JP2018179783A - Target substance detection chip, target substance detection device and target substance detection method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a target substance detection chip, device, and method, which enable detection of a target substance using magnetic particles, improve target substance detection accuracy, and allow for manufacturing a compact target substance detection device at low cost.SOLUTION: A target substance detection chip 1 of the present invention comprises a concavo-concave structure consisting of a plurality of protrusions periodically arranged on a light-transmissive substrate 2.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a target substance detection chip capable of optically observing the movement of a target substance accompanying the application of a magnetic field, a target substance detection device, and a target substance detection method.

  In recent years, methods have been developed for detecting and quantifying minute substances present in a solution, particularly biologically relevant substances such as DNA, RNA, proteins, viruses, and bacteria. As such a method, for example, a method using an evanescent field by total reflection, a method using surface plasmon resonance, excitation using a waveguide mode (also called an optical waveguide mode, a waveguide mode, an optical waveguide mode, etc.) The way to do it is known.

  As a method of utilizing the evanescent field by the total reflection, there is a total reflection illumination fluorescence microscope. Total reflection illumination fluorescence microscope is a technology that totally reflects incident light at the interface between a sample and a cover glass or slide glass, uses the evanescent field generated thereby as excitation light, and performs fluorescence observation with a small amount of background light as noise. (See Patent Document 1). The technology is a technology capable of achieving super resolution, and enables single molecule observation.

As a method of using the surface plasmon resonance, for example, surface plasmon resonance excitation-enhanced fluorescence spectroscopy is known.
This method excites surface plasmon resonance on the gold thin film by total reflection of incident light at the interface between the gold thin film layer on the glass surface in contact with the prism and the liquid sample using an optical arrangement called a Kretschmann arrangement. Forming an enhanced electric field on the surface of the gold thin film. Using the light enhanced in the vicinity of the surface of the gold thin film by the surface plasmon resonance as excitation light, the fluorescent molecules present in the enhanced electric field are excited to generate strong fluorescence and perform fluorescence observation with less background light (See Patent Document 2).

In the method using excitation of the waveguide mode, a detection chip in which a silicon layer (semiconductor layer) and an SiO ₂ layer are laminated in this order on a silica glass substrate is placed on a silica glass trapezoidal prism. Then, light is irradiated from the side of the trapezoidal prism under the condition of total reflection on the surface of the detection chip to obtain an enhanced electric field (see Non-Patent Document 1). In this method, when the light is irradiated at a specific incident angle while satisfying the total reflection condition from the back surface side (silica glass substrate side) to the detection chip, the light of a specific wavelength propagates in the detection chip Coupled with the guided mode, the guided mode is excited. When the guided mode is excited, the enhanced electric field is generated near the surface of the detection chip. Thereby, the fluorescent molecule present in the enhanced electric field is excited, and fluorescence observation with less background light can be performed (see Non-Patent Document 2). The semiconductor layer may be formed of a metal layer, and the waveguide mode excited in the detection chip in which the semiconductor layer is formed of the metal layer may be called leaky mode, leaky mode, etc. Non-Patent Document 3).

However, in the fluorescence observation method using the enhanced electric field generated in the vicinity of the surface of the detection chip, the detection accuracy has a problem, and further improvement of the detection accuracy is desired.
That is, in the light signal detected by the fluorescence observation method, in addition to the fluorescence from the fluorescent molecule, the scattered light due to the stains or scratches on the surface of the detection chip, the autofluorescence generated from the components of the detection chip, Since the noise signal based on the light emission from the impurities contained is included, the fluorescence observation method is required to exclude the noise signal that is a cause of the decrease in the detection accuracy.

From the above, as the target substance detection method in which the noise signal is eliminated, the present inventors bind a labeling substance such as a fluorescent labeling substance or a light scattering substance to a target substance and a magnetic particle, and By performing comparative observation before and after application of a magnetic field by a magnetic field application unit (for example, a magnet), a method is proposed in which detection is performed excluding noise signals contained in an optical signal before application of the magnetic field.
According to this method, while the target substance combined with the labeling substance and the magnetic particles is moved by the application of the magnetic field, the noise signal caused by a flaw or the like on the surface of the detection chip is It is possible to perform the detection excluding the noise signal by using the fact that it does not move by application (see Non-Patent Documents 4 and 5).

  However, even in this proposal, the conjugate may adsorb nonspecifically to the surface of the detection chip and may not move before and after application of the magnetic field, and the light signal from the conjugate which does not move is the noise signal In the same manner as in the above, depending on the adsorption situation of the conjugate and the detection chip, improvement in the detection accuracy may not be expected. On the other hand, in order to solve this problem, if a strong magnetic field is applied to move the combination reliably, the apparatus becomes large-scaled and the manufacturing cost increases.

JP 2002-236258 A International Publication 2015/194663

M. Fujimaki et al. Optics Express, Vol. 23 (2015) pp. 10925-10937 K. Nomura et al. J. Appl. Phys. Vol. 113, (2013) pp. 143103-1-143103-6 R. P. Podgorsek, H. Franke, J. Woods, and S. Morrill, Sensor. Actuat. B51 pp. 146-151 (1998) Masato Yasuura, Makoto Fujimaki "Development of a guided-wave mode image sensor for trace detection" Research Institute of Electrical Engineers of Japan Material Sensor and Micromachine Division General Research Group (June 29, 2016), pp. 45-52, The Institute of Electrical Engineers of Japan (2016) M. Yasuura and M. Fujimaki, Sci. Rep. Vol. 6, pp. 39241-1-39241-7 (2016)

  The present invention solves the above-mentioned problems in the prior art and can be used for detection of a target substance using magnetic particles, improves the detection accuracy of the target substance and makes the target substance detection device smaller and less expensive. An object of the present invention is to provide a target substance detection chip that can be manufactured, a target substance detection device, and a target substance detection method.

The means for solving the problems are as follows. That is,
<1> A target substance detection chip having a concavo-convex structure configured by periodically arranging a plurality of convex portions on a light transmitting substrate.
<2> A light transmitting substrate having a smooth surface, and a concavo-convex structure providing layer which is laminated on the smooth surface of the light transmitting substrate and whose surface opposite to the surface on the light transmitting substrate side is a concavo-convex surface The target substance detection chip according to <1>, wherein the concavo-convex structure is formed by the concavo-convex surface.
<3> An electric field enhancing layer is formed on the light transmitting substrate, on which at least one surface is irradiated with light under total reflection conditions so that an enhanced electric field is formed on the other surface. When the light is irradiated under total reflection conditions to the one surface of the electric field enhancing layer from the back surface side with the surface as the back surface, the enhanced electric field can be present in the vicinity of the surface from <1> to <2 The target substance detection chip according to any one of the above.
<4> A light transmitting substrate having a smooth surface, a smooth electric field enhancing layer laminated on the smooth surface of the light transmitting substrate, and a concavo-convex structure imparting layer laminated on the electric field enhancing layer The target substance detection chip according to <3>, wherein a concavo-convex structure is formed on the concavo-convex surface of the concavo-convex structure providing layer.
<5> A light transmitting substrate having a smooth surface, and a concavo-convex structure in which the light transmitting substrate is laminated on the smooth surface and the surface opposite to the surface on the light transmitting substrate side is a first uneven surface A second layer having a shape obtained by laminating the application layer and the first concavo-convex surface of the concavo-convex structure application layer and the surface opposite to the surface on the concavo-convex structure application layer side to which the concavo-convex pattern of the first concavo-convex surface is transferred The target substance detection chip according to <3>, wherein the target substance detection chip is formed of an electric field enhancing layer which is a concavo-convex surface, and a concavo-convex structure is formed by the second concavo-convex surface.
<6> A light transmitting substrate having a first uneven surface, and a surface stacked on the first uneven surface of the light transmitting substrate and opposite to the light transmitting substrate side is the first uneven surface The target substance detection chip according to <3>, wherein the target substance detection chip is formed of an electric field enhancing layer, which is a second uneven surface having a shape to which the uneven pattern is transferred, and is formed of the second uneven surface.
A <7> convex part is formed in two or more types of shapes, and at least 1 type of the said shape is made into either of 2-fold rotational symmetry shape and line symmetry shape to either of said <1> to <6> The target substance detection chip described above.
<8> Total reflection condition from the back surface side with the target substance detection chip according to any one of <1> to <7> and the surface opposite to the surface on which the concavo-convex structure of the target substance detection chip is formed A light irradiating section capable of irradiating light, and moving a magnetic particle contained in a liquid sample introduced onto the surface of the target substance detection chip in a direction parallel to the surface or in a direction away from the surface A first magnetic field application unit to which a magnetic field can be applied, and a second magnetic field application unit that is disposed on the back surface side of the target substance detection chip and draws the magnetic particles in the liquid sample introduced onto the surface onto the surface A target substance detection device comprising: a magnetic field application unit formed by at least one of a second magnetic field application unit to which a magnetic field can be applied.
<9> A second magnetic field application unit is provided, and the second magnetic field application unit applies a second magnetic field in a direction having a vector component in a direction parallel to the in-plane direction of the target substance detection chip surface. The target substance detection device according to <8>, which is movable.
The light irradiation which irradiates light on total reflection conditions from the said back surface side by making the surface opposite to the surface in which the uneven structure of the target substance detection chip in any one of <10> said <1> to <7> is formed into a back surface Moving the combination of the target substance and the magnetic particles contained in the liquid sample introduced onto the surface of the target substance detection chip in a direction parallel to the surface or in a direction away from the surface by application of a first magnetic field And at least a second combination moving step of drawing the combination in the liquid sample on the surface by application of a second magnetic field from the magnetic field application unit disposed on the back surface side. And a conjugate transfer step performed in any one of the above.
<11> A vector component in a direction parallel to the in-plane direction of the target substance detection chip surface, including the second combined body moving step, and the second combined body moving step applies the second magnetic field, and the magnetic field application unit The target substance detection method according to <10>, wherein the target substance is moved in a direction having the following equation to move the combined body following the movement of the magnetic field application unit.

  According to the present invention, the above-mentioned problems in the prior art can be solved, which can be used for detection of a target substance using magnetic particles, and the detection accuracy of the target substance can be improved and the target substance detection device can be miniaturized. It is possible to provide a target substance detection chip, a target substance detection device, and a target substance detection method which can be manufactured at low cost.

It is an explanatory view showing a schematic structure of a 1st embodiment. It is explanatory drawing (1) explaining a mode that adsorption | suction of a coupling body is suppressed by uneven structure. It is explanatory drawing (2) explaining a mode that adsorption | suction of a coupling body is suppressed by uneven structure. It is explanatory drawing which shows schematic structure of 2nd Embodiment. It is explanatory drawing which shows schematic structure of 3rd Embodiment. It is explanatory drawing which shows schematic structure of 4th Embodiment. It is a perspective view which shows the example of formation of an uneven structure. It is a top view which shows the example of formation of an uneven structure. It is a top view which expands and shows one convex part. It is a figure which added each side of a longitudinal direction and a transverse direction in addition to the upper surface. It is explanatory drawing which shows schematic structure of 5th Embodiment. It is a figure which shows the mode on the surface in front of the application of a magnetic field. It is a figure which shows the mode on the surface after the application of a magnetic field. It is explanatory drawing which shows schematic structure of 6th Embodiment. It is a figure which shows the mode on the surface in front of the application of a magnetic field. It is a figure which shows the mode on the surface after the application of a magnetic field. It is explanatory drawing which shows schematic structure of 7th Embodiment. It is a figure which shows the mode on the surface in front of the movement of a 2nd magnetic field application part. It is a figure which shows the mode on the surface after the movement of a 2nd magnetic field application part.

(Target substance detection chip)
The target substance detection chip of the present invention has a light transmitting substrate, and as necessary, an electric field enhancing layer and a concavo-convex structure providing layer.

<Light transmission substrate>
The light transmitting substrate is a member that transmits light.
There is no restriction | limiting in particular as said transparent substrate, According to the objective, it can select suitably, For example, well-known transparent substrates, such as a glass substrate and a plastic substrate, can be used.
Moreover, when forming an uneven surface in the said transparent substrate, there is no restriction | limiting in particular as a formation method of the said uneven surface, Well-known methods, such as an injection molding method, a nanoimprint method, an etching method, can be mentioned.
In the present specification, “light transmissive” indicates that the visible light transmittance is 0.5% or more.

<Field enhancement layer>
The electric field enhancing layer is a layer in which an enhancing electric field is formed on another surface when light is irradiated to one surface under total reflection conditions.
There is no restriction | limiting in particular as said electric field enhancement layer, According to the objective, it can select suitably, A well-known surface plasmon excitation layer and a waveguide mode excitation layer can be applied.

Examples of the surface plasmon excitation layer include metal layers containing at least one of gold, silver, platinum and aluminum.
In the metal layer, surface plasmon resonance is excited on the other surface by the light irradiated to the one surface, and the enhanced electric field is obtained on the other surface.
As the thickness of the metal layer, an optimum value is determined according to the constituent material and the wavelength of light to be irradiated, but it is known that this value can be calculated from calculation using the Fresnel equation. In general, when the surface plasmon resonance is excited in the near ultraviolet to near infrared region, the thickness of the metal layer is several nm to several tens nm.

The method of forming the metal layer is not particularly limited, and known methods such as vapor deposition, sputtering, CVD, PVD, spin coating, etc. may be mentioned, but the material for forming the light transmitting substrate is plastic In the case of a material or a glass material, if the metal layer is formed directly on the light transmitting member, the adhesion may be lowered and it may be easily peeled off.
Therefore, from the viewpoint of improving adhesion, it is preferable to form an adhesive layer made of nickel or chromium on the surface of the light transmitting substrate, and to form the metal layer on the adhesive layer.

When light emission from the target substance or the labeling substance for labeling the target substance is observed, when the target substance and the labeling substance are in proximity to the metal layer, the target substance and the fluorescent substance are obtained from the excitation light Energy may be transferred to the metal layer to cause a phenomenon called quenching in which the luminous efficiency is reduced.
In this case, if a covering layer is formed on the surface of the metal layer for the purpose of separating the target substance and the labeling substance from the surface of the metal layer, the quenching is suppressed and the decrease in light emission efficiency is suppressed. Can.
There is no restriction | limiting in particular as said coating layer, It can form with the transparent material of several nm-several dozen nm thickness formed with glass materials, such as a silica glass, an organic polymer material, etc.

There is no restriction | limiting in particular as said waveguide mode excitation layer, The laminated body of the thin film layer formed with a metal material or a semiconductor material, and the dielectric material layer formed with a transparent dielectric material is mentioned.
In the waveguide mode excitation layer, the light irradiated to the one surface excites the waveguide mode in the dielectric layer, and the enhanced electric field is obtained on the other surface.
In the waveguide mode excitation layer, the thin film layer constitutes a layer on the one surface side, and the dielectric layer constitutes the other surface side.

There is no restriction | limiting in particular as said metal material, For example, gold | metal | money, silver, copper, platinum, aluminum etc. are mentioned.
The semiconductor material is not particularly limited, and examples thereof include semiconductor materials such as silicon and germanium or known compound semiconductor materials. Among these, silicon is preferable because of low cost and easy processing.
The thickness of the thin film layer is the same as that of the surface plasmon excitation layer, and the optimum value is determined by the constituent material and the wavelength of the light to be irradiated, and this value can be calculated from the calculation using the Fresnel equation. It is known. In general, when light in a wavelength band of near ultraviolet to near infrared is used, the thickness of the thin film layer is several nm to several hundreds nm.

The light transmitting dielectric material is not particularly limited, and examples thereof include resin materials such as silicon oxide, silicon nitride and acrylic resin, metal oxides such as titanium oxide, and metal nitrides such as aluminum nitride. It is preferable to use silicon oxide which is easy to use and has high chemical stability.
In addition, as a formation method of the said thin film layer and the said dielectric material layer, it can select suitably from the well-known formation method according to material.

<Convex-convex structure application layer>
The said uneven | corrugated structure provision layer is a layer which has the said light transmittance | permeability, and makes one surface an uneven surface.
There is no restriction | limiting in particular as a forming material of the said uneven structure provision layer, According to the objective, it can select suitably, For example, an acrylic resin, polyester resin, polyolefin resin, general purpose plastics, engineering plastics, super engineering plastics, synthetic high Known light transmitting materials such as molecular resin, natural resin, ultraviolet curable resin, thermosetting resin, thermoplastic resin and the like can be used.
The method of forming the uneven surface is not particularly limited and may be appropriately selected according to the purpose. For example, etching method, electron beam drawing method, laser drawing method, laser interference method, cutting method, self-organization Known formation methods such as In addition, a method of transferring asperities of the mold and the plate by using the material formed by the above-mentioned forming method as a mold and a plate, pouring the molten various materials into the mold and the plate, curing after peeling is mentioned. Can.

<Layer composition>
The target substance detection chip may be constituted by the light transmitting substrate itself, or the uneven structure imparting layer may be laminated on the light transmitting substrate, or the electric field enhancing layer may be formed on the light transmitting substrate. Alternatively, the electric field enhancing layer and the concavo-convex structure imparting layer may be laminated on the light transmitting substrate.
In the case where the electric field enhancing layer is not formed in the target substance detection chip, an evanescent field is generated when light is irradiated from the back surface side on the back surface side with the surface opposite to the surface on which the uneven structure is formed as the back surface. It is possible to exist on the surface.
In the case where the electric field enhancing layer is formed in the target substance detection chip, the enhanced electric field can be present on the surface when the light is irradiated from the back surface side under total reflection conditions.
A specific example of the layer configuration will be described later with reference to the drawings.

Irregular structure
The target substance detection chip has a concavo-convex structure configured by periodically arranging a plurality of convex portions for the purpose of suppressing adsorption of a combined body formed by binding at least magnetic particles to the target substance. It is formed to have on the surface.
The said uneven structure is formed based on the uneven surface formed in either of the said transparent substrate and the said uneven | corrugated structure provision layer.

There is no restriction | limiting in particular as a shape of the said convex part, It can be set as arbitrary shapes, such as prismatic shape, trapezoid shape, semicircle shape, semielliptical shape, by the cross sectional view of the minor axis direction.
The concavo-convex structure is not particularly limited. For example, when it is estimated that the combined body has a diameter of about 1 μm, the height of the convex part is 1 μm to 1.55 μm in whole or in part. In addition, it is preferable that a periodic structure in which the pitch interval between adjacent convex portions is either a small interval of 0.45 μm to 0.55 μm or a large interval of 1.45 μm to 1.55 μm.
In addition, the specific example of the said uneven structure is later mentioned with drawing.

First Embodiment
A first embodiment of the present invention will be described with reference to the drawings. The first embodiment is an embodiment according to the target substance detection chip of the present invention.
As shown in FIG. 1, the target substance detection chip 1 has a light transmitting substrate 2 and a concavo-convex structure providing layer 3. FIG. 1 is an explanatory view showing a schematic configuration of the first embodiment.
The light transmitting substrate 2 has a smooth surface, and the concavo-convex structure providing layer 3 is stacked on the smooth surface.
The light transmitting substrate 2 has a side wall portion so as to form a box-like body having the smooth surface as the bottom, and the box-like body is used as a liquid storage portion to store a liquid sample for verifying the presence of the target substance. It may be configured to
Further, in the present specification, “smooth” means optically smooth, and means that the surface precision of the surface to be smooth is λ / 2 or less.

  The concavo-convex structure imparting layer 3 is laminated on the light transmitting substrate 2 and a surface opposite to the surface on the light transmitting substrate 2 side is a concavo-convex surface. In addition, there is no restriction | limiting in particular as an uneven | corrugated structure provision layer 3, It fixes on the transparent substrate 2 by adhesion, adhesion | attachment, melt | fusion, bonding etc.

In the target substance detection chip 1, a concavo-convex structure is formed on the surface by the concavo-convex surface of the concavo-convex structure providing layer 3.
The target substance detection chip 1 has a structure in which the concavo-convex structure application layer 3 is stacked on the light transmitting substrate 2, and the concavo-convex structure is viewed from the back surface side with the surface opposite to the surface on which the concavo-convex structure is formed. When the light is irradiated to the application layer 3 under total reflection conditions, the evanescent field can be present near the surface.

  The uneven structure has a role of suppressing adsorption of the combined body in the liquid sample introduced onto the surface to the surface, and a plurality of convex portions are periodically arranged. A state in which the adsorption of the combined body is suppressed by this uneven structure will be described with reference to FIGS. 2 (a) and 2 (b). 2 (a) and 2 (b) are explanatory diagrams for explaining how adsorption of the combined body is suppressed by the concavo-convex structure.

As shown in FIG. 2 (a), the relief structure, the pitch spacing _{P 1} between the convex portions adjacent to each, to the particle diameter R of the conjugate M, 9 / 20R~11 / 20R length of, That is, the distance is set smaller than the particle diameter R of the combined body M. Further, the height H of the convex portion is set to a length of 1 R to 2 μm with respect to the particle diameter R of the combined body M, and the width W of the convex portion with respect to the particle diameter R of the combined body M , 9 / 20R to 3 / 2R are set.
Having such a concavo-convex structure reduces the contact area and reduces the work of adhesion, whereby the conjugate M is adsorbed to the surface of the target substance detection chip 1 (the concavo-convex surface of the concavo-convex structure imparting layer 3). Can be suppressed.
Here, from the viewpoint of performing movement observation of the target substance accompanying the application of the magnetic field, it can be suitably used in the case where it can be regarded as, for example, a sphere having a diameter of about 1 μm as the combined body M. As such an example, a case where a protein of several nm in size is used as the target substance, a fluorescent dye of several nm in size is used as the labeling substance, and magnetic particles of 1 μm in particle diameter are used as the magnetic fine particles is mentioned. Be In this example, the target substance and the target substance have a size equal to or smaller than one-hundredth of the size of the magnetic fine particles, and thus the conjugate M can be regarded as a sphere having a particle diameter of 1 μm, and the uneven structure is formed By doing this, the convenience of the target substance detection chip 1 can be improved.
In the case of such an example, the pitch interval P1 is preferably 0.45 μm to 0.55 μm, the height H is preferably ₁ μm to 1.55 μm, and the width W is preferably 0.45 μm to 1.5 μm is preferred.

In the example shown in FIG. 2 (a), it was smaller interval than the particle size R of the coupling body M the pitch P _1, as shown in FIG. 2 (b), the particles of the conjugate M the pitch interval P ₁ With respect to the diameter R, the lengths of 29 / 20R to 31 / 20R, that is, the particle diameter R of the combined body M may be set larger. As the pitch _{P 1,} on the assumption that the detection conjugate having a diameter of about 1 [mu] m, 1.45Myuemu～1.55Myuemu are preferred.
When such a concavo-convex structure is provided, one combined body may be attached and stabilized, but the target substance detection chip 1 is obtained by reducing the amount of work of adhesion for a plurality of attached and stable, and aggregated. It can suppress that the coupling body M adsorb | sucks to the said surface (The said uneven surface of uneven | corrugated structure provision layer 3 ') of the.
The height H and the width W are the same as those described with reference to FIG.
Moreover, although the shape of the said convex part was demonstrated as cross-sectional square pillar shape in FIG. 2 (a), (b), as a shape of the said convex part, arbitrary shapes, such as cross-sectional trapezoidal shape, semicircle shape, semielliptical shape, etc. The settings can be applied to the pitches P ₁ , the height H and the width W in these shapes with the maximum length.

As described above, in the target substance detection chip 1 according to the first embodiment, the adsorption of the combined body can be suppressed by the uneven structure.
Moreover, in the target substance detection chip 1 according to the first embodiment, the application of the magnetic field onto the surface is not restricted, and the movement of the combined body is possible. It can be used to detect substances, and the target substance detection device to be applied can be manufactured in a small size and at low cost.

The target substance detection chip 1 according to the first embodiment has a structure in which the concavo-convex structure imparting layer 3 is laminated on the light transmitting substrate 2, but in the light transmissible substrate 2 itself, the concavo-convex structure imparting layer 3 is By forming the uneven surface similar to the uneven surface, it is possible to configure the target substance detection chip from the light transmitting substrate itself.
Second Embodiment
A second embodiment of the present invention will be described with reference to the drawings. The second embodiment is an embodiment according to the target substance detection chip of the present invention.
As shown in FIG. 3, the target substance detection chip 10 has a light transmitting substrate 12, a concavo-convex structure providing layer 13, and an electric field enhancing layer 14. FIG. 3 is an explanatory view showing a schematic configuration of the second embodiment.

  The light transmitting substrate 12 has a smooth surface, and the electric field enhancing layer 14 is stacked on the smooth surface. Further, the light transmitting substrate 12 is formed with a side wall portion so as to form a box having the above-mentioned smooth surface at the bottom, and a liquid sample for verifying the presence of the target substance as the liquid reservoir 15 in the box. It is configured to be stored.

The electric field enhancement layer 14 is a layer in which an enhanced electric field is formed on the other surface when light is irradiated from one surface under total reflection conditions, and is laminated on the smooth surface of the light transmitting substrate 12. It is a smooth layer. The electric field enhancing layer 14 is configured according to a known surface plasmon excitation layer and a waveguide mode excitation layer.
The concavo-convex structure imparting layer 13 is stacked on the electric field enhancing layer 14 and a surface opposite to the surface on the electric field enhancing layer 14 side is a concavo-convex surface. The concavo-convex structure imparting layer 13 is not particularly limited, but is fixed on the electric field enhancing layer 14 by adhesion, adhesion, fusion, or bonding. When the surface plasmon excitation layer known as the electric field enhancing layer 14 is used, the concavo-convex structure providing layer 13 has the enhancing electric field on the surface so that the enhancing electric field can be present in the vicinity of the surface of the target substance detection chip 10. Need to be thin enough to reach Therefore, in this case, it is preferable that the thickness of the thinnest portion of the concavo-convex structure providing layer 13, that is, the portion where the convex portion is not formed is 200 nm or less. In order to form such a thin concavo-convex structure imparting layer 13, it is preferable to apply a resist on the electric field enhancing layer 14 and develop the resist after exposure to give a concavo-convex structure. On the other hand, in the case of using a known waveguide mode excitation layer as the electric field enhancing layer 14, the concavo-convex structure providing layer 13 is integrated with the dielectric layer in the electric field enhancing layer 14 to form a waveguide layer. There is no particular limitation on the thickness 13, and the enhanced electric field can be present near the surface of the target substance detection chip 10.

In the target substance detection chip 10, a concavo-convex structure is formed on the surface by the concavo-convex surface of the concavo-convex layer providing layer 13 constituting the outermost layer.
Further, in the target substance detection chip 10, the electric field enhancing layer 14 and the concavo-convex structure imparting layer 13 are stacked in this order on the light transmitting substrate 12, and the surface opposite to the surface on which the concavo-convex structure is formed. When the light is irradiated from the back surface side to the one surface of the electric field enhancing layer from the back surface side under total reflection conditions, the enhanced electric field can be present in the vicinity of the surface. As described above, in the case of using a known waveguide mode excitation layer as the electric field enhancing layer 14, the concavo-convex structure providing layer 13 is integrated with the dielectric layer in the electric field enhancing layer 14 to form a waveguide layer. The surface on which the light is totally reflected is the surface of the concavo-convex structure providing layer 13.

As described above, in the target substance detection chip 10 according to the second embodiment, the adsorption of the combined body can be suppressed by the uneven structure.
Further, in the target substance detection chip 10 according to the second embodiment, the application of the magnetic field onto the surface is not restricted, and the movement of the combined body is possible. It can be used for fluorescence observation of substances, and the target substance detection device to be applied can be manufactured in a small size and at low cost.
The other matters are the same as those of the target substance detection chip 1, and therefore, the redundant description will be omitted.

Third Embodiment
A third embodiment of the present invention will be described with reference to the drawings. The third embodiment is an embodiment according to the target substance detection chip of the present invention.
As shown in FIG. 4, the target substance detection chip 20 has a light transmitting substrate 22, a concavo-convex structure imparting layer 23, and an electric field enhancing layer 24, and the light transmitting substrate 22 has a liquid as an optional configuration. Reservoir 25 is formed. FIG. 4 is an explanatory view showing a schematic configuration of the third embodiment.

In the target substance detection chip 20, unlike the target substance detection chip 10, the concavo-convex structure imparting layer 23 is formed on the light transmitting substrate 22 and the electric field enhancing layer 24 is formed on the concavo-convex structure imparting layer 23.
That is, in the target substance detection chip 20, the light transmitting substrate 22 having a smooth surface, and the surface that is stacked on the smooth surface of the light transmitting substrate 22 and opposite to the surface on the light transmitting substrate 22 side is The concavo-convex structure imparting layer 23, which is a concavo-convex surface, and the concavo-convex pattern of the first concavo-convex surface are laminated on the first concavo-convex surface of the concavo-convex structure imparting layer 23 And the electric field enhancing layer 24 which is a second concavo-convex surface of the transferred shape, and the concavo-convex structure is formed by the second concavo-convex surface.
The concavo-convex structure imparting layer 23 is not particularly limited, but is fixed on the light transmitting substrate 22 by adhesion, adhesion, fusion, or bonding.

The said 1st uneven surface in the uneven | corrugated structure provision layer 23 is formed similarly to the said uneven surface in the uneven | corrugated structure provision layer 3 (and 3 ').
When the electric field enhancing layer 24 is formed with a uniform thickness on the first uneven surface of the uneven structure imparting layer 23, the second uneven surface having the shape to which the uneven pattern of the first uneven surface is transferred is formed on the electric field enhancing layer 24. A concavo-convex structure can be formed on the surface by the second concavo-convex surface of the electric field enhancing layer 24 that can be formed and that forms the outermost layer.

In the target substance detection chip 20 configured as described above, since the electric field enhancement layer 24 is the outermost layer, the target substance detection chip 10 is formed on the surface from the electric field enhancement layer 14 through the concavo-convex structure imparting layer 13. The enhanced electric field can be present in a wider range on the surface than the enhanced electric field, and it is easier to observe an optical signal of the target substance or the like in the enhanced electric field.
That is, the range to which the enhanced electric field extends is a range separated by about 400 nm to about 1,200 nm in both the electric field enhancing layer 14 and the electric field enhancing layer 24. In the target substance detection chip 20 in which the enhanced electric field is present in a wide area on the surface with the enhancement layer 24 as the outermost layer, the detection range of the target substance or the like by the enhanced electric field is set wider than the target substance detection chip 10 Can.
The other matters are the same as those of the target substance detection chip 10, and therefore redundant description will be omitted.

Fourth Embodiment
A fourth embodiment of the present invention will be described with reference to the drawings. The fourth embodiment is an embodiment according to the target substance detection chip of the present invention.
As shown in FIG. 5, the target substance detection chip 30 has a light transmitting substrate 32 and an electric field enhancing layer 34, and the liquid storing portion 35 is formed in the light transmitting substrate 32. FIG. 5 is an explanatory view showing a schematic configuration of the fourth embodiment.

In the target substance detection chip 30, the electric field enhancement layer 34 is made the outermost layer similarly to the target substance detection chip 20, but unlike the target substance detection chip 20, the electric field enhancement layer 34 is laminated on the light transmitting substrate 32. Configured
That is, in the target substance detection chip 30, a first uneven surface similar to the uneven surface in the uneven structure imparting layer 3 (and 3 ') is formed on the light transmitting substrate 32 itself, and uniform on the first uneven surface. By forming the electric field enhancing layer 34 with a certain thickness, the second uneven surface having the shape to which the uneven pattern of the first uneven surface is transferred is formed in the electric field enhancing layer 34, and the electric field enhancing layer 34 constituting the outermost layer is formed. An uneven structure is formed on the surface by the second uneven surface.
The light transmitting substrate 32 having the first concavo-convex surface can be formed by the same method as the method of forming the concavo-convex structure providing layer 3 (and 3 ′).

  In the target substance detection chip 30 configured in this manner, the enhanced electric field can be present in a wide range on the surface, and in addition to facilitating observation of the light signal of the target substance or the like in the enhanced electric field, Since the application layer is not provided, the number of parts can be reduced and the cost can be reduced.

[Example of formation of uneven structure]
About the said uneven structure in 1st Embodiment-4th Embodiment, a further suitable example of formation is demonstrated, referring to drawings.
As shown in FIGS. 6A and 6B, in the example of the concavo-convex structure forming example 50, a plurality of convex portions 51 are formed on the smooth portion 52. 6 (a) is a perspective view showing an example of formation of the concavo-convex structure, and FIG. 6 (b) is a top view showing an example of formation of the concavo-convex structure.

In the concavo-convex structure forming example 50, the plurality of convex portions 51 are formed in two or more types of shapes. That is, in the example of the concavo-convex structure formation, the protrusions 51 are formed in a plurality of shapes having different lengths in the longitudinal direction (the “Y” direction in the drawing).
Each of the plurality of convex portions 51 has either a two-fold rotational symmetry shape or a line symmetry shape, as an example is shown enlarged in FIGS. 7A and 7B. 7 (a) is a top view showing one convex portion in an enlarged manner, and FIG. 7 (b) is a view in which each side surface in the longitudinal direction and the short direction is added to the upper surface.

As for the plurality of convex portions 51, those having the longest length in the longitudinal direction are disposed at the center, and when viewed from this as the axis of symmetry, the lengths of adjacent ones in the lateral direction ("X" direction in the figure) It has a rhombic unit periodic structure in top view, in which the length of the direction is shortened three in order in a direction away from the one disposed at the center.
Further, the plurality of convex portions 51 are arranged so as to share the shortest one in the longitudinal direction at the end in the longitudinal direction of the unit periodic structure with the other unit periodic structure adjacent in the lateral direction. Are arranged such that the longest length in the longitudinal direction in one of the unit period structures is opposed to the shortest length in the other adjacent unit period structures in the longitudinal direction. And the whole has a periodic structure in which a plurality of the unit periodic structures are periodically arranged.
In the example 50 of forming a concavo-convex structure configured as described above, each of the unit periodic structures is made to have a structure similar to the scaly flakes possessed by fish in the natural world, and the whole is made scaly. It is possible to suppress the adsorption of foreign matter including the above-mentioned combined body on the surface on which 51 is formed.
In the present example, the unit periodic structure is in the shape of a rhombus, but may be in the form of an isosceles triangle or in the form of a parallelogram.
When the uneven structure of the target substance detection chip is formed by the uneven structure forming example 50, the uneven structure forming example 50 is formed on the uneven surface and the first uneven surface serving as the base of the second uneven surface. The shape may be applied.
Further, the length in the short direction in the example 50 of forming the concavo-convex structure corresponds to the "width W" described using Figs. 2 (a) and 2 (b).
Moreover, as a formation method of uneven | corrugated structure formation example 50, the matter of Unexamined-Japanese-Patent No. 2015-160342 can be applied.

(Target substance detection device)
The target substance detection device of the present invention comprises the target substance detection chip of the present invention, a light irradiation part, and a magnetic field application part, and has a light detection part as necessary.
The target substance detection device detects the target substance using the magnetic particles bound to the target substance. When the target substance is a substance which hardly generates fluorescence or scattered light by the evanescent field or the enhanced electric field, a labeling substance for labeling the target substance is used.
The labeling substance is not particularly limited, and examples thereof include a fluorescent labeling substance and a light scattering substance, which specifically adsorb or bind to the target substance to label the target substance.
As the fluorescent labeling substance, for example, known fluorescent substances such as fluorescent dyes, quantum dots, fluorescent stains and the like can be used.
Moreover, as said light-scattering substance, well-known light-scattering substances, such as a nanoparticle, for example, a polystyrene bead and a gold nanoparticle, can be used, for example.
The method for binding the target substance to the labeling substance is not particularly limited, and known binding methods such as physical adsorption, antigen-antibody reaction, DNA hybridization, biotin-avidin bond, chelate bond, amino bond and the like may be used. It can apply.

<Light irradiator>
The light irradiator is capable of emitting light under the condition of total reflection from the back surface side with the surface opposite to the surface on which the uneven structure of the target substance detection chip is formed as the back surface.
There is no restriction | limiting in particular as a light source of the said light irradiation part, According to the objective, it can select suitably, A well-known lamp, LED, a laser, etc. are mentioned. In the present invention, an evanescent field or an enhanced electric field is formed on the surface by irradiating light from the back side of the target substance detection chip under total reflection conditions, and the object wherein the evanescent field or the enhanced electric field is excitation light An optical signal is generated from the combination comprising the substance and the magnetic particles. Therefore, the role required of the light irradiation unit is only to irradiate light from the back surface side of the target substance detection chip under total reflection conditions, and selection of a light source is limited if it plays such a role. There is no

When a radiation light source such as a lamp or an LED is used, the back surface side of the target substance detection chip is irradiated with the emitted light to avoid leakage of the irradiation light from the surface side of the target substance detection chip. It is necessary for the light in all directions to satisfy the condition of total reflection. From such a thing, when using a radiation light source, you may use guide parts, such as a collimating lens which controls the irradiation direction of irradiation light to a specific direction.
When fluorescence is used as the light signal, a monochromatic light source having a wavelength capable of exciting the fluorescence is used, or light from a light source having a wide wavelength band such as a lamp or an LED is transmitted through an optical filter such as a band pass filter. It is preferable that irradiation is performed from the back surface side of the target substance detection chip after taking out only a wavelength capable of exciting fluorescence and extracting fluorescence.

  Here, when the front surface and the back surface of the target substance detection chip are parallel plates, the light irradiated from the back surface side is not totally reflected if a liquid is present on the surface. Therefore, in such a case, by forming a diffraction grating on the back surface portion of the target substance detection chip, the light is diffracted by the diffraction grating when the diffraction grating is irradiated with light at a specific angle. Light is introduced into the target substance detection chip, and the light introduced into the target substance detection chip is irradiated on the surface under total reflection conditions to form the evanescent field or the enhanced electric field on the surface Alternatively, the target substance detection chip may be configured. Or you may form so that the said surface and the said back may not become parallel. Alternatively, light emitted from the light source may be irradiated to the back surface of the target substance detection chip through a known prism. The prism can be optically bonded to the back surface of the target substance detection chip with a refractive index adjustment oil, an optical adhesive, or the like. In addition, when the same forming material as the forming material of the light transmitting substrate is selected as the forming material of the prism, it is also possible to use one in which the light transmitting substrate and the prism are integrally molded.

<Magnetic field application unit>
The magnetic field application unit is formed of at least one of a first magnetic field application unit and a second magnetic field application unit.

<First magnetic field application unit>
A first magnetic field application unit moves a first magnetic field for moving the magnetic particles contained in the liquid sample introduced onto the surface of the target substance detection chip in a direction parallel to the surface or in a direction away from the surface It is a portion that can be applied, and a magnetic field to move the magnetic particles away from the surface of the target substance detection chip, or a magnetic field acting in a direction to move parallel to the surface on the surface of the target substance detection chip Can be applied.
The target substance and the labeling substance that constitute the conjugate together with the magnetic particles generate an optical signal only in the evanescent field or the enhanced electric field. In addition, the electric field strengths of the evanescent field and the enhanced electric field attenuate as they move away from the surface of the target substance detection chip. Therefore, when the combination is moved away from the surface by the application of a magnetic field, the light signal is attenuated, and further, the combination is a distance to such an extent that the electric field strength of the evanescent field or the enhanced electric field can be regarded as zero. The optical signal of the combined body disappears when it is moved away from the surface. In addition, when two-dimensional image information can be acquired by using an imaging device for the light detection unit, the light signal emitted from the combined body that has fluctuated on the surface by the application of the first magnetic field is fluctuation of the light signal. It becomes possible to measure over time. The target substance detection device detects the target substance by detecting such attenuation (including extinction) or fluctuation (which may be accompanied by attenuation or extinction) of the light signal.
The first magnetic field application unit is not particularly limited as long as the combination can be moved by application of a magnetic field, and can be appropriately selected according to the purpose. Any one or more can be used.

<Second magnetic field application unit>
The second magnetic field application unit can apply a second magnetic field that is disposed on the back surface side of the target substance detection chip and draws the magnetic particles in the liquid sample introduced onto the surface onto the surface. It is a section that
There is no restriction | limiting in particular as said 2nd magnetic field application part, According to the objective, it can select suitably, For example, it can comprise using a well-known electromagnet and permanent magnet.
When such a second magnetic field application unit is provided, the combined body floating in the liquid sample can be drawn to the surface of the target substance detection chip, and the target substance is detected in a short time. Can.

The second magnetic field application unit is a unit capable of moving the magnetic particles in a direction having a vector component in a direction parallel to the in-plane direction of the surface in a state where the second magnetic field is applied. Is preferred.
As the second magnetic field application unit, for example, the electromagnet or the permanent magnet is held on a slide member, and the light from the light irradiation unit on the back surface side of the target substance detection chip is irradiated. The electromagnet or the permanent magnet in an initial state in which the electromagnet or the permanent magnet is positioned in the vicinity of the target region (detection region), and the electromagnet or direction having a vector component in a direction parallel to the in-plane direction of the surface of the target substance detection chip It can comprise by carrying out movement control between the state to which the said permanent magnet was moved. In addition, when using the said electromagnet, it is set as the state made to excite continuously or intermittently during the said movement control. Further, the intensity of excitation may be changed during the movement control.
In addition, a configuration is also provided in which the movement control is performed by holding the electromagnet or the permanent magnet on the slide member by arranging a plurality of electromagnets or permanent magnets and controlling the application state of the magnetic field in each member. The same effect can be obtained.
Further, the second magnetic field application unit is not particularly limited, but a through hole is formed, or an incomplete ring such as a U shape or a plurality of members are arranged in a ring or an incomplete ring The configuration may be modified.
The second magnetic field application unit is a unit capable of moving the magnetic particles in a direction having a vector component in a direction parallel to the in-plane direction of the surface in a state where the second magnetic field is applied. The signal can be eliminated.
That is, while the target substance combined with the magnetic magnetic particles moves following the movement of the second magnetic field application unit, the noise caused by a flaw or the like on the surface of the target substance detection chip is applied to the second magnetic field Since the movement does not follow the movement of the unit, the noise signal can be eliminated by performing detection based on the moving light signal.

<Light detection unit>
The light detection unit is disposed on the front surface side of the target substance detection chip, and can detect an optical signal emitted from the combined body.
There is no restriction | limiting in particular as said light detection part, According to the objective, it can select suitably, Photodetectors, such as a well-known photodiode and a photomultiplier tube, can be used.
If information of an optical signal can be acquired as two-dimensional image information, position information of the optical signal in the two-dimensional image information appearing as a light spot, size information observed in two dimensions, light signal intensity at the light spot By observing the increase / decrease information in time series, the light spot is due to the target substance, or indicates information related to the target substance, or foreign matter, fluctuation of light source output, detection plate surface It is possible to distinguish whether it is information that does not relate to the target substance such as scratches. In order to enable acquisition of such two-dimensional image information, an imaging device may be selected as the light detection unit. There is no restriction | limiting in particular as said imaging device, According to the objective, it can select suitably, Image sensors, such as a well-known CCD image sensor and a CMOS image sensor, can be used.

(Target substance detection method)
The target substance detection method of the present invention includes at least a light irradiation step and a conjugate transfer step.

<Light irradiation process>
The light irradiation step is a step of irradiating light under total reflection conditions from the back surface side with the surface opposite to the surface on which the uneven structure of the target substance detection chip of the present invention is formed as the back surface.
The said light irradiation process can be implemented by the said light irradiation part in the said target substance detection apparatus of this invention.

<Conjugate transfer process>
In the conjugate transfer step, the conjugate of the target substance and the magnetic particles contained in the liquid sample introduced onto the surface of the target substance detection chip is applied in a direction parallel to the surface or from the surface by the application of a first magnetic field. A second combined body moving step in a direction to move away, and a second combined body for drawing the combined body in the liquid sample on the surface by application of a second magnetic field from a magnetic field application unit disposed on the back side It is a process carried out in any of the transfer processes.
In the second combined body moving step, the magnetic field applying unit is further moved in a direction having a vector component in a direction parallel to the in-plane direction of the surface in a state where the second magnetic field is applied, It is preferable to be a step of moving the combined body in accordance with the movement of
The combined body transfer step can be performed by the magnetic field application unit in the target substance detection device of the present invention.

Fifth Embodiment
Next, a fifth embodiment of the present invention will be described with reference to the drawings. The fifth embodiment is an embodiment according to the target substance detection device of the present invention.
As shown in FIG. 8, in the target substance detection device 100, an optical system is configured according to the configuration of a known total reflection illumination fluorescent microscope, and light irradiation is configured with the target substance detection chip 1, the light source 110 and the optical prism 120. And a first magnetic field application unit 130 and a light detection unit 140 (imaging device). The imaging device is configured by, for example, a known CCD image sensor or the like, and can acquire a two-dimensional image. FIG. 8 is an explanatory view showing a schematic configuration of the fifth embodiment.

The target substance detection chip 1 is irradiated with the light L from the back surface side, and the evanescent field can be present on the surface (surface on which the concavo-convex structure imparting layer 3 is formed). In addition, the target substance detection chip 1 holds the liquid sample A introduced onto the surface with a cover glass G.
In the light irradiator, the light L irradiated from the light source 110 is incident on the light transmitting substrate 2 through the optical prism 120, passes through the light transmitting substrate 2, and is totally reflected on the surface of the uneven structure imparting layer 3 Irradiation is possible under the following conditions.
In addition, the first magnetic field application unit 130 is applied to a detection region on the surface of the target substance detection chip 1 (a region where the evanescent field is formed on the surface upon receiving the light L from the light irradiation unit). Of the liquid sample A introduced on the surface toward the first magnetic field application unit 130 by application of a magnetic field, in a direction away from the surface of the target substance detection chip 1 It is configured to move. The first magnetic field application unit 130 in the present example is configured of an electromagnet.

In the target substance detection device 100 configured as described above, first, the liquid sample A is introduced onto the surface of the target substance detection chip 1.
Next, the condition in which the combined body floating in the liquid of the liquid sample A is gravitationally settled on the surface of the target substance detection chip 1 and then totally reflected on the surface of the uneven structure providing layer 3 via the optical prism 120 The light L is emitted from the light source 110 in step S12, and the light detection unit 140 acquires the light signal S based on the evanescent field formed on the surface of the target substance detection chip 1.

Next, the electromagnet as the first magnetic field application unit 130 is excited to draw the combined substance in the liquid sample A toward the first magnetic field application unit 130 by applying a magnetic field, and from the surface of the target substance detection chip 1 Move in the direction to move away.
Here, in the target substance detection device 100, adsorption of the combined body and the surface of the target substance detection chip 1 is suppressed by the uneven structure formed on the surface of the target substance detection chip 1, and application of the magnetic field is performed. The conjugate can be easily moved back and forth.
Next, an optical signal detection unit 140 acquires an optical signal on the surface of the target substance detection chip 1 after moving the combination while maintaining the observation field of view.

In the target substance detection device 100 configured as described above, optical signals before and after application of the magnetic field (before and after movement of the combined body) are obtained as shown in FIGS. 9A and 9B, and the target substance is obtained. The light signals a and c based on are detected clearly from noise signals b such as flaws on the surface of the target substance detection chip 1, adsorption on the surface or contaminants present on the surface, and fluctuations in light source output. Ru. FIG. 9A is a view showing the appearance on the surface before application of the magnetic field, and FIG. 9B is a view showing the appearance on the surface after the application of the magnetic field. Although not shown, the appearance of an optical signal based on movement from outside the observation field can also be detected.
As described above, according to the target substance detection device 100, the combination can be easily moved by the uneven structure of the target substance detection chip 1, and the target substance constituting the combination is detected with high accuracy. be able to. Further, even when the contaminants are adsorbed on the surface of the target substance detection chip 1, the detection can be performed ignoring the presence thereof, so that the surface does not have to be washed every time the detection is performed. Efficient detection can be performed.

Sixth Embodiment
Next, a sixth embodiment of the present invention will be described with reference to the drawings. The sixth embodiment is an embodiment according to the target substance detection device of the present invention.
As shown in FIG. 10, the target substance detection apparatus 200 has an optical system according to a known surface plasmon resonance sensor and a waveguide mode sensor, and is configured of a target substance detection chip 10, a light source 210 and an optical prism 220. It comprises a light irradiation unit, a first magnetic field application unit 230, and a light detection unit 240 (the imaging device). FIG. 10 is an explanatory view showing a schematic configuration of the sixth embodiment.

The target substance detection chip 10 is irradiated with the light L from the back surface side, and the enhanced electric field can be present on the surface (the surface on which the concavo-convex structure imparting layer 13 is formed). Further, in the target substance detection chip 10, the liquid sample A is introduced into the liquid storage unit 15.
The light irradiator enables the light L emitted from the light source 210 to be irradiated to the electric field enhancing layer 14 through the optical prism 220 and the light transmitting substrate 12 under total reflection conditions.
In addition, the first magnetic field application unit 230 is applied to a detection region on the surface of the target substance detection chip 10 (a region in which the enhanced electric field is formed on the surface upon receiving the light L from the light irradiation unit). Of the liquid sample A introduced into the liquid storage unit 15 toward the first magnetic field application unit 230 by application of a magnetic field, while moving away from the surface of the target substance detection chip 10 Configured to be moved to The first magnetic field application unit 230 in the present example is configured of an electromagnet.

In the target substance detection device 200 configured as described above, first, the liquid sample A is introduced into the liquid storage unit 15.
Next, after the combined body floating in the liquid of the liquid sample A gravity settles on the surface of the target substance detection chip 10, the light L emitted from the light source 210 is used as the optical prism 220 and the light transmitting substrate 12 The light is irradiated to one surface of the electric field enhancing layer 14 under total reflection conditions, and the light detection unit 240 obtains an optical signal S based on the enhanced electric field formed on the surface of the target substance detection chip 10.

Next, the electromagnet as the first magnetic field application unit 230 is excited to draw the combined body in the liquid sample A in the liquid storage unit 15 toward the first magnetic field application unit by applying a magnetic field, and the target substance detection chip 10 Move away from the surface of the
Here, in the target substance detection device 200, the adsorption between the combined body and the surface of the target substance detection chip 10 is suppressed by the uneven structure formed on the surface of the target substance detection chip 10, and the application of the magnetic field is performed. The conjugate can be easily moved back and forth.
Next, an optical signal detection unit 240 acquires an optical signal on the surface of the target substance detection chip 10 after moving the combination while maintaining the observation field of view.

In the target substance detection device 200 configured as described above, optical signals before and after application of the magnetic field (before and after movement of the combined body) are obtained as shown in FIGS. 11A and 11B, and the target substance is obtained. The light signals d and f based on are detected clearly from noise signals e such as flaws on the surface of the target substance detection chip 10, adsorption on the surface or contaminants present on the surface, and fluctuations in the light source output. Ru. FIG. 11A is a view showing the appearance on the surface before the application of the magnetic field, and FIG. 11B is a view showing the appearance on the surface after the application of the magnetic field.
As shown in FIGS. 11 (a) and 11 (b), the background of the light signal is turned to a dark field by the attenuation of the enhanced electric field, and the target substance detection device 200 detects the target substance based on the light signal of the light spot. To detect. Although not shown, the appearance of an optical signal based on movement from outside the observation field can also be detected.
According to the target substance detection device 200, the combination can be easily moved by the uneven structure of the target substance detection chip 10, and the target substance constituting the combination can be detected with high accuracy. In addition, even when the contaminants are adsorbed on the surface of the target substance detection chip 10, detection can be performed ignoring the presence thereof, so the liquid storage portion 15 needs to be washed every time it is detected. And efficient detection can be performed.

Seventh Embodiment
Next, a seventh embodiment of the present invention will be described with reference to the drawings. The seventh embodiment is an embodiment according to the target substance detection device of the present invention.
As shown in FIG. 12, in the target substance detection device 300 according to the seventh embodiment, an optical system is configured according to a known surface plasmon resonance sensor and waveguide mode sensor, and a target substance detection chip 10, a light source 310, and It is comprised by the light irradiation part comprised by the optical prism 320, the 2nd magnetic field application part 330, and the light detection part 340 (the said imaging device). FIG. 12 is an explanatory view showing a schematic configuration of the seventh embodiment.
The light irradiation unit and the light detection unit 340 can be configured in the same manner as the light irradiation unit and the light signal detection unit 240 in the target substance detection device 200 according to the sixth embodiment, and the seventh embodiment The target substance detection device 300 is different from the target substance detection device 200 according to the seventh embodiment in that a second magnetic field application unit 330 is provided instead of the first magnetic field application unit 230. The differences will be described below.

The second magnetic field application unit 330 is disposed on the back surface side of the target substance detection chip 10 and applies the magnetic field to the combined body in the liquid sample A introduced to the liquid storage part 15 as the target substance detection chip 10 It can be drawn on the surface and can be moved in a direction having a vector component in a direction parallel to the in-plane direction of the surface of the target substance detection chip 10 in the state where the magnetic field is applied. Here, the second magnetic field applying unit 330 is formed out with sliding member for sliding said permanent magnet and the permanent magnet in the direction of the X ₁ or X ₂ (not shown).
In the movement of the combined body, the combination in the liquid sample A of the target substance detection chip 10 was once drawn to the surface of the target substance detection chip 10 by the application of the magnetic field from the second magnetic field application unit 330. After that, with the magnetic field applied, the second magnetic field application unit 330 is moved in a direction (direction of X ₁ or X ₂ ) having a vector component in a direction parallel to the in-plane direction of the surface of the target substance detection chip 10 The movement of the second magnetic field application unit 330 is made to follow.
In the case of using the second magnetic field application unit 330, the combination in the liquid sample A floats because the combination in the liquid sample A is drawn on the surface of the target substance detection chip 10 by the application of the magnetic field. There is no need to wait for gravity to settle on the surface of the target substance detection chip 10.

  Further, in the target substance detection device 300 configured as described above, optical signals before and after the movement of the second magnetic field application unit are obtained as shown in FIGS. 13A and 13B, and an optical signal based on the target substance is obtained. h can be clearly distinguished from noise signals i such as flaws on the surface of the target substance detection chip 10, impurities adsorbed on the surface or contaminants present on the surface, and fluctuations of the light source output. FIG. 13 (a) is a view showing the state on the surface before movement of the second magnetic field application unit, and FIG. 13 (b) is a view showing the state on the surface after movement of the second magnetic field application unit. FIG.

1, 10, 20, 30 Target substance detection chip 2, 12, 22 Light transmitting substrate 3, 3 13 13, 23 Irregular structure application layer 14, 24, 34 Electric field enhancing layer 15, 25, 35 Liquid reservoir 50 Irregular structure Formation example 51 Convex part 52 Smooth surface 100, 200, 300 Target substance detection device 110, 210, 310 Light source 120, 220, 320 Optical prism 130, 230 First magnetic field application unit 140, 240, 340 Light detection unit 330 Second magnetic field Applying unit

Claims (11)

  A target substance detection chip having a concavo-convex structure configured by periodically arranging a plurality of convex portions on a light transmitting substrate.   A light transmitting substrate having a smooth surface, and a concavo-convex structure providing layer which is laminated on the smooth surface of the light transmitting substrate and whose surface opposite to the surface on the light transmitting substrate side is a concavo-convex surface The target substance detection chip according to claim 1, wherein an uneven structure is formed on the uneven surface.   On the light transmitting substrate, an electric field enhancing layer is formed in which an enhanced electric field is formed on at least one surface when light is irradiated under total reflection conditions, and the surface on the light transmitting substrate side is the back surface The enhanced electric field according to any one of claims 1 to 2, wherein the enhanced electric field can be present in the vicinity of the surface when the light is irradiated from the back surface side to the one surface of the electric field enhancing layer under total reflection conditions. Target substance detection chip.   A light transmitting substrate having a smooth surface, a smooth electric field enhancing layer laminated on the smooth surface of the light transmitting substrate, and a concavo-convex structure imparting layer stacked on the electric field enhancing layer, 4. The target substance detection chip according to claim 3, wherein a concavo-convex structure is formed on the concavo-convex surface of the concavo-convex structure providing layer.   A light transmitting substrate having a smooth surface, and a concavo-convex structure providing layer which is laminated on the smooth surface of the light transmitting substrate and whose surface opposite to the surface on the light transmitting substrate side is a first uneven surface A second concavo-convex surface having a shape in which the concavo-convex pattern of the first concavo-convex surface is transferred onto the first concavo-convex surface of the concavo-convex structure providing layer and the surface opposite to the surface on the concavo-convex structure providing layer side; 4. The target substance detection chip according to claim 3, wherein the target substance detection chip is configured by an electric field enhancing layer to be formed, and a concavo-convex structure is formed by the second concavo-convex surface.   A light transmitting substrate having a first uneven surface, and a surface which is laminated on the first uneven surface of the light transmitting substrate and opposite to the surface on the light transmitting substrate side has an uneven pattern of the first uneven surface 4. The target substance detection chip according to claim 3, wherein the target substance detection chip is formed of an electric field enhancing layer which is a second concavo-convex surface of a transferred shape, and the concavo-convex structure is formed by the second concavo-convex surface.   The target substance detection chip according to any one of claims 1 to 6, wherein the convex portion is formed in two or more types of shapes, and at least one of the shapes is made into either a 2-fold rotational symmetry shape or a line symmetry shape. . The target substance detection chip according to any one of claims 1 to 7.
A light irradiation unit capable of emitting light under the condition of total reflection from the back surface side with the surface opposite to the surface on which the concavo-convex structure of the target substance detection chip is formed as the back surface;
A first magnetic field application unit capable of applying a first magnetic field for moving magnetic particles contained in a liquid sample introduced onto the surface of the target substance detection chip in a direction parallel to the surface or in a direction away from the surface; A second magnetic field application unit disposed on the back surface side of the target substance detection chip and capable of applying a second magnetic field for attracting the magnetic particles in the liquid sample introduced onto the surface onto the surface A magnetic field application unit formed by at least one of
A target substance detection device comprising:   A second magnetic field application unit, and movable in a direction having a vector component in a direction parallel to the in-plane direction of the surface of the target substance detection chip in a state where the second magnetic field application unit applies a second magnetic field; The target substance detection device according to claim 8. A light irradiation step of irradiating light under total reflection conditions from the back surface side with the surface opposite to the surface on which the concavo-convex structure of the target substance detection chip according to any one of claims 1 to 7 is formed,
First, a combination of a target substance and magnetic particles contained in a liquid sample introduced onto the surface of the target substance detection chip is moved in a direction parallel to the surface or in a direction away from the surface by application of a first magnetic field At least one of a combined body moving step, and a second combined body moving step of drawing the combined body in the liquid sample on the surface by applying a second magnetic field from the magnetic field application unit disposed on the back surface side A conjugate transfer step to be performed;
A target substance detection method characterized by including. A direction including a second combined body moving step and a vector component in a direction parallel to the in-plane direction of the target substance detection chip surface in the state where the second combined body moving step applies a second magnetic field The target substance detection method according to claim 10, wherein the combined substance is moved following the movement of the magnetic field application unit.