the presence or absence of the target substance, detection of the abundance of the target substance (quantitative measurement), real-time observation of the existence state of the target substance, and the like.

Further, the optical signal detection unit is generated from the magnetic particles when the conjugate receives the propagating light or the near-field light among the optical signals based on the propagating light or the near-field light, and one of the above. Only the optical signal based on the conjugate in which two or more of the magnetic particles are bound to the target substance can be detected as the target of the signal change.
That is, in the present invention, when an optical signal generated from the magnetic particles is detected as a signal change target, only the conjugate in which two or more of the magnetic particles are bound to one target substance is the target of the signal change. The core of the technique is to eliminate the noise signal based on the magnetic particle alone shown in FIG. 8 by detecting and not subjecting the optical signal generated from one magnetic particle to the signal change.
The configuration of the optical signal detection unit that performs such detection is not particularly limited and may be appropriately selected depending on the intended purpose. For example, one target substance rather than an optical signal generated from one magnetic particle. Taking advantage of the fact that the optical signal generated from the combined body to which two or more of the magnetic particles are bonded has a larger signal size, a size larger than the signal size of the optical signal generated from one of the magnetic particles is set as a threshold value. By doing so, a configuration in which only an optical signal equal to or higher than a threshold value is detected, or an optical signal generated from the conjugate in which two or more of the magnetic particles are bonded to one target substance rather than an optical signal generated from one of the magnetic particles. Taking advantage of the fact that the signal strength is stronger, the configuration is such that only the optical signal above the threshold is detected by setting the signal strength stronger than the signal strength of the optical signal generated from one said magnetic particle as the threshold, or both of these. For example, a configuration having a configuration may be used. As for the signal intensity, when the optical signal is detected as an optical point, the one with high luminance is regarded as a strong signal, and when the optical signal is detected as a dark spot, the one with low luminance is regarded as a strong signal.
Further, instead of the configuration by setting the threshold value, by adjusting the light source output in the light irradiation unit, adjusting the exposure time of the optical signal detection unit, and the like, the optical signal detection unit is one of the magnetic particles. The optical signal generated from the above may not be detected as a signal below the detection limit.
Further, as information such as the signal size, signal strength, and detection limit of the optical signal generated from one of the magnetic particles necessary for adopting these configurations, for example, only the magnetic particles are dispersed in water or the like in advance. It can be grasped by detecting the sample.

Next, the detection in the optical signal detection unit due to the movement of the coupled body by the magnetic field application unit will be described.
The optical signal based on the propagated light detected by the optical signal detection unit includes the optical signal acquired by the known transmission type microscope or the epi-illumination type microscope, and is above the surface of the liquid sample introduction plate. Light signal 1 of transmitted light or reflected light of the propagated light to the liquid sample and generated when the propagated light is irradiated to the conjugate in the liquid sample, and can be distinguished from the optical signal 1. The optical signal 2 and the optical signal 3 generated when the propagating light is irradiated to the contaminants in the liquid sample and distinguishable from the optical signal 1 are present on the surface of the liquid sample introduction plate. There is an optical signal 4 generated when the propagating light is applied to the scratches and the contaminants adsorbed on the surface. Further, the optical signal also includes a noise signal caused by fluctuation of the light source output and the like.
When 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 deteriorated.
However, in the target substance detection device, the conjugate is changed based on the magnetic field application portion formed by the first magnetic field application portion and the second magnetic field application portion of the conjugate, and the variation is caused by the variation. 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, whereas the optical signal 2 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 conjugate containing the magnetic particles. By detecting the signal change of the optical signal based on light, it is possible to detect the conjugate and, by extension, the target substance constituting the conjugate with high accuracy.

Here, as the mode of the optical signal 2 of interest as the changing optical signal, various modes can be taken depending on the type of the magnetic particles and the type of the optical system of the target substance detection device. That is, the optical signal 2 includes scattered light, reflected light, phase difference, transmitted light based on differential interference, fluorescence of the magnetic particles, phosphorescence, and the like, which are emitted when the magnetic particles are irradiated with the propagating light. , And an optical signal based on the light absorption of the magnetic particles. When the transmitted light based on the phase contrast and the differential interference contrast is detected as the optical signal 2, each of the liquid sample holding unit, the light irradiation unit, and the optical signal detection unit can be detected by a known phase contrast microscope, known. It is constructed according to the optical system in the differential interference microscope.
Examples of the mode of change of the optical signal 2 include increase / decrease in intensity, phase change, position movement, shape rotation, focus shift, and appearance / disappearance.

Further, the optical signal based on the near-field light detected by the optical signal detection unit includes the optical signal acquired by a known surface plasmon resonance sensor or a known optical waveguide mode sensor in the liquid sample. The optical signal 5 generated when the conjugate is irradiated with the near-field light, the optical signal 6 generated when the contaminants in the liquid sample are irradiated with the near-field light, and the liquid. There are scratches existing on the surface of the sample introduction plate, optical signals 7 generated when the near-field light is applied to the contaminants adsorbed on the surface, and the like. Further, the optical signal also includes a noise signal caused by fluctuation of the light source output and the like.
That is, even when the near-field light is used, as in the case of using the propagated light, 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 portion formed by the first magnetic field application portion and the second magnetic field application portion of the conjugate, and the change is changed. Since it is detected as a signal change of the optical signal based on the close 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 conjugate containing the magnetic particles. By detecting the signal change of the optical signal based on the field light, it is possible to detect the conjugate and, by extension, the target substance constituting the conjugate with high accuracy.

Here, as the mode of the optical signal 5 of interest as the changing optical signal, various modes can be taken depending on the type of the magnetic particles and the type of the optical system of the target substance detection device. That is, the optical signal 5 includes scattered light emitted when the magnetic particles are irradiated with the near-field light, light emission such as fluorescence of the conjugate, and an optical signal based on the light absorption of the magnetic particles. Can be mentioned.
Further, examples of the mode of change of the optical signal 5 include increase / decrease in intensity, position movement, rotation of shape, and appearance / disappearance.

<Other parts>
The other parts are not particularly limited and may be appropriately selected depending on the intended purpose. For example, a third magnetic field application part, a known transmission electron microscope, a known epi-illumination microscope, and a known surface plasmon resonance sensor. , Any part used in known optical waveguide mode sensors and the like can be mentioned.

-Third magnetic field application part-
When the magnetic field application portion is formed by the first magnetic field application portion, the third magnetic field application portion is further arranged on the back surface side of the liquid sample introduction plate and on the liquid sample introduction plate. It is a portion capable of attracting 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 portion is formed by the second magnetic field application portion, the conjugate in the liquid sample is attracted to 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 by focusing on the surface of the liquid sample introduction plate or its vicinity thereof, the fluctuation state of the conjugate attracted to the surface is detected. be able to.
However, when the magnetic field application unit is formed by the first magnetic field application unit, when the optical signal detection unit detects the optical signal by focusing on the surface of the liquid sample introduction plate or its vicinity. The combined body is not necessarily attracted to the surface of the liquid sample introduction plate, and for example, immediately after the liquid sample is introduced into the liquid sample introduction plate, the combined body is the liquid sample. It is considered to be suspended in the liquid layer. When the floating body is 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 bond is not detected. Become.
Therefore, when the optical signal detection unit detects the optical signal by focusing on the surface of the liquid sample introduction plate or its vicinity, 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 settle by gravity on the surface of the liquid sample introduction plate, which takes time to prepare for detection. In particular, if the specific gravity of the conjugate is small, it will take a longer time.
Therefore, by applying the magnetic field by the third magnetic field application unit, the conjugate suspended in the liquid layer of the liquid sample is attracted to the surface side of the liquid sample introduction plate, thereby shortening the preparation time for detection. It is possible to perform more efficient detection.

The third magnetic field application unit is not particularly limited and may be appropriately selected depending on the intended purpose. For example, a known electromagnet and permanent magnet can be used.
The third magnetic field application unit attracts the coupling body to the surface side of the liquid sample introduction plate, and then the coupling body does not hinder the movement of the coupling body by the first magnetic field application unit. On-off control is required to stop the application state of the magnetic field that attracts the magnetic field. In this regard, when the permanent magnet is used, for example, the permanent magnet is held in a moving member, and the magnetic field generated by the permanent magnet is in close proximity to the liquid layer of the liquid sample and the magnetic field generated by the permanent magnet is the liquid. It is possible to control the movement of the sample introduction plate from the separated state that does not reach the liquid layer of the liquid sample, and to turn on / off the applied state of the magnetic field. Further, when the electromagnet is used, on-off control of the applied state of the magnetic field can be performed through the 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 bonded body and a shielded state in which the magnetic field attracted to the surface is not applied to the bonded body. It can be controlled by and to turn on / off the applied state of the magnetic field.
The third magnetic field application portion is not particularly limited, but a through hole is formed, an incomplete annular shape such as a U-shape, or a plurality of members are arranged in an annular shape or an incomplete annular shape. It is preferable that the configuration is as follows. When the third magnetic field application portion 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 annular in the back surface side light irradiation portion. Is possible. The members arranged in the annular shape are not particularly limited as long as they are arranged so as not to obstruct the optical path of the light irradiation or the optical signal, and may be individually capable of controlling the applied state of the magnetic field.
Further, when the third magnetic field application portion is provided, the conjugate can be gathered and 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 bond fluctuation step, and an optical signal detection step, and includes other steps as necessary.

<Liquid sample introduction and holding process>
In the liquid sample introduction and holding step, a liquid sample containing a target substance and magnetic particles forming a bond with the target substance is introduced onto the front surface, and transmitted light of light emitted from the back surface side or the front surface side is propagated. As a light-transmitting plate capable of propagating to the surface side opposite to the side to be irradiated with the light, the propagating light is the reflected light of the light emitted from the surface side while 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 an introduction plate into which the liquid sample is introduced onto the surface and the surface is irradiated under all reflection conditions. A liquid sample introduction plate formed by any of the detection plates capable of generating near-field light by the light is arranged on the surface, and the liquid sample can be held on the surface of the liquid sample introduction plate. This is a step of introducing and holding the liquid sample on the surface of the liquid sample introduction plate with respect to the liquid sample holding portion.
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, in general, the magnetic particles are dispersed and stored in a solution or stored in the form of powder, and are added to and mixed with the liquid sample at the time of use.
The method for mixing the liquid sample is not particularly limited and may be appropriately selected depending on the intended purpose. For example, (1) the liquid sample to which the magnetic particles are not added is held in the liquid sample holding portion. A method in which the magnetic particles are added to the liquid sample and mixed, (2) the liquid sample in a state where the magnetic particles are not added after the magnetic particles are held in the liquid sample holding portion. A method of introducing into the liquid sample holding portion and mixing, (3) before introducing into the liquid sample holding portion, the magnetic particles are added to the liquid sample to the liquid sample in a state where the magnetic particles are not added. A method such as a mixing method (pre-mixing method) can be mentioned.
Above all, according to the premixing method of (3), the magnetic particles in the mixing container and the combined body containing the magnetic particles are collected by a magnet through the mixing container, and these are prevented from flowing down by the magnet. By separating a part of the mixed liquid, contamination of the liquid sample introduced into the liquid sample holding portion can be suppressed, and the mixture introduced into the liquid sample holding portion can be suppressed. 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, which has been made solid by drying or the like, with a solution in which the magnetic particles, the labeling substance and the weight substance are dispersed. good.

Further, it is important that the liquid sample is prepared so as to contain the magnetic particles in a content of twice or more the content of the target substance.
That is, the optical signal based on the conjugate in which one magnetic particle is bound to the one target substance shown in FIG. 6 is regarded as an optical signal not detected, which leads to omission of detection of the target substance.
Therefore, by preparing the liquid sample so as to contain the magnetic particles in a content more than twice the content of the target substance, one said magnetism with respect to one said target substance shown in FIG. By 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 the one target substance shown in FIG. 7. , It is preferable to suppress the detection omission of the target substance.
Since the magnetic particles may bind to one target substance in an amount of three or more, the liquid sample contains the magnetic particles three times or more as much as the content of the target substance. It may be prepared to contain particles, and further, it may be prepared to contain the magnetic particles in a content equal to or higher than the content that causes saturation in binding to the target substance with respect to the content of the target substance. ..

Although the number of the target substances present in the liquid sample is unknown at the stage before detection, it is possible to introduce an excessive amount of the magnetic particles in view of the number of the target substances assumed from the empirical rule. Two or more of the magnetic particles can be bound to one of the target substances. In addition, the magnetic particles are introduced into the liquid sample in a plurality of times, and detection is performed for each introduction to the conjugate in which two or more of the magnetic particles are bound to one target substance. By stopping the introduction of the magnetic particles when the number of generated optical signals based on the light signal is saturated, two or more of the magnetic particles can be bound to one target substance without waste.

<Light irradiation process>
The light irradiation step is a back-side light irradiation step of irradiating the light from the back surface side of the liquid sample introduction plate when the liquid sample introduction plate is formed of the light-transmitting plate, and the liquid sample introduction plate is the liquid sample introduction plate. A surface-side light irradiation step of irradiating the light from the surface side of the liquid sample introduction plate when formed by either the light-transmitting plate or the reflection plate, 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 with the light 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. This is either a side light irradiation step or a total reflected light irradiation step of irradiating the surface with the light under total reflection conditions when the liquid sample introduction plate is formed by the detection plate.
The back surface side light irradiation step can be carried out by the back surface side light irradiation unit described in the target substance detection device.
Further, the surface side light irradiation step can be carried out by the surface side light irradiation unit described in the target substance detection device.
Further, the side surface side light irradiation step can be carried out by the side surface side light irradiation unit described in the target substance detection device.
Further, the total reflected light irradiation step can be carried out by the total reflected light illumination unit described in the target substance detection device.

<Combination fluctuation process>
In the combined body fluctuation step, the combined body in the liquid sample introduced onto the surface of the liquid sample introducing plate is applied with a magnetic field in a direction parallel to the in-plane direction of the surface of the liquid sample introducing plate. On the back surface side of the liquid sample introduction plate and the first bond change step of moving the liquid sample introduction plate in either the direction having the vector component or the direction away from the liquid sample introduction plate or changing the posture of the bond. The combined body in the liquid sample introduced onto the surface of the liquid sample introduction plate is attracted to the surface of the liquid sample introduction plate by the application of the magnetic field from the arranged magnetic field application unit, and the magnetic field is applied. In this state, the magnetic field application portion 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 coupled body is moved by following the movement of the magnetic field application portion. Alternatively, it is any step of the second bond variation step of changing the posture of the bond.
The first bond fluctuation step can be carried out by the first magnetic field application unit described in the target substance detection device.
Further, the second bond fluctuation step can be carried out by the second magnetic field application unit described in the target substance detection device.
Further, the detection accuracy can be improved by repeating the first bond variation step and the second bond 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 portion and the second magnetic field applying portion, the first combined body changing step and the second combined body changing step are interwoven. Can be carried out.
Further, in the combined variable step, the same effect is obtained by moving the liquid sample introduction plate 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 when a magnetic field is applied. You may.

<Optical signal detection process>
The optical signal detection step is the propagating light or the propagating light in any of the contexts before and after the application of the magnetic field by the first conjugate fluctuation step and before and after the movement of the magnetic field application portion by the second conjugate variation step. This is a step of detecting a signal change of an optical signal based on the near-field light.
Further, the optical signal detection step is generated from the magnetic particles when the conjugate receives the propagating light or the near-field light among the optical signals based on the propagating light or the near-field light, and one of the above. This is a step of detecting only an optical signal based on the conjugate in which two or more of the magnetic particles are bound to a target substance as a target of signal change.
The optical signal detection step can be carried out by the optical signal detection unit described in the target substance detection device.

<Other processes>
The other steps are not particularly limited and may be appropriately selected depending on the intended purpose. For example, a bond attracting step can be mentioned.

-Combination pulling process-
The combined attraction step is described by applying an attractive magnetic field when the combined variable step is the first combined variable step, and after the liquid sample introduction and holding step and before the combined variable change step. This is a step of temporarily pulling all or a part of the conjugate in the liquid sample onto the surface of the liquid sample introduction plate.

When the conjugate variation step is carried out by the first conjugate variation step, when the optical signal is detected by focusing on the surface of the liquid sample introduction plate or its vicinity in the optical signal detection step. The combined body is not necessarily attracted to the surface of the liquid sample introduction plate, and for example, immediately after the liquid sample is introduced into the liquid sample introduction plate, the combined body is the liquid sample. It is considered to be suspended in the liquid layer. If the floating conjugate is outside the imageable range in which the optical signal can be detected in the optical signal detection step or outside the near-field light generation region, the conjugate is not detected. Become.
Therefore, when the detection of the optical signal by the optical signal detection step is performed by focusing on the surface of the liquid sample introduction plate or its 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 settle by gravity on the surface of the liquid sample introduction plate, which takes time to prepare for detection. In particular, if the specific gravity of the conjugate is small, it will take a longer time.
Therefore, when the conjugate variation step is the first conjugate variation step, the conjugate attraction step can be further carried out to shorten the preparation time for detection and perform more efficient detection. preferable.
The combined attraction step can be carried out by the third magnetic field application unit described in the target substance detection device.

There is no particular limitation, but there is no particular limitation in the case where the bond pulling step is carried out and the first bond changing step is carried out by moving the bond in a direction away from the liquid sample introduction plate. After the liquid sample introduction and holding step, it is preferable to repeat the bond pulling step, the bond fluctuation step, and the optical signal detection step a plurality of times in this order (alternate magnetic field application).
By applying the alternating magnetic field, the optical signal caused by the same coupled body is repeatedly detected, so that the accuracy of detection can be improved. Further, it is also possible to amplify this optical signal by periodically applying the alternating magnetic field and applying a known lock-in amplifier to the frequency of the optical signal due to the same conjugate. Therefore, the sensitivity of detection can be improved.

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

[First Embodiment]
The target substance detection device according to the first embodiment of the present invention is configured according to the target substance detection device according to the study described above with reference to FIG. 1, and the optical signal detection unit 5 is among the optical signals. The conjugate is generated from the magnetic particles when the conjugate is irradiated with the propagating light or the near-field light, and the conjugate is bound to one target substance by two or more of the magnetic particles (FIG. 7). It is characterized in that only an optical signal based on (see) can be detected as a signal change target, and an optical signal based on one of the magnetic particles (see FIGS. 6 and 8) is not detected as a signal change target.

Here, in the optical signal detection unit 5, for example, the optical signal generated from the conjugate in which two or more of the magnetic particles are bonded to one target substance is a signal rather than the optical signal generated from one of the magnetic particles. Utilizing the large size, a configuration in which only an optical signal equal to or larger than the threshold value is detected by setting a size larger than the signal size of the optical signal generated from one said magnetic particle as a threshold, or a configuration generated from one said magnetic particle. Utilizing the fact that the optical signal generated from the conjugate in which two or more of the magnetic particles are bonded to one target substance has a stronger signal intensity than the optical signal, the signal of the optical signal generated from one of the magnetic particles It is configured to detect only optical signals above the threshold by setting a signal intensity stronger than the intensity as the threshold, or to have a configuration having both of these configurations.

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

According to the target substance detection apparatus according to the first embodiment, only the optical signal based on the conjugate (see FIG. 7) in which two or more of the magnetic particles are bound to one target substance is signal-changed. If it is detected as the target of the above, it is possible to detect the target substance with higher accuracy, ignoring the existence of the magnetic particles alone (see FIG. 8).
In the following, in the optical signal detection unit, among the optical signals, two of the optical signals are generated from the magnetic particles when the conjugate is irradiated with the propagating light or the near-field light, and two for one target substance. Only the optical signal based on the coupled body (see FIG. 7) to which the magnetic particles are bonded can be detected as the target of the signal change, and the optical signal based on one of the magnetic particles (see FIGS. 6 and 8) is signaled. On the premise that it is not detected as an object of change, other modifications and embodiments capable of performing high-precision detection by such an optical signal detection unit will be described.
Further, in the following description, since the target substance detection device according to the first embodiment has a configuration similar to that of the target substance detection device according to the study shown in FIG. 1, the reference numerals shown in FIG. 1 are used for convenience of explanation. The target substance detection device according to the first embodiment will be described with reference to the device.

First, the target substance detection device according to the first modification of the target substance detection device 1 according to the first embodiment will be described with reference to FIG. Note that FIG. 11 is an explanatory diagram of the target substance detection device according to the first modification.
As shown in FIG. 11, the target substance detection device 1A 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. Will be done. Other than this, since it is the same as the target substance detection device according to the first embodiment, the description thereof will be omitted.

The third magnetic field application unit 6 is arranged on the back surface 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 is made possible to be attracted to the surface, and here, it is formed by an annular electromagnet having a through hole, and the light irradiation unit 3 can irradiate light from the back surface 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, after the liquid sample introduction and holding step, the liquid sample is suspended in the liquid layer. The combined body is attracted by the third magnetic field application unit 6 after the liquid sample introduction holding step and before the combined body fluctuation step without waiting for the liquid sample introduction plate 2 to gravitationally settle on the surface. By applying a magnetic field, all or part of the conjugate in the liquid sample can be once attracted onto the surface of the liquid sample introduction plate 2 (combined attracting step).
Therefore, according to the target substance detection device 1A, in addition to the advantages of the target substance detection device 1 according to the first embodiment, the time required for detection is shortened, and the target substance can be detected more efficiently. It can be carried out.

Next, the target substance detection device according to the second modification of the target substance detection device 1 according to the first embodiment will be described with reference to FIG. Note that FIG. 12 is an explanatory diagram of the target substance detection device according to the second modification.
As shown in FIG. 12, in the target substance detection device 1B according to the second modification, the first magnetic field application unit is replaced with the first magnetic field application unit 4 in the target substance detection device according to the first embodiment. It is composed by arranging 7. Other than this, since it is the same as the target substance detection device 1 according to the first embodiment, the description thereof will be omitted.

The first magnetic field application unit 7 is composed of an electromagnet and is irradiated with light by the light irradiation unit 3 on the front surface of the liquid sample introduction plate 2 (on the back surface side, the light propagates above the surface). The combined body in the liquid sample, which is arranged diagonally upward with respect to the region where light is generated) and introduced onto the surface of the liquid sample introduction plate 2, 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 bond fluctuation step).

FIG. 13 schematically shows the state of the liquid sample introduction plate 2 in the observation field of view observed by the image pickup device 5a on the surface after the first bond fluctuation step carried out using the first magnetic field application unit 7. Shown in. The state before the first bond change step is the same as in FIG.

As can be understood through comparison between FIG. 2 showing the state before the first bond change step and FIG. 13 showing the state after the first bond change step, the optical signals a and b are described above. The optical signal changes before and after the first conjugate variation step, and the optical signals c and d do not change before and after the first conjugate variation step.
Therefore, according to the target substance detection device 1B, the substances a'and b'that generate the optical signals a and b include the target substance and the optical signal is similar to the target substance detection device 1 according to the first embodiment. It can be determined that c and d are noise signals such as scratches on the surface of the liquid sample introduction plate 2, impurities adhering to the surface or existing on the surface, and fluctuations in the light source output.

After the first conjugate fluctuation step carried out 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 attached to 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 the line AA in FIG. Further, the arrow B in FIG. 14 indicates an imageable range in which an optical signal can be acquired.
As shown in FIG. 14, the substance a'and the substance b'are directed in parallel to the in-plane direction of the surface of the liquid sample introduction plate 2 by the magnetic field attracted from diagonally above by the first magnetic field application unit 7, respectively. It moves in the direction having the vector components x ₁ and x ₂ of the above and the vector components y ₁ and y ₂ in the direction away from the liquid sample introduction plate 2.
Therefore, in the target substance detection device 1B, the target substance detection device 1 according to the first embodiment, which moves the substance a'and the substance b'only in the direction away from the liquid sample introduction plate 2, and the first combination variation. The state after the process is different.

This difference leads to a reduction in the detection burden of the target substance.
That is, when an attempt is made to detect the target substance based on the optical signals a and b while comparing FIGS. 4 and 13, the optical signal b is the result of the disappearance of the optical signal, which is different. However, regarding the optical signal a, in the case shown in FIG. 4, only the target substance can be detected based on the size change, and the target substance cannot be detected based on the movement, whereas in the case shown in FIG. 13, the size can be detected. In addition to the detection of the target substance based on the change, the target substance can be detected based on the movement, and the case shown in FIG. 13 is easier to detect the target substance.
Therefore, the target substance detection device 1B can detect the target substance with even higher accuracy.

[Second Embodiment]
Next, the target substance detection device according to the second embodiment of the present invention will be described with reference to FIG. Note that 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 type microscope, and includes a liquid sample introduction plate 12, a light irradiation unit 13, and a second magnetic field application. It is composed of a unit 18 and an optical signal detection unit 15 composed of an image pickup 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 device 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 arranged in that the second magnetic field application unit 18 is arranged in place of the first magnetic field application unit 4. It is different from the substance detection device 1. The differences will be described below.

The second magnetic field application unit 18 is arranged on the back surface side of the liquid sample introduction plate 12, and the combined body in the liquid sample introduced on the front surface of the liquid sample introduction plate 12 is liquid by applying a magnetic field. It is made possible to be attracted to the surface of the sample introduction plate 12 and to be 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 12 in a state where the magnetic field is applied. .. Here, the second magnetic field application unit 18 is formed of an annular permanent magnet having a through hole and a slide moving member (not shown) that slides the permanent magnet in the direction of X ₁ or X ₂ . The light irradiation unit 13 can irradiate light from the back surface side of the liquid sample introduction plate 12 through the through hole.
The movement of the combined body is performed by using the second magnetic field application unit 18 as the magnetic field application unit and introducing the liquid sample onto the surface of the liquid sample introduction plate 12 by applying the magnetic field from the second magnetic field application unit 18. The combined body inside is attracted onto the surface of the liquid sample introduction plate 12, and the second magnetic field application portion 18 is placed in a direction parallel to the in-plane direction of the surface of the liquid sample introduction plate 12 in a state where the magnetic field is applied. This is performed by moving the bond in the direction having the vector component and following the movement of the second magnetic field application unit 18 to move the bond (second bond variation step). Further, when the second magnetic field application unit 18 is composed of a plurality of members arranged in an annular shape, the second combined body change step without using the slide moving member by controlling the magnetic field application state for each member. It is also possible to do.
When the second magnetic field application unit 18 is used, in the second bond fluctuation step, all or part of the bond in the liquid sample is placed on the surface of the liquid sample introduction plate 12 by applying the magnetic field. Therefore, it is not necessary to wait for the conjugate floating in the liquid layer of the liquid sample to undergo gravity settling on the surface of the liquid sample introduction plate 12 after the liquid sample introduction and holding step.

FIG. 16 schematically shows the state on the surface of the liquid sample introduction plate 12 in the observation field of view observed by the image pickup device 15a after the second bond fluctuation step. The state before the second bond change step is the same as in FIG. Further, FIG. 17 schematically shows the state on the surface before the second bond change step when anisotropy can be confirmed in the shape of the optical signal. Further, FIG. 18 schematically shows the state on the surface after the second bond change step in this case.
As can be understood through the comparison between FIG. 2 and FIG. 16 showing the state after the second conjugate change step, or the comparison between FIGS. 17 and 18, the optical signals a and b are the first. The optical signal changes before and after the conjugate fluctuation step of No. 2, 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 an adsorbed substance on the surface, a contaminant existing on the surface, or a fluctuation of the light source output.

19 and 20 show the state of the substance a'that generates the optical signal a and the substance b'that generates the optical signal b when viewed from the side surface of the liquid sample introduction plate 12 after the second bond variation step. Shown in. 19 is a sectional view taken along line AA in FIG. 16, and FIG. 20 is a sectional view taken along line AA in FIG. The arrow B in FIGS. 19 and 20 indicates an imageable range in which an optical signal can be acquired.
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 the application of the magnetic field from the second magnetic field application unit 18, and then the second magnetic field is applied. The second magnetic field application part 18 is based on the movement of the liquid sample introduction plate 12 of the part 18 in the direction having the vector component in the direction parallel to the in-plane direction of the surface (direction X ₁ or X ₂ in FIG. 15). The liquid sample introduction plate 12 moves or rotates in a direction parallel to the in-plane direction of the surface of the liquid sample introduction plate 12 following the movement of the liquid sample introduction plate 12.
Although FIGS. 16 and 19 show an example in which the substance a'and the substance b'move in the observation field, the second magnetic field application portion 18 is directed to the in-plane direction of the surface of the liquid sample introduction plate 12. When the substance is moved in a direction having a vector component in a parallel direction and in a direction parallel to the direction of any one side that regulates the rectangular observation field, at a distance longer than the length of the side, the observation field is within the observation field. The substance a'and the substance b'can be moved out of the observation field, and high-precision detection can be performed based on the disappearance of the optical signals a and b.
Further, in the target substance detection apparatus according to the first and second embodiments, the optical system is irradiated with light from the back surface side of the liquid sample introduction plates 2 and 12 according to the configuration of a known upright microscope, and the front surface thereof. The optical signal based on the propagating light transmitted to the side is detected by the optical signal detection units 5 and 15, but the light is irradiated from the surface side of the liquid sample introduction plate according to the configuration of a known inverted microscope. Then, the optical signal based on the propagating light transmitted to the back surface side may be detected by the optical signal detection unit arranged on the back surface side.

[Third Embodiment]
Next, the target substance detection device according to the third embodiment of the present invention will be described with reference to FIG. 21. Note that FIG. 21 is an explanatory diagram of the target substance detection device according to the third embodiment.
As shown in FIG. 21, the target substance detection device 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, and an image pickup device 25a. It is composed of an optical signal detection unit 25 composed of an objective lens 25b and a half mirror (dichroic mirror or the like) 25c. The image pickup device 25a is composed of, 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.

The liquid sample introduction plate 22 is formed of a reflector plate capable of propagating the liquid sample onto the surface and using the reflected light RL of the light _L emitted from the surface side as the propagating light. Ru. Further, the liquid sample introduction plate 22 constitutes the liquid sample holding portion by itself, and after the liquid sample is introduced onto the surface, a cover glass or the like is arranged so as to cover the liquid sample. Hold the sample.
The light irradiation unit 23 is configured as a surface side irradiation unit capable of irradiating light L from the surface side of the liquid sample introduction plate 22 by the reflected light from the half mirror 25c.
Further, 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 combined body in the liquid sample introduced on 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 passes through the through hole to the liquid sample introduction plate 22. The light signal based on the reflected light RL of the light _L can be irradiated and can be detected 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 propagated light before and after the application of the magnetic field by the first magnetic field application unit 24. To.
The liquid sample introduction plate 22, the light irradiation unit 23, and the optical signal detection unit 25 (imaging device 25a, objective lens 25b, half mirror 25c) can be configured according to a known epi-illumination 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 has gravity settled on the surface of the liquid sample introduction plate 22, the light L emitted from the light irradiation unit 23 is liquid through the half mirror 25c. The surface side of the sample introduction plate 22 is irradiated (light irradiation step), the irradiation objective lens 25b is adjusted to put the surface or its vicinity within the imageable range, and the image pickup device 25a of the light L on the surface. An optical signal based on the reflected light _RL is acquired (optical signal detection step).

Next, by applying a magnetic field to the combined body in the liquid sample introduced onto the surface of the liquid sample introduction plate 22 by exciting the electromagnet of the first magnetic field application unit 24, the first magnetic field application unit 24 The combined body is moved toward the liquid sample introduction plate 22 in a direction away from the liquid sample introduction plate 22 (first combined body variation step).
Next, the image pickup device 25a acquires an optical signal on the surface of the liquid sample introduction plate 22 after moving the conjugate in a direction away from the liquid sample introduction plate 22 while maintaining the imageable range and the observation field of view. (Optical signal detection step).

In the target substance detection device 20 configured as described above, the optical signals before and after the first bond fluctuation step in the optical signal detection step are obtained as shown in FIGS. 2 and 4 above, and the light based on the target substance is obtained. The signal can be clearly distinguished from noise signals such as scratches on the surface of the liquid sample introduction plate 22, impurities adsorbed on or 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, detection can be performed ignoring the presence thereof, so that the cleaning treatment for the liquid sample introduction plate 22 is not necessarily performed for each detection. It is not necessary to perform it, and efficient detection can be performed. 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. Further, as a mode of change in the optical signal, a phenomenon of disappearance in addition to defocus can be used, so that the change in the optical signal can be clearly captured.

Next, the target substance detection device according to the modified example of the target substance detection device according to the third embodiment will be described with reference to FIG. 22. Note that FIG. 22 is an explanatory diagram of the target substance detection device according to the modified example of the target substance detection device according to the third 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, and the first The first magnetic field application unit 27 is arranged in place of the magnetic field application unit 24. Other than this, since it is the same as the target substance detection device 20 according to the third embodiment, the description thereof will be omitted.

The third magnetic field application unit 26 is formed of an electromagnet and is arranged on the back surface side of the liquid sample introduction plate 22. The combined body in the liquid sample introduced into the liquid sample introduction plate 22 is liquid by applying a magnetic field. It can be attracted to the surface of the sample introduction plate 22.
According to the third magnetic field application unit 26, as in the case of using the target substance detection device 20, after the liquid sample introduction and holding step, the conjugate floating in the liquid layer of the liquid sample is the liquid sample introduction plate. Without waiting for gravity to settle on the surface of 22 All or part of the combined body can be once attracted onto the surface of the liquid sample introduction plate 22 (combined body attracting 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.

Further, the first magnetic field application unit 27 is composed of an electromagnet and is exposed to a detection region on the surface of the liquid sample introduction plate 22 (on the surface side, the light irradiation unit 23 is irradiated with light, and is above the surface. The combined body in the liquid sample, which is arranged diagonally upward with respect to the region where the propagating light is generated and introduced onto the surface of the liquid sample introduction plate 22, is applied to the liquid sample introduction plate 2 by applying a magnetic field. The surface is moved in a direction having a vector component in a direction parallel to the in-plane direction (first bond variation step).
When the first magnetic field application unit 27 is used instead of the first magnetic field application unit 24, the optical signals before and after the first conjugate fluctuation step in the optical signal detection step are obtained as shown in FIGS. 2 and 13 above. In addition to the detection of the target substance based on the size change 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 even higher accuracy. Can be detected.

[Fourth Embodiment]
Next, the target substance detection device according to the fourth embodiment of the present invention will be described with reference to FIG. 23. Note that 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. It is composed of a unit 38 and an optical signal detection unit 35 composed of an image pickup 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 is arranged in that the second magnetic field application unit 38 is arranged in place of the first magnetic field application unit 24. It is different from the substance detection device 20. The differences will be described below.

The second magnetic field application unit 38 is arranged on the back surface side of the liquid sample introduction plate 32, and the combined body in the liquid sample introduced on the front surface of the liquid sample introduction plate 32 is liquid by applying a magnetic field. It is made possible to be attracted to the surface of the sample introduction plate 32 and to be 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 32 in a state where the magnetic field is applied. .. Here, the second magnetic field application unit 38 is formed of a permanent magnet and a slide moving member (not shown) that slides the permanent magnet in the direction of X ₁ or X ₂ .
The movement of the coupled body is performed by using the second magnetic field application unit 38 as the magnetic field application unit and introducing the liquid sample onto the surface of the liquid sample introduction plate 32 by applying the magnetic field from the second magnetic field application unit 38. The combined body inside is attracted onto the surface of the liquid sample introduction plate 32, and the second magnetic field application portion 38 is placed in a direction parallel to the in-plane direction of the surface of the liquid sample introduction plate 32 while the magnetic field is applied. This is performed by moving the bond in the direction having the vector component and following the movement of the second magnetic field application unit 38 to move the bond (second bond variation step). Further, when the second magnetic field application unit 38 is composed of a plurality of members arranged in an annular shape, the second combined body change step without using the slide moving member by controlling the magnetic field application state for each member. It is also possible to do.
When the second magnetic field application unit 38 is used, in the second bond fluctuation step, all or part of the bond in the liquid sample is placed on the surface of the liquid sample introduction plate 32 by applying the magnetic field. Therefore, it is not necessary to wait for the conjugate floating in the liquid layer of the liquid sample to undergo gravity settling on the surface of the liquid sample introduction plate 32 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 bond fluctuation step in the optical signal detection step are obtained as shown in FIGS. 2 and 16 above, and the light based on the target substance is obtained. The signal can be clearly distinguished from noise signals such as scratches on the surface of the liquid sample introduction plate 32, impurities adsorbed on or present on the surface, and fluctuations in the output of the light source.
Although FIG. 16 shows an example in which the substance a'and the substance b'move in the observation field, the second magnetic field application portion 38 is parallel to the in-plane direction of the surface of the liquid sample introduction plate 32. When the substance a'of the observation field is moved in a direction having a vector component of the direction and in a direction parallel to the direction of any one side that regulates the rectangular observation field, at a distance longer than the length of the side. And the substance b'can be moved out of the observation field, and high-precision detection can be performed based on the disappearance of the optical signals a and b.

As an example of the optical signal based on the combined body, FIGS. 2, 4, 13, 16 and 18 have been given as examples, and it has been described that the optical signal is caused by the scattered light, the reflected light, fluorescence and the like. However, this is for the convenience of drawing display, and the mode of the optical signal may be an optical signal caused by the transmitted light due to the phase difference, the differential interference, or the like.
Further, as an example of the mode of change of the optical signal based on the coupled body, FIGS. 4, 13, 16 and 18 are given as the position movement, the focus shift, the disappearance, and the rotation (posture change of the coupled body). As described above, the modes of change in the optical signal include increase / decrease in intensity (decrease in intensity due to the defocusing, etc.), phase change (phase change after position movement), and appearance (position movement from outside the observation field). Can also be mentioned.

[Fifth Embodiment]
Next, the target substance detection device according to the fifth embodiment of the present invention will be described with reference to FIG. 24. Note that FIG. 24 is an explanatory diagram of the 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, has a liquid sample introduction plate 42, a light irradiation unit composed of a light source 43a and an optical prism 43b, and a first unit. It is composed of a light source application unit 47 and an optical signal detection unit 45 (imaging device). The image pickup device is composed of, for example, a known CCD image sensor or the like, and can acquire a two-dimensional image.

The liquid sample introduction plate 42 is capable of generating near-field light above the surface by being irradiated with light L in which the liquid sample E is introduced onto the surface and the surface is irradiated with light L under total reflection conditions. It is formed of a detection plate. Further, the liquid sample introduction plate 42 constitutes the liquid sample holding portion by itself, and after the liquid sample E is introduced onto the surface, the cover glass G is arranged so as to cover the liquid sample. Hold the liquid sample E.
The light irradiation unit is configured as a total reflection light irradiation unit capable of irradiating the surface of the liquid sample introduction plate 42 with light L emitted from the light source 43a via an optical prism 43b under total reflection conditions. .. In addition, the total reflected light irradiation unit introduces light L emitted from the light source 43a under total reflection conditions to 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.
Further, the first magnetic field application unit 47 receives the light L irradiation by the light irradiation unit on the front surface of the liquid sample introduction plate 42 (on the back surface side, the near-field light is generated on the surface. The combined body in the liquid sample, which is arranged diagonally upward with respect to the region to be introduced and introduced onto the surface of the liquid sample introduction plate 42, is applied with a magnetic field in the in-plane direction of the surface of the liquid sample introduction plate 42. It is configured to move in a direction that has a vector component in a 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 has gravity settled on the surface of the liquid sample introduction plate 42, the light L emitted 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 full reflection conditions (light irradiation step), and the optical signal detection unit 45 acquires an optical signal S based on the near-field light on the surface (optical signal detection step).

Next, by applying a magnetic field to the combined body in the liquid sample introduced onto the surface of the liquid sample introduction plate 42 by exciting the electromagnet of the first magnetic field application unit 47, the first magnetic field application unit 47 The combined body is moved toward the surface by applying a magnetic field 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 42 (first combined body variation step).
Next, the optical signal detection unit 45 acquires an optical signal on the surface of the liquid sample introduction plate 42 after moving the conjugate while maintaining the observation field (optical signal detection step).

In the target substance detection device 40 configured as described above, the optical signals before and after the first bond fluctuation step in the optical signal detection step 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 clearly distinguished from the scratches on the surface of the liquid sample introduction plate 42, the contaminants adsorbed on or present on the surface, and the noise signal f such as the fluctuation of the light source output. .. Note that FIG. 25 is a diagram showing the state of the liquid sample introduction plate 42 on the surface before the bond change step, and FIG. 26 is a view showing the state of the liquid sample introduction plate 42 on the surface after the bond change step. It is a figure which shows.
As shown in FIGS. 25 and 26, the optical signal obtained by using the near-field light has a dark field background due to the attenuation of the near-field light, and the target substance detection device 40 uses the optical signal at the light spot. Based on this, the target substance is detected. Further, although not shown, the appearance of an optical signal based on movement from outside the observation field of view can also be detected.
According to the target substance detection device 40, 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 42, the presence thereof can be ignored for detection. Therefore, the cleaning treatment for the liquid sample introduction plate 42 is not necessarily performed for each detection. It is not necessary to perform it, and efficient detection can be performed. Further, an optical signal generated based on a phenomenon such as the scattered light or the fluorescence can be treated as an identification signal. Further, as an aspect of the change of the optical signal, in addition to the movement of the position, the phenomenon of appearance / disappearance / rotation of the shape can be used, so that the change of the optical signal can be clearly grasped.

[Sixth Embodiment]
Next, the target substance detection device according to the sixth embodiment of the present invention will be described with reference to FIG. 27. Note that 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. It is composed of a light irradiation unit, a second light source application unit 58, and an optical signal detection unit 55.
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 arranged in that the second magnetic field application unit 58 is arranged in place of the first magnetic field application unit 47. It is different from the substance detection device 40. The differences will be described below.

The second magnetic field application unit 58 is arranged on the back surface side of the liquid sample introduction plate 52, and the combined body in the liquid sample introduced on the front surface of the liquid sample introduction plate 52 is liquid by applying a magnetic field. It is possible to attract the sample introduction plate 52 onto the surface thereof and to move the liquid sample introduction plate 52 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 in a state where the magnetic field is applied. .. Here, the second magnetic field application unit 58 is formed of a permanent magnet and a slide moving member (not shown) that slides the permanent magnet in the direction of X ₁ or X ₂ .
The fluctuation of the combined body is caused by the liquid sample introduced on the surface of the liquid sample introduction plate 52 by applying the magnetic field from the second magnetic field application unit 58 with the second magnetic field application unit 58 as the magnetic field application unit. The combined body inside is attracted onto the surface of the liquid sample introduction plate 52, and the second magnetic field application portion 58 is placed in a direction parallel to the in-plane direction of the surface of the liquid sample introduction plate 52 while the magnetic field is applied. It is performed by moving in the direction having the vector component and following the movement of the second magnetic field application unit 58 to move the combined body or by changing the posture of the combined body (second combined body variation step). .. Further, when the second magnetic field application unit 18 is composed of a plurality of members arranged in an annular shape, the second combined body change step without using the slide moving member by controlling the magnetic field application state for each member. It is also possible to do.
When the second magnetic field application unit 58 is used, in the second bond fluctuation step, all or part of the bond in the liquid sample is placed on the surface of the liquid sample introduction plate 52 by applying the magnetic field. Therefore, it is not necessary to wait for the conjugate floating in the liquid layer of the liquid sample to undergo gravity settling 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, the optical signals before and after the second bond fluctuation step in the optical signal detection step are obtained as shown in FIGS. 28 and 29, and the optical signals based on the target substance are obtained. h can be clearly distinguished from the noise signal i such as scratches on the surface of the liquid sample introduction plate 52, impurities adhering to the surface or existing on the surface, and fluctuation of the light source output. Note that FIG. 28 is a diagram showing the state of the liquid sample introduction plate 52 on the surface before the bond change step, and FIG. 29 is a view showing the state of the liquid sample introduction plate 52 on the surface after the bond change step. It is a figure which shows.

The target substance detection device according to the fifth and sixth embodiments is configured according to the configuration of the surface plasmon resonance sensor, but as a modification of these embodiments, the liquid sample introduction plates 42 and 52 Is the detection plate used in the optical waveguide mode sensor, and the optical system is the optical system used in the optical waveguide mode sensor. Detection can be performed. Further, it is also possible to use an optical system that utilizes near-field light generated by total internal reflection for illumination, such as an optical system of a known total internal reflection microscope.

It should be noted that the examples shown in the above embodiments and the like are described for the sake of easy understanding of the invention, and are not limited to this embodiment.

In the above-described embodiments, the liquid sample introduction plate is configured by using the light-transmitting plate, the reflection plate, and the detection plate, but the introduction plate may be used. In this case, in the optical signal detection unit arranged on the front surface side or the back surface side of the liquid sample introduction plate by adopting the side surface side light irradiation unit as the light irradiation unit, scattered light from the conjugate. It can be configured to detect reflected light and the like. Alternatively, in the optical signal detection unit arranged on the side surface of the liquid sample introduction plate (the side surface of the liquid sample introduction plate opposite to the side on which the side surface side light irradiation unit is arranged), of the combined body. It can be configured to detect light absorption, transmitted light, and the like.

The 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. 24. Hereinafter, 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 in 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. board. A 600 μm core diameter optical fiber with a collimating lens attached to the emission end is connected to the red LED light source (Thorlabs, model number M625F2) to the light source 43a, and a 650 nm short path filter and a polarizing filter are arranged at the end of the emission end. Was used. A prism 43b made of SiO ₂ glass is optically in close contact with 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 embodiment corresponds to the fifth embodiment. As the target substance, virus-like particles of norovirus were selected. As the magnetic particles, particles in which an anti-norovirus antibody was bound to fluorescent magnetically labeled beads (manufactured by Tamagawa Seiki Co., Ltd., FF beads Cy5 Streptavidin, model number TAB8851N2170) were used.
The fluorescent magnetic labeled beads are mixed with the solution containing the target substance to prepare a mixed solution (liquid sample), and then 100 μL of the mixed solution is placed on the liquid sample introduction plate 42 with a through hole having a diameter of 8 mm and a thickness of 2 mm. It was introduced into the liquid sample holding part formed by installing the silicon rubber sheet of.
After introducing the mixed solution, a cover glass G was arranged, the liquid sample holding portion was covered, and the magnetic particles were gravity-settled on the surface of the liquid sample introduction plate 42.
After gravitational settling, a near field was formed on the surface of the liquid sample introduction plate 42 by irradiation with the incident light, and the optical signal was measured.
The optical signal was observed with a cooled CCD camera (BITRAN, model number BU-59LIR) via a 650 nm long pass filter using an optical microscope equipped with a 5x objective lens. That is, a CCD camera equipped with a 5x objective lens and a 650 nm long pass filter is used as the optical signal detection unit 45 (imaging device). The magnetic particles contain a fluorescent dye that receives the light of the light source 43a and emits fluorescence in a wavelength range having a peak in the range of 660 nm to 670 nm, and the imaging device observes only the fluorescence from the magnetic particles. be able to.

FIG. 30 shows an image observed with an exposure time of 5 seconds before the bond change step. In FIG. 30, the background is black and the optical signal detection position is white. The field of view in the figure is approximately 2.5 mm × 2.0 mm. In the figure, tens to hundreds of light spots can be confirmed in the entire field of view.
Next, FIG. 31 shows an image observed with an exposure time of 0.5 seconds before the conjugate change step. The field of view in the figure is the same as that in 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 location is observed. In this way, by controlling the exposure time, it is possible to distinguish between the light spots formed by the magnetic particles alone and the light spots composed of the plurality of magnetic particles.
Further, observation is performed after applying a magnetic field having a vector component in a parallel direction in the plane of the surface of the liquid sample introduction plate 42 using a permanent magnet as the first magnetic field application unit 47 (after the bond fluctuation step). The image is shown in FIG. 32. The field of view in the figure is the same as that in FIGS. 30 and 31. The solid line circle and the dotted line circle in the drawings of FIGS. 31 and 32 are added for convenience of explanation.
When the magnetic particles are moved from the surface of the liquid sample introduction plate 42 by applying the magnetic field, three of the light spots in FIG. 31 observed by the CCD camera before the magnetic field is applied (solid circles in the figure). It can be seen that (exists in the circled with) but disappeared in FIG. 32 (not present in the circled with a dotted line in the figure).
Here, the extinguished light spot is due to the fact that the combined body of the target substance and the magnetic particles is moved away from or moved from the surface of the liquid sample introduction plate 42 in the form shown in FIG. 14 by the application of the magnetic field. .. It is probable that the disappearance of the light spot observed in this embodiment is due to the fact that the conjugate at the position of the light spot moves out of the range of the near field or out of the observation field of view.
Further, it is considered that the light spots in which movement was not observed in FIGS. 31 and 32 caused non-specific adsorption on the surface of the liquid sample introduction plate 42, and a plurality of the magnetic particles were targeted at the same location. Even when non-specific adsorption occurs without the intervention of a substance, it is possible to distinguish it from the conjugate via the target substance by utilizing the movement of the light spot due to the application of a magnetic field as in this embodiment.

In this way, by controlling the exposure time and taking the difference between the optical signals before and after the application of the magnetic field, the detection of the target substance with suppressed erroneous detection is carried out by utilizing the fact that only the conjugate can be detected. can.

1,1A, 1B, 10,20,20A,30,40,50 Target substance detector 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 application part 18, 38, 58 Second magnetic field application part 43a, 53a Light source 43b, 53b Optical prism _L light TL transmitted light _RL reflected light a to i light signal a', b'material X ₁ , X ₂ directions x ₁ , x ₂ , y ₁ , y ₂ Vector component G Cover glass E Liquid sample S Optical signal

Claims (6)

A liquid sample containing a target substance and magnetic particles that form a bond with the target substance and have photoresponsiveness is introduced onto the front surface, and transmitted light of light emitted from either the back surface side or the front surface side. A translucent plate capable of propagating to the surface side opposite to the side on which the light is irradiated, the reflected light of the light emitted from the surface side while the liquid sample is introduced onto the surface. A reflecting plate capable of propagating above the surface as the propagating light, an introduction plate into which the liquid sample is introduced onto the surface, and a total reflection of the liquid sample on the surface and with respect to the surface. A liquid sample introduction plate formed by any of the detection plates capable of generating near-field light by the light irradiated under the conditions is arranged on the surface, and the liquid sample is placed on the surface of the liquid sample introduction plate. With a liquid sample holder that can be held in
When the liquid sample introduction plate is formed of the translucent plate, the back side light irradiation portion capable of irradiating the light from the back surface side of the liquid sample introduction plate, and the liquid sample introduction plate is the translucent plate. And the surface side light irradiation portion capable of irradiating the light from the surface side of the liquid sample introduction plate when formed by any of the reflection plates, and the liquid sample introduction plate are formed by the introduction plate. The side light irradiation unit capable of irradiating the liquid sample held on the liquid sample introduction plate with the light from the side surface side of the liquid sample introduction plate, and the liquid sample introduction plate are described above. A light irradiation unit formed by any of the total reflected light irradiation units capable of irradiating the surface with the light under total reflection conditions when formed by the detection plate.
The conjugate in the liquid sample, which is arranged on the back surface side of the liquid sample introduction plate and introduced on 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 magnetic field application unit that is attractable and can move 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 in a state where the magnetic field is applied.
Either the propagating light or the near-field light arranged on any of the front surface side, the back surface side, and the side surface side of the liquid sample introduction plate and before and after the movement of the magnetic field application portion. It has an optical signal detection unit that can detect a signal change of an optical signal based on light and can acquire a state of a detection region on the surface of the liquid sample introduction plate as a two-dimensional image.
Among the optical signals, the optical signal detection unit is generated from the magnetic particles when the conjugate is irradiated with either the propagating light or the near-field light, and becomes one target substance. On the other hand, only an optical signal based on the conjugate in which two or more of the magnetic particles are bonded can be detected as a signal change target.
The surface of the liquid sample introduction plate is surface-treated with an adsorption inhibitor that suppresses adsorption of the conjugate .
The combined body in which the magnetic field application unit exists within the image formation range of the two-dimensional image is made to follow the movement of the magnetic field application unit, and the position is either within the image formation range or outside the image formation range. A target substance detector characterized by being movable to. A liquid sample containing a target substance and magnetic particles that form a bond with the target substance and have photoresponsiveness is introduced onto the front surface, and transmitted light of light emitted from either the back surface side or the front surface side. A translucent plate capable of propagating to the side opposite to the side on which the light is irradiated, the reflected light of the light emitted from the surface side while the liquid sample is introduced onto the surface. A reflector that can propagate above the surface as the propagating light, an introduction plate into which the liquid sample is introduced onto the surface, and a total reflection condition for the surface when the liquid sample is introduced onto the surface. A liquid sample introduction plate formed by any of the detection plates capable of generating near-field light by the light irradiated in is arranged on the surface, and the liquid sample is placed on the surface of the liquid sample introduction plate. A liquid sample introduction and holding step of introducing and holding the liquid sample on the surface of the liquid sample introduction plate with respect to the liquid sample holding portion that can be held.
Backside side light irradiation step of irradiating the light from the back surface side of the liquid sample introduction plate when the liquid sample introduction plate is formed by the translucent plate, the liquid sample introduction plate is 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, and the liquid sample introduction when the liquid sample introduction plate is formed by the introduction plate. The side light irradiation step of irradiating the liquid sample held on the plate with the light from the side surface side of the liquid sample introduction plate, and when the liquid sample introduction plate is formed by the detection plate. A light irradiation step, which is one of the total reflected light irradiation steps of irradiating the surface with the light under all reflection conditions, and
The combined body in the liquid sample introduced onto the front surface of the liquid sample introduction plate by applying a magnetic field from the magnetic field application portion arranged on the back surface side of the liquid sample introduction plate is used in the liquid sample introduction plate. A bond moving step of pulling the liquid sample onto the surface and moving the magnetic field application portion in a state of applying the magnetic field 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.
By the optical signal detection unit capable of acquiring the state of the detection region on the surface of the liquid sample introduction plate as a two-dimensional image, the propagating light and the propagating light before and after the movement of the magnetic field application unit by the conjugate movement step. Including an optical signal detection step of detecting a signal change of an optical signal based on any of the near-field light based on the two-dimensional image.
The optical signal detection step is generated from the magnetic particles when the conjugate is irradiated with either the propagating light or the near-field light among the optical signals, and becomes one target substance. On the other hand, it is a step of detecting only an optical signal based on the combined body in which two or more of the magnetic particles are bonded as a signal change target.
The surface of the liquid sample introduction plate is surface-treated with an adsorption inhibitor that suppresses adsorption of the conjugate .
The bond moving step causes the coupled body existing within the imageable range of the two-dimensional image to follow the movement of the magnetic field application portion, and is either within the imageable range or outside the imageable range. A target substance detection method characterized in that it is a step of moving to a position. The target substance detection method according to claim 2, wherein the magnetic particles are particles that generate scattered light by being irradiated with either propagating light or near-field light. The target substance detection method according to claim 2, 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 2, wherein the magnetic particles contain a fluorescent dye. The second aspect of claim 2, wherein 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 substance that is irradiated with the propagating light to cause light absorption. Target substance detection method.

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